A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis

von Willebrand factor (vWf) deficiency causes severe von Willebrand disease in humans. We generated a mouse model for this disease by using gene targeting. vWf-deficient mice appeared normal at birth; they were viable and fertile. Neither vWf nor vWf propolypeptide (von Willebrand antigen II) were detectable in plasma, platelets, or endothelial cells of the homozygous mutant mice. The mutant mice exhibited defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events in ≈10% of neonates. As in the human disease, the factor VIII level in these mice was reduced strongly as a result of the lack of protection provided by vWf. Defective thrombosis in mutant mice was also evident in an in vivo model of vascular injury. In this model, the exteriorized mesentery was superfused with ferric chloride and the accumulation of fluorescently labeled platelets was observed by intravital microscopy. We conclude that these mice very closely mimic severe human von Willebrand disease and will be very useful for investigating the role of vWf in normal physiology and in disease models.     

Localized reduction of atherosclerosis in von Willebrand factor-deficient mice

To examine the role of the platelet adhesion molecule von Willebrand factor (vWf) in atherogenesis, vWf-deficient mice (vWf-/-) were bred with mice lacking the low-density lipoprotein receptor (LDLR-/-) on a C57BL/6J background. LDLR-/-vWf+/+ and LDLR-/-vWf-/- mice were placed on a diet rich in saturated fat and cholesterol for different lengths of time. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules in both genotypes, indicating an increase in P-selectin-mediated adhesion to the endothelium. After 8 weeks on the atherogenic diet, the fatty streaks formed in the aortic sinus of LDLR-/-vWf-/- mice of either sex were 40% smaller and contained fewer monocytes than those in LDLR-/-vWf+/+ mice. After 22 weeks on the atherogenic diet (early fibrous plaque stage), the difference in lesion size in the aortic sinus persisted. Interestingly, the lesion distribution in the aortas of LDLR-/-vWf-/- animals was different from that of LDLR-/- vWf+/+ animals. In vWf-positive mice, half of all lesions were located at the branch points of the renal and mesenteric arteries, whereas lesions in this area were not as prominent in the vWf-negative mice. These results indicate that the absence of vWf primarily affects the regions of the aorta with disturbed flow that are prone to atherosclerosis. Thus, vWf may recruit platelets/leukocytes to the lesion in a flow-dependent manner or may be part of the mechano-transduction pathway regulating endothelial response to shear stress.     

Cytokines in sickle cell disease

Sickle red cells express adhesion molecules including integrin alpha4beta1, CD36, band 3 protein, sulfated glycolipid, Lutheran protein, phosphatidylserine and integrin-associated protein. The proadhesive sickle cells may bind to endothelial cell P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD36 and integrins leading to its activation. Monocytes also activate endothelium by releasing proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). Sickle monocytes also express increased surface CD11b and cytoplasmic cytokines TNFalpha and IL-1beta indicating activated state. Polymorphonuclear leukocytes (PMNs) are also activated with reduced L-selectin expression, enhanced CD64 expression and elevated levels of sL-selectin, sCD16 and elastase resulting in increased adhesiveness to the endothelium. Platelets are also activated and secrete thrombospondin (TSP) and cytokine IL-1. They also form platelet- monocytes aggregates causing endothelial cell P-selectin expression. Endothelial cell activation by these multiple mechanisms leads to a loss of vascular integrity, expression of leukocyte adhesion molecules, change in the surface phenotype from antithrombotic to prothrombotic, excessive cytokine production and upregulation of HLA molecules. Furthermore, contraction of these activated endothelial cells leads to exposure of extracellular matrix proteins, such as TSP, laminin, and fibronectin and their participation in adhesive interactions with bridging molecules from the plasma such as von Willebrand factor (vWf) released from endothelial cells, ultimately culminating in vasoocclusion and local tissue ischemia, the pathognomonic basis of vasoocclusive crisis.     

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.     

Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type IIA von Willebrand disease. The patient's EC, compared with those from normal individuals, produced vWf that had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features characteristic of plasma vWf from patients with type IIA von Willebrand disease. The type IIA EC did produce a full spectrum of vWf multimers in both cell lysates and postculture medium, although the relative amounts of the largest species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites that contained approximately equal to 170-kDa proteolytic fragments, suggesting that this patient's functional defect is due to abnormal proteolysis and not to a primary failure of vWf subunit oligomerization. Moreover, the observed degradation appears to result from an abnormal vWf molecule and not elevated protease levels. These results suggest that this patient's von Willebrand disease phenotype is caused by increased proteolytic sensitivity of his vWf protein.     

von Willebrand factor mediates platelet adhesion to virally infected endothelial cells.

von Willebrand factor is an adhesive glycoprotein critical to normal hemostasis. It is stored in the Weibel-Palade body of endothelial cells and upon release may mediate platelet adhesion. Herpesvirus-infected endothelium is known to be prothrombotic and to support enhanced platelet adherence. We previously identified P-selectin as a monocyte receptor that is translocated from the Weibel-Palade body to the endothelial cell surface in response to the local generation of thrombin on herpesvirus infected cells. In this study, we show that viral injury to vascular endothelial cells induces secretion of von Willebrand factor which mediates enhanced platelet adhesion to these cells.     

CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin

The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.     

Activated platelets induce Weibel-Palade-body secretion and leukocyte rolling in vivo: role of P-selectin

The presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin-/- platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Endothelial P-selectin is stored with von Willebrand factor (VWF) in Weibel-Palade bodies. The release of Weibel-Palade bodies on infusion of activated platelets was indicated by both elevation of plasma VWF levels and by an increase in the in vivo staining of endothelial P-selectin. We conclude that the presence of activated platelets in circulation promotes acute inflammation by stimulating secretion of Weibel-Palade bodies and P-selectin-mediated leukocyte rolling.     

Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules

Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P- selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P- selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.     

Endothelial dysfunction in subjects with subclinical hyperthyroidism

Numerous clinical and experimental reports suggest that a high von Willebrand factor (vWf) level reflects endothelial damage or endothelial dysfunction. The present study was designed to evaluate vWf in subclinical hyperthyroidic subjects compared with euthyroidic subjects. We selected 20 subclinical hyperthyroidic subjects and 20 euthyroidic control subjects matched for age, gender and body mass index. The level of vWf was significantly higher in the subclinical hyperthyroidic group than in the euthyroidic group (42.9+/-9.6 vs 37.6+/-6.4%, p=0.026). In conclusion, our results suggest that subjects with subclinical hyperthyroidism tend to have an endothelial dysfunction. Endothelial dysfunction could contribute to increasing the cardiovascular risk in subclinical hyperthyroidism.     

Leukocyte count and vascular function in Type 2 diabetic subjects with treated hypertension

BACKGROUND: Traditional cardiovascular risk factors may only partially explain abnormal vascular function in Type 2 diabetic patients. This study examined the associations between vascular function and markers of inflammation in Type 2 diabetic subjects with treated hypertension. METHODS: Flow-mediated dilatation (FMD) and glyceryl-trinitrate mediated dilatation (GTNMD) of the brachial artery were used to assess endothelium-dependent and -independent function, respectively, in 29 hypertensive Type 2 diabetic subjects (HbA1c <9%), and 17 healthy control subjects. Plasma C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and leukocyte count were used as markers of inflammation. Soluble L-selectin, P-selectin, and von Willebrand factor (vWf) were measured to assess leukocyte, platelet and endothelial cell activation, respectively. RESULTS: Compared with controls, diabetic subjects had impaired FMD (3.9+/-3.0 vs. 5.5+/-2.4%, P=0.07) and GTNMD (11.4+/-4.8% vs. 15.4+/-7.1%, P=0.04). They also had higher levels of CRP (2.7+/-2.6 vs. 1.4+/-1.1 mg/l, P=0.03), fibrinogen (3.4+/-0.7 vs. 2.7+/-0.3 g/l, P<0.001) and TNF-alpha (20.9+/-13.4 vs. 2.5+/-1.7 pg/l, P<0.001). In diabetic subjects, after adjustment for age and gender, leukocyte count was an independent predictor of FMD (P=0.02), accounting for 17% of total variance. Similarly, leukocyte count (P<0.001) accounted for 23% and IL-6 (P=0.03) for 12% of the variance in GTNMD. vWf was correlated with leukocyte count (r=0.38, P=0.04), FMD (r=-0.35, P=0.06) and GTNMD (r=-0.47, P=0.009), whilst P-selectin correlated with fibrinogen (r=0.58, P=0.001). CONCLUSION: These cross-sectional observations are consistent with the hypothesis that reduced FMD and GTNMD in Type 2 diabetes is at least in part secondary to increased inflammation, with associated endothelial and platelet activation.     

A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.     

von Willebrand factor, fibrinogen, and soluble P-selectin levels after mitral valve replacement versus mitral valve repair

Patients with mitral valve disease undergoing surgery are at an increased risk of thromboembolism. We hypothesized that this may be due in part to abnormalities in platelet activation, endothelial damage or dysfunction, and plasma fibrinogen in such patients. To test this hypothesis, we measured indexes of platelet activation (soluble P-selectin), endothelial damage or dysfunction (von Willebrand factor [vWf], enzyme-linked immunosorbent assay) and fibrinogen (modified Clauss) in 56 consecutive patients (35 women, mean age 65 years) admitted for isolated mitral valve repair (n = 39) or replacement (using mechanical implants, n = 17). Samples were taken from a peripheral vein before and at 3 months after valve surgery. Baseline results were compared with 56 healthy age- and sex-matched controls. Compared with controls, patients with mitral valve disease had higher levels of vWf (mean +/- SD 132 +/- 28 vs 101 +/- 35 IU/dl; p <0.001), but there were no significant differences in mean fibrinogen (p = 0.418) or soluble P-selectin (p = 0.855) levels between cases and controls. There was a significant increase in plasma vWf after mitral valve replacement: 142 +/- 25 IU/dl preoperatively, increasing to 161 +/- 33 IU/dl at 3 months after surgery (p = 0.0261). However, there were no significant changes in plasma fibrinogen (p = 0.306) or soluble P-selectin levels (p = 0.191). Patients undergoing mitral valve repair did not have any significant changes in mean vWf (p = 0.25), soluble P-selectin (p = 0.77), or fibrinogen (p = 0.22). There was a significant negative correlation (Spearman, r = -0.4, p = 0.003) in postoperative plasma vWf levels and the size of valve prosthesis used. Thus, patients with mitral valve disease have increased plasma vWf levels when compared with healthy controls, suggesting endothelial damage or dysfunction, with a further increase in levels after mitral valve replacement. Conversely, patients undergoing mitral valve repair do not demonstrate any significant changes in fibrinogen, or indexes of endothelial dysfunction or platelet activation.     

Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.     

Is mild essential hypertension without obvious organ complications and risk factors associated with increased levels of circulating markers of endothelial dysfunction? Effect of ACE inhibitor therapy

The objective of the investigation was to assess whether circulating adhesion molecules, von Willebrand factor (vWf) and endothelin-1 are elevated in patients with mild uncomplicated essential hypertension without further risk factors of atherosclerosis and whether they could serve as indicators of endothelial dysfunction in this form of hypertension. Furthermore the authors investigated the effect of ACE inhibitor treatment (ACEI), quinapril, on the level of these markers of endothelial dysfunction. The level of adhesion molecules [intercellular cytoadhesion molecule-1 (ICAM-1), E-selectin, P-selectin], von Willebrand s factor (vWf) and endothelin-1 were assessed in patients with mild essential hypertension without further cardiovascular risk factors or clinical manifestations of atherosclerosis before and after quinapril treatment (n = 25) and compared with normotensive controls (n = 29). The results of the examinations provided evidence that contrary to controls the hypertensive subjects had significantly higher ICAM-1 levels (237.8 vs. 207.8 ng/ml, P = 0.02) vWf (118 vs. 106 IU/dl, p < 0.05) and endothelin-1 (5.81 vs. 5.15 fmol/ml, p < 0.05). Three-month treatment of hypertensive patients with ACEI led to a significant drop of endothelin-1 levels (5.81 vs. 5.26 fmol/ml, p = 0.01). The authors proved also an unequivocal declining trend of other cytoadhesion molecules and vWf after ACEI treatment, the changes however were not statistically significant. From the investigation it may be concluded that also patients with uncomplicated essential hypertension without other cardiovascular risk factors or clinical manifestations of atherosclerosis have significantly elevated plasma levels of ICAM-1, vWf and endothelin-1. Higher concentrations of these factors suggest endothelial dysfunction already in mild forms of essential hypertension without further risk factors or cardiovascular complications. A significant drop of endothelin-1 and declining trend of the other investigated indicators suggest that ACEI treatment can favourably influence endothelial dysfunction in hypertensive patients also independently on reduction of the BP.     

Relationships between von Willebrand factor and hemorheology in sportsmen

It is well established that exercise performance in athletes is related to improved blood fluidity. However, training effects on functional state of endothelium and relations of endothelial cell functions with hemorheology are poorly known. Circulating levels of von Willebrand factor may serve as a good marker of endothelial cell functions, its activation, and damage. Relationships between von Willebrand factor antigen (vWf) and blood rheology in 30 endurance sportsmen were investigated. Athletes were divided according to vWf into tertile groups. Compared to 16 controls, all subgroups of sportsmen had a lower erythrocyte rigidity index Tk (p<0.05-0.01), explained by lower values of mean corpuscular hemoglobin concentration (MCHC) (p<0.05-0.01), and a higher W170. The lower tertile group (<0.35 U/ml) had a lower blood viscosity (p<0.05), explained by a lower Tk, and a lower erythrocyte aggregability index (microscopy of diluted blood samples) (p<0.05). The upper tertile group of athletes demonstrated higher levels of plasma viscosity, explained by higher total globulins (p<0.01), and higher vWf levels (p<0.01) than controls, and a lower W170 compared to athletes from the lower tertile subgroup. In the entire group of athletes, log (vWf) was positively correlated to plasma viscosity (r=0.478, p=0.007), total serum globulins (r=0.430, p=0.018), erythrocyte aggregability index (r=0.427, p=0.019), and negatively to log (W170) (r=-0.449, p=0.013). Multiple stepwise regression analysis revealed that plasma viscosity was the primary correlate of vWf. These data shown that the higher von Willebrand level reflecting strong activation of endothelial cells in a part of athletes was closely correlated with increased plasma viscosity levels. We suggest that these hemorheological and endothelial disturbances in these athletes might be a result of exercise overloads.     

Prevention of Cytokine Accumulation in Platelets Obtained with the COBE Spectra Apheresis System

Background and Objectives: Febrile nonhemolytic transfusion reactions frequently accompany platelet trasfusions and may be due to accumulation of cytokines mediating inflammation during storage of platelet concentrates (PCs). We wished to determine whether PCs collected using the COBE® spectra? Apheresis System (Version4) were sufficiently leukocyte reduced (LR) to limit cytokine accumulation during storage. Materials and Methods: Cytokine accumulation-interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α)- and release of platelet α-granule - P-selectin, transforming growth factor-β1 (TGF-β1), platelet-derived growth factor AB (PDGF-AB), von Willebrand factor (vWf)- or dense granule (serotonin) markers were investigated during a 7-day storage period comparing apheresis-collected, LR PCs (LR PCs) and random donor platelets prepared from whole blood (WB). Results: Leukocyte counts were reduced 99.95% comparing LR PCs (5.7×10⁵/1) and WB PCs (1.09×10⁹/1). Little or no accumulation of leukocyte-derived cytoknies was observed in LR PCs during storage in contrast to WB PCs. A reduction in the release of platelet α-granule proteins, such as P-selectin, TGF-β1 and PDGF-AB, was observed on day 0 for LR PCs compared to WB PCs with little or no difference observed from day 3 to 7. Plasma vWf levels were higher in LR PCs compared to WB PCs on days 0–7. Conclusion: Leukocyte levels in PCs collected with the COBE Spectra Apheresis System are sufficiently low to limit cytokine production during 7 days of storage.     

Blood cell dynamics in P-selectin-deficient mice

P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin- deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P-selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury.     

Circulating intercellular cell adhesion molecule-1, endothelin-1 and von Willebrand factor-markers of endothelial dysfunction in uncomplicated essential hypertension: the effect of treatment with ACE inhibitors

The aim of the study was to examine whether the circulating cell adhesion molecules, von Willebrand factor (vWf) and endothelin-1, are elevated in patients with essential hypertension with no other risk factors for atherosclerosis and thus may serve as a markers of endothelial dysfunction in uncomplicated hypertension. Furthermore, the effect of treatment with the ACE inhibitor, quinapril, on levels of endothelial dysfunction markers were studied. The levels of adhesion molecules (intercellular cell adhesion molecule-1 [ICAM-1], E-selectin, P-selectin), von Willebrand factor (vWf) and endothelin-1 were measured in patients with hypertension without any other risk factors of atherosclerosis before and after treatment with quinapril (n = 22) and in normotensive controls (n = 22). Compared with normotensive subjects, the hypertensive patients had significantly higher levels of ICAM-1 (238 vs 208 ng/ml, P = 0.02), vWf (119 vs 105 IU/dl, P < 0.05) and endothelin-1 (5.76 vs 5.14 fmol/ml, P < 0.05). Three-month treatment of hypertensive patients with quinapril led to a significant decrease in the levels of endothelin-1 (5.76 vs 5.28 fmol/ml, P < 0.01). We did not observe significant changes in the levels of adhesion molecules and vWf after ACE inhibitor treatment, although a trend toward a decrease was apparent with all these parameters. Patients with uncomplicated hypertension with no other risk factors of atherosclerosis had significantly elevated levels of ICAM-1, vWf, and endothelin-1. Our data suggest that these factors may serve as markers of endothelial damage even in uncomplicated hypertension. In hypertensive patients, treatment with the ACE inhibitor quinapril resulted in a significant decrease in endothelin-1 levels. These findings indicate a beneficial effect of ACE inhibitors on endothelial dysfunction in hypertensive patients.