Arterial tracing-based technique for rapid purification of pulmonary arterial smooth muscle cells from rat lungs

Primary pulmonary arterial smooth muscle cells (PASMCs) of pre-capillary vessels are used to study the molecular mechanism of hypoxic pulmonary vasoconstriction (HPV). Cytochrome c oxidase subunit IV isoform 2 (COX4i2) is highly expressed in these PASMCs and an essential player in HPV, as its knockout abolishes the response. However, the exact molecular mechanism is not fully understood; therefore, fast and reliable methods to isolate PASMC from small pulmonary vessels that retain their phenotype during culture are essential. In the current study, we aimed to develop a fast and user-friendly method to isolate and culture homogeneous PASMCs from rat lungs. PASMCs were isolated utilizing agar mixed with a food-safe blue dye to delineate pulmonary arteries (PAs) within the lung, facilitating the tissue dissection process. Cox4i2 expression was determined by qPCR and mass spectrometry. Highly enriched populations of PASMCs expressing the classic smooth muscle cell markers α-SMA (alpha-smooth muscle actin) and SM-MHC (smooth muscle myosin heavy chain) were obtained for rat lungs. The cells were cultured up to passage 4, remained homogeneous, and maintained their identity. Additionally, the cells expressed Cox4i2 under normoxia and demonstrated an increase in Cox4i2 expression after exposure to chronic hypoxia, a known PASMC response. PASMCs were easily and rapidly obtained from rat lungs and provide a cell culture model to study mechanisms of HPV in which a key player, COX4i2, is retained during the cell culture process.