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 -smooth muscle actin in culture and in myocardium, as confirmed by FACS. Of the primary MVEC, ~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 ~100% of aortic endothelial cells (AEC) expressed NOS III. In AEC, NOS III activity (pmol citrulline · mg protein¹ · min¹) 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 -smooth muscle actin, show low-grade NOS III activity, and provide a suitable in vitro system for the study of endothelial pathophysiology.

nitric oxide synthase; pig heart; aortic endothelium

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