Isolation, culture and identification of pulmonary arterial smooth muscle cells from rat distal pulmonary arteries

Abstract

The culture of pulmonary arterial smooth muscle cells (PASMCs) is one of the most powerful tools for exploring the mechanisms of pulmonary hypertension (PH). Both pulmonary vasoconstriction and remodeling occur predominantly in distal pulmonary arteries (PA). In this study, we provide our detailed and standardized protocol for easy isolation and culture of PASMCs from rat distal PA to supply every investigator with a simple, economical and useful method in studying PH. The protocol can be divided into four stages: isolation of distal PA, isolation of cells, growth in culture and passage of cells. Rat distal PASMCs were characterized by morphological activity and by immunostaining for smooth muscle α-actin and smooth muscle myosin heavy chain, but not for CD90/Thy-1 or von Willebrand factor. Furthermore, functional assessments were performed, confirming the presence of voltage-dependent Ca channels and physiological characteristic of response to hypoxia. In conclusion, we have developed a detailed and simple protocol for obtaining rat distal PASMCs. These PASMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to further explore the pathophysiological mechanisms of PH.

Long non‑coding RNA MALAT1 sponges miR‑124‑3p.1/KLF5 to promote pulmonary vascular remodeling and cell cycle progression of pulmonary artery hypertension

“…Using a light microscope, the distal PAs were microdissected from lung explant tissues in a dissection dish containing cold PBS. The separation steps were performed according to a previously published study (18). The HPASMCs were subsequently transferred to culture plates and incubated in SmGM-2 Smooth Muscle Growth medium-2 BulletKit media (Lonza Group, Ltd.) containing 10% (volume/volume) heat-inactivated FBS (Gibco; Thermo Fisher Scientific, Inc.), 2 ng/ml human recombinant fibroblast growth factor, 0.5 ng/ml human recombinant epidermal growth factor, 50 µ g/ml gentamicin and 5 µ g/ml insulin.…”

Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic‐to‐pulmonary shunt

“…As previously reported by Peng et al (24), distal pulmonary arteries (PAs) (greater than fourth generation) were dissected from lungs removed from healthy rats (8-wk-old male Sprague-Dawley rats) or rats from SOG, CSG-8W, and CSG-SL. After denuding from adventitia and endothelium, PASMCs were enzymatically digested from the tissue, harvested, and cultured in complete smooth muscle cell growth medium containing 2% fetal bovine serum (FBS), 1% smooth muscle cell growth supplement, and 1% penicillin-streptomycin solution at 37°C in a humidified atmosphere with 5% CO 2 -95% air.…”

“…For cell cycle analysis, PASMCs were harvested and fixed in 70% ethanol for at least 24 h at 4°C, then washed and incubated in 500 ml Rnase A of 200 mg/ml at 37°C for 20 min; after that, PI (25 mg/ ml) was added and the samples were analyzed on a fluorescenceactivated cell sorter (FACSCalibur; BD Biosciences, San Jose, CA, USA) within 3 h. Proliferation ELISA kit (11647229001; Roche) according to the manufacturer's instructions. Briefly, PASMCs were seeded in 96well plates at a density of 1.5 3 10 3 cells per well in 100 ml CSMCM, allowed to attach overnight, and then starved in basic smooth muscle cell medium (0.4% FBS) for 48 h. Thereafter, arresting PASMCs were stimulated with recombinant rat OPN (P08721; R&D Systems, Minneapolis, MN, USA) of different concentrations (0, 1, 2, 5, and 10 mg/ml; 0 mg/ml was control group) for 24 h or optimally proliferative concentration for different time intervals (12,24,48, and 72 h). BrdU (10 ml) was added to each well 6 h before the end of incubation, and then PASMCs were fixed with fixing solution, incubated with anti-BrdU antibody, and quantified by measuring the absorbance at 450 nm using a microplate reader (Mode 680; Bio-Rad).…”

Chronic hypoxia promoted pulmonary arterial smooth muscle cells proliferation through upregulated calcium‐sensing receptorcanonical transient receptor potential 1/6 pathway

“…As we previously described, 17,21 primary PASMCs were isolated from rat distal (>4th generation) intrapulmonary arteries and grown in DMEM supplemented with 10% FBS, 100 U ml −1 of streptomycin, and 0.1 mg ml −1 of penicillin. The cells at 40%-50% confluence were incubated in DMEM containing 0.5% FBS for 24 h. Chronic hypoxic and normoxic PASMCs were then respectively cultured for 60 h under 4% O 2 /5% CO 2 and 21% O 2 /5%CO 2 in DMEM containing 10% FBS at 37°C.…”