Effects of nifedipine on endothelial function and endothelial

Document Sample
Effects of nifedipine on endothelial function and endothelial Powered By Docstoc
					                            Online data supplement

                Nifedipine Improves Endothelial Function:

                   Role of Endothelial Progenitor Cells

         Tomonori Sugiura,1,2,3 Takahisa Kondo,1 Yasuko Kureishi-Bando,1

Yasushi Numaguchi,1 Osamu Yoshida,1 Yasuaki Dohi,2             Genjiro Kimura,2

            Ryuzo Ueda,3 Ton J. Rabelink,4 and Toyoaki Murohara,1

    Department of Cardiology, Nagoya University Graduate School of Medicine,

 Department of Cardio-Renal Medicine and Hypertension, and 3Department of

    Internal Medicine and Molecular Science, Nagoya City University Graduate

                    School of Medical Science, Nagoya, Japan

     Department of Nephrology and Hypertension, Leiden University Medical

                         Center, Leiden, The Netherlands

Address for correspondence:
Toyoaki Murohara, MD, PhD, FAHA, or Takahisa Kondo, MD, PhD.
Department of Cardiology, Nagoya University Graduate School of Medicine, 65
Tsurumai, Showa-ku, Nagoya 466-8550, Japan
Phone: +81-52-744-2149; Fax: +81-52-744-2138

E-mail: murohara@med.nagoya-u.ac.jp or takahisa@med.nagoya-u.ac.jp
Online supplement

Materials and methods

Quantification of Circulating CD34+CD133+ Progenitor Cells and

Endothelial Progenitor Cells

Circulating CD34+CD133+ mononuclear cells were defined as circulating

progenitor cells (CPCs) and were quantified by flow cytometry as described

previously. Endothelial progenitor cells (EPCs) were isolated from hypertensive

subjects and analyzed by the cell culture method as reported previously.1

Isolated EPCs were characterized as spindle-shaped adherent cells that showed

uptake of DiI-acetylated LDL (Molecular Probes, Oregon) and binding of

FITC-UEA-1 lectin (Sigma, St. Louis, MO). Double positive adherent cells were

visualized by fluorescence microscopy (Biozero BZ-8000; Keyence, Osaka,

Japan) and the number of these cells was counted in 5 randomly selected

microscopic fields. To confirm whether or not these adherent cells had an

endothelial phenotype, expression of endothelium-specific surface markers such

as CD31 and vascular endothelial growth factor receptor 2 (VEGFR2 or Flk-1)

was assessed by immunocytochemistry and by the reverse

transcription-polymerase chain reaction (RT-PCR).

Biochemical Analysis

Peripheral blood (10 ml) was collected from patients before and after 4 wks of

nifedipine treatment. The serum malondialdehyde-LDL (MDA-LDL) level was

measured by ELISA according to the manufacturer’s instructions (Daiichi, Tokyo,


Nifedipine Preparation

Nifedipine was kindly provided by Bayer Japan Co., Ltd. (Osaka, Japan). It was

dissolved in a solvent (15% ethanol, 15% polyethylene glycol 400, and 70%

distilled water) in a darkened room.3 The final concentration of the solvent was

< 1% (vol/vol %).

Reverse Transcriptation-Polymerase Chain Reaction

Total RNA was extracted from EPCs and subjected to RT-PCR analysis as

previously described.4 The primer sets for endothelial nitric oxide synthase

(eNOS), VEGFR2, and CD31 were as follows,









The expected PCR products for eNOS, VEGFR2, CD31, and GAPDH had a size

of 422 bp, 802 bp, 700 bp, and 557 bp, respectively.

Detection of Oxidative Stress

Oxidative stress was detected by an intracellular reactive oxygen species (ROS)

assay using the fluorescent dye 5-6-chloromethyl-2’,

7’-dichlorodihydrofluorescein diacetate (Molecular Probes, Oregon).5 Isolated

EPCs were loaded with the dye and changes of fluorescence were examined

using fluorescence microscopy and a fluorescent plate reader (Fluoroskan

Ascent FL; Labsystems, Helsinki, Finland) with excitation and emission at

wavelengths of 485 nm and 527 nm.

Cell Proliferation and Viability Assay

Proliferation and viability of EPCs were analyzed using a previously validated

colorimetric [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium] (MTS) assay with the electron coupling reagent

phenazine methosulfate (Cell Titer 96 AQ; Promega, Madison, Wisconsin)

according to the manufacturer’s protocol.4

Detection of Apoptosis

Apoptotic cell death was detected using a terminal dUTP nick end labeling

(TUNEL) assay kit (In situ cell death detection kit; Roche, Basel, Switzerland)

according to the manufacturer’s instructions.5 TUNEL-positive cells and all

cells were counted in 5 randomly selected fields, and the results were expressed

as the ratio of TUNEL-positive cells to total cells.

Cell Migration Assay

The migratory activity of EPCs was assessed using a modified Boyden chamber

apparatus (Neuroprobe, Gaithersburg, MD), as described previously.4 Briefly,

culture medium (25 l) supplemented with 1% fetal bovine serum (FBS) and

nifedipine and/or recombinant vascular endothelial growth factor (VEGF)

(rhVEGF; R&D systems, Mineapolis) was placed in the lower chamber of the

apparatus. After a polyvinylpyrolidone-free polycarbonate-coated filter was

placed upon the lower chamber, EPCs suspended in 50 l of culture medium

containing 1% FBS and nifedipine, were placed in the upper chamber, and

incubated for 4 hours at 37°C in a humidified incubator. After removal of

non-migrated EPCs, the filters were fixed with methanol and stained with

May-Gruenwald’s solution (MERCK, Darmstadt, Germany) and Giemsa’s stain

solution (Sigma, St. Louis, MO). Then the number of migrating EPCs was

counted in 5 randomly selected microscopic fields. All experiments were

performed in triplicate.


1. Kondo T, Hayashi M, Takeshita K, Numaguchi Y, Kobayashi K, Iino S, Inden

Y, Murohara T. Smoking cessation rapidly increases circulating progenitor cells

in peripheral blood in chronic smokers. Arterioscler Thromb Vasc Biol.


2. Kitano S, Kanno T, Maekawa M, Sakurabayashi I, Kotani K, Hisatomi H, Hibi

N, Kubono K, Harada S. Improved method for the immunological detection of

malondialdehyde-modified low-density lipoproteins in human serum. Analytica

Chimica Acta. 2004;509:229-235.

3. Kitakaze M, Asanuma H, Takashima S, Minamino T, Ueda Y, Sakata Y,

Asakura M, Sanada S, Kuzuya T, Hori M. Nifedipine-induced coronary

vasodilation in ischemic hearts is attributable to bradykinin- and NO-dependent

mechanisms in dogs. Circulation. 2000;101:311-317.

4. Lee M, Aoki M, Kondo T, Kobayashi K, Okumura K, Komori K, Murohara T.

Therapeutic angiogenesis with intramuscular injection of low-dose recombinant

granulocyte-colony stimulating factor. Arterioscler Thromb Vasc Biol.


5. Brunt KR, Fenrich KK, Kiani G, Tse MY, Pang SC, Ward CA, Melo LG.

Protection of human vascular smooth muscle cells from H2O2-induced apoptosis

through functional codependence between HO-1 and AKT. Arterioscler Thromb

Vasc Biol. 2006;26:2027-2034.


Shared By: