METHODS
Rat carotid artery angioplasty model
Angioplasty was performed as previously described.1 Briefly, male Wistar rats (350-375g,
Harlan) were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg, i.p.) A 2-F
Fogarty balloon catheter (Baxter, Irvine, CA) was inserted into the left common carotid
through the left external carotid artery and angioplasty performed by retracting the
catheter with an inflated balloon 3 times. Upon catheter removal, the left external carotid
artery ligated, and blood flow to the common and internal carotid arteries restored. As
controls, intact un-operated and sham-operated rats were used. The sham operation
consisted of the same procedure as angioplasty, including tying off the external carotid
artery, but without insertion of the catheter and angioplasty itself. As controls, intact un-
operated and sham-operated rats were used, as indicated. To test the effect of NPY on
medial hypertrophy, in a separate group of rats (n=6), an NPY pellet (10 g/14 days) was
placed on the side of an un-injured artery for 14 days. Following surgery, rats were
returned to their cages with free access to food and water. All procedures were approved
by the Animal Care and Use Committees of Georgetown University and conducted in
according to the National Institutes of Health guidelines.
Drug delivery
The Y1 (H409/22 acetate) and Y5 antagonist (CGP71683A) were gifts from AstraZeneca,
Sweden, and their structures are identical to BIBP32232 and to the Novartis compound3,
respectively. Their specificity for Y1 and Y5 receptors, respectively, has been
established previously in many studies.2-4 Antagonists (60 mg/ml) or vehicle (in 60%
PEG-400, n=6 each group), were delivered at 0.02 mol/kg/min/14 days into the right
jugular vein via osmotic minipumps (Alzet Durect Corporation, Cupertino, CA)
implanted subcutaneously. At these concentrations (0.1 M), both Y1 and Y5
antagonists blocked proliferation of rat aortic VSMC in response to 0.01 M NPY in
vitro. 5
NPY- (1-10 g/pellet/14 days) or placebo-containing pellets (Innovative
Research of America, Sarasota, FL) (n=6 each) were placed during angioplasty laterally
at the site of the left carotid artery and secured in place with a neighboring muscle.
Measurement of neointima and medial thickening
After 14 days, in re-anesthetized and artificially respired rats, hearts were perfused at 100
mmHg with saline to clear them of circulating blood, and then with 4%
paraformaldehyde in saline to fix the carotid arteries (common left and right). Tissue
slices were paraffin-embedded and stained with hematoxylin-eosin (H-E) or Masson’s
Trichrome. Neointimal and medial thickening was measured as areas from slides stained
with either H-E or Masson’s Trichrome (which allows for better visualization of internal
and external laminas as borders). Intima was calculated as the area luminal from the
internal lamina, and media as area between external and internal lamina. Each area was
averaged from 15 sections cut along the length of the common carotid artery
(approximately 1 cm), and imaged using the Optimax-Nikon computer system with NIH
imaging software (developed at the U.S. National Institutes of Health and available on
the Internet at http://rsb.info.nih.gov/nih-image/).
2
Histo- and immunocytochemistry
Tissue sections were de-paraffinized and stained with primary antibodies against
endothelial markers: cd31 (Santa Cruz Biotech., Santa Cruz, CA) and von Willebrand
factor (vWf, Novocastra Laboratories Ltd, UK), macrophage scavenger receptor cd68
(Dako, Carpinteria, CA), hyaluronan (HA, kindly provided by Dr. C. Underhill,
Georgetown University), a marker for matrix deposition.6 Additionally, sections were
stained with Red Oil O (Sigma, St. Louis, MO) to identify lipid deposition and Masson’s
Trichrome to identify matrix and neovascularization.
RT-PCR analysis
Both common carotid arteries were harvested at 0, 6 and 24 hrs and 14 days after
angioplasty (n=6 each) and samples immediately snap frozen. Total RNA was isolated
using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio) and cDNA
synthesized with random hexamer and MMLV reverse transcriptase (Stratagene, La Jolla,
CA). 18s rRNA served as an internal control (Ambion Inc., Austin, TX) but amplified for
20 cycles, due to the overabundance of this RNA relative to the mRNA in question. The
NPY/NPY receptor primer sequences were as follows: Y1 (5’-CTC TTG CTT ATG GRG
ATG TGA-3’, 5’-CTG GAA GTT TTT GTT CAG GAA YCC A-3’); Y2 (5’-CCT ACT
GCT CCA TCA TCT TGC-3’, 5’-GTA GTT GCT GTT CAT CCA GCC-3’); Y5 (5’-
ATG GAG TTT AAG CTT GAG GAG C-3’, 5’-TGT GTA GGC AGT GGA TAA
GGG-3’); DPPIV (5’-GTC CTG GAG GAC AAT TCT GC-3’, 5’-TGG AGA TCT GAG
CTG ACT GC-3’); NPY (5’-TAC CCC TCC AAG CCG GAC AA-3’, 5’-TCT CAT
TTC CCA TCA CCA CAT G-3’). PCR was performed as described7 using Taq DNA
polymerase (Promega, Madison, WI) in the following conditions: 94°C for 1 min, 52°C
3
for 1 min, 72°C for 1.5 min, 35 cycles. After electrophoresis on a 2% agarose gel, the
products were visualized by ethidium bromide staining.
Western blotting analysis
Carotid arteries were harvested at time 0 (from un-operated intact and sham-operated rats)
and at 24 hrs after angioplasty. They were then homogenized in RIPA buffer containing
10% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, PMSF 0.5 l/ml, Leupeptin
10l/ml, Antipain 5l/ml, Aprotinin 1l/ml in 1x PBS, centrifuged two times and
supernatants from the second centrifugation collected for Western blotting. The protein
samples (15 g) were resolved on a 4-20% Tris-Glycine gel (Novex, San Diego, CA) and
transferred onto a nitrocellulose membrane. The blots were blocked with 5% nonfat milk
in TBST buffer (50 mM Tris buffered saline with 0.05% Tween 20, pH 8.0) at 4C
overnight and incubated with rabbit polyclonal anti-human Y1, Y2 or Y5 antibodies (8
g/ml, AstraZeneca, Sweden). The antibodies were selected based on their selectivity
using cells transfected with the appropriate receptors, as performed and provided by
AstraZeneca. The membranes were then washed with TBST buffer and incubated with
horseradish peroxidase conjugated anti-rabbit IgG antibody (1:2000, Amersham
Pharmacia Biotech, NJ). The signal was detected using ECL reagents (Amersham
Pharmacia Biotech, NJ) and visualized by autoradiography.
Measurement of NPY-immunoreactivity (-ir)
NPY-ir was measured using ELISA (Peninsula Lab, CA)8, 9 in platelet-poor-plasma (PPP)
and platelet-rich plasmas (PRP), carotid arteries, and neighboring skeletal muscle,
4
derived from 3 groups of rats: intact rats and rats 14 days after angioplasty, with and
without NPY pellet. Tissues were harvested after the circulation was cleared of blood by
perfusing with heparinized saline at 100 mm Hg pressure, similarly to the procedure for
the morphometric studies; this minimized the effect platelet NPY content8, 9 on tissue
peptide levels. Plasma NPY levels achieved by local administration of the NPY pellet
were additionally compared with those achieved by exposure to cold stress. A separate
group of rats was instrumented with indwelling femoral arterial catheters (24 hrs prior to
testing) and exposed to a 2-hour stress consisting of placing the rats in 1-cm 4oC water, as
previously described.9 Blood was collected from stressed rats while they were conscious
and from control intact or subjected to angioplasty while they were anesthetized.
Collection was made by a free flow from the femoral artery into tubes containing 1.6
mg/ml EDTA and 50 units/ml heparin PRP and PPP were prepared by sequential
centrifugation at 380 g for 6 min and 10,000 g for 2 min, respectively.8 Tissue were
prepared by boiling in 1 M acetic acid followed by homogenization, sonication and
centrifugation (20,000 g/60 min at 4 oC), then dried and stored at -80 oC until assayed.
Statistical analysis
Results were analyzed by one- and two-way analysis of variance and where appropriate, a
post hoc Dunnett’s t-test was used. Data are presented as mean SEM for indicated
number of repetitions and were considered significant at p < 0.05.
5
REFERENCES
1. Jenkins GM, Crow MT, Bilato C, Gluzband Y, Ryu WS, Li Z, Stetler-Stevenson
W, Nater C, Froehlich JP, Lakatta EG, Cheng L. Increased expression of
membrane-type matrix metalloproteinase and preferential localization of matrix
metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries.
Circulation. 1998;97:82-90.
2. Doods HN, Wienen W, Entzeroth M, Rudolf K, Eberlein W, Engel W, Wieland
HA. Pharmacological characterization of the selective nonpeptide neuropeptide Y
Y1 receptor antagonist BIBP 3226. J Pharmacol Exp Ther. 1995;275:136-142.
3. Criscione L, Rigollier P, Batzl-Hartmann C, Rueger H, Stricker-Krongrad A,
Wyss P, Brunner L, Whitebread S, Yamaguchi Y, Gerald C, Heurich RO, Walker
MW, Chiesi M, Schilling W, Hofbauer KG, Levens N. Food intake in free-
feeding and energy-deprived lean rats is mediated by the neuropeptide Y5
receptor. J Clin Invest. 1998;102:2136-2145.
4. Zukowska-Grojec Z, Marks ES, Haass M. Neuropeptide Y is a potent
vasoconstrictor and a cardiodepressant in rat. Am J Physiol. 1987;253:H1234-
1239.
5. Pons J, Kitlinska J, Ji H, Lee EW, Zukowska Z. Neuropeptide Y and vascular
growth: role of Y1 and Y5 receptors and adrenergic activation. Can. J. Phys.
Pharm. 2003;81:177-185.
6. Underhill CB, Zhang L. Analysis of hyaluronan using biotinylated hyaluronan-
binding proteins. Methods Mol Biol. 2000;137:441-447.
6
7. Zukowska-Grojec Z, Karwatowska-Prokopczuk E, Rose W, Rone J, Movafagh S,
Ji H, Yeh Y, Chen WT, Kleinman HK, Grouzmann E, Grant DS. Neuropeptide Y:
a novel angiogenic factor from the sympathetic nerves and endothelium. Circ Res.
1998;83:187-195.
8. Myers AK, Farhat MY, Vaz CA, Keiser HR, Zukowska-Grojec Z. Release of
immunoreactive-neuropeptide by rat platelets. Biochem Biophys Res Commun.
1988;155:118-122.
9. Zukowska-Grojec Z, Dayao EK, Karwatowska-Prokopczuk E, Hauser GJ, Doods
HN. Stress-induced mesenteric vasoconstriction in rats is mediated by
neuropeptide Y Y1 receptors. Am J Physiol. 1996;270:H796-800.
7