The vesiculo-vacuolar organelle _VVO_ a distinct endothelial cell

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The vesiculo-vacuolar organelle _VVO_ a distinct endothelial cell Powered By Docstoc
					The vesiculo-vacuolar     organelle (VVO): a distinct endothelial
   cell structure that provides a transcellular  pathway for
   macromolecular     extravasation
                        A. M. Dvorak, Sarah Kohn,*                                                Ellen         S. Morgan,                    Patricia              Fox, Janice                 A. Nagy,
                        and Harold F. Dvorak
                        Departments              ofPathology,                  Beth Israel             Hospital        and         Harvard          Medical          School,        Boston,       Massachusetts;                  and the
                        *Technjon4srael                Institute              of Technology,               Hafa,           Israel



Abstract:                  The vesiculo-vacuolar                                      organelle              (YVO)                is         taller    than                 capillary             endothelium         and   suggest                               that
a recently               described     organelle                          found           in the          cytoplasm                          upregutated                      YVO             function       accounts     for    the                             well-
of endothelial           cells    that                     line         tumor       microvessels           and                               known              hyperpermeabulity                              of tumor              blood            vessels.         J.
normal          venules.        VVOs                         are          grape-like         clusters        of                              Leukoc.                Biol.         59:       100-115;                 1996.
interconnecting                uncoated                                 vesicles       and       vacuoles,
bounded       by trilaminar                           unit membranes,           that span the                                                Key          Words:               macromolecular                  electron-dense               tracers         transen-
entire    thickness         of                     vascular        endothelium,        thereby                                               dothelial           transport          .   tumor     vasculature              .    vascular        permeability
                                                                                                                                             cytokines           . electron        microscopy
providing        a potential                           trans-endothelial          connection
between       the vascular                            lumen       and    the extravascular
space.           Macromolecular                            tracers     preferentially                                cross
                                                                                                                                             INTRODUCTION
hyperpermeable                            tumor           microvessels          through                             VVOs.
The         present                investigation                   was        undertaken                       to    eluci-
                                                                                                                                             It is now well established           that the blood vessels          supplying
date        further         the ultrastructure            and function       of YVOs
                                                                                                                                             transplantable          animal    tumors         are  hyperpermeable            to
in a murine               ovarian      carcinoma          (MOT)      and in normal
                                                                                                                                             plasma         proteins   and  other      circulating      macromolecules
venules.                Morphometry            revealed        that     VVOs       were
                                                                                                                                             relative     to the blood vessels      that supply     comparable        normal
enormous                  cytoplasmic           structures         (median        area,
                                                                                                                                             tissues            [1-13].     The hyperpermeability                                   of tumor    vessels      is
0.12-0.14                  pm2       in single       electron       micrographs).
                                                                                                                                             substantial               (4- to 10-fold   greater                       than         in comparable        nor-
Moreover,                    the       individual                 vesicles              and       vacuoles                 that
                                                                                                                                             mal         tissues)         and      likely       has     contributed                importantly              to such
comprised             VVOs     were                  on average     substantially      larger
                                                                                                                                             success            as has          been      obtained             in tumor     imaging    and therapy
than        capillary       caveolae                    and followed         a non-normal
                                                                                                                                             with          monoclonal                   antibodies               and    other    macromolecules
distribution            that was                    skewed     to the right.      Specimen
                                                                                                                                             [14].            Though            disorganized,                  the    leaky          vessels            supplying
tilting         provided        conclusive      evidence                                        that   individual
                                                                                                                                             several            different              animal         tumors          resembled                post-capillary
YVO           vesicles      and vacuoles       communicated                                             with    each
                                                                                                                                             venules   and small    veins                             more       closely          than        any      other      type
other           and    with    the endothelial          cells’                                   plasma       mem-
                                                                                                                                             of normal   vessel  [6, 15].
branes            by       stomata,                some            of     which               were         closed             by
                                                                                                                                                    A major            interest          of our       laboratory           has       been        to determine
diaphragms                    composed                 of         a single              membrane.                     Stud-
                                                                                                                                             the       structural basis of tumor    microvessel                                            hyperpermeabil-
ies with two tracers,           ferritin    (FE,    diameter           1 1 nm)
                                                                                                                                             ity.      To that end we have injected      a variety                                         of macromolecu-
and horseradish          peroxidase        (HRP,      diameter       5    nm),
                                                                                                                                             lar     tracers           intravenously                  into     mice or guinea                    pigs bearing
revealed     that      passage         of macromolecules             through
                                                                                                                                             any         of     several        well-defined                  transplantable                    syngeneic      tu-
\rV05    was     regulated         at the     level      of stomatal       dia-
                                                                                                                                             mors     and have followed       the extravasation      of these  tracers
phragnis,                thereby        demonstrating        a mechanism         for
                                                                                                                                             from tumor      and normal     vessels   over time by both light and
controlling                 the    passage       of macromolecules          across
                                                                                                                                             electron    microscopy     [6, 15]. These       studies led to the iden-
endothelial                 cells.    Thus,      compared      with   tumor      mi-
                                                                                                                                             tification             of a previously                   undescribed                organelle             present           in
crovessels,                little        circulating                FE and               HRP           entered              the
VVOs        of          normal              venular               endothelium                        because               sto-
mata          joining              vesicles         and       vacuoles                  to each            other           and
to the lumen           and ablumen             were      closed.       VVOs    and
their      component        vesicles/vacuoles              were      readily   dis-
                                                                                                                                                 Abbreviations:        VVO, vesiculo-vacuolar        organelle;      MOT, murine     ovarian    carci-
tinguished        from    endosomal           organelles         such as coated                                                              noma;     FE, ferritin;     HRP, horseradish        peroxidase;       MVB, multivesicular        bodies;
vesicles      and multivesicular              bodies,       which      also accu-                                                            VPF, vascular        permeability    factor.
                                                                                                                                                 Reprint     requests:    Ann M. Dvorak,      Department        of Pathology,  Beth Israel Hospi-
mulated                 FE         and    HRP.     Our                       findings              indicate                that
                                                                                                                                             tal, 330 Brookline         Ave., Boston,     MA 02215.
VVOs           provide               a major   pathway                         for      the       extravasation                                  Received       May 31, 1995; revised         September        1, 1995; accepted      September        6,
of        circulating                   mac      romolecules                          across             endotheia                           1995.




100          Journal          of     Leukocyte            Biology            Volume       59,        January        1996
Fig.         1   .   Tumor          vessels       and         their      lining      endothelial                   cells       10 s after          i.v.     injection            of FE      illustrate             overall         VVO structure.                     (A) low magnification                        view       that
includes                -25%           of a vessel             circumference.                      The       vascular            lumen         contains           a red          blood       cell    and         the       plasma            appears            denser        than      normal          because             of the
large         amount               of circulating                FE.      Arrows            indicate               several        VVOs.            (B-D)         higher-magnification                              views         show         VVOs              to consist           of grape-like               clusters        of
interconnecting                       vesicles          and      vacuoles           in abluminal                     (B),      luminal         (C),       and    lateral          (D) portions              of endothelial                    cell      cytoplasm.              Individual            vesicles         exhibit
pale         circular          stomata           (one      is indicated               by an open                   arrow        in panel           B).      Interconnecting                   vesicles           are       constricted                focally       at stomatal              attachment             points       of
diaphragms                   that     close      stomata              between        vesicles              (closed           arrow,       panel       B). In (C),           a VVO adjacent                    to the           FE-filled             vascular        lumen           (L) shows         a VVO          vacuole
(V) attached                   to two vesicles                by narrow             necks          (one       indicated               by a closed            arrow).       Other          portions          of this          cluster         reveal        the     trilaminar            unit      membrane-bound
vesicular               stomata         viewed          in cross          section           (one         indicated            by an open              arrow).          Several          stomata          exhibit           central,          dense        knobs.         In (D),        a vesicular            component
of a YVO                 is attached             to the        endothelial             cell’s            lateral       plasma            membrane               by a neck                (arrow).         Note         the      numerous                stomata,         bounded                by a tril.aminar              unit
membranes                    and      often      containing               central       dense              knobs,           in the       cluster          of vesicles            that     comprise           this        VVO.          (B)     is of an unstained                      section.        Bars:        A, 2 jim;
B,     0.4       jim;      C, D, 77 nm.




                                                                                                                                                                        Dvorak           et aL       YVO:              basis          of tumor              vessel           hyperpermeability                               101
Fig.       2.    Medium-                 to high-magnification                         electron            micrographs              illustrate           YVO         structure            in uninjected                 normal          venular          endothelium                from       tumor-bearing
(A, C, D) and                 normal            control        (B)      mice         as well        as from          tumor       vessel          endothelium                 30     mm        after      i.v.     FE    injection          (E).        In (A),     two        interconnected                vesicles
span       a narrowed                 segment          of endothelial                 cytoplasm               from       vascular         lumen           (L) to basal              lamina.           At their         point     of contact,             both      vesicles          are     constricted            and
are     separated             from        each       other      by a diaphragm                       (arrow).        In (B),        a vacuole             exhibits           a stomata            with          a central       dense        knob         that     is enveloped                by a trilaminar
unit      membrane                (closed          arrow);        in addition,                the     vacuole         communicates                      openly        with        an adjacent              vesicle          by means             ofa     narrow        neck        (open       arrow).        In (C),
two       vesicles          (above          attach        below          to a larger               vacuole         which,        in turn,          connects            to still         another           vacuole           beneath.         Note         constriction              and      diaphragm              that
separates             the     two        vacuoles            (arrow).          (D)     illustrates              a VVO         situated           near      the       abluminal             plasma               membrane.              Several          constituent             vesicles         and       vacuoles
exhibit         stomata           with       prominent               dense           knobs          (one      indicated          by an arrow).                   In (E),          two    vesicular              constituents            of a VVO             interconnect                  by a narrow             neck
(arrow);          the       fenestra          of one         vesicle         is guarded               by a diaphragm                     at attachment                 sites        to the        abluminal             membrane                 above       the      basal       lamina         (B),      which          is
devoid          of FE        label.        Bars:      A, 70          nm;       B, 77         nm;      C, 57        nm;       D, 240        nm;      E, 135           nm.




the        cytoplasm    of endothelial                                      cells   lining     tumor                            microves-                             define              the          barriers                that        might              regulate               tracer              passage
sels,        which   we have named                                       the vesiculo-vacuolar                                  organelle                             through    YVOs.     To that end, we performed           high-magnifica-
or      VVO               [15].           VVOs               provide              a trans-cytotic                            pathway               by                 tion transmission       electron    microscopy       with specimen         tilt-
wFich             soluble     macromolecular                                            tracers     extravasate                           from                        ing on the leaky       vessels   of a representative        transplantable
leaky           tumor     blood   vessels.                                 Study          of normal      mice                       revealed                          mouse    tumor    (the mouse     ovarian    tumor    or MOT) at defined
that         morphologically                              similar              structures                    were         present,               and                  intervals                after            intravenous                (i.v.)         injection               of macromolecu-
with          equal  frequency,                               in venules     and                           small    veins                 of the                      lar tracers.     Two soluble   macromolecular          tracers      of differing
skin         and subcutaneous                                 tissue.   However,                            in contrast                  to yes-                      size, ferritin     (FE) and horseradish       peroxidase         (HRP),       were
sels         supplying                     tumors,              these            normal     vessels   leaked    only                                                  employed.        In addition, we performed        size and shape            meas-
small            amounts                    of     circulating                     macromolecules         and their                                                   urements       on VVOs and on the individual             vesicles/vacuoles
VVOs              contained                      only     small                  amounts      of macromolecular                                                       that comprised          YVOs  in both      tumor     and adjacent,            non-
tracers.                                                                                                                                                              leaky,            venular                 endothelium.                     Together,               these            studies          extend
    The              goal of               the      present   study    was                                   to investigate                       the                 earlier             findings                 that VVOs    provide                               the primary    route     by
detailed               structure                 and function     of VVOs                                    and particularly                       to                which              both FE                  and HRP    exit tumor                                 vessels.  In addition,



102             Journal             of     Leukocyte                   Biology           Volume              59,     January          1996
they           provide                new               quantitative                               data regarding     VVO                                              struc-
                                                                                                                                                                                                                                             Area                                        Perimeter
ture          in both               tumor               and normal                                vessels  and characterize                                                 the
                                                                                                                                                                                                                                 MaSE:             13*0.2                  .   2.3*0.2
stomata                 linking                 vesicles                     and            vacuoles                      to each                 other               and           to                                           Medial.:          1.2                          1.9

the       endothelial                           cells’             luminal                   and            abluminal                          plasma                  mem-




                                                                                                                                                                                                                          JkL.1L.
branes.
                                                                                                                                                                                                                         10                                                                                                       (5

MATERIALS                                     AND                  METHODS

Tumors                   and tracers



                                                                                                                                                                                                                         ‘#{176}JILLM
Mouse            ovarian               tumor              (MOT)              was          passaged                   weekly              in      ascites              form           in
syngeneic                C3HeB/FeJ                        mice            [1].      Solid           tumors             were         studied               5 days             after
injection              of 3 x i0                 ascites             tumor             cells/site              into        the       subcutaneous                           space
of other          C3HeB/FeJ                       mice;            at this             interval,             tumors              weighed                 -20           mg and                                                    M*5E..
                                                                                                                                                                                                                                 M&1a              1.4
                                                                                                                                                                                                                                                   1.7*0.2                     2.4*0.2
                                                                                                                                                                                                                           0
had       become                vascularized.                                                                                                                                                                                    0    1        2      3      4A                0     1      2         3     4      5*

        FE      (type           I cadmium-free,                              from           horse            spleen,             450 kDa;
                                                                                                                                  mol.          mass                                                                                                                                  nfl, (xlO            4)

Einstein-Stokes                         radius,            5.5 nm) and                            HRP         type VI (mol. mass, 40 kDa;
Einstein-Stokes                         radius,            2.5-3             nm)          were           obtained                from          Sigma            Chemical
                                                                                                                                                                                              Fig.          3.    Architectural                   properties              of     VVOs            in       endothelial            cells     lining
Company,                  St.       Louis,           MO.            Tumor-bearing                            and          normal               mice            received                 a
                                                                                                                                                                                              hyperpermeable               MOT tumor microvessels   and in venular endotheium      of
single          i.v.      (tail         vein)          injection                  containing                   40-60               mg         ferritin           or        5,000
                                                                                                                                                                                              normal             subcutis.  A total of 43 MOT VVOs and 39 control        VVOs were
units          HRP           in 0.4-0.6                   ml        saline              [15].          Animals                were            killed           and         tumor
                                                                                                                                                                                              measured               and compared   (see Methods). No statistical differences were
and          normal           tissues            sampled                   for      electron                microscopy                      at 30         s and             30       m
                                                                                                                                                                                              found         between            tumor        and      normal          vessel        VVOs           with          respect      to overall        area
after        injection            of FE           and         at 10 s, 1, 5, and                             15 mm               after        injection               of HRP
                                                                                                                                                                                              or perimeter.              Means          ± standard                error        (M ± SE)           and medians                are indicated.
[15].         When            tissues            were           harvested                   at 1            mm after                 i.v.       tracer           injection,
animals               were        killed          by decapitation;                                at later           intervals,                mice        were            killed
with         ether-C02.

                                                                                                                                                                                              hum            (Fig.             1)1.     VYOs                of similar                   structure                   and        equivalent
Electron                     microscopy
                                                                                                                                                                                              frequency    are also present       in the endothelium           lining     nor-
Tumors            from          mice          injected               with          FE       were           rapidly           isolated              with         surround-                     mal venules     of the subcutaneous         space     (Fig.    2B). Within
ing      connective                   tissue,          cut         into          1-mm-thick                   slices,            fixed          by immersion                         in
                                                                                                                                                                                              endothelial    cell cytoplasm      VVOs      may be situated           in close
a mixture                of 2.0%              paraformaldehyde-2.5%                                            glutaraldehyde-0.025%                                             cal-
cium           chloride               in 0.1            M sodium                     cacodylate                    buffer,            pH         7.4,          for     2 h, at
                                                                                                                                                                                              proximity   to inter-endothelial       cell junctions       or distant     from
       and
20#{176}C, were processed                                           for electron                    microscopy                     [15].                                                      such           junctions.                In favorable                       electron                microscopic                      sections,
        Tumors               from          animals                 injected                 with           HRP            were           similarly                   isolated,                VVOs               span           the      entire              thickness                   of endothelial                          cell      cyto-
fixed,          rinsed            overnight                   in      0.1          M        sodium              cacodylate                      buffer,              pH          7.4;         plasm              from          luminal              to abluminal                         surfaces.
40-jim            sections              were           cut         with           a tissue               chopper              (Sorvall             TC-2,               Dupont
Instruments)                      and           were          prepared                   for        cytochemical                         detection                    of     HRP
                                                                                                                                                                                              Morphometric  analysis of tumor and normal                                                                                           vessel
using          3-3’          diaminobenzidine                                (Sigma)                as      substrate                [16].         Subsequently,
sections               were         processed                   routinely                   for      electron              microscopy                      [15].            Some
                                                                                                                                                                                              VVOs and their component    vesicles/vacuoles
sections              were        stained              with         uranyl             en       bloc        and        with        lead         citrate              on grids;
other          sections               were        not         stained              with           either          metal.            Thin          sections                  (pale-
                                                                                                                                                                                              Quantitative                       measurements                              were             performed                      on     VVOs           as
gold,         70-80             nm       thick)           were            examined                   in a Philips                    400         electron                  micro-             viewed      in random                               electron            micrographs      (Fig.   3). In both
scope.                                                                                                                                                                                        tumor      vessels      and                          normal             venules     YVOs    were enormous
                                                                                                                                                                                              structures         (median                             area          ---O.12-O.14      im2)     whose   areas
Quantitative                            analyses                                                                                                                                              and           perimeters                 conformed                     to a non-normal                               distribution              that
A total           of 82           VVOs              (43       from           tumor              vessels,             39       from            vessels           of normal                     was            skewed               to     the right.                    However,      the                           VVOs         of        tumor
subcutaneous                       tissue)             were          photographed                           at a magnification                             of x42,000                         vessels              and         normal               venules               did         not       differ            statistically              with
in the          electron               microscope                    and           were           printed            at a final                 magnification                        of       respect               to area or perimeter           (Fig. 3).
x104,000.                    YVOs             and         the        individual                     vesicles              and         vacuoles                  that         com-
                                                                                                                                                                                                  The             individual      vesicles      and vacuoles           that                                               together      com-
prised           them           were          then         outlined                 manually                  with         an        ultra-fine-tip                        photo-
                                                                                                                                                                                              prised               VVOs      were     uncoated,        circular-to-ovoid                                                        structures
marking                 pen          and         the          images                were            captured                  with            a video                 camera,
digitized              in a computer                         equipped                   with         tM-series                version              3.46p             software                 bounded                    by       a trilaminar                            unit           membrane.                         Quantitative
(Analytical                   Imaging                Concepts).                     Once             digitized,               the         areas,           perimeters,                        analysis                of      individual                       vesicles             and vacuoles                             revealed                 a
and          longest            and        shortest                dimensions                       of VVOs                and           of their              individual                     continuum                     of sizes and                       shapes             that followed                           a non-normal
vesicles-vacuoles                             were           measured                    and           subjected                 to statistical                       analysis
                                                                                                                                                                                              distribution                      that was skewed          to the right     (Fig.     4). The
using           the      t-test          or      Mann-Whitney                               non-parametric                           test,         as      appropriate
                                                                                                                                                                                              areas       and                  perimeters        of individual      tumor       VVO       yes-
[17].
                                                                                                                                                                                              icles/vacuoles                        were slightly    larger    than their    counterparts
                                                                                                                                                                                              in normal                    subcutaneous                           venules.               Thus,                  median           and      mean
                                                                                                                                                                                              areas              of tumor               vessel               endothelial                        vesicles/vacuoles                          were
RESULTS

As originally                        described                              [15], YVOs                          are grape-like       clusters
                                                                                                                                                                                                        specimens
                                                                                                                                                                                                     ‘All              illustrating        HRP cytochemistry       (Figs. 7-12) are of unstained
of vesicles                       and vacuoles                                deployed                         at discrete     intervals      in                                              material.    Other unstained          sections      include Figs. lB. 6A and B, and 13A. Figures
the          cytoplasm                        of hyperpermeable                                             tumor                vascular                      endothe-                       of FE-injected      material      include      Figs. 1, 2E, 5, 6, and 13.




                                                                                                                                                                                            Dvorak          et al.       VVO:          basia          of     tumor             veseel           hyperpermeability                              103
                                                                                                                                   (5              Fig.      4. Architecturalproperties                                    of the          individual                 vesicles
                                                                                                                                                   and      vacuoles            that     comprise             YVOs              in MOT          tumor           vs. normal
                                                                                                                                                   venular           endothelium.                  A total         of 521           vesicles              and        vacuoles
                                                                                                                                                   from       MOT             endothelium                   and         487         from         control              venular
                                                                                                                                                   endothelium                  were      measured                and      compared                  (see       MATERIALS
                                                                                                                                                   AND       METHODS).                  Means          ±      standard               error           (M      ± SE)           and
                                                                                                                                                   medians            are       indicated.            The      mean              areas         and        perimeters               of
                                                                                                                                                   MOT        vesicles-vacuoles                        were        significantly                 larger          than      their
                                                                                                                                                   normal            vascular           endothelial                cell         counterparts                    (P         0.03
                                                                                                                                                   and       0.01,          respectively).                 Also,          the      vesicles            and           vacuoles
                                                                                                                                                   that      comprise              MOT          VVOs           were           significantly                  more         ovoid
                                                                                                                                                   (less       round)            than        their         normal               venule          endothelial                  cell
                                                                                                                                                   counterparts                 (P <         0.001).




7,900         nm2 and               10,300          nm2, respectively;     corresponding                                 sometimes          opened    to the lateral          (junctional)        surface         of
areas        in normal              control         venules  were smaller:       7,500   nm2                             plasma         membranes,      joining      these      surfaces      either      above
and        9,000        nm2        (P            0.03).         Though          achieving             statistical        (luminal        to) or below     (abluminal       to) zones       of close,       inter-
significance,              absolute              differences             were      small     and are un-                 endothelial               cell       junctional                     apposition                         (Fig.    1D). This     ob-
likely   to have accounted                          for the very                large functional        dif-             servation           has          significance                    for        interpreting                     the pathways       of
ferences    observed     in                      macromolecular                    transport     between                 macromolecular                        tracer             transport                 across                vascular    endothe-
tumor  and              normal         vessel VVOs    (see                  below).                                      hum (see below).
   In both             tumor         and normal    venular                    endothelium,                  a sub-          We hypothesized                                     that         the           interconnections                                      of       yes-
stantial          fraction           of vesicles/vacuoles                        had areas    that fell                  icles/vacuoles                    with          the           plasma              membrane                          and            with         each
within          the range             (2,500-6,000        nm2)                  expected   for typical                   other       might          provide                 likely           control               points                for     regulation                       of
60-80    nm uncoated                      caveolae             of capillary         endothelial    cells;                tracer     passage.     Therefore,      we performed      high-magnifica-
i.e., 30%   of tumor                      and 34%                of normal          vessel      VVO yes-                 tion electron        microscopy       to define   the structure       of these
icles/vacuoles.                      However,                  the     majority             of VVO     yes-              interconnections.          We found       that vesicles    (and   less com-
icles/vacuoles                      were     larger               than     6,000             nm2,  and      a            monly      vacuoles)     joined    the luminal    or abluminal      endothe-
substantial             number           (-.- 15%         in tumor          endothelium,                 -5%        in   hal     surface            by way               of stomata                    similar                  to those                  previously
normal           venular    endothelium)                        had areas     that                    exceeded           described      in capillary   caveolae     [18,   19].                                                                 Stomata                  were
32,000          nm2, corresponding                        to vacuolar   diameters                      of >200           either    open or were closed      by a diaphragm                                                                     composed                   of a
nm.                                                                                                                      single     membrane                      [18]. Typically,       stomata    closed  by a dia-
   The width/length                      ratio      was calculated       to provide   a meas-                            phragm      appeared                     constricted      (Fig. 1B, and 2A, C). In some
ure of vesicle/vacuole                           roundness;      a value    of 1.0 describes                             instances,      vesicles                   joined    the plasma      membrane     by way of
a perfect       circular        structure,      whereas      values     below                              1.0 in-       a narrow      neck,      the stomata     of which       were either  open      or
dicate        progressive              degrees       of ovalness.           The                              mean        closed    (Fig. 2A, E). Central          knobs     (Figs.   1C, D; 2B, D) of
width/length              ratio       of tumor       vessel      endothelial                                   yes-      the type described           in caveolae      that open to the surface         of
icles/yacuoles           was 0.73           compared        with 0.79        for                           control       capillary      endothelial       cells   [18]     were    sometimes    visible
vessel       endothelium                     (P       <          0.001);          thus,         tumor   yes-             within  stomatal   diaphragms.       Commonly,                                                                  when      theii    sto-
icles/vacuoles          were              slightly             more ovoid           than       their normal              mata were closed     by a diaphragm,      vesicles                                                               attached       to the
venular            counterparts.                                                                                         luminal   surface had contents     that appeared                                                                 less dense       than
                                                                                                                         plasma.      Because     the electron      density                                                 of plasma     is largely
                                                                                                                         attributable      to its content      of plasma                                                  proteins,   this finding
Interconnections                         of individual vesicles/vacuoles                                                 implies        a lower               protein              concentration                            within               vesicles                 than
with endothelial                        cell plasma membranes         and with                                           in plasma,       presumably      because       the diaphragm       closing     the
each other                                                                                                               vesicles     serves     as a barrier      that limits    the inward        flow of
                                                                                                                         plasma     proteins.
Some of the individual                           vesicles      (and less                 commonly     vacu-                  In addition      to opening     to the endothelial       cell surface,     the
oles)    that comprised                        VYOs       in tumor   and                  normal  vascular               vvO vesicles and vacuoles                commonly      formed     connections
endothelium      abutted                       on or communicated                          openly with the               with each other,   generally       by means     of short narrow          necks
luminal      or abluminal      surfaces     of endothelial       cells.   In addi-                                       (Figs.  1C and 2E), resembling          those that joined        vesicles      to
tion, when        VVOs     were situated       adjacent      to inter-endothe-                                           the endothelial   cell’s     plasma     membrane.        Individual         yes-
hal     cell    junctions,       individual         vesicles       or vacuoles                                           ides   commonly    displayed         two or three      stomata,       with or




104         Journal         of     Leukocyte           Biology       Volume        59,      January       1996
Fig.       5.   High-magnification                    micrographs            of portions               of tumor         vessel       VVOs          30     mm         after     i.v.    injection              of FE        illustrate          FE transit          via       VVOs         from       the
vascularlumen     to the ablumen with discharge into the underlying                                                          basal       lamina.          (A,    B, C)         Portions             of VVOs          near         or opening          to the      vascular            lumen          (L).
(A) and (B) illustrate FE-containing    vesicles attached to the plasma                                                           membrane              but apparently                 closed           from       it by a stomatal               diaphragm.                 FE    particles         are
evident in the vascular    lumen (L). In (C), also illustrating portions                                                         of a YVO located                    near the vessel lumen                          (L), several            vesicles        contain           FE; FE            is also
noted       within      a narrow         neck     (arrow)        that     connects          two of the vesicular                   structures.            In (D),       portion           of a VVO              located          at mid-level          of endothelial                cytoplasm,
contains        large     numbers          of FE particles;                several        FE    particles            are located         within narrow                 necks          that         interconnect             adjacent           vesicles.         (E)     Portion          of a VVO
deep  in endothelial cell                   cytoplasm,            facing        on    the      basal        lamina       beneath.         Multiple              FE     particles             are      present        within         vesicles         and    a few         are      also        present
within the basal lamina.                    Bars:       A, 30       nm;      B, 83     nm;        C, 77 nm;            D, 80 nm;           E, 111          nm.




without          diaphragms,                    and     larger          vacuoles               exhibited               as many                      within            vascular  lumens    and had                                       already    entered                        portions    of
as      eight       distinct             stomata.           Exact            right    angle                  sections              re-              YVOs              that openly     communicated                                            with vascular                           lumens;
vealed           that     these             stomata          were            completely                      bounded                by              however,                   at      this           early         time,            FE        was         not         yet        present                in
t,rilaminar             unit       membranes                 (Figs.          1 C,D             and       2 B,D).                                    vesicles   or vacuoles     interfacing    with the abluminal      endo-
                                                                                                                                                    thelial  surface.    However,       at 30 mm after tracer    injection
Functional                  anatomy       of WOs   as determined                                                      by                            (Fig. 5), FE was found in VVO vesicles              and vacuoles        at
transcytosis                  of circulating   macromolecular                                                      tracers                          all     levels              of the                cytoplasm,                   and         FE had              in addition     ex-
                                                                                                                                                    travasated                      from            vessels               into      and         beyond              the underlying
                                                                                                                                                    basal    lamina.
Studies          withferritin                   (FE)
                                                                                                                                                        Higher      magnification                                      study    revealed                     that FE particles
Animals              were       killed          at 30       s and          30        mm        after        i.v.      injection                     entered     vesicles       that                             were       open   to the                   vascular  lumen;    in
of FE.          In tumor           vessels            at 30 s, FE was                       abundantly                  present                     the      case            of closed                 vesicles,                 FE attached                to central                 portions



                                                                                                                                                 Drorak          et zL         VVO:            basis        of tumor               vessel        hyperpermeability                                 105
Fig.        6.      High-magnification                            electron           micrographs                   illustrate          tumor           vessel       endothelial               cell     VVO         vesicles           with      stomata           that     contain           FE,      30     mm       after      i.v.
injection            of FE.           In (A),           stomata         are     adjacent             to the         vascular           lumen           (L),     in (B) at mid-level                     of endothelial                 cell     cytoplasm,               and        in (C) at the            basal      lamina
front       (B).        (A,     B),        both         unstained             with        heavy          metals,          show        a central            rounded             collection             of FE        particles           within         vesicular            stomata;            (C),        from      a stained
section,            also      shows          a central            stomata            filled       with      FE particles.                 In all three              vesicles           illustrated          in (A-C),           the      vesicle        chambers               contain          little      FE.      Note     that
the     vesicle            in (C) is bounded                        by a trilaminar                 unit      membrane                and       is connected               to the underlying                      plasma        membrane                by an elongate,                  narrow            neck      (arrows).
Several            FE       particles             are     present          in the         basal       lamina             (B).     Bars:      A, 51            nm;     B, 27       nm;        C, 63         nm.




of      the          covering                     diaphragm.                         In       some            instances,                     FE          was                   lated         to resolve               overlap                 in sections                 as thin             as 40 nm and                        of
found    within    vesicles                                       that were closed   to the endothelial                                                                        a potential                    overlap   space    of < 8 nm                                           [20].       These  studies
lumen    by diaphragms                                            (Fig. 5, A and B); not uncommonly,                                                                           confirmed                    that adjacent     vesicles-vacuoles                                                were in fact in-
however,      such caveolae                                          exhibited other  stomata  that were                                                                       terconnected                       and          were           not       separate                    discontinuous                       struc-
open             (Fig.          5, A and                    B),        suggesting                    that          FE           gained           access                        tures.
to these  apparently    closed                                                caveolae       by an alternate         route;                                                        In no case did FE particles      cross     vascular     endothelium
i.e., by way of attachments                                                    to vesicles/vacuoles          present        in                                                 by way of inter-endothelial     cell juctions.       In both tumor and
other   planes       of section.      Alternatively,       FE could      have en-                                                                                              normal                vessels,              adjacent.                 endothelial                       cells             were         always
tered   such      vesicles      at a time when        they were open to the                                                                                                    joined        together     by zones                               of close                approximation                            that were
luminal      surface;      i.e.,   prior     to their    closure    with     a dia-                                                                                             insufficient          to accommodate                                   the               passage      of FE                       particles.
phragm.                                                                                                                                                                        However,                 focal         dilatations                    were         observed                  both           luminal            and
    FE and                    accompanying                              plasma                proteins      commonly    filled                                                 abluminal        to zones of close approximation                                                            and these                    some-
the        entire             lumens     of the                         individual                 vesicles    and vacuoles                                                    times     contained      FE particles   [15]. As                                                        noted   above,                       VVO
that together     comprised           VVOs     (Fig.     5E). In some         cases,                                                                                           vesicles              not     uncommonly            directly       into these focally
                                                                                                                                                                                                                                                 opened
however,      FE filled      vacuolar      lumens        except     for their     sto-                                                                                         dilated            regions  of the lateral   endothelial         surface;      discharge
matal    openings     (Fig. 5D). In other            instances,       the opposite                                                                                             of FE             (or other    tracer)   to such     dilatations          abluminal      to
pattern     was observed         such     that clusters         of FE particles                                                                                                zones    of close    membrane                                       approximation       could    lead to the
were observed        exclusively        within      stomata      and not in the                                                                                                mistaken     conclusion      that                                 tracer    had traversed     the endothe-
remainder  of the vacuolar   chamber.                                                               Such            FE-filled                 stomata                          hum          by way               of junctions.
were found   in VVO vesicles      and                                                             vacuoles             at all               levels    of
the        cytoplasm                       from          luminal               to abluminal                        surfaces                (Fig.          6).                  Studies               with        horseradish                    peroxidase                      (HRP)
In favorable                          sections,                   FE      particles                  were sometimes       found
in single-file                         alignment,                       traversing                   the narrow     necks    that                                              HRP           was        also        transported                      across              tumor           vessel             endothelial
interconnected                               adjacent                   vesicles-vacuoles                                  (Fig.          5C);           FE-                   cells        by way               of VYOs               but       more          rapidly               than          FE.       In animals
labeled                    necks             and           stomata               provided                    hard               evidence                 that                  killed          as early             as 10 s after                     i.v.     injection,                 HRP              had       already
such             vesicles-vacuoles                                  were        in fact             in open               communication                                        filled   YVOs                     at all levels     of tumor    vessel                                          endothelial                    cell
with         each             other          and           that        particles               the diameter                        of FE (-11                                  cytoplasm.                        Both   individual      HRP-labeled                                                vesicles                   and
nm) were    able   to pass     through                                                            stomata                 from            one          VVO                     HRP-labeled                          vesicles                  that        were            clearly              joined              to other
vesicle or vacuole    to another.                                                                                                                                              vvo            vesicles-vacuoles                               were           found             in     continuity                   with the
    Specimen       tilting      was carried      out to determine       whether                                                                                                basal    lamina      (Figs.      7, 8 A, and 9 A, C-E);          moreover,
adjacent     vesicular         structures    were indeed      in communica-                                                                                                    the basal       lamina      immediately    underlying       such  openings
tion with each           other      or could   be resolved      into separate,                                                                                                 was   focally     peroxidase-positive      (Figs.     7 and 9 C-E).     HRP
non-communicating                 structures    in our sections      (-70-      to                                                                                             labeling               of tumor    vessel                       endothelial                  VVOs               was maximal       in
80-nm                thick).               Specimens                       were            tilted           through                a full          range                       animals                killed   between                          10 s and                   5 mm                after i.v. tracer
         in
of 70#{176} two                         perpendicular                           directions,                    a procedure                       calcu-                        injection               (Figs.              10     and            1 1). Intensity                         was          somewhat                 re-



106              Journal              of      Leukocyte                    Biology            Volume               59,      January             1996
Fig.       7. Tumor             vessel        endothelial                 cell    VVOs          at      10 s after           injecting                HRP            i.v.    Sections          were        reacted          for        HRP          reaction           product         by cytochemistry                      (see
MATERIALS             AND       METHODS)            but         were        otherwise           unstained.                In both              (A)     and           (B),     continuity           of VVO             vesicles           with         the      blood       vascular            space        (L)     is made
evident         by HRP           reaction         product.               As indicated                by arrows,             reaction             product              also     extends           to the      basal         lamina            (B),     focally          in (A) and           massively             in (B).       In
(B),      HRP       reaction         product             also      extends           beyond            the     basal       lamina              into     the      surrounding                  connective              tissue.          Also         in (B),       a large,         HRP-positive                   cluster          of
vesicles        opens          to an adjacent                    HRP-positive                  vesicle           (to the         left)     by means                     of a narrow              channel         filled           with       HRP            reaction         product           (black        arrow);          the
remainder             of this      VVO is HRP-negative.                                 Note         that    the       interendothelial                       cell      junction            in (A) does              not     contain           HRP           (arrowhead).              An      individual             vesicle
(arrowhead)             of a      partially           HRP-positive                   VVO         in (B)          is fused          with         the        abluminal                plasma        membrane                  and        has     released              a cloud       of HRP              adjacent         to the
basal      lamina        and       tissue        front          (white       arrow       pointing             left).     Bars:           A, 260         nm;           B, 154          nm.




duced            at      15       mm,            by        which                 time          the          basal          lamina                    and                    interconnections                               with          each            other            and         with         the        vascular
surrounding                     extravascular                            stroma           were               diffusely                   and          in-                   lumen   and ablumen       (Figs.    7-12).     HRP staining         permitted
tensely           labeled                (Fig.           12).                                                                                                               clear distinction   between      the stomata      interconnecting           mdi-
       Cell     membranes,                       and            therefore               stomata!                morphology,                           are                   vidual   VVO vesicles-vacuoles             that were open         (contained
not        well-visualized                        in the                 unstained                   electron               microscope                                      HRP             product,            hence                  intense               black          staining)               or that                 were
sections              required      for studies      of HRP                                           as tracer.               However,                                     closed            (no HRP     product,      hence      lack of cytochemical           de-
positive              HRP      staining     provided       an                                         effective               means     for                                 posits)           (Figs. 7 A, and 8-1 1). HRP staining                 also permitted
identifying               the individual        components                                              of VVOs                and their                                    clear           visualization     of patent       necks    interconnecting         adja-



                                                                                                                                                                       Dvorak           et al.      VVO:         basis            of     tumor              vessel        hyperperineability                                 107
                                                                                                                       Fig          8.        Tumor             vessel             VVOs             harvested                     10
                                                                                                                       S    after         i.v.        HRP.           (A) The               full         thickness                 of
                                                                                                                       an       endothelial                       cell           is depicted                    from          the
                                                                                                                       intensely                      HRP-positive                           vascular                   lumen
                                                                                                                       (top)             to      the          HRP-stained                           basal             lamina
                                                                                                                       (bottom).                    Clusters              of vesicles                  representing
                                                                                                                       portions                  of     a YVO                    span         the            cytoplasm;
                                                                                                                       their         contents                  are       variably             dense,                depend-
                                                                                                                       ing          on         their           variable                 content                 of       HRP.
                                                                                                                       Structures                     at mid-cytoplasm                                 are      intensely
                                                                                                                       positive;                    faintly           positive                VVOs                  (arrows)
                                                                                                                       extend                 to the          HRP-containing                                  basal        lam-
                                                                                                                       ma. Some                       of the         stomata               in one              portion            of
                                                                                                                       the          VVO               in the              mid-cytoplasm                               (arrow-
                                                                                                                       head)             contain               dense              HRP            (i.e.,        are open)
                                                                                                                       and others                       do not (i.e., are closed).                                            The
                                                                                                                       latter            round,               electron-lucent                             stomata              are
                                                                                                                       outlined                  by dense                 HRP           in the surrounding
                                                                                                                       vesicle                chambers                    that      comprise                    the      VVO.
                                                                                                                       (B-E)              Illustrate                 individual                    HRP-positive
                                                                                                                       (open)                 and         HRP-negative                                 (closed)               sto-
                                                                                                                       mata              and          HRP-filled                     intervesicular                          con-
                                                                                                                       necting                 channels.                  In (B, top),                  two adjacent
                                                                                                                       vesicles                  are      illustrated;                     one          (arrow,            at 1)
                                                                                                                       contains                     HRP           reaction              product                 only          in a
                                                                                                                       stomata                  (the        round,               electron-dense                          struc-
                                                                                                                       ture)             within             the       electron                 lucent                 vesicle,
                                                                                                                       whereas                   the second                      vesicle           (arrow              at 2) is
                                                                                                                       filled            with          HRP           except             for a stomata                         (the
                                                                                                                       central,                round,             electron-lucent                             structure),
                                                                                                                       which              is devoid                  of electron-dense                                  tracer.
                                                                                                                       In (C),                at mid-endothelial                              cell           cytoplasm,
                                                                                                                       several                  interconnecting,                              variably                   HRP-
                                                                                                                       positive                     vesicles               are       illustrated,                      two          of
                                                                                                                       which                  are       interconnected                                 by       a narrow
                                                                                                                       channel                   or neck                  (arrow)             which                 contains
                                                                                                                       HRP. In (D), illustratingportions                                                            ofaVVO
                                                                                                                       at the level of mid-endothelium,                                                             HRP          re-
                                                                                                                       action             product                 fills          vesicles,                stomata             and
                                                                                                                       interconnecting                               channels.                    In         (E),      also         at
                                                                                                                       the       level           of mid-endothelium,                                        dense         HRP
                                                                                                                       reaction                  product              fills         several                 stomata              but
                                                                                                                       is present                      at lower                  concentrations                          in the
                                                                                                                       surrounding                        vesicle                chambers.                    Two        HRP-
                                                                                                                       positive                     stomata                 are         connected                       by        an
                                                                                                                       HRP-containing                                 narrow               channel                    (arrow).
                                                                                                                       Bars:A,274nm;B,                                            124nm;C,95nm;                                      D
                                                                                                                       and          E, 67             nm.



cent vesicles       and vacuoles     (Figs.   7B; 8C and E; 1OA and               open      connections       between       vesicular-vacuolar                                                      structures
F; 1 1B). As was the case              with    FE, YVO     vesicles    and        rather     than individual,      separate      structures                                          that          were not                       in
vacuoles     exhibited    three  different      patterns of HRP     label-        continuity.
ing: 1) complete       filling;   2) filling   of the entire       chambers          In contrast       to the much larger                           FE, HRP was                                able             to cross
except   for stomata;       and 3) filling    of stomata      only (Figs.     8   both tumor         and control   vascular                           endothelium                                by             passing
B and 11 C).                                                                       through   apparently     closed  portions     of inter-endothelial                                                                 cell
    As with      FE,  HRP-stained          YVOs     were     examined       by    junctions      [15]. However,    this was a much            slower                                                            process
specimen     tilting   through     a full range      of 700,     confirming        than transport      by way of VVOs.       Whereas       HRP had                                                              crossed



108     Journal   of   Leukocyte   Biology    Volume   59,   January   1996
endothelial       cells by way of VVOs        as early as 10 s after i.v.                                                 tumor            and normal      vascular      endothelium                              (Fig.    13). Thus,
injection     (Fig. 7A), HRP did not appear            in junctions until                                                 typical            coated   vesicles      were     readily                          found     both   free in
5 mm but persisted            through     at least    15 mm when      the                                                endothelial                     cytoplasm                and attached             to the plasma                mem-
concentration          of HRP   in vessel   lumens     and in VVOs     re-                                               brane       of            the      luminal              front,  though            generally   not             to the
mained            high         (Fig.       12).                                                                          abluminal                  endothelial                 surface         [19].    However,           in the      endo-
                                                                                                                          thelial           cell         populations                 we studied,     coated             vesicles        were
                                                                                                                          less        frequent              than     the            smooth    membrane-bound                       vesicles
Distinction of VVOs from                                       endosomal  organelles                                      and         vacuoles              that            comprised           YVOs.        Coated         vesicles        are
that also became   labeled                                     with FE and HRP                                            known             to traffic                 to     and       fuse     with     multivesicular               bodies
                                                                                                                         (MVBs)   [19, 21, 22] (Fig. 13). Like coated                                                vesicles,         MVBs
VVOs            were       clearly              distinguishable           from    several  compo-                        were also less frequent    than VVOs  in the                                              endothelial          cells
nents         of the      endosomal                  pathway        which    were also present    in                     we          studied             and       were             readily       distinguished             from       VVOs.




Fig.        9. Tumor      vessel         YVOs    at 10 s after i.v. HRP injection. In (A), HRP-positive                            VVO connects         the vascular lumen (top, strongly           HRP-positive)      with
the    focally      positive           underlying    basal   lamina. (B) Illustrates a mid-cytoplasmic                             level       VVOwith HRP.negative,           electron-lucent      stomata    (therefore,
presumably           closed)           within      otherwise      HRP-positive      vesicles.    In   (C),    many     endothelial          cell vesicles are filled with dense reaction product similar in
intensity        to the   overlying             HRP-fihled      vessel    lumen.   One   HRP-Ioaded          vesicle    is releasing         HRP focally     into the underlying       basal   lamina (arrow). (D) and
(E) show clusters               of HRP-containing VVOs releasing                     dense reaction          product    directly        into the underlying      HRP-positive        basal lamina.     Bars:  A, 140 nm;
B, 91 nm; C, 147                 nm; D, 104 nm; E, 71 nm




                                                                                                                       Drorak         et al.       VVO:        bail.          of tumor         vessel   hyperpermeability                  109
Fig.       1 0.     High-magnification                          micrographs            of tumor            vessel       VVOs           harvested            5 mm          after     i.v. injection                of HRP          to illustrate             patent         communication                   between
adjacent          vesicles.            (A-C)        and      (F) represent             mid-endothelium                       whereas          (D) and             (E) represent              basal       cytoplasm.                   In (A),      two adjacent                vesicles          are connected
by a narrow               HRP-filled                channel.            One    vesicle           (1, arrow)            has       HRP        within          its chamber,                  outlining            an     HRP-negative                   (presumably                    closed)       stomata;              the
second        vesicle          (2, arrow)           does        not have      HRP           within     its chamber               but     is connected                   by the HRP-positive                       channel             to an HRP-positive                      (therefore,             presumably
open)       stomata.           In (B),         vesicles          (1, arrow)          with      a faintly        positive          stomata          and      (2, arrow)             an HRP-negative                      stomata,            outlined            by the        dense        reaction           product
in the      vesicle           lumen,         are     connected              by an HRP-negative                         channel           (open          arrow).          In (C),       a VVO          vesicle          contains            dense          reaction          product,           outlining          three
electron-lucent                 (closed)           stomata.          In (D),     the        VVO       (arrowhead)                shows       electron-lucent,                     closed        stomata             outlined           by HRP             present          within       vesicle         chambers.
In (E),       the      VVO        (arrowhead)                   shows       a mixture           of electron-lucent                      (closed)          and       electron-dense                    (open)         stomata            with       very      little        HRP        reaction          product           in
the     surrounding              vesicle           chamber.             The    abluminal              plasma           membrane              is faintly             stained         with      HRP         (closed           arrow);            however,             the    underlying             basal        lamina
is HRP-negative                    (open           arrow).         In (F),     the     outer         limits         of portions           of a VVO                are     delineated               by a dashed                 line     (compare             with         routinely           processed           VVO
images        in Figs.          1 and          2 and        femtin-filled             VVO         images        in Figs.          13C       and         5, C-E).           Note      that     HRP         fills      some         stomata          as well           as a narrow              channel         (arrow)
that     interconnects                 two      vesicles.           Compare           the      HRP-positive                  channel          illustrated                here      with      the      FE-positive                channel           of Fig.          5C).      Bars:       A, 67         nm;     B and
C, 83       nm;       D, 91       nm;        E, 87         nm;      F, 91     nm.




Thus,     MVBs      were                           large    membrane-bound                                  structures                      that                   within            Weibel-Palade       bodies   (not                                          shown).   FE was not                                ob-
contained      variable                              numbers      of small                           vesicles,        each                   ap-                   served            free in the cytoplasm      outside                                           of membrane-bounded
proximately                    one-third                     the size           of plasmalemmal                                   vesicles                         YVOs.
(caveolae).                   The space                      between            these   small   MVB                               vesicles                             As with FE,                       MYB              accumulated                           HRP     but               the small   yes-
was filled    with variably                                          dense,     homogeneous                                   contents,                            ides   enclosed                       within             MVB did                       not    contain                  reaction  prod-
whereas    the MVB vesicles                                            themselves      appeared                                electron                            uct. Weibe!-Palade           bodies      were intermittently           positive      for
lucent.                                                                                                                                                            HRP reaction       product,       indicating    uptake       of tracer      by these
    Coated                vesicles                 and MVBs                    in endothelial                          cells    lining                             secretory   granules,       presumably       by a process         similar       to that
venules                 in normal                     subcutaneous                   tissue                         were     labeled                               of uptake    of HRP       by the secretory         granules        of guinea        pig
strongly              with        FE         and           FE      actually            became                 concentrated                         in              basophils    [231.
MVBs              (Fig.         13A).              In contrast,                fewer            MVBs                were         found             in
tumor   vessel   endothelial     cells                                         and these   contained                                    lesser
amounts     of FE than       in normal                                           vessels (Fig.    13B).                                 Thus,                      DISCUSSION
different   patterns       of FE labeling       were found      in tumor     and
normal    venular       endothelium;      i.e., strong   labeling    of VVOs                                                                                       The data presented      here extend earlier  work demonstrating
in tumor       vessels,       strong  labeling       of MVBs       in normal                                                                                       that YVOs     provide     the major anatomic     pathway     for                                                                                 the
venules     (Fig.      13, A-C).     FE was occasionally             observed                                                                                      extravasation    of both FE and HRP across       the hyperperme-



1 10          Journal            of      Leukocyte                  Biology           Volume           59,      January            1996
                                                                                                 Fig. 1 1. Tumor vessel endothelial                                              cell YVOs connected                                to     the     abluminal
                                                                                                 surface         at 5 rain           after         i.v.      HRP.            In (A),        HRP-positive                        vesicles,            outlining
                                                                                                 negative            stomata,          open           into        the        largely       HRP-negative                          basal           lamina        area
                                                                                                 (B). In (B), portions                         of an HRP-containing                                   VVO communicate                                   with      the
                                                                                                 basal      lamina           (B), which                   is also HRP-positive.                           (C) shows                   an open,              HRP-
                                                                                                 containing              fenestra          of a vesicle                 in contact             with     the         plasma           membrane.                  The
                                                                                                 stomata           contains           dense HRP,                    and         the vesicle             lumen              is    faintly          HRP-posi-
                                                                                                 tive. The abluminal                          surface             of the endothelial                         cell      plasma               membrane                   is
                                                                                                 focally        stained            with       HRP            beneath              his     vesicle,            but      the        basal    lamina (B)
                                                                                                 remains           unstained.                Compare                the HRP-positive                         stomata               of this abluminal
                                                                                                 vesicle        with       the      FE-positive                   stomata              of a similar           abluminal                    vesicle         in Fig.
                                                                                                 6C. These                images          indicate             passage             of tracers            by open                  fenestra              in conti-
                                                                                                 nuity        with        other       vesicles               of the           YVO         at other            levels             before           the     vesicle
                                                                                                 lumen          itself      becomes                extensively                  tracer-positive                     (or,        alternatively,                 after
                                                                                                 the     vesicle          chamber             proper              has        emptied            of tracer).            Such           images              support
                                                                                                 interconnected,                    patent           passages                throughout               VVOs           that        span        tumor          vessel
                                                                                                 endothelia.               Bars:       A, 80          nm;         B, 87          nm;       C, 80        nm.




                                                                                                 of some                 importance                       in that             the thickness                          of routine                    electron
                                                                                                 microscope                        sections                  (70-100                     nm)          is substantially                                  greater
                                                                                                 than          the diameter                     of stomata     (< 50 nm); therefore,                                                                      speci-
                                                                                                 men           tilting was                   required     to prove  that the observed                                                                      inter-
                                                                                                 connections                          did     not reflect                                   overlap     of                        adjacent                     but
                                                                                                 unconnected                         vesicles/vacuoles                                     [20]. Finally,                          stomata                  link-
                                                                                                  ing       vesicles                 with            each               other            and          with           the           plasma                  mem-
                                                                                                 brane               were             found                  to         be        either               open                 or       closed                  with
                                                                                                 diaphragms                        resembling                       those that close                                the caveolae                          found
                                                                                                 in capillary                      endothelium.                         These stomata                                  and their                        closing
                                                                                                 diaphragms                         likely            provide                   the        structural                      basis            for regula-
                                                                                                  tion of tracer     passage      across                                               microvascu!ar    endothelium.
                                                                                                      The  substantial       difference                                                  in the permeability         of                                         mi-
                                                                                                  crovessels                  supplying      tumor     and                                     normal                tissues    was                        borne
                                                                                                  out in the                 present    study.     Within                                      seconds                 to minutes                          of i.v.
                                                                                                  injection,                 electron                     dense              tracers             had          entered                     VVOs                 that
                                                                                                  opened                 to the           luminal                  surface                of tumor                   microvascular                               en-
                                                                                                  dothelium,    were visualized                                                  in YVO vesicles                                    and vacuoles
                                                                                                  at all levels   of endothelia!                                                 cell cytoplasm,                                  and were also
                                                                                                  found   opening                       to the abluminal         surface,    spilling    both trac-
                                                                                                  ers into the                        underlying      basal      lamina.     In contrast       to its
                                                                                                  ready   passage                       across   tumor      vascular      endothelium        by way
                                                                                                  of VVOs,                   FE           (diameter                             1 1 nm)                was            not           found               to exit
                                                                                                  tumor           vessels                 through                 inter-endothelial                                  cell junctions    that
                                                                                                  were           regularly                   closed                 in tumor        vessels                              [15].  A smaller
                                                                                                  tracer,            HRP (diameter                                 -5 nm), did exit tumor                                                  vessels   to a
                                                                                                  limited             extent through                                closed inter-endothelial                                                 cell junc-
                                                                                                  tions         but only                   after           a delay       of several                              minutes,                      at a time
                                                                                                  when          extensive                   HRP             extravasation        had                            already                    taken   place
                                                                                                  through                YVOs.      YVOs                       of similar                        size, shape,    and                                 vesicle-
                                                                                                  vacuole                composition                          were   also                       identified    in the                                 venular
                                                                                                  endothelium          of normal       skin and subcutaneous           tissue      of
                                                                                                  tumor-bearing          mice,     but, in contrast,        these   VVOs      were
                                                                                                  labeled      minimally       with FE and to only a slightly              greater
able    endothelium             that    lines
                                     tumor                microvessels.        They have          extent    with HRP;        also, as in tumor     vessels,      HRP did belat-
permitted        us                VVOs
                         to represent       in             schematic      fashion      (Fig.     edly exit                  from           normal                  vessels                by way               of inter-endothelial
14)     and     have also provided      new               quantitative       information         junctions.
regarding        VVO structure.    The use                 of specimen        tilting    pro-            Our         findings                are          relevant                to one              of the               more            chronically
vided       conclusive           evidence         that   individual       YVO     vesicles        disputed     problems                              in vascular      biology,                                  namely,       that of de-
and vacuoles             communicated              with each       other  and with        the     fining    the anatomic                               pathways      by which                                    circulating       macro-
endothelial           cells’  plasma            membranes.        This last point        was      molecules       cross                            blood     microvessels.                                      Physiologists         have



                                                                                                Dvorak         et a!.       YVO:             basis           of tumor                  vessel         hyperpermeability                                        111
Fig.       12.   Tumor           vessels          at 15 mm after              HRP           injection            show        lesser       amounts              of HRP      product           in vessel       lumens          (L) than          at 10 s (compare                with      Figs.      7-9);
the    basal      laminae            and       extravascular           connective                     tissues       now      contain            more     concentrated                  HRP      than      do vascular          lumens.            VVO        vesicles        and      vacuoles             are
now     also     less     heavily          labeled        with      HRP        reaction               product         than      at 10 s; however,                   HRP-labeled                VVOs        continue          to demonstrate                 a clear     pathway            across          the
endothelial             cytoplasm,              interconnecting                the     blood            vascular          lumen          (L) with          tissue       fronts     (A,       B, C).      Even      at this     late      interval,          and    despite         the     continued
presence          of tracer           in the         vascular        lumen           (L),        an     interendothelial                     cell    junction          is devoid         of detectable                HRP     reaction            product         in its elongate,                narrow
mid-portion              [(A),      arrows];          however,         HRP           is focally             present          within          the    junction        at both        luminal         and      abluminal           ends       (A).      A portion          of the        VVO        labeled
in (A)        by an arrowhead                   is shown          at higher           magnification                   in (B).          Bars:        A, 700       nm;     B, 320          nm;     C, 240         nm.




long        known            that          with       each         passage              through                    capillary                 beds                formed            to the          size         criteria        required                for the          large           pore        sys-
a fraction               of circulating                         plasma           and plasma                            proteins     ex-                          tem. Studies      with circulating           FE suggested       that this tracer
travasates               into the tissues                         [24].        This fraction                          varies    in dif-                          extravasated      from capillaries            by means       of plasmalemmal
ferent     vascular                    beds,             is      regulated                  in     part by intra-                              and               vesicles     that shuttled       back     and forth across         the endothe-
extra-vascular                      pressure                  relationships,                     and is dependent                                on              hum from blood          to tissue     fronts    of endothelia       [29]. Others
the size and other                                properties        of the tracer        being     investi-                                                      were            not         convinced                that      these              vesicles             were             motile             or
gated.   Historically,                            physiologists         accounted       for such trans-                                                          formed   interconnections          that                               spanned    capillary                        endothelial
port   by postulating                                that      endothelial        cells     behaved         as                                                   cells from lumen          to ablumen                                   [30, 31].
semi-permeable               membranes            with       pores      of two sizes,       a                                                                        Initial electron    microscopic         studies                                         were not able to pro-
small      pore system         (4- to 9-nm           diameter)         and a large pore                                                                          vide a structural      correlate      of the small                                          pore system  that was
system        (25-     to 70-nm         diameter)         [25-27].         This proposal                                                                         acceptable       to all investigators.         Early                                         work with the rela-
stimulated         extensive      ulirastructural           investigations      aimed      at                                                                    tively small tracer,  HRP (-5-nm                                                 diameter),    suggested      that
elucidating          the anatomical            basis     of the postulated           small                                                                       the small pore system    corresponded                                                to the closely     apposed
and   large pores.                                                                                                                                               portions              of inter-endothelial                           junctions                and       did       not       involve
   The prominent                            60-80               nm plasmalemmal                                    and cytoplas-                                 endothelial          cell                 vesicles            [32]        .     More  recently,       however,
mic vesicles     [28]                       found             in many   capillary                               endothelia     con-                              other     investigators                      have           made              use of 2-nm       heme-peptide



1 12           Journal           of Leukocyte                    Biology             Volume              59,     January              1996
Fig.       13.     Comparison              of MVBs           (A, B) and             a YVO         (C) 30 mm         after     i.v.     injection         of tracer           FE.      In (A),        an MVB            (closed       arrowhead)              within       an endothelial
cell     from      a control          vessel       is packed       with           dense      FE    particles.      A coated            vesicle         (open     arrowhead)                 connects            with      the    plasma       membrane                 and     opens      to the
vascular          lumen          (L); it and the vascular                  lumen           also contain         FE particles.              This       section     was         not subjected               to staining            with     heavy          metals.       Basal     lemma,           B.
(B) MV (arrowhead)                      from a MOT tumor vessel endothelial                                     cell contains              strikingly          less FE than the MYB in (A). (C) illustrates                                              portions        of a YVO close
to the      vascular          lumen        (L) in tumor           vessel           endothelium.            FE    particles           are    present        in at least             four    of the       vesicles          comprising              this    YVO.        Note      the    striking
morphological               differences            between        the      MVBs           of(A)   VVO of(C). VVOs
                                                                                                   and     (B) and      the                                          are comprised                   ofclusters           ofinterconnected                   60-      to 80-nm         vesicles
and      larger        vacuoles        that are characterized         by stomata;  in contrast,  MVBs are comprised                                                   of single            vacuole-sized                  containers         (-380-            to 400-nm          diameter)
within        which        reside       multiple  smaller     vesicles     (-30-nm    diameter).  Bars: A, 182 nm;                                                B, 100             nm; C,          59 nm.




tracers  along with                            specimen            tilting     to demonstrate      that the                                           circulating   macromolecules         exit readily                                                    from tumor vessels
small pore system                              of muscle           capillaries       could    very well be                                            whose dimensions        and lining      endothelial                                                    cells more closely
at the level of the                            constrictions                  10-nm
                                                                           (-S-         diameter;    range,                                           resemble    venules      than  capillaries          [6,                                               15]. Second,    they
8-15             nm)      that      join        interconnecting                       plasmalemmal                    vesicles                        indicate               that,        despite              the        much          greater            cytoplasmic                  thick-
in capillary                     endothelium                   [20].        At        present,             therefore,                the              nesses            of       tumor          endothelial                      cells       compared                   with           normal
possible                relative           contributions                   of cell            junctions              [32]       and                   capillary  endothelium,       a somewhat                                                 similar             anatomic     path-
constrictions                    in      transcellular                  channels    [20] to the small                                                 way exists for transendothelial        cell                                            transport              of macromole-
pore system                    and       to the transport                  of macromolecules    across                                                cules;             i.e.,            a system                   of     interconnected                            yesicles              and
normal    capillary     endothelia     is not settled.                                                                                                vacuoles.      However,     in contrast    to the yesicles       of capillary
    Our findings      add several     new dimensions                                                      to this discus-                             endothelium,       which     tend to be individual         or clustered        in
sion.  First,     they support     further   our earlier                                                    findings   that                           groups     of two or three,     those found    in tumor     vascular     endo-




                                                                                                                                                  Dvorak        et al.         VVO:          ba.ia        of      tumor          vessel      hyperpermeability                               113
                                                                                                                                                           bathed             in interstitial               fluid         and        it is tempting               to speculate
                                                                                                                                                           that one               or more of the cytokines                             present          in tumor  intersti-
                                                                                                                                                           tial fluid              is involved in activating                           VVOs           so that they facili-
                                                                                                                                                           tate  the extravasation            of circulating         macromolecules.
                                                                                                                                                           Among    the many       tumor-secreted         cytokines,      only one,                                               vas-
                                                                                                                                                           cular           permeability                  factor          (VPF)            is    at    present          known             to
                                                                                                                                                           enhance     the permeability         of normal    vessels.      VPF is a po-
                                                                                                                                                           tent microvascular       permeabilizing        agent,    acting    at concen-
                                                                                                                                                           trations               below         1O’3M             and       with          a potency             some        50,000
                                                                                                                                                           times            that of histamine                        on a molar     basis                       [3, 4, 7, 12,
                                                                                                                                                           34-37].             Although   VPF                     possesses substantial                          vascular per-
                                                                                                                                                           meabilizing                    activity          and         is synthesized                 by       MOT        and        by
                                                                                                                                                           many other   tumor   cells,   both                                  in vitro          and in vivo, there                      is
                                                                                                                                                           at present no direct   evidence                                    indicating            that VPF regulates
                                                                                                                                                           VVO            function.   However,    immunocytochemical                                               localization
                                                                                                                                                           of VPF             to VYOs    in tumor    microvessels                                      strongly         suggests
Fig.        14.         Schematic                 diagram        showing          two VVOs               within          an endothelial
cell.      The        complex              trans-cytoplasmic                pathway            linking          the     vascular              lumen
                                                                                                                                                           that        this is a realistic                  possibility     [38].
with the endothelial                          cell basal           lamina       is illustrated                 in both;         one,        that      on          In     summary,     the                data     presented       here                  provide           new         in-
the       left,       opens          to     the     interendothelial                cell      junction            above            its      narrow         sights          into     the mechanisms        by which                              macromolecules                cross
mid-portion.                  Also         shown       within       some        vesicles          and      vacuoles             are       stomata          vascular               endothelium      to exit blood                               vessels.   Most           prior ul-
( small           circular       structures);               some       of these      exhibit            dense         central            knobs.
                                                                                                                                                           trastructural                   studies          of microvascular                          permeability               have
                                                                                                                                                           investigated                   capillaries.         The inherently                        low permeability                of
                                                                                                                                                           normal             capillaries             has     made            it difficult            to define           the    ana-
thelium                (and in normal                            venules)       are               organized                   into       much              tomical            pathways               of molecular                 extravasation,                even      with     the
larger              and more    complex                             structures,                   YVOs,                that           may in-              use         of labeled             tracers,  and                has      contributed      importantly        to
dude               as many                 as 20         individual               vesicles                and         vacuoles                    in a     the controversy                     and confusion                     regarding      the nature     of these
single               planar section.   The overall     size and complexity                                                                            of   pathways.                In the        present           study         it would           have       been      difficult
YVOs                 and the fact that,   in favorable     sections, they                                                                     have         to identify               YVOs    as a major                        pathway             for extravasation                 of
been found      to traverse      the entire     thickness           of the endothe-                                                                        circulating               macromolecular                          tracers            had we confined                    our
hum     suggest     that they       are neither         transient        nor motile;                                                                       attention               to normal             venules.                We       suggest,          therefore,           that,
rather,    they   likely    represent       long-lived,           fixed    structures                                                                      because                of their       high       intrinsic             level      of permeability,       tumor
specialized    for transendothelial           cell                                           transport.     In addition,                                   vessels             may          provide          an         attractive            model     for studies       of
the individual    vesicles     and vacuoles                                                  that together     comprise                                    macromolecular                         transcytosis                   and       one that will yield          in-
YVOs are, on average,           significantly                                                larger     than the caveo-                                    sights  that are                  useful         for a general                 understanding                  of vascu-
lae       described                       in capillary      endothelium                             [19, 20, 33].                                          lar permeability.
        Third,      the                   much      greater     transport                           of macromolecular
tracers    across   tumor   vessel                                     endothelium    compared      with the
endothelium       of comparably                                         sized venules   in normal     tissues                                              ACKNOWLEDGMENTS
of the same animal        suggests                                        that VVO transport    function      is
subject                 to regulation.                      Single-membrane                               diaphragms                          were
found               to close                 a fraction                of the         stomata                   interconnecting                                We thank       Peter    K. Gardner       for editorial     assistance        in
vesicles                with each other and the                                    plasma       membrane.                             Open-                the preparation          of the manuscript.         This    work      was sup-
ing and                 closing  of stomata and                                    interconnections                                between                 ported      by USPHS       NIH grants      AI-33372,       CA-50453,           and
abutting                 vesicles-vacuoles                             likely       provides                   an anatomic        ba-                      CA-58845        and      by the Beth       Israel    Hospital        Pathology
sis       for regulating                          VVO function.                   In support                    of this interpre-                          Foundation,      Inc.
tation,  it was not uncommon            to find tracer-filled         vesicles
luminal     to a closed   inter-vesicular       connection           with     ab-
sence   of tracer-labeled    vesicles      and vacuoles       distal      (ablu-                                                                           REFERENCES
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