Cardiac microvascular endothelial cells express α-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 α-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 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 α-smooth muscle actin, show low-grade NOS III activity, and provide a suitable in vitro system for the study of endothelial pathophysiology.