Geert W. Schmid-Schönbein

In vivo evidence for microvascular oxidative stress in spontaneously hypertensive rats. Hydroethidine microfluorography

The factors that predispose to the accelerated organ injury that accompanies the hypertensive syndrome have remained speculative and without a firm experimental basis. Indirect evidence has suggested that a key feature may be related to an enhanced oxygen radical production. The purpose of this study was to refine and use a technique to visualize evidence of spontaneous microvascular oxidative stress in vivo in the spontaneously hypertensive rat (SHR) compared with its normotensive control, the Wistar-Kyoto rat (WKY). We investigated the effects of adrenal glucocorticoids on the microvascular oxidative stress sequence. The mesentery was superfused with hydroethidine, a reduced, nonfluorescent precursor of ethidium bromide. In the presence of oxidative challenge, hydroethidine is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by virtue of its red fluorescence. The fluorescent light emission from freshly exteriorized and otherwise unstimulated mesentery microvessels was recorded by digital microscopy. The number of ethidium bromide-positive nuclei along the arteriolar and venular walls in SHR was found to be significantly increased above the level exhibited by WKY. The elevation in ethidium bromide fluorescence in SHR arterioles could be attenuated by a synthetic glucocorticoid inhibitor and in rats subjected to adrenalectomy. The administration of glucocorticoids after adrenalectomy by injection of dexamethasone restored the oxidative reaction in SHR arterioles. Treatment with dimethylthiourea and with a xanthine oxidase inhibitor attenuated the superoxide formation. Although a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) enhanced the ethidium bromide staining in WKY, it did not affect that in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Passive mechanical properties of human leukocytes

Micropipette experiments are used to determine the rheological properties of human leukocytes. Individual cells in EDTA are subjected to a known aspiration pressure via a micropipette, and their surface deformation from the undeformed spherical shape is recorded on a television monitor. The cells are mathematically modeled as homogeneous spheres, and a standard solid viscoelastic model is found to describe accurately the deformation of the cell for small strains. These experimental and theoretical studies provide the basis for further investigations of leukocyte rheology in health and disease.

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension

Background Oxygen free radicals may play roles in hypertension both in arteriolar constriction and in formation of lesions. Objective To quantify free radical production in blood plasma of genetic hypertensives. Design Hydrogen peroxide levels were measured, because it is one of the most stable reactive oxygen species. Methods An electrode technique was used to determine plasma hydrogen peroxide levels after blockade of endogenous catalase with sodium azide. This method was validated by an independent spectrophotometric technique. Results Members of the essential hypertensive group (n = 21) had higher plasma hydrogen peroxide levels (3.16 ± 0.14 versus 2.50 ± 0.16 mmol/l, P = 0.005) than did members of the normotensive group (n = 29). Furthermore, within the normotensive group, those with a family history of hypertension (n = 15) exhibited higher hydrogen peroxide levels (2.83 ± 0.27 versus 2.14 ± 0.13 μmol/l, P = 0.03) than did those without such a family history (n = 14). Plasma hydrogen peroxide levels in these 50 subjects were correlated to their mean arterial pressures (r = 0.54, P < 0.001). When hypertensives were grouped with normotensives without a family history of hypertension, the correlation improved (r = 0.70, P < 0.001). Statistical analysis (two-way analysis of variance) revealed that a family history of hypertension was a better predictor of plasma hydrogen peroxide production than was blood pressure status (P = 0.003 versus P = 0.093). Further investigations showed that superoxide is produced in plasma and that one of its sources is xanthine oxidase. Conclusions Hydrogen peroxide is produced in blood plasma and elevation of its level could constitute a pathogenic factor in vascular organ damage attendant upon systemic hypertension. J Hypertens 16:291-303 (c) 1998 Rapid Science Ltd.

Plasma Hydrogen Peroxide Production in Human Essential Hypertension Role of Heredity, Gender, and Ethnicity

Oxygen free radicals, including hydrogen peroxide, may mediate oxidative stress in target organ tissues and contribute to cardiovascular complications in hypertension. To examine heritability of hydrogen peroxide production, we investigated this trait in a family-based cohort consisting of family members (n=236) ascertained through probands (n=57) with essential hypertension. Significant effects on hydrogen peroxide production were found for gender and ethnicity, with men having greater values than women (P <0.001) and white subjects having greater values than black subjects (P =0.025). Hydrogen peroxide production correlated directly with plasma renin activity (P =0.015), suggesting an important interaction between circulating oxygen radicals and the renin-angiotensin system and a potential mechanism for lower hydrogen peroxide values observed in blacks. Heritability estimates from familial correlations revealed that approximately 20% to 35% of the observed variance in hydrogen peroxide production could be attributed to genetic factors, suggesting a substantial heritable component to the overall determination of this trait. Hydrogen peroxide production negatively correlated with cardiac contractility (r =−0.214, P =0.001) and renal function (r =−0.194, P =0.003). In conclusion, these results indicate that hydrogen peroxide production is heritable and is related to target organ function in essential hypertension. Genetic loci influencing hydrogen peroxide production may represent logical candidates to investigate as susceptibility genes for cardiovascular target organ injury.

Oxidative Stress in the Dahl Hypertensive Rat

Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction, resistance to blood flow, and organ damage. The aim of this study was to determine whether oxygen free radicals are involved in the development of salt-induced hypertension. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a high salt (6.0% NaCl) or low salt (0.3% NaCl) diet for 4 weeks. The high salt diet caused the development of severe hypertension in Dahl-S animals and had no effect on blood pressure in Dahl-R animals. A tetranitroblue tetrazolium dye was used to detect superoxide radicals in microvessels of the mesentery. Light absorption measurements revealed enhanced staining along the endothelium of arterioles and venules in hypertensive Dahl-S animals, with significantly lower values in normotensive animals. In addition, a Clark electrochemical electrode was used to measure hydrogen peroxide levels in fresh plasma. Hypertensive Dahl-S animals had a higher plasma hydrogen peroxide concentration compared with their normotensive counterparts (2.81+/-0.43 versus 2.10+/-0.41 micromol/L), while no difference was detected between high- and low salt-treated Dahl-R animals (1.70+/-0.35 versus 1.56+/-0.51 micromol/L). The plasma hydrogen peroxide levels of all groups correlated with mean arterial pressure (r=.77). These findings demonstrate an enhanced production of oxygen free radicals in the microvasculature of hypertensive Dahl-S rats.

Cell distribution in capillary networks

Abstract The distribution of red and white blood cells at diverging capillary branches in the rabbit ear chamber has been studied by means of high-speed microcinephotography. The experimental results are summarized in the form of a cell distribution function which is defined as the flux of cells as a function of the bulk flow into the daughter vessels. In capillaries the cell distribution function is nonlinear and strongly dependent on the eccentric position of the blood cells at the upstream entrance to the branch. If one daughter vessel carries the majority of the entering flow, all red blood cells are collected into the same channel. The cell distribution function is readily incorporated into a numerical network analysis to simulate the path velocity, pressure drop, and concentrations of red and white cells as a function of time throughout a capillary bed. The computational results show that, due to nonlinear cell distribution functions and nonuniform flow rates, the concentration of blood cells in a capillary network will be generally nonuniform. The flow fluctuations are the consequence of the particle nature of blood in capillaries without active changes in their diameters. Vasomotion would alter the hemodynamic conditions in upstream vessels and the influx of cells into the capillary network; therefore its effect would be superimposed on the flow fluctuations considered here.

Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock.

Recent evidence shows that circulating granulocytes play an important role in capillary stasis and tissue injury. We investigated two aspects of the problem in a Wiggers hemorrhagic shock model of the rat: the survival rate and the microvascular no-reflow phenomenon. A conventional group of rats with normal blood cells and a neutropenic group of rats pretreated with intraperitoneal antigranulocyte antibody were used to evaluate the effects of granulocytes. Two hemorrhagic shock protocols (HSP) were carried out. In HSP-1, the rats were subjected to 40 mm Hg mean arterial pressure for 3 hours. The conventional group (n = 11) showed a 36% survival rate compared with 100% in the neutropenic group (n = 6). In HSP-2, the hypotension was more severe, 30 mm Hg mean arterial pressure for 7 hours. There were no survivors in the conventional group (n = 8), compared with a 100% survival rate in the neutropenic group (n = 6). The extent, location, and mechanism of the no-reflow phenomenon was investigated by examining histological sections from several organs after infusion of a contrast medium to mark vessels with flow in a control group without shock and in the HSP-2 model 2 hours after blood replacement. The arterioles and venules uniformly contained contrast medium in all three groups; only capillaries showed no-reflow. A significantly higher percentage of no-reflow was observed in the capillaries of the conventional shock group than in the neutropenic shock group. We concluded that the obstruction of capillaries was largely due to trapped granulocytes, suggesting that these leukocytes play a key role in the capillary no-reflow phenomenon and survival from hemorrhagic shock.

Leukocyte counts and activation in spontaneously hypertensive and normotensive rats.

The etiology for the progressive organ injury in hypertension is largely speculative. Recent studies have shown that leukocytes play a key role in several cardiovascular diseases. As an initial step toward investigating the role of leukocytes in hypertension, we measured leukocyte counts and spontaneous activation of granulocytes of freshly drawn unseparated blood samples in spontaneously hypertensive rats and in their normotensive counterpart, Wistar-Kyoto rats. The animals were derived from one breeder in the United States and from two breeders in Europe. Total leukocyte counts in young, mature, and old hypertensive rats were 50-100% above the controls. The number of granulocytes in mature and old spontaneously hypertensive rats is more than 100% elevated compared with control rats. In young hypertensive rats the mean granulocyte count was only slightly elevated. The number of spontaneously activated granulocytes, as detected by the nitroblue tetrazolium reduction, increases with age in both species; in mature spontaneously hypertensive rats, it is more than 300% above the values in the controls. Furthermore, in mature hypertensive rats the number of monocytes, activated monocytes, and the lymphocyte count are also significantly elevated over the values in the normotensive controls. It is proposed that these elevated leukocyte counts may constitute an enhanced risk for organ injury in the spontaneously hypertensive rat

Biomechanics: Cell Research and Applications for the Next Decade

With the recent revolution in Molecular Biology and the deciphering of the Human Genome, understanding of the building blocks that comprise living systems has advanced rapidly. We have yet to understand, however, how the physical forces that animate life affect the synthesis, folding, assembly, and function of these molecular building blocks. We are equally uncertain as to how these building blocks interact dynamically to create coupled regulatory networks from which integrative biological behaviors emerge. Here we review recent advances in the field of biomechanics at the cellular and molecular levels, and set forth challenges confronting the field. Living systems work and move as multi-molecular collectives, and in order to understand key aspects of health and disease we must first be able to explain how physical forces and mechanical structures contribute to the active material properties of living cells and tissues, as well as how these forces impact information processing and cellular decision making. Such insights will no doubt inform basic biology and rational engineering of effective new approaches to clinical therapy.

The Inflammatory Aspect of the Microcirculation in Hypertension: Oxidative Stress, Leukocytes/Endothelial Interaction, Apoptosis

Evidence is increasing in hypertensive models for an inflammatory reaction in the microcirculation with abnormal leukocyte counts and adhesion to the endothelium, enhanced arteriolar tone, and microvascular and tissue apoptosis. The spontaneous form of hypertension (SHR) is accompanied by a glucocorticoiddependent increase in circulating leukocyte count with elevated levels of activation and at the same time depressed leukocyte–endothelial interaction and endothelial P‐selectin function. The SHR exhibits immune suppression with lymphocyte apoptosis in the thymus. Generation of reactive oxygen species (ROS) in and around microvascular endothelial cells may regulate signal transduction pathways responsible for controlling gene expression and protein modification and thereby cause an elevation of vascular tone and, in excess, may form an injury mechanism for cells and tissue. A series of enzyme systems such as xanthine oxidase, reduced nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide oxidase, and cytochrome P450 monooxygenases in conjunction with suppression of ROS scavengers seem to be involved in the oxidative stress responses in hypertension. The increase in ROS generation contributes to vascular remodeling, apoptosis, and proliferation of vascular smooth muscle, whereas gaseous monoxides such as nitric oxide and carbon monoxide have the ability to modulate elevated vascular tone and proliferative cell responses. Such biological actions of gases not only regulate activation of soluble guanylate cyclase but could also be attributable to inhibition of cytochrome P450 monooxygenases. We examine here the molecular basis of signal transduction by ROS, NO, and CO and functional alterations in their sensor molecules. An inflammatory reaction may underlie the pathogenesis of hypertension and its associated lesion formation and organ dysfunction.