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.

To the Editor: Patients with chronic heart failure (CHF) due to left ventricular (LV) systolic dysfunction develop skeletal muscle alterations that contribute to lower peak aerobic capacity (1). Patients with type 2 diabetes develop skeletal muscle alterations similar to those of patients with CHF (2). To test the hypothesis that diabetes may further reduce peak aerobic capacity in patients with CHF, we prospectively measured peak oxygen uptake (peak VO 2 ) in 156 diabetic and nondiabetic patients with CHF who were matched for age and gender.A total of 156 patients met inclusion/exclusion criteria and agreed to participate in the study. We first identified 204 patients with CHF and diabetes among the 689 patients with LV ejection fraction Ͻ40% in our CHF clinic and then attempted to match these 204 diabetic patients for age and gender to the 485 remaining nondiabetic patients. We found age and gender matches for 106 of the 204 patients. Informed consent and complete data were obtained in 78 of the 106 matched patients. Inclusion criteria included: steady clinical state, ability to perform a maximal exercise test, and therapy consistent with current CHF guidelines. Exclusion criteria were: exertional angina or arrhythmias; systolic or diastolic blood pressure Ͼ160 and 90 mm Hg, respectively; joint, pulmonary, or peripheral arterial disease; participation in a training program; and active tobacco use. Glycosylated hemoglobin was measured in all patients. They had all undergone coronary angiography. Chronic kidney disease was defined by a creatinine clearance Ͻ50 ml/min (3). All patients were familiar with exercise testing and measurement of expired gas.Patients who met inclusion and exclusion criteria underwent evaluation of baseline physical activity. Plasma B-type natriuretic peptide (BNP) level and peak VO 2 were determined within one week of evaluation of physical activity. The average number of daily steps was used to quantify baseline physical activity (4). It was recorded daily and averaged over seven consecutive days. Ejection fraction was assessed by echocardiography. Plasma levels of BNP were measured using the triage immunoassay (Biosite Inc., San Diego, California). Peak VO 2 was measured during a symptomlimited treadmill exercise test. Patients who discontinued exercising for other than shortness of breath and fatigue or did not reach a respiratory exchange ratio Ͼ1.0 were excluded. Peak VO 2 was compared in diabetics and nondiabetics using the t test for two independent samples. Multiple linear regression analysis was performed to assess the effect of diabetes on peak VO 2 controlling for hypertension, coronary artery disease (CAD), chronic kidney disease, BNP plasma level, LV ejection fraction, physical activity, and glycosylated hemoglobin. We aimed to have a minimum of 15 patients for each parameter that we anticipated to include in our regression model.Baseline characteristics and medications are summarized in Table 1. Hypertension and CAD were more prevalent in diabetics than in nondiabetics. Pla...