100 mm dishes

We isolated CFs from the hearts of six 2–3-day-old Wistar rats. Briefly, hearts were harvested and cut into 1 mm² small pieces. The tissues were digested with 0.05% (w/v) tryptase (Gibco, Amarillo, TX, USA) and 0.05% (w/v) collagenase II (Worthington, Lakewood, NJ, USA) at 37 °C with gentle agitation. After 5 digestions, the supernatants were pooled and centrifuged for 5 min at 1,000 rpm. The cell suspensions were filtered through a 100 µm cell strainer and plated in 100 mm dishes (Eppendorf, Hamburg, Germany) with Dulbecco’s modified Eagle medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin for 2 h, and then the medium was replaced. The attached cells were considered to be CFs, which were identified by discoidin domain receptor 2 (DDR2) immunostaining. Cells before passage 5 were used for further study.

VSMCs were isolated from 6-week-old male Sprague-Dawley rats and grown in 100 mm dishes (Eppendorf) with Dulbecco’s modified eagle’s medium (DMEM) (LM001-05; Welgene) and 10% Hyclone fetal bovine serum (FBS) (GE Healthcare Life Sciences) up to passages 2–7. The rat VSMC cell line, A10 (CRL-1476; ATCC), was cultured in DMEM (LM001-05) with 10% FBS (Welgene). Human coronary artery smooth muscle cells (SMCs) were grown in medium 231 and SMC growth or differentiation supplement and were used at passages 4–8. The animal experiment was conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). All animal experiment protocols were reviewed and approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee.