Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium. J Appl Physiol 92: 1331-1338, 2002; 10.1152/japplphysiol.00680. 2001.-Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans. vascular biology VASCULAR ENDOTHELIAL DYSFUNCTION plays a major role in the pathogenesis of metabolic and cardiovascular diseases. Indirect measurement of reduced nitric oxide (NO) availability in the coronary or in the peripheral circulation is the most frequently assessed parameter of vascular endothelial dysfunction in patients with hypercholesterolemia, diabetes mellitus, hypertension, coronary artery disease, and chronic heart failure (CHF) (1,16,21,22,24,29). However, the vascular endothelium mediates several other physiological and pathological processes besides NO-mediated control of the vasomotor tone. Inflammation, hemostasis, and angiogenesis are all modulated by the vascular endothelium through transitions between quiescent and activated states (30). These nonvasomotor functions of the vascular endothelium are not routinely characterized in patients, primarily because of limited access to the vascular endothelium.Thus the aim of the present investigation was to develop a novel approach to further characterize the vascular endothelial abnormalities that accompany metabolic and cardiovascular diseases. A new minimally invasive technique was designed to safely collect 200-1,000 endothelial cells from either a superficial forearm vein or from the radial artery in human subjects. Measurements of protein expression were performed using quantitative immunofluorescence analysis, an innovative technique that requires only a small number of endothelial cells to accurately quantify intracellular protein levels. Because of our interest in the cellular mechanisms of endothelial dysfunction in patients with CHF, we initially applied this novel approach to quantify oxidative stress by nitrotyrosine formation, endothelial cell activation by nuclear factor-B (NF-B) nuclear translocation and cyclooxygenase-2 (COX-2) expression, and NO synthesis by endothelial NO synthase (eNOS) expression in the vascular endothelium of pati...
Clinical decompensation in CHF is associated with activation of the venous endothelium. Return to a compensated state after short-term inotropic therapy results in a significant reduction in endothelial nitrotyrosine formation, COX-2, and iNOS expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.