Isolation and In Vitro Culture of Vascular Endothelial Cells from Mice

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidate the disease mechanism. Although the mouse model has many advantages for in vivo and in vitro research, efficient procedures for the isolation and propagation of primary mouse EC have been problematic. We describe a high yield process for isolation and in vitro culture of primary EC from mouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle of Willis). Mouse arteries were carefully dissected without damage under a light microscope, and small pieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growth supplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetal calf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity of the cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized by a monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers for von-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Our simple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

Apoptosis and Inflammation Associated Gene Expressions in Monocrotaline-Induced Pulmonary Hypertensive Rats after Bosentan Treatment

BACKGROUND AND OBJECTIVES: Vascular wall remodeling in pulmonary hypertension can be caused by an aberration in the normal balance between proliferation and apoptosis of endothelial cell in the pulmonary artery. The objective of this study was to evaluate the effect of bosentan on apoptosis in monocrotaline (MCT)-induced pulmonary hypertension. MATERIALS AND METHODS: Sprague-Dawley rats were divided into three groups: control (C) group, M group (MCT 60 mg/kg) and B group (MCT 60 mg/kg plus bosentan 20 mg/day orally). Gene expressions of Bcl (B cell leukemia/lymphoma)-2, caspase-3, complement component (C)-6, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha) were analyzed by real time polymerase chain reaction and western blot analysis. RESULTS: The messenger ribonucleic acid (mRNA) expressions of caspase-3 and VEGF were significantly increased in the M group compared with the C group, and significantly decreased in the B group compared with the M group in week 4. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 in the B group compared with the M group. mRNA expression of TNF-alpha was significantly decreased on day 5 and in weeks 1 and 2 in the B group compared with the M group. CONCLUSION: Bosentan may have potential for preventing apoptosis and inflammation.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 microM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.