Endothelial cell basal medium

An in vitro scratch assay with HAECs and HASMCs was performed as described previously (Liang, Park, & Guan, 2007). Cells were seeded in six‐well cell culture plates and left to adhere until confluent in usual cell growth media. For the HAEC experiments, cells were then sustained with endothelial cell basal medium (PromoCell) without growth or supplement factors but containing 10% FBS (F9665, Sigma‐Aldrich) to allow cell survival but limiting proliferation. For the HASMC experiments, once confluent, cells were switched to smooth muscle cell basal medium (PromoCell) without growth or supplement factors but supplemented with 0.1% FBS. After 24 hr, a scratch was marked on each well with a P200 pipette tip, washed with PBS, and replaced with media alone, or media containing ucOCN (10 ng/ml). Images of the scratch were captured at baseline and at various time points to monitor cell migration. The area of the scratch wound at baseline and at each time point was measured using the ImageJ software.

Normal Rat Kidney (NRK; Leibniz Institute DSMZ GmbH, Germany) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L D-glucose and 3.7 g/L NaHCO₃ supplemented with 5% (v/v) fetal calf serum (Biochrom, Germany), 2 mM L-glutamine, 100 µg/mL penicillin and 100 µg/mL streptomycin. RAT1 rat fibroblasts were purchased from DSMZ (Germany) and cultured in high glucose DMEM as detailed above supplemented with 10% fetal calf serum (Gibco, Life Technologies, USA). Human Umbilical Vein Endothelial Cells (HUVEC; PromoCell, Germany) were cultured in Endothelial Cell Basal Medium supplemented with Endothelial Cell Growth Medium Supplement Mix (both from PromoCell). The cells were cultured until reaching 80–90% confluency before subculture. Routinely, cell culture substrates were not pre-coated with adhesive proteins except for HUVEC cells. HUVECs were grown in culture dishes coated with 0.45 µg/cm² collagen IV (mouse, purchased from Corning, USA).

Endothelial cell basal medium and pericyte growth medium were purchased from PromoCell (Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM) low glucose was from Lonza (Cologne, Germany). Human TNF-α was from Provitro (Berlin, Germany). MA was purchased from Santa Cruz Inc. (Heidelberg, Germany). Fluorescein isothiocyanate-labeled dextran 150,000, rhodamine 6 G and peroxidase-labeled-streptavidin were purchased from Sigma-Aldrich (Munich, Germany). Mayer’s hemalaun solution was from Merck (Darmstadt, Germany). 3-Amino-9-ethylcarbazole was obtained from Abcam (Cambridge, UK). Ketamine (Ursotamin) was from Pharmacia GmbH (Erlangen, Germany) and xylazine (Rompun) was from Bayer (Leverkusen, Germany).

N-Acetyl-d-penicillamine (NAP), sodium nitrate, vanadium (III) chloride (VCl₃), sulfanilamide, N-(1-naphthyl) ethylene diamine dihydrochloride (NEDD), PCL (Mn = 80,000), BioUltra grade PEG (Mn = 4000), dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium were purchased from Sigma-Aldrich (St. Louis, MO). Poly caprolactone (PCL) filament (Mn ≈ 100,000 g/mol, white, diameter 1.75 mm) under the commercial name Resomer^® C was supplied by Evonik Corp. (USA). Hydroxyl (OH) functionalized multiwalled carbon nanotubes (MWCNTs, OD: 8–15 nm, L: 10–50 µm) were purchased from IoLiTec (Germany). Sulfuric acid (H₂SO₄), hydrochloric acid (HCl), ortho-phosphoric acid (H₃PO₄), methanol (MeOH), tetrahydrofuran (THF), toluene, tryptic soy broth (TSB), Luria broth (LB), and sodium nitrite (NaNO₂) were obtained from Merck (Darmstadt, Germany). Endothelial cell basal medium, FCS and supplement pack endothelial cell obtained from PromoCell (Heidelberg, Germany). Calcium and magnesium free Dulbecco’s phosphate buffered saline (PBS, pH 7.4), Dulbecco’s modified Eagle’s medium (DMED), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Scotland, UK). Live/Dead viability/cytotoxicity kit, Alexa Fluor™ 594 Phalloidin, and DAPI were supplied by Thermo Fisher, Invitrogen™ and BD Pharmingen, respectively.

HUVEC (Promocell, Heidelberg, Germany) were cultured on gelatin-coated tissue culture plastics with endothelial cell basal medium (Promocell, Heidelberg, Germany), supplemented with 10% foetal calf serum, 4 μl/ml endothelial cell growth supplement, 0.1 ng/ml human epidermal growth factor, 1 ng/ml human basic fibroblast growth factor, and 1 μg/ml hydrocortisone (all Promocell, Heidelberg, Germany). Media were replaced every two days and cells subcultured just before confluence. HUVEC used in experiments were between passages 3-5. For co-culture of HUVEC and SMC, HUVEC (as above) and Human Vascular Smooth Muscle Cells (Promocell, Heidelberg, Germany) were seeded at a 50:50 ratio into 8-chamber slides, 1 × 10⁵ cells per 400 μl per chamber in media as above. Cultures grew for 3 days.