348
Augusto C. Montezano et al.
times in order to a successful primary culture. Concentrations and incubation times suggested here were optimized for murine aortas. 6. After the cells are attached and growing, one should proceed with the endothelial cell characterization to ensure that the cells obtained are positive for endothelial cell markers, such as platelet endothelial cell adhesion molecule (PECAM) and von Willebrand’s factor (vWF). 7. To ensure that the phenotype of cells is maintained with passaging, it is important to characterize cells at all passages. At some point in passaging (usually around passage 6–7 in our experience), the phenotype changes and expression of receptors and ion channels change. We suggest that cells beyond this point of passage are not used for further experiments. References 1. Mensah GA (2007) Healthy endothelium: the scientific basis for cardiovascular health promotion and chronic disease prevention. Vascul Pharmacol 46(5):310– 314 2. Yetik-Anacak G, Catravas JD (2006) Nitric oxide and the endothelium: history and impact on cardiovascular disease. Vascul Pharmacol 45(5):268–276 3. Manconi F, Markham R, Fraser IS (2000) Culturing endothelial cells of microvascular origin. Methods Cell Sci 22:89–99 4. Michiels C (2003) Endothelial cell functions. J Cell Physiol 196:430–443 5. Sumpio BE, Riley JT, Dardik A (2002) Cells in focus: endothelial cell. Int J Biochem Cell Biol 34(12):1508–1512 6. Vita JA, Keaney JF (2002) Endothelial function: a barometer for cardiovascular risk? Circulation 106:640–642
7. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373–376 8. Forstermann U, Munzel T (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113:1708–1714 9. Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH (2002) EDHF: bringing the concepts together. Trends Pharmacol Sci 3:374–380 10. Garlanda C, Dejana E (1997) Heterogeneity of endothelial cells specific markers. Arterioscler Thromb Vasc Biol 17:1193–1202 11. Suh SH, Vennekens K, Manolopoulos VG, Freichel M, Schweig U, Prenen J, Flockerzi V, Droogmans G, Nilius B (1999) Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling. Eur J Physiol 438:612–620
