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

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

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

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

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

A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo.

Using an in vivo test system, the role of the β1 integrin very late activation antigen-4 (VLA-4) in eosinophil accumulation in allergic and nonallergic inflammatory reactions was investigated. Eosinophil infiltration and edema formation were measured as the local accumulation of intravenously injected 111In-labeled eosinophils and 125I-human serum albumin. The inflammatory reactions investigated were a passive cutaneous anaphylaxis (PCA) reaction and responses elicited by intradermal soluble inflammatory mediators (platelet-activating factor, leukotriene B4, C5a des Arg), arachidonic acid, and zymosan particles. The in vitro pretreatment of 111In-eosinophils with the anti-VLA-4 monoclonal antibody (mAb) HP1/2, which crossreacts with guinea pig eosinophils, suppressed eosinophil accumulation in all the inflammatory reactions investigated. Eosinophil accumulation was inhibited to the same extent when mAb HP1/2 was administered intravenously. It is interesting that HP1/2 had no effect on stimulated edema formation. These results suggest a role for VLA-4 in eosinophil accumulation in vivo and indicate a dissociation between the inflammatory events of eosinophil accumulation and edema formation

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis

Mycophenolate mofetil inhibits lymphocyte binding and the upregulation of adhesion molecules in acute rejection of rat kidney allografts.

Mycophenolate mofetil (MMF) interacts with purine metabolism and possibly with the expression of adhesion molecules. In the present study, we analysed the expression of these molecules in transplanted kidney allografts treated with RS LBNF1 kidneys were orthotopically transplanted into Lewis rats and either treated with RS (20 mg/kg/day) or vehicle. Rats were harvested 3, 5 and 7 days following transplantation. For binding studies, fresh-frozen sections of transplanted kidneys were incubated with lymph node lymphocytes (LNL) derived from transplanted rats. Additionally, immunohistology was performed with various monoclonal antibodies. In general, MMF resulted in better preservation of graft structure by 7 days. Cellular infiltration and tubular atrophy were less pronounced. At day 3, macrophages were diminished in MMF-treated animals to a high extent, while the number of T cells was almost identical to that of controls. In addition, the number of cells positive for MHC class II and LFA-1 was reduced in the MMF-treated animals. These findings correlated with the binding results. Three days following engraftment, LNL bound to MMF-treated kidneys to a lesser extent compared to controls. In conclusion, MMF resulted in a markedly reduced leucocytic infiltrate, presumably based on a reduced expression of lymphocytic adhesion molecules and an interaction with macrophages