Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression.

Vascular cell adhesion molecule-1 (VCAM-1) plays important roles in development and inflammation. Tumor necrosis factor-α (TNF-α) and focal adhesion kinase (FAK) are key regulators of inflammatory and integrin-matrix signaling, respectively. Integrin costimulatory signals modulate inflammatory gene expression, but the important control points between these pathways remain unresolved. We report that pharmacological FAK inhibition prevented TNF-α-induced VCAM-1 expression within heart vessel-associated endothelial cells in vivo, and genetic or pharmacological FAK inhibition blocked VCAM-1 expression during development. FAK signaling facilitated TNF-α-induced, mitogen-activated protein kinase activation, and, surprisingly, FAK inhibition resulted in the loss of the GATA4 transcription factor required for TNF-α-induced VCAM-1 production. FAK inhibition also triggered FAK nuclear localization. In the nucleus, the FAK-FERM (band 4.1, ezrin, radixin, moesin homology) domain bound directly to GATA4 and enhanced its CHIP (C terminus of Hsp70-interacting protein) E3 ligase-dependent polyubiquitination and degradation. These studies reveal new developmental and anti-inflammatory roles for kinase-inhibited FAK in limiting VCAM-1 production via nuclear localization and promotion of GATA4 turnover

Circulating adhesion molecules and arterial stiffness

Aim: VCAM-1 and ICAM-1 are two important members of the immunoglobulin gene superfamily of adhesion molecules, and their potential role as biomarkers of diagnosis, severity and prognosis of cardiovascular disease has been investigated in a number of clinical studies. The aim of the present study was to determine the relationship between circulating ICAM-1 and VCAM-1 levels and aortic stiffness in patients referred for echocardiographic examination. Methods: Aortic distensibility was determined by echocardiography using systolic and diastolic aortic diameters in 63 consecutive patients referred for echocardiography. Venous samples were collected in the morning after a 12-hour overnight fast, and serum concentrations of ICAM-1 and VCAM-1 were measured using commercial enzyme immunoassay kits. Results: Data of a total of 63 participants (mean age 55.6 ± 10.5 years, 31 male) were included in the study. Circulating levels of adhesion molecules were VCAM-1: 12.604 ± 3.904 ng/ml and ICAM-1: 45.417 ± 31.429 ng/ml. We were unable to demonstrate any correlation between indices of aortic stiffness and VCAM-1 and ICAM-1 levels. Conclusion: The role of soluble adhesion molecules in cardiovascular disease has not been fully established and clinical studies show inconsistent results. Our results indicate that levels of circulating adhesion molecules cannot be used as markers of aortic stiffness in patients

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα

Cytoadherence and virulence - the case of Plasmodium knowlesi malaria.

BACKGROUND: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36. METHODS: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3. RESULTS: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36. CONCLUSIONS: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions

Attenuation of leukocyte sequestration by selective blockade of PECAM-1 or VCAM-1 in murine endotoxemia

Background: Molecular mechanisms regulating leukocyte sequestration into the tissue during endotoxemia and/or sepsis are still poorly understood. This in vivo study investigates the biological role of murine PECAM-1 and VCAM-1 for leukocyte sequestration into the lung, liver and striated skin muscle. Methods: Male BALB/c mice were injected intravenously with murine PECAM-1 IgG chimera or monoclonal antibody (mAb) to VCAM-1 ( 3 mg/kg body weight); controls received equivalent doses of IgG2a ( n = 6 per group). Fifteen minutes thereafter, 2 mg/kg body weight of Salmonella abortus equi endotoxin was injected intravenously. At 24 h after the endotoxin challenge, lungs, livers and striated muscle of skin were analyzed for their myeloperoxidase activity. To monitor intravital leukocyte-endothelial cell interactions, fluorescence videomicroscopy was performed in the skin fold chamber model of the BALB/c mouse at 3, 8 and 24 h after injection of endotoxin. Results: Myeloperoxidase activity at 24 h after the endotoxin challenge in lungs (12,171 +/- 2,357 mU/g tissue), livers ( 2,204 +/- 238 mU/g) and striated muscle of the skin ( 1,161 +/- 110 mU/g) was significantly reduced in both treatment groups as compared to controls, with strongest attenuation in the PECAM-1 IgG treatment group. Arteriolar leukocyte sticking at 3 h after endotoxin (230 +/- 46 cells x mm(-2)) was significantly reduced in both treatment groups. Leukocyte sticking in postcapillary venules at 8 h after endotoxin ( 343 +/- 69 cells/mm(2)) was found reduced only in the VCAM-1-mAb-treated animals ( 215 +/- 53 cells/mm(2)), while it was enhanced in animals treated with PECAM-1 IgG ( 572 +/- 126 cells/mm(2)). Conclusion: These data show that both PECAM-1 and VCAM-1 are involved in endotoxin-induced leukocyte sequestration in the lung, liver and muscle, presumably through interference with arteriolar and/or venular leukocyte sticking. Copyright (C) 2004 S. Karger AG, Basel

Oxylipins in triglyceride-rich lipoproteins of dyslipidemic subjects promote endothelial inflammation following a high fat meal.

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype

Pathogenetic role of tissue factor in graft-versus-host disease

Graft-versus-host disease (GVHD) is a serious complication after allogeneic stem cell transplantation, the mechanism of it is still not elucidated. Recent findings suggest that host endothelial cells are a target of alloreactive donor cytotoxic T lymphocytes in GVHD and tissue factor (TF) plays an important role not only in coagulation-inflammation cycle, but also in transplant immunology. We postulate TF expression in vascular endothelial cells(VEC) may play an pivotal role in the pathogenesis of GVHD. TF gene andprotein expression in target organs of GVHD in aGVHD mice was significantly elevated compared to that of controls as determined by real-time PCR and Western blotting. Allogeneic CD4^+^T cell and CD8^+^T cells enhanced TF, VCAM-1, TNF-[alpha], IFN-[gamma] and IL-6 expression in TNF-[alpha] prestimulated HUVECs compared to controls as determined by flowcytometry and real-time PCR. JNK and p38MAPK mediated allogeneic T cells-induced TF expression in HUVECs. These effects were largely prevented by monoclonal antibody against TF, SB203580 and SP600125. In concert, these data provide strong evidence that upregulated TF expression is related to tissue damage caused by GVHD, TF isthe key factor in GVHD mediated by endothelial cells and allogeneic T cells-induced TF and consecutive proinflammatory cytokines expression in VEC contribute to the pathogenesis of GVHD

The isolation of VCAM-1+ endothelial cell-derived extracellular vesicles using microfluidics

Background: Vascular cell adhesion molecule-1 (VCAM-1+) endothelial cell-derived extracellular vesicles (EC-EVs) are augmented in cardiovascular disease, where they can signal the deployment of immune cells from the splenic reserve. Endothelial cells in culture activated with pro-inflammatory tumor necrosis factor-α (TNF-a) also release VCAM-1+ EC-EVs. However, isolating VCAM-1+ EC-EVs from conditioned cell culture media for subsequent in-depth analysis remains challenging. Aim: We utilized the extracellular vesicles (EV) microfluidics herringbone chip (EVHB-Chip), coated with anti-VCAM-1 antibodies, for selective capture of VCAM-1+ cells and EC-EVs. Methods and Results: Engineered EA.hy926 endothelial cells overexpressing VCAM-1 (P < 0.001 versus control) showed increased binding to the VCAM-1- EVHB-Chip versus an IgG device. TNF-α-stimulated human umbilical cord vein endothelial cells (HUVECs) exhibited elevated VCAM-1 protein levels (P < 0.001) and preferential binding to the VCAM-1- EVHB-Chip versus the IgG device. HUVECs stimulated with TNF-α showed differential gene expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.001) and VCAM-1 (P < 0.001) by digital droplet PCR versus control cells. HUVEC-derived EC-EVs were positive for CD9, CD63, HSP70, and ALIX and had a modal size of 83.5 nm. Control and TNF-α-stimulated HUVEC-derived EC-EV cultures were captured on the VCAM-1- EVHB-Chip, demonstrating selective capture. VCAM-1+ EC-EV were significantly enriched for ICAM-1 (P < 0.001) mRNA transcripts. Conclusion: This study presents a novel approach using the EVHB-Chip, coated with anti-VCAM-1 antibodies and digital droplet PCR for the study of VCAM-1+ EC-EVs. Isolation of VCAM-1+ EC-EV from heterogeneous sources such as conditioned cell culture media holds promise for subsequent detailed characterization, and may facilitate the study of VCAM-1+ EC-EVs in cardiovascular and metabolic diseases, for disease monitoring and therapeutic insights