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)

In vivo studies of the rheological behavior of blood in the microcirculation were conducted by direct in situ measurements in cat mesentery. Upstream to downstream pressure drops were measured in unbranched arterioles, capillaries, and venules, with diameters from 7 to 58 micrometer. Simultaneous measurements of red cell velocity and vessel geometry facilitated computation of bulk velocity, pressure gradient, apparent viscosity, wall shear stress, and resistance. Arteriovenous distributions of these parameters revealed the following. Maximum pressure gradient (0.015 cm H20/micrometer) occurs in the true capillaries (7 micrometer in diameter); intravascular wall shear stress averaged 47.1 dynes/cm2 in arterioles and 29.0 dynes/cm2 in venules. Extreme values as great as 200 dynes/cm2 were observed in a few shunting arterioles. Apparent viscosity averaged 3.59 cP in arterioles, 5.15 cP in venules, and 4.22 cP overall. Intravascular resistance per unit length of microvessel varied with luminal diameter as a power law function with exponents of -4.04 for arterioles, -3.94 for venules, and -3.99 for all vessels combined. This apparent maintenance of Poiseuille's law is attributed to the opposing processes of hematocrit reduction and decreasing shear rate as blood is dispersed in successive arteriolar segments, and the converse action of these processes in the venous confluences which lessen the extent of network variations in apparent viscosity. Reductions in bulk velocity from the normal flow state to below 0.5 mm/sec resulted in increases in apparent viscosity by a factor of 2 to 10, which are attributed primarily to obstruction of the lumen by leukocyte-endothelium adhesion.

Leukocyte migration from a hemopoietic pool across marrow endothelium requires active pseudopod formation and adhesion. Leukocytes rarely show pseudopod formation while in circulation. At question then is the mechanism that serves to minimize leukocyte pseudopod formation in the circulation. We tested the hypothesis that fluid shear stress acts to prevent pseudopod formation. When individual human leukocytes (neutrophils, monocytes) spreading on glass surfaces in vitro were subjected to fluid shear stress (Ϸ1 dyn͞cm 2 ), an instantaneous retraction of pseudopods was observed. Removal of the fluid shear stress in turn led to the return of pseudopod projection and cell spreading. When steady shear stress was prolonged over several minutes, leukocyte swelling occurs together with an enhanced random motion of cytoplasmic granules and a reduction of cytoplasmic stiffness. The response to shear stress could be suppressed by K ؉ channel blockers and chelation of external Ca 2؉ . In rat mesentery microvessels after occlusion, circulating leukocytes project pseudopods in free suspension or when attached to the endothelium, even though immediately after occlusion only few pseudopods were present. When flow was restored, pseudopods on adhering leukocytes were retracted and then the cells began to roll and detach from the endothelium. In conclusion, plasma shear stress in the circulation serves to reduce pseudopod projection and adhesion of circulating leukocytes and vice versa reduction of shear stress leads to pseudopod projection and spreading of leukocytes on the endothelium.Under physiological conditions, most circulating leukocytes in the blood stream exhibit low levels of pseudopod projection or expression of membrane molecules for adhesion to endothelium. Projection of pseudopods is associated with F-actin formation and cytoplasm stiffening (1). Leukocytes with pseudopods show enhanced resistance to flow in microvessels (2) to the point of complete capillary arrest (3, 4). In contrast, leukocytes require activation and projection of pseudopods (also referred to as lamellipods or microvilli) to migrate from their hemopoietic compartment of origin across marrow endothelium into the circulation (5). Migration across the peripheral vascular endothelium also requires active pseudopod formation and adhesion. Rheological studies of leukocytes under static conditions have yielded higher values for the cytoplasmic properties than under in vivo conditions, when they are subject to physiological shear stress prior to deformation (6). The evidence thus suggests that in the circulation a mechanism exists for shifting leukocytes from active pseudopod projection to a more passive state without pseudopods.Endothelial cells (7,8), erythrocytes (9), and platelets (10) are shear sensitive. Shear stress and shear stress gradients (11) serve to modulate endothelial cell shape as well as orientation of the cytoskeletal actin and tubulin network, membrane ion transport, endocytosis, and gene expression (12-15) and ␤ 2 integrin-med...

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Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g͞kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (؊)BOF4272 [(؊)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-␣)-1,3,5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive f luoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was Ϸ3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (؊)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.