Proteolysis in septic shock patients: plasma peptidomic patterns are associated with mortality.

BACKGROUND: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome. METHODS: Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects. RESULTS: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation. CONCLUSIONS: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients. CLINICAL TRIAL REGISTRATION: NCT02141607.

THE AUTODIGESTION HYPOTHESIS AND RECEPTOR CLEAVAGE IN DIABETES AND HYPERTENSION.

One of the key features of cardiovascular complications, such as hypertension or diabetes, is that they often appear at the same time in the same individual together with other forms of co-morbidities. While clinically a recognized phenomenon, no molecular mechanism for such co-morbidities has received universal acceptance. We propose a new hypothesis that provides a molecular basis for co-morbidities in hypertension due to unchecked proteolytic activity and receptor destruction. Testing of the hypothesis in the spontaneously hypertensive rat reveals an unchecked matrix metalloproteinase and serine protease activity in plasma and on several cardiovascular and parenchymal cells. The elevated proteolytic activity causes extracellular cleavage of multiple receptor types, such that cleavage of one receptor type leads to loss of the function carried out by this receptor. Proteolytic cleavage of the extracellular domain of the ß(2) adrenergic receptor in arteries and arterioles causes vasoconstriction and elevation of the central blood pressure while cleavage of the extracellular domain of the insulin receptor leads to insulin resistance and lack of transmembrane glucose transport. A diverse set of cell dysfunctions in the spontaneously hypertensive rat are accompanied by cleavage of the membrane receptors that are involved in these functions. Chronic inhibition of the unchecked protease activity in the spontaneously hypertensive rat serves to restore the extracellular receptor density and alleviates the corresponding cell dysfunctions. The mild unchecked proteolytic activity in the spontaneously hypertensive rat points towards a chronic autodigestion process as a contributor to the end organ injury encountered in this rat strain. The presence of various soluble receptors, which consist of extracellular fragments of membrane receptors, in the plasma of hypertensive and diabetic patients suggest that the autodigestion process may also be present in man.

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.

Mechanisms in experimental venous valve failure and their modification by Daflon 500 mg.

OBJECTIVES: To characterize the acute response of the vein wall to venous hypertension and associated altered fluid shear stress and to test the effect of micronized purified flavonoid fraction (MPFF, Daflon 500), on this response. MATERIAL AND METHODS: A femoral arteriovenous fistula was created in Wistar rats (n=48). A cohort of 24 rats received oral treatment with MPFF (100 mg/kg/day body weight), 24 rats underwent the arteriovenous fistula procedure and received no treatment. At days 1, 7 and 21 the animals (n=8 at each time point) were killed. Experimental parameters measured included limb circumference, blood flow at the sapheno-femoral junction, leukocyte infiltration and gelatinase activity (matrix metalloproteinase, MMP). RESULTS: The acute rise in venous hypertension was accompanied by limb edema and venous reflux together with an eventual loss of valve leaflets in the saphenous vein. There was an increase in granulocyte and macrophage infiltration into the venous wall and the surrounding tissue, and a lesser increase in T- and B-lymphocyte infiltration. These changes were accompanied by a local increase in the proteolytic enzymes, MMP-2 and MMP-9. Administration of MPFF reduced the edema and lessened the venous reflux produced by the acute arteriovenous fistula. Decreased levels of granulocyte and macrophage infiltration into the valves were also observed compared with untreated animals. CONCLUSIONS: Venous hypertension caused by an arteriovenous fistula resulted in the development of venous reflux and an inflammatory reaction in venous valves culminating in their destruction. MPFF was able to delay the development of reflux and suppress damage to the valve structures in this rat model of venous hypertension.

Temporal correlation between maximum tetanic force and cell death in postischemic rat skeletal muscle.

To gain insight into the mechanisms responsible for muscle dysfunction after ischemia-reperfusion, a rat spinotrapezius muscle preparation was developed which enabled sequential measurements of in vivo maximum tetanic force production and cell death assessed using digital microfluorographic determination of propidium iodide (PI) staining. After 60 min of no-flow ischemia, maximum tetanic force fell significantly during 90 min of reperfusion compared with control, nonischemic muscles. The most striking fall was evident within 30 min of reperfusion and occurred concomitant with an explosive increase in PI-positive myocyte nuclei. Treatment with the oxygen radical scavenger, dimethylthiourea, attenuated both the fall in force and increased PI staining. Indeed, the rise in PI-positive nuclei correlated closely (r= 0.728) with the reduction of maximum tetanic force developed following ischemia and reperfusion under all conditions. Superoxide dismutase also attenuated the rise in PI-positive nuclei. Assessment of mitochondrial inner membrane potential (deltapsi) using Rhodamine 123 fluorescence revealed that myocytes with the lowest initial mitochondrial membrane potential were subject to the greatest injury after 90 min of reperfusion (r= 0.828). These results support the hypothesis that myocyte injury, as visualized by PI-staining, reflects an impaired contractile function in fibers with a low oxidative potential which is likely mediated by oxygen radicals.

Preliminary profiling of blood transcriptome in a rat model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger's shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients.

Mechanisms for regulation of fluid shear stress response in circulating leukocytes.

We have shown that leukocytes retract their pseudopods and detach from substrates after exposure to physiological fluid shear stresses ( approximately 1.5 dyn/cm(2)). In inflammation, however, pseudopod projection during spreading and firm adhesion on endothelium is observed even in microvessels with normal blood flow and fluid shear stresses. Thus, we examined mechanisms that may serve to regulate the shear stress response of circulating leukocytes. In the presence of inflammatory mediators (platelet-activating factor [PAF] f-met-leu-phe), a subgroup of cells ceases to respond to shear stress. cGMP analogs and nitric oxide (NO) donors enhance the shear stress response and reverse the inhibitory effect of inflammatory mediators on the shear stress response, whereas depletion of cGMP leads to cessation of the shear stress response even in unstimulated leukocytes. The ability of cGMP to enhance the shear stress response is not associated with CD18 expression, because cGMP has no effect on CD18 expression in response to shear stress. The shear stress response of leukocytes in endothelial nitric oxide synthase (-/-) mice, in which NO level in blood is decreased, is attenuated compared with that in wild-type mice. In rat mesentery venules stimulated by PAF under normal blood flow, a cGMP analog diminishes pseudopod projection of leukocytes, whereas inhibition of NO leads to enhanced pseudopod projection and spreading. The evidence suggests that inflammatory mediators suppress the shear stress response of leukocytes leading to spreading even under normal physiological shear stress, whereas cGMP may serve to maintain shear stress response even in inflammation.

Control of overweight and obesity in childhood through education in meal time habits. The 'good manners for a healthy future' programme.

OBJECTIVE: Our aim is to determine the effect of paced eating, exposure to an educational programme that promotes healthy eating habits and allowing the satiety reflex to limit food intake in controlling weight gain in healthy adolescents. METHODS: Fifty-four healthy individuals consisting of 18 adolescent girls and 36 boys aged 12 ± 2 years were given recommendations for reducing eating rate without changing diet or meal size according to the educational programme 'good manners for a healthy future'. Each participant was provided with a 30-s portable hourglass to pace time between bites. Individuals using and not using the hourglass were placed either into an 'adhering' or a 'non-adhering' group, respectively. Control data were obtained from a similar population. RESULTS: Initially, the adhering group had higher weight compared with the non-adhering group (64.1 ± 13.2 vs. 56.2 ± 11.7 kg). Control group weight was no different from the study group at baseline (56.3 ± 10.3 kg). Weight in the adhering group decreased after the first semester of participation by 2.0 ± 5.7% and after a year by 3.4 ± 4.8%, while the non-adhering group gained weight by 5.8 ± 4.5% and 12.6 ± 8.3%. The control group increased weight after a year by 8.2 ± 6.5%. In total, 18 non-adhering and 14 adhering adolescents completed the study. CONCLUSIONS: This 1-year study shows a statistically significant association between rate of food intake and weight control in adherence to an educational programme directed at developing healthy eating habits. The proposed behavioural training may serve as an option for weight control in adolescents.

Flow dynamics of QW7437, a new dodecafluoropentane ultrasound contrast agent, in the microcirculation: microvascular mechanisms for persistent tissue echo enhancement.

OBJECTIVES: The purpose of this study was to test the hypothesis that a subgroup of QW7437 microbubbles, dodecafluoropentane-based ultrasound contrast microspheres, resides for prolonged periods in the microvasculature. BACKGROUND: QW7437 produces echo enhancement in myocardium which may persist relatively longer than opacification in the left ventricular cavity. The mechanism for this persistent enhancement remains unknown. METHODS: The transit of fluorescently labeled erythrocytes was examined by fluorescence intravital microscopy in the microvessels in five rat mesenteries. Ten rats were used to observe the behavior of fluorescently labeled QW7437 microbubbles in the mesenteric microcirculation. RESULTS: There was no significant change in erythrocyte velocity in the arterioles and venules after the administration of QW7437 microbubbles (0.05 ml/kg) preactivated by negative hydrodynamic pressure. Of 552 microbubbles observed in four arterioles and five capillaries, 549 (99.5%) passed without stoppage (> or = 0.1 s stoppage); only one stopped transiently in arteriole and two in capillaries, each for <0.5 s. Sixty-five of 478 microbubbles (13.6%) observed in six postcapillary venules 11 to 30 microm in diameter and 24 of 408 microbubbles (5.9%) in four venules 31 to 50 microm in diameter stopped transiently (0.1 to 180 s) with an attachment to venular endothelium; the remaining microbubbles passed through the venules without stoppage. CONCLUSIONS: Prolonged survival as microbubbles in the circulation and transient stoppage of a subgroup of microbubbles in the microvasculature, particularly in venules, are potential mechanisms for the persistent tissue echo enhancement by QW7437 microbubbles during contrast echocardiography.

Leukocyte contribution to parenchymal cell death in an experimental model of inflammation.

The relationship between leukocyte migration and parenchymal cell death in vivo remains poorly documented. Accordingly, cell killing in the rat mesentery, as recorded by propidium iodide staining, was investigated with an intravital approach. Superfusion of platelet-activating factor (PAF, 10(-8) M) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) led to extensive leukocyte extravasation but no significant cell death. In contrast, pretreatment with 10(-8) M PAF or fMLP for 1 h, followed by superfusion of PAF in combination with fMLP (both at 10(-8) M) led to an increase in cell death. Mesenteric parenchymal cells but no endothelial cells were killed. Some of the dead cells were identified as granulocytes/monocytes that were already in the tissue at the start of the experiment. The incidence of cell death was lower but not eliminated when leukocyte migration was blocked with a monoclonal antibody against CD18. A xanthine oxidase inhibitor, BOF-4272, failed to diminish cell death, whereas a hydroxyl radical scavenger, dimethylthiourea, attenuated cell killing without an effect on the number of adhering and migrating leukocytes. These observations demonstrate that leukocytes serve as a factor in the killing of extravascular cells only after the development of a level of stimulation that differs from that required to induce a migratory stimulus into the extravascular space.

Modification of leukocyte adhesion in spontaneously hypertensive rats by adrenal corticosteroids.

Leukocyte adhesion is a key factor in the pathogenesis of organ injury following a variety of stimuli. In this study we have addressed the role of leukocyte adhesion in hypertensives as a risk factor for organ injury. In the spontaneously hypertensive rat (SHR), the number of circulating leukocytes and their level of activation are significantly increased compared with its normotensive control, the Wistar-Kyoto rat (WKY). We have demonstrated that elevated levels of glucocorticoid in SHR suppress P-selectin-mediated leukocyte-endothelial interaction in the microcirculation. It is possible that the disturbance in leukocyte-endothelial interactions may result in an elevated number of leukocytes in the circulation. The aim of the present study was to investigate the contribution of the adrenal glands to the disturbance in leukocyte behavior in SHR by subjecting the animals to bilateral adrenalectomy and investigating the effect of hydrocortisone. In addition, we have studied by immunohistochemistry the expression of the endothelial adhesion molecule, P-selectin, in response to histamine in the mesenteric venules of normal and adrenalectomized SHR and WKY. The elevated blood pressure, above-normal leukocyte counts, and elevated number of activated neutrophils (nitroblue tetrazolium test) in SHR were blunted after adrenalectomy. The blunted histamine-induced leukocyte-endothelial interaction in the mesenteric venules of SHR was restored after adrenalectomy. Treatment with hydrocortisone significantly attenuated the elevated leukocyte adhesion in the adrenalectomized SHR as well as in WKY. The suppressed P-selectin expression in SHR mesentery was restored after adrenalectomy. In conclusion, the subnormal leukocyte-endothelial interaction in response to an inflammatory stimulation in SHR is abolished after adrenalectomy, suggesting a relationship between the altered leukocyte adhesiveness and the adrenal corticosteroids in hypertensives.

Mechanisms and consequences of cell activation in the microcirculation.

Cells undergo activation in response to a wide range of stimuli. In vascular cells (leukocytes, endothelial cells, and platelets), the different forms of activation include degranulation, oxygen free radical formation, expression of membrane adhesion proteins, and biophysical changes such as pseudopod formation and increased cytoplasmic viscosity. Cell activation and low flow are common features of many cardiovascular diseases. There is evidence that plasma from patients contains an activating factor for neutrophils as well as other vascular cells. Activated neutrophils have the ability to impair microcirculatory transit by elevation of endothelial permeability, leukocyte adhesion to the endothelium, leukocyte capillary plugging, release of vasoactive products, and capillary deformation and compression due to oxygen-radical-mediated interstitial edema and cell dysfunction. In addition to reduced organ perfusion, cell activation can also cause cell dysfunction via release of cytotoxic mediators. A lower degree of neutrophil activation prior to acute circulatory challenge (i.e., low preactivation) correlates with improved survival rates after challenge and suggests that elevated levels of in vivo cell preactivation is a risk factor for cell injury and organ failure. Under conditions of low in-vivo cell preactivation (e.g., as is the case in endotoxin-tolerant animals), there is reduced tissue injury and lower mortality after challenge. We hypothesize that in-vivo cell preactivation due to everyday activity (infection, diet, smoking) may be a mechanism for microvascular low blood flow with leukocyte accumulation and may represent a risk factor for various cardiovascular diseases.

Effects of induced tolerance to bacterial lipopolysaccharide on myocardial infarct size in rats.

OBJECTIVES: Induced tolerance to bacterial lipopolysaccharide (LPS) by pretreatment with sublethal doses of LPS has been shown to reduce the inflammatory response of monocytes, circulating PMNs and PMN adhesion to endothelial cells in response to subsequent stimuli, and also to increase cellular and organ tolerance to stress by other mechanisms. Therefore, we undertook to determine whether or not LPS desensitization is associated with reduced myocardial infarct size at 3 days after reperfusion following coronary occlusion. METHODS: Rats were randomized to either daily intraperitoneal LPS injections to provide LPS tolerance, or to equal volumes of saline (controls). In both groups at day 7 nontransmural infarction was produced by a 45 min coronary occlusion followed by 3 days of reperfusion during which LPS injections were continued. Histologic infarct size was assessed as percent of the left ventricle and as a percent of the risk zone (determined by fluorescent microspheres). RESULTS: Myocardial infarct size as percent of the left ventricle and of the risk zone were significantly reduced in the LPS-tolerant group (n = 14) compared to control rats (n = 12), the latter being reduced by 37% (33.6 +/- 18.4 vs. 54.1 +/- 8.6% of the risk zone, P < 0.002). The percentages of activated circulating PMN after LPS desensitization and saline pretreatment were not different prior to coronary occlusion (at 7 days), but 3 days after coronary occlusion and reperfusion the percent of activated PMNs in the treated group was markedly reduced compared to controls (2.9 +/- 1.6 vs. 11.4 +/- 7.2%, respectively, P < 0.02). CONCLUSIONS: LPS desensitization in rats for 1 week prior to coronary occlusion inhibited activation of circulating PMNs 3 days after reperfusion following 45 min of coronary occlusion. LPS also is well-known to induce heat stress proteins and may affect other protective mechanisms. These actions are associated with a significant reduction in myocardial infarct size in LPS-tolerant animals compared to untreated controls.

Vasodilator response of mesenteric arterioles to histamine in spontaneously hypertensive rats.

Recent evidence suggests that spontaneously hypertensive rats (SHR) exhibit an impaired response to inflammatory mediators. We designed this study to analyze the response of arterioles of SHR after stimulation with a proinflammatory agent, histamine, compared with the response of arterioles of normotensive Wistar-Kyoto (WKY) controls. We observed mesenteric arterioles by intravital microscopy in rats under general anesthesia and measured their lumen diameters after histamine superfusion. To compare the concentration-response curve with histamine, we also studied the effect of an endothelium-dependent vasodilator, acetylcholine, and an independent vasodilator, sodium nitroprusside. At the end of each experiment we applied papaverine topically to determine the maximal diameter for each vessel, from which we computed arteriolar tone. Arteriolar tone in SHR is set at a higher steady state level than in WKY. The concentration required for a 50% dilator response (EC50) of histamine in SHR was significantly higher than that in WKY. In SHR the arteriolar response showed the same refractory pattern to histamine as to acetylcholine. In contrast, the EC50 of sodium nitroprusside in SHR was similar to that in WKY. Our results indicate that SHR exhibit an impaired dilator response to histamine that is due to a blunted endothelium-dependent vasodilation.

Glucocorticoid modulates vasodilator response of mesenteric arterioles in spontaneously hypertensive rats.

We previously reported that the response of the arterioles in spontaneously hypertensive rats (SHR) to histamine is blunted compared with that in normotensive control rats (Wistar-Kyoto rats [WKY]). The present study was designed to analyze the extent to which this blunted arteriolar response may be attributed in SHR to the concurrent elevation of circulating glucocorticoids through the use of adrenalectomy with and without dexamethasone supplementation. Mesenteric arterioles were observed by intravital microscopy under general anesthesia, and their lumen diameters were measured after histamine superfusion. The concentration-response curve with histamine was compared with that of an endothelium-independent vasodilator, sodium nitroprusside. At the end of each experiment, papaverine was applied topically to determine the maximal diameter for each vessel, from which a measure of arteriolar tone could be computed. The arteriolar tone in sham-operated SHR is set at a higher steady-state level than in sham-operated WKY. The concentration required for a 50% dilator response (EC50) of histamine in adrenalectomized SHR was restored to the level of WKY. Adrenalectomy did not significantly affect the EC50 of histamine in WKY. When adrenalectomized SHR received a supplement of dexamethasone, the arteriolar response was found to show the same refractory pattern to histamine as sham-operated SHR. In contrast, the EC50 of sodium nitroprusside in sham-operated and adrenalectomized SHR was similar to that in sham-operated WKY. Our results indicate that the impaired dilator response to histamine in SHR is related to an enhanced adrenal glucocorticoid secretion.

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.

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)

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 in 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.

Microvascular tone in a skeletal muscle of spontaneously hypertensive rats.

We studied the degree of arteriolar smooth muscle constriction in the spinotrapezius muscle microcirculation of spontaneously hypertensive rats and their normotensive controls, Wistar-Kyoto rats. The constriction was expressed in the form of a nondimensional tone as the difference between steady state and dilated diameter (after papaverine treatment) divided by the dilated diameter. Both animal strains showed on average a progressive increase of tone toward the more distal arterioles, with a peak tone being reached in the transverse arterioles. Tone values in the hypertensive animals were consistently elevated. The number of arterioles that had more than 5% tone (so-called responder arterioles) was higher in the hypertensive animals. These studies suggest that, besides the anatomical adjustments documented earlier in our laboratory in the arteriolar network of this muscle, functional adjustments in the form of an elevated microvascular tone are associated with the elevated resistance in spontaneously hypertensive rats.

Impaired leukocyte-endothelial cell interaction in spontaneously hypertensive rats.

Hypertension is associated with a progressive organ injury whose etiology remains largely speculative. An increasing database shows that activated leukocytes, while affording an important immune protection, may be a contributing factor to several of the pathogenetic features of the hypertension syndrome. The purpose of this study was to determine the extent to which the glucocorticoid pathway may be involved in the atypical kinetics of leukocytes in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. The typical venular leukocyte adhesion induced by histamine application was significantly lower in SHR, and a comparison of normalized leukocyte rolling velocity (VWBC/VRBC) showed the values to be significantly higher in SHR relative to WKY controls. This abnormal trend in adherent leukocyte numbers and in VWBC/VRBC values could be counteracted when SHR were pretreated with RU 486, a synthetic glucocorticoid inhibitor, and restored to the levels observed in WKY rats. Anti-P-selectin monoclonal antibody (PB1.3) attenuated in SHR and WKY rats the increment of adherent leukocyte numbers as well as the decrement of VWBC/VRBC value that developed under combined histamine and RU 486 superfusion. Furthermore, an anti-intercellular adhesion molecule-1 monoclonal antibody (1A29) served to attenuate the increment of adherent leukocyte number induced by a combination of histamine and RU 486 superfusion in WKY rats and SHR. The results indicate that the deficient leukocyte-endothelial cell interaction in SHR can be circumvented by a glucocorticoid inhibitor.