title: Cardiac microvascular endothelial cells express alpha-smooth muscle actin and show low NOS III activity.
abstract: We established a culture system of porcine coronary microvascular endothelial cells (MVEC) with high cellular yield and purity >98%. Endothelial origin was confirmed by immunostaining, immunoblotting and fluorescence-activated cell sorter (FACS) analysis using low-density lipoprotein uptake, CD31, von Willebrand factor, and the lectin Dolichos biflorus agglutinin. MVEC were positive for alpha-smooth muscle actin in culture and in myocardium, as confirmed by FACS. Of the primary MVEC, approximately 30% expressed nitric oxide synthase (NOS) III in numbers decreasing from the first passage (6 +/- 1%) to the second passage (4 +/- 1%; P < 0.001 vs. primary isolates), whereas approximately 100% of aortic endothelial cells (AEC) expressed NOS III. In AEC, NOS III activity (pmol citrulline. mg protein-1. min-1) was 80 +/- 10 and was nearly abolished in the absence of calcium (5 +/- 1, P < 0.001). In primary MVEC, however, NOS III activity in the presence and absence of calcium was 20 +/- 4 and 25 +/- 5, respectively. We conclude that cardiac MVEC, in contrast to AEC, contain alpha-smooth muscle actin, show low-grade NOS III activity, and provide a suitable in vitro system for the study of endothelial pathophysiology.
mesh_terms: D000185:Actinin / Q000032:analysis / Q000276:immunology; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001011:Aorta / Q000737:chemistry / Q000201:enzymology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000737:chemistry / Q000201:enzymology; D003893:Desmin / Q000032:analysis / Q000276:immunology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000201:enzymology*; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008833:Microcirculation / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis / Q000276:immunology; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D013552:Swine; D014335:Tropomyosin / Q000032:analysis / Q000276:immunology; D014746:Vimentin / Q000032:analysis / Q000276:immunology; D014841:von Willebrand Factor / Q000032:analysis / Q000276:immunology