Effects of nifedipine on endothelial function and endothelial

							                            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

 1
    Department of Cardiology, Nagoya University Graduate School of Medicine,

2
 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

     4
     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).




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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,


Japan).2




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,


eNOS;          5’-GTGATGGCGAAGCGAGTGAAG-3’




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               3’-CAAGACACACAAGCCCGAGCC-5’


VEGFR2         5’-CTGACCTTGGAGCATCTCATC-3’


               3’-CACCATACCAAGAACGGAGTC-5’


CD31           5’-GCTGTTGGTGGAAGGAGTGC-3’


               3’-CTCGTGGAGGTCGGTTGAAG-5’


GAPDH          5’-CTTCACCACCATGGAGGAGG-3’


               3’-CCACCAGAGGAGACTGAAGT-5’.


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.




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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




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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.



References

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.


2004;24:1442-1447.


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



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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.


2005;25:2535-2541.


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.




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