Markers Of Endothelial Integrity Research Articles

Comparative Study on the Chronic Vascular Responses Induced by Regular Versus Occasional Waterpipe Smoke Inhalation in Mice.

Waterpipe smoke (WPS) is the second most prevalent form of smoking in the world. There are ample evidences about the vascular alterations caused by regular WPS (Reg-WPS). Nonetheless, comparison of the chronic vascular response induced by regular versus occasional WPS (Occ-WPS) exposure is very scarce. We investigated, in BALB/c mice, the effects of Occ-WPS (30 minutes/day, 1 day/week) versus Reg-WPS (30 minutes/day, 5 days/week) for 6 months on thrombogenicity and platelet aggregation in vivo and in vitro. Moreover, various markers of endothelial integrity, inflammation and oxidative stress were assessed by enzyme-linked immunosorbent assay and colorimetric assay. Control mice were exposed to air. Our results showed that either Occ-WPS or Reg-WPS exposure shortened the thrombotic time in pial microvessels in vivo. Moreover, in pial venules, this effect was more marked in Reg-WPS group (-47%) compared with Occ-WPS (-34%). Similarly, exposure to either Occ-WPS or Reg-WPS reduced the prothrombin time and activated partial thromboplastin time. Platelet count was increased only in Reg-WPS exposure. Exposure to either Occ-WPS or Reg-WPS induced platelet aggregation in vitro. In addition, there was a statistically significant difference between Occ-WPS and Reg-WPS groups in platelet count and aggregation. Plasma concentration of tissue factor (+98%), P-selectin (+14%) and E-selectin (+16%) were significantly increased in Occ-WPS group compared with air exposed group. Likewise, compared with air group Reg-WPS caused an increase in concentration of tissue factor (+193%), P-selectin (+21%) and E-selectin (+42%). Nevertheless, only Reg-WPS induced a decrease (-38%) in the plasma concentration of tissue plasminogen activator. Notably, our results showed a statistically significant difference between Occ-WPS and Reg-WPS groups in the concentration of tissue factor. Erythrocyte numbers, hemoglobin concentration, hematocrit and lactate dehydrogenase activity were augmented only in Reg-WPS group compared with either control or Occ-WPS groups. Likewise, only Reg-WPS induced an increase in proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β compared with either control or Occ-WPS groups. However, markers of oxidative stress including 8-isoprostane and total antioxidants were enhanced in both Occ-WPS and Reg-WPS compared with control group. Our data confirm the vascular toxicity of the chronic Reg-WPS exposure and shows that even occasional chronic exposure to WPS caused thrombosis, platelet aggregation, endothelial alterations and oxidative stress. The latter findings are an additional cause of concern about the long-term toxicity of occasional waterpipe smoking.

Upregulation of Class II HLA as a Marker of Antibody-Mediated Rejection in Heart Allografts

In 2013, the ISHLT introduced the working classification for pathological changes associated with antibody-mediated rejection of the heart allograft, known as p-AMR. With two components H, for histologic changes, and I for immunolabeled markers associated with AMR, the proposed classification also suggests to use Class II HLA as a marker of endothelial integrity. We hypothesized that Class II HLA rather than a marker of mere endothelial presence, is a marker of endothelial activation, and therefore could be associated with the diagnosis of AMR. 838 heart allograft biopsies, collected from January 2016 to September 2018 at a single institution were evaluated for both morphologic (H/E) and immunolabeled changes of AMR. Biopsies were immunolabeled with immunofluorescence with antibodies against C4d, and for immunohistochemistry with antibodies against C3d, CD68 and Class II HLA. ISHLT criteria was used to classify the biopsies, and for Class II HLA, both the percentage and the stain intensity were recorded. 74.8% of biopsies showed either a H, I or a combined evidence of AMR. The percentage of endothelial HLA Class II staining was significantly correlated with the diagnosis of AMR (r=0.39). HLA Class II showed a spectrum of intensity, quantified as negative (0) to strongly positive (+3). The diagnosis of AMR significantly correlated with moderate (+2) and strong (+3) staining intensity for Class II HLA, as follow: H+I (OR 28.3, p 0.0001); H alone (OR 22.73, p 0.0001) and I alone (32.63, p 0.0001). Our study also confirmed the value of the other AMR markers proposed by the ISHLT, such as C4d, C3d and CD68 (all p 0.0001), but importantly, and in contrast to prior observations, we showed that the C4d focally positive category (10-50%) is also significantly correlated to the diagnosis of AMR (OR 12.25, p 0.0005). We confirmed our hypothesis that in heart allograft biopsies there is a spectrum of both percentage and intensity of HLA Class II in endothelial cells, and that its staining by IHC is a marker significantly correlated with the diagnosis of AMR. In addition, the group of focally positive C4d biopsies should be considered positive for the I component of the 2013 ISHLT classification, as this group of biopsies also correlated with the diagnosis of AMR.

Upregulation of Endothelial HLA Class II is a Marker of Antibody-Mediated Rejection in Heart Allograft Biopsies

In 2013, the International Society of Heart and Lung Transplant (ISHLT) introduced the working classification for pathologic changes associated with antibody-mediated rejection (AMR) of the heart allograft, known as pathologic AMR (pAMR). With 2 components associated with AMR, histopathologic changes) and immunopathologic markers, the proposed classification also suggests the use of class II HLA as a marker of endothelial integrity. It is known that during allograft rejection, endothelial cells are activated, therefore, we hypothesized that endothelial class II HLA rather than a marker of mere endothelial presence, is a marker of endothelial activation and becomes upregulated in AMR.Eight hundred thirty-eight heart allograft biopsies, collected from January 2016 to September 2018 at a single institution from patients with a current or recent diagnosis of AMR, were evaluated for both histopathologic and immunopathologic changes of AMR. Biopsies were labeled with immunofluorescence with antibodies against C4d and for immunohistochemistry with antibodies against C3d, CD68, and class II HLA. ISHLT criteria were used to classify the biopsies, and for class II HLA, both the percentage and the stain intensity were evaluated.Biopsies (74.8%) from our cohort showed either histopathologic pAMR-1, immunopathologic pAMR-1, or combined histopathologic and immunopathologic pAMR-2 evidence of AMR. Expression of endothelial HLA class II was significantly correlated with the diagnosis of AMR by percentage area (P < .0001) and intensity of staining (P < .0001). The diagnosis of AMR significantly correlated with moderate (+2) and strong (+3) staining intensity for class II HLA as follows: histopathologic and immunopathologic pAMR-2 with odds ratio (OR) = 28.3 (P < .0001);histopathologic pAMR-1 alone with OR = 22.7 (P < .0001); and immunopathologic pAMR-1 alone with OR = 32.6 (P < .0001). Interestingly, our study also suggested that the inclusion of C4d focally positive cases also significantly correlates with the diagnosis of AMR (P < .003).We confirmed our hypothesis that in heart allograft biopsies, there is a spectrum of both percentage and intensity of HLA class II expression due to endothelial activation, and that class II HLA by immunohistochemistry is a marker significantly correlated with the diagnosis of AMR. In addition, the group of focally positive C4d biopsies (10%-50%) should be considered positive for the immunopathologic component of the 2013 ISHLT classification, as this group of biopsies also correlated with the diagnosis of AMR.

Tubastatin A prevents hemorrhage-induced endothelial barrier dysfunction.

Microvascular hyperpermeability resulting from endothelial barrier dysfunction (EBD) is associated with worse clinical outcomes in trauma-induced hemorrhagic shock. We have previously shown that treatment with Tubastatin A (TubA), a histone deacetylase 6 inhibitor, improves outcomes in animal models of shock. In this study, we investigate whether TubA treatment may prevent trauma-related EBD. Wistar-Kyoto rats subjected to 40% hemorrhage were treated with TubA or vehicle control. Acute lung injury (ALI) was assessed histologically from tissues harvested 6 hours posthemorrhage. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured in EGM BulletKit medium. Medium was exchanged for glucose-free Dulbecco's Modified Eagle Medium (0.5% fetal bovine serum) with or without TubA, and cells were placed in an anoxic chamber (5% CO2, 95% N2, 20-48 hours). Expression of acetylated tubulin and hypoxia-inducible factor 1α was measured by Western blot. Soluble Intercellular Adhesion Molecule-1 concentration within the medium, a marker of endothelial integrity, was determined using enzyme-linked immunosorbent assay. Monolayers were assessed for permeability via transwell assays using fluorescein isothiocyanate-labeled albumin. Rats treated with TubA had significantly reduced ALI relative to vehicle control. In vitro, TubA significantly attenuated anoxia-induced hyperpermeability, hypoxia-inducible factor 1α expression, and glycocalyx shedding. Our findings demonstrate that TubA prevents hemorrhage-induced ALI in rats. Additionally, we have shown that TubA prevents anoxia-induced EBD in vitro. Taken together, these results suggest that TubA could attenuate microvascular hyperpermeability related to hemorrhagic shock.

Endothelial alterations in a canine model of immune thrombocytopenia

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.

Abstract P046: Association of Stromal Derived Factor 1 (SDF1) and Transforming Growth Factor ß1 (TGFß1) with Incident Hypertension: The Multi-Ethnic Study of Atherosclerosis (MESA)

Incident hypertension varies across races/ethnicities. Mechanisms implicated in the pathogenesis are endothelial dysfunction with reduction of nitric oxide and alterations of the Renin-Angiotensin-Aldosterone System (RAAS). Stromal Derived Factor 1 (SDF1) and Transforming Growth Factor β1 (TGFβ1) are essential in these processes. SDF1 is a chemotactic protein that regulates migration of endothelial progenitor cells from the bone marrow to the arterial wall to repair endothelial damage; TGFβ1 promotes extra-cellular matrix deposition within arterial vessels, inhibits nitric oxide production and stimulates renin release. In order to investigate the association of circulating levels of these proteins with incident hypertension across different ethnicities, an ethnic stratified random sample of 2,880 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) was used. Of those, 988 participants had available plasma samples and were without hypertension, defined as blood pressure ≥ 140/90 mmHg, or current use of antihypertensive medications, at baseline. During a median follow up of 8.5 years, 388 new cases of hypertension (101 in non-Hispanic whites, 94 in Chinese, 101 in African Americans and 92 in Hispanics) were observed. Time to hypertension incidence was estimated using Cox proportional hazards regression. Hazard Ratios were adjusted for age, sex, diabetes, LDL, HDL, triglycerides, BMI, smoking status and current alcohol use. The race/ethnicity by protein interaction p-values were 0.055 and 0.066 for TGFβ1 and SDF1 respectively, suggesting heterogeneity across races/ethnicities. In Chinese, a difference of one standard deviation of plasma TGFβ1 was associated with a 22% increase in incident hypertension (HR (95% CI) 1.22 (1.02, 1.47), p-value=0.03); and a difference of one standard deviation of plasma SDF1 was associated with a 31% decrease in incident hypertension (HR (95% CI) 0.69 (0.52, 0.91), p-value 0.008). No significant association was found between these proteins and incident hypertension among non-Hispanic whites, African or Hispanic Americans. In conclusion, soluble markers of endothelial integrity and of RAAS activation were associated with the incidence of hypertension among Chinese Americans. These results suggest heterogeneity in the pathophysiology of hypertension according to race/ethnicity.

Dietary flavanol intervention lowers the levels of endothelial microparticles in coronary artery disease patients

Current evidence suggests that regenerative v. degenerative endothelial responses can be integrated in a clinical endothelial phenotype, reflecting the net result between damage from risk factors and endogenous repair capacity. We have previously shown that a cocoa flavanol (CF) intervention can improve endothelial function and increase the regenerative capacity of the endothelium by mobilising circulating angiogenic cells in patients with coronary artery disease (CAD). The aim of the present study was to investigate whether CF can lower the levels of circulating endothelial microparticles (EMP), markers of endothelial integrity, along with improvements in endothelial function. The levels of EMP in the frozen plasma samples of CAD patients were measured along with endothelial function (flow-mediated vasodilation, FMD); n 16, FMD data published previously), and these data were compared with those of young (n 12) and age-matched (n 12) healthy control subjects. The CAD patients exhibited significantly increased levels of EMP along with impaired FMD when compared with the healthy control subjects. The levels of CD144⁺ and CD31⁺/41⁻ EMP were inversely correlated with FMD (r -0.67, P=0.01 and r -0.59, P=0.01, respectively). In these CAD patients, the levels of EMP were measured after they had consumed a drink containing 375 mg of CF (high-CF intervention, HiFI) or 9 mg of CF (macro- and micronutrient-matched low-CF control, LoFl) twice daily over a 30-d period in a randomised, double-blind, cross-over study. After 1 month of HiFI, the levels of CD31⁺/41⁻ and CD144⁺ EMP decreased (-25 and -23%, respectively), but not after LoFl. Our data show that flavanols lower the levels of EMP along with higher endothelial function, lending evidence to the novel concept that flavanols may improve endothelial integrity.

Coagulation activation, depletion of platelet granules and endothelial integrity in case of uraemia and haemodialysis treatment

BackgroundDuring haemodialysis (HD) treatment, increase of platelet (PLT) activation and induction of procoagulant activity is demonstrated. Although the role of the endothelium and its direct interaction with coagulation and homeostasis is known, it is not elucidated how PLT activation markers and activation of coagulation coincide with markers of endothelial integrity during HD treatment. In the present study uraemia and HD induced changes, with particular emphasis on PLT granules depletion, activation of coagulation and endothelial integrity were investigated.MethodsTo detect depletion of PLT granules, peripheral blood slide smears were screened by light microscopy for qualitative evaluation of PLT granule containing cytoplasm, as indicated by its granules staining density. Activation of coagulation was investigated by establishement of thrombin-antithrombin (TAT) and fibrinogen concentrations. To evaluate endothelial integrity proendothelin (proET-1) plasma concentrations were established.ResultsResults of our study demonstrate that proET-1 plasma concentrations were obviously increased in the subjects’ group with end-stage chronic kidney disease (CKD) and renal failure if compared with a group of apparently healthy subjects. The amount of depleted PLT granules was obviously increased in the subjects’ group with end-stage CKD if compared with the group with renal failure. Mean plasma concentrations of TAT and fibrinogen revealed results within the reference range.ConclusionsIt is demonstrated that uraemia is associated with endothelial damage and aberrations in PLT granules morphology in subjects with HD treatment. We hypothesize that increased proET-1 concentrations reflect ongoing stress on endothelial cells amongst others due to uraemia. Biomarkers like proET-1 and aberrations in PLT granules morphology assist in the early detection of procoagulant activity of the endothelium.

Adhesion Molecules, Altered Vasoreactivity, and Brain Atrophy in Type 2 Diabetes

OBJECTIVETo investigate the effects of inflammation on perfusion regulation and brain volumes in type 2 diabetes.RESEARCH DESIGN AND METHODSA total of 147 subjects (71 diabetic and 76 nondiabetic, aged 65.2 ± 8 years) were studied using 3T anatomical and continuous arterial spin labeling magnetic resonance imaging. Analysis focused on the relationship between serum soluble vascular and intercellular adhesion molecules (sVCAM and sICAM, respectively, both markers of endothelial integrity), regional vasoreactivity, and tissue volumes.RESULTSDiabetic subjects had greater vasoconstriction reactivity, more atrophy, depression, and slower walking. Adhesion molecules were specifically related to gray matter atrophy (P = 0.04) and altered vasoreactivity (P = 0.03) in the diabetic and control groups. Regionally, sVCAM and sICAM were linked to exaggerated vasoconstriction, blunted vasodilatation, and increased cortical atrophy in the frontal, temporal, and parietal lobes (P = 0.04–0.003). sICAM correlated with worse functionality.CONCLUSIONSDiabetes is associated with cortical atrophy, vasoconstriction, and worse performance. Adhesion molecules, as markers of vascular health, have been indicated to contribute to altered vasoregulation and atrophy.

Hemoglobin-Induced Oxidative Stress Contributes to Matrix Metalloproteinase Activation and Blood–Brain Barrier Dysfunction in vivo

Hemoglobin (Hb) released from extravasated erythrocytes is implicated in brain edema after intracerebral hemorrhage (ICH). Hemoglobin is a major component of blood and a potent mediator of oxidative stress after ICH. Oxidative stress and matrix metalloproteinases (MMPs) are associated with blood-brain barrier (BBB) dysfunction. This study was designed to elucidate whether Hb-induced oxidative stress contributes to MMP-9 activation and BBB dysfunction in vivo. An intracerebral injection of Hb into rat striata induced increased hydroethidine (HEt) signals in parallel with MMP-9 levels. In situ gelatinolytic activity colocalized with oxidized HEt signals in vessel walls, accompanied by immunoglobulin G leakage and a decrease in immunoactivity of endothelial barrier antigen, a marker of endothelial integrity. Administration of a nonselective MMP inhibitor prevented MMP-9 levels and albumin leakage in injured striata. Moreover, reduction in oxidative stress by copper/zinc-superoxide dismutase (SOD1) overexpression reduced oxidative stress, MMP-9 levels, albumin leakage, and subsequent apoptosis compared with wild-type littermates. We speculate that Hb-induced oxidative stress may contribute to early BBB dysfunction and subsequent apoptosis, partly through MMP activation, and that SOD1 overexpression may reduce Hb-induced oxidative stress, BBB dysfunction, and apoptotic cell death.

Role of glutathione redox cycle in TNF-α-mediated endothelial cell dysfunction

Modulation of the glutathione redox cycle may influence tumor necrosis factor-α (TNF)-mediated disturbances of endothelial integrity. To test this hypothesis, normal endothelial cells or cells with either increased or decreased glutathione levels were exposed to 100 ng (500 U) TNF/ml. Increased glutathione levels were achieved by exposure to 0.2 mM N-acetyl-L-cysteine (NAC) and decreased glutathione levels by exposure to 25 μM buthionine sulfoximine (BSO). Several components of the glutathione redox cycle as well as markers of endothelial integrity, such as cytoplasmic free calcium and transendothelial albumin transfer, were measured in the treated cells. Exposure to TNF for 3 and 6 h decreased total glutathione levels, which was followed by an increase at later time points. Moreover, treatment with TNF resulted in an increase in the ratio of oxidized to reduced glutathione, intracellular free calcium, albumin transfer across endothelial monolayers and lipid hydroperoxides. However, an increase in lipid hydroperoxides was seen only when endothelial cell cultures were supplemented with iron. BSO treatment increased susceptibility of endothelial cells to TNF-mediated metabolic disturbances. On the other hand, NAC partially protected against TNF-induced injury to endothelial monolayers. Our results demonstrate the important role of the glutathione redox cycle in TNF-mediated disturbances of the vascular endothelium and indicate that modulation of glutathione levels may potentiate the injurious effects of this inflammatory cytokine.

Increased vascular permeability in pancreas of diabetic rats: detection with high resolution protein A-gold cytochemistry.

The role of the pancreatic microcirculation in the pathogenesis of Type 1 (insulin-dependent) diabetes mellitus remains poorly understood. Herein, a method is described for the ultrastructural investigation of the integrity of the pancreatic microvasculature. The method consists of histochemical detection and isolation of the islets followed by albumin and protein A-gold immunocytochemistry, whereby the distribution of endogenous albumin is used as a marker of endothelial integrity. This technique, applied to the study of spontaneously diabetic rats, reveals a selective increase in permeability of islet capillaries and post-capillary venules at the onset of diabetes, while acinar capillaries and arterioles remain intact. At 50 days of age, before the onset of diabetes, the microvasculature of diabetes-prone rats shows no alterations in permeability to albumin. When used in conjunction with morphometric analyses, this methodological approach may be useful for further studies in pathologic or experimental conditions involving the pancreatic microvasculature.