The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal. IntroductionThrough the elaboration of numerous biological substances, ECs actively regulate fundamental physiological processes, such as regulation of blood coagulation, homing of immune cells, and barrier function. Studies over the past several decades have also identified key physiologic and pathologic phenotypic modulators of ECs. For example, stimulation of the endothelium with proinflammatory cytokines renders the endothelium dysfunctional, inducing a proadhesive and prothrombotic phenotype. In contrast, laminar flow induces critical genes that confer potent antithrombotic, antiadhesive, and antiinflammatory properties. The significance of fluid shear stress is evidenced by the observation that segments of the arterial tree exposed to laminar flow (e.g., straight regions of the vasculature) are less prone to the development of atherosclerotic lesions than are regions exposed to nonlaminar/disturbed flow (e.g., branch points). These observations have led to the current view that the balance of biochemical and biomechanical stimuli is the central determinant of vascular function under physiologic and pathologic conditions. Given the importance of the endothelium in vessel homeostasis, there is great interest in identifying molecular pathways that mediate the effects of both biochemical and biomechanical stimuli. Prior studies from our group and others have identified 2 members of the Kruppel-like factor (KLF) family of transcription factors, KLF2 and KLF4, as being of particular interest. Both KLF2 and KLF4 are induced by laminar flow and in in vitro stud-
The heterogeneity of biological processes driving the severity of nonalcoholic fatty liver disease (NAFLD) as reflected in the transcriptome and the relationship between the pathways involved are not well established. Well-defined associations between gene expression profiles and disease progression would benefit efforts to develop novel therapies and to understand disease heterogeneity. We analyzed hepatic gene expression in controls and a cohort with the full histological spectrum of NAFLD. Protein-protein interaction and gene set variation analysis revealed distinct sets of coordinately regulated genes and pathways whose expression progressively change over the course of the disease. The progressive nature of these changes enabled us to develop a framework for calculating a disease progression score for individual genes. We show that, in aggregate, these scores correlate strongly with histological measures of disease progression and can thus themselves serve as a proxy for severity. Furthermore, we demonstrate that the expression levels of a small number of genes (~20) can be used to infer disease severity. Finally, we show that patient subgroups can be distinguished by the relative distribution of gene-level scores in specific gene sets. While future work is required to identify the specific disease characteristics that correspond to patient clusters identified on this basis, this work provides a general framework for the use of high-content molecular profiling to identify NAFLD patient subgroups.
Objective Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. Methods and Results We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-κB were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-κB activity and VCAM-1 expression under conditions of atheroprone flow. Conclusion These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-κB–mediated gene expression.
Dash A, Simmers MB, Deering TG, Berry DJ, Feaver RE, Hastings NE, Pruett TL, LeCluyse EL, Blackman BR, Wamhoff BR. Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro. Am J Physiol Cell Physiol 304: C1053-C1063, 2013. First published March 13. 2013 doi:10.1152/ajpcell.00331.2012.-In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma Cmax levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4␣ (HNF-4␣)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ϳ4-fold and urea ϳ5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 Ϯ 10.3; CYP1A2: 64.0 Ϯ 15.1; CYP2B1: 15.2 Ϯ 2.9; CYP2B2: 2.7 Ϯ 0.8; CYP3A2: 4.0 Ϯ 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 Ϯ 2.41 vs. 0.42 Ϯ 0.015; CYP1B: 3.47 Ϯ 1.66 vs. 0.4 Ϯ 0.09; CYP3A: 11.65 Ϯ 4.70 vs. 2.43 Ϯ 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A ϭ 27.33 and CYP3A ϭ 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.hemodynamics; hepatocyte; metabolism; organotype; phenotype HEPATOTOXICITY AND BIOAVAILABILITY issues comprise over 60% of drug failures during clinical trials (45) and are a major cause of postmarketing withdrawal (23), pointing to the need to develop more efficient and predictive preclinical test systems. Simple cellular and subcellular assays used to screen compound libraries offer the advantage of higher throughput but are often unable to capture complex biological effects that may require a physiological context for drug interactions with cells. Primary in vitro hepatocyte models widely used to study liver disease, drug metabolism, and toxicity are extensively reviewed in the literature (16,42). The ability to test the metabolic f...
Key Words: hemodynamics Ⅲ atherosclerosis Ⅲ fibronectin A therosclerosis is a focal inflammatory disease of the vasculature, marked by chronic activation of the endothelium. 1 In regions of disease, inflammatory signaling is continuously stimulated by the local environment to promote disease progression. It is known that atherogenic environments tend to develop in bulbous, bending, or bifurcating locations in the vasculature that generally have low and oscillatory shear stress patterns compared to atheroprotective regions where shear stress is generally unidirectional and high. 2 Indeed, atheroprone and atheroprotective in vitro shear stress induce distinct endothelial phenotypes, and shear stress is among the early endothelial activators that lead to focal atherogenesis. 3,4 Atherogenic shear stress patterns activate the inflammatory transcription factor nuclear factor (NF)-B, whose downstream targets include multiple cytokines (monocyte chemoattractant protein [MCP]1, interleukin [IL]-8) and adhesion molecules (vascular cell adhesion molecule [VCAM], E-selectin), which together mediate recruitment of leukocytes necessary for lesion formation. 4 -6 NF-B activation is mediated by a shear stress mechanosensor, platelet-endothelial cell adhesion molecule (PECAM)-1. 7,8 PECAM is an intercellular junction protein that forms a mechanosensory complex with VE-Cadherin and VEGF receptor 2. This complex initiates many early signaling events in response to shear stress, including activation of MAP kinases and integrins. 7 PECAM is necessary for the activation of NF-B, lesion formation, and vascular remodeling in atherogenic regions in PECAM Ϫ/Ϫ mouse models of atherosclerosis. 8 -11 Therefore, shear stress activates endothelial inflammation via mechanotransduction in response to local atheroprone shear stress.Atheroprone regions are also marked by differences in the extracellular matrix. The endothelium normally resides on an extracellular matrix composed primarily of collagen IV and Collectively, atheroprone regions have heightened inflammation, which is regulated by PECAM mechanotransduction and enhanced by local FN deposition. The goal of the present study was to determine whether fluid shear stress mediates the deposition of FN beneath the endothelium at atheroprone regions of arteries. These data reveal a positive feedback mechanism that could play a prominent role in the chronic inflammation and hence plaque formation in these regions. Interrupting this loop may therefore provide a novel approach for the prevention, regression or stabilization of atherosclerosis. Methods Mouse ModelsAll mouse studies were conducted with the approval of the University of Virginia Animal Care and Use Committee (ACUC no. 3597), and in accordance with the NIH Guide for the Care and Use of Laboratory Animals. To study the role of PECAM in FN deposition, tissue sections from atheroprone aortic arches of both ApoE Ϫ/Ϫ (nϭ3) and ApoE Ϫ/Ϫ PECAM Ϫ/Ϫ (nϭ3) double knockout (DKO) mice (C57BL6; previously described 8 ) and were euthanized ...
Objective-The initiation of atherosclerosis is in part dependent on the hemodynamic shear stress environment promoting a proinflammatory phenotype of the endothelium. Previous studies demonstrated increased expression of ER stress protein and unfolded protein response (UPR) regulator, GRP78, within all vascular cells in atherosclerotic lesions and its regulation in the endothelium by several atherosclerotic stressors; however, regulation of GRP78 by shear stress directly has not been established. Method and Results-Using an in vitro model to simulate human arterial shear stress waveforms, atheroprone or atheroprotective flow was applied to human endothelial cells. GRP78 was found to be significantly upregulated (3-fold) in a sustained manner under atheroprone, but not atheroprotective flow up to 24 hours. This response was dependent on both sustained activation of p38, as well integrin ␣21. Increased GRP78 correlated with the activation of the ER stress sensing element (ERSE1) promoter by atheroprone flow as a marker of the UPR. Shear stress regulated GRP78 through increased protein stability when compared to other flow regulated proteins, such as connexin-43 and vascular cell adhesion molecule (VCAM)-1. Increased endothelial expression of GRP78 was also observed in atheroprone versus atheroprotective regions of C57BL6 mice. Conclusions-This study supports a role of the hemodynamic environment in preferentially inducing GRP78 and the UPR in atheroprone regions, before lesion development, and suggests a potential atheroprotective (ie, prosurvival), compensatory effect in response to ER stress within atherosclerotic lesions. Key Words: endothelial Ⅲ GRP78 Ⅲ shear stress Ⅲ atherosclerosis Ⅲ unfolded protein response A therosclerosis is a focal inflammatory disease that develops preferentially in areas of disturbed flow, where variations in shear stress have been shown to alter the phenotypes of endothelial cells toward either an atheroprone or atheroprotective state in vitro and in vivo. 1,2 Therefore, hemodynamic-induced shear stress provides a major mechanical signal, which causes the overlying endothelium to become at risk for the promotion of atherosclerosis.Of many proteins of interest, the chaperone protein, glucose regulated protein 78 (GRP78), a common marker for endoplasmic reticulum (ER) stress, is preferentially expressed in advanced atherosclerotic lesions 3 and on the fibrous cap surface in ApoE-KO mice. 4 Further, cell-surface associated GRP78 has been speculated to serve a protective role in atheroprone environments by inhibiting tissue factor through direct binding to the endothelium overlying the plaque. 5 Hyperhomocysteinemia is associated with increased risk of cardiovascular disease possibly by limiting the antioxidant activity and causing ER stress, leading to the activation of GRP78. 4 ER stress is further linked to oxidative stress through peroxynitrite-induced GRP78 expression. 3 ER stress is present at every stage of atherosclerosis, even preceding free cholesterol accumulation. 6 This su...
Haemodynamic variations are inherent to blood vessel geometries (such as bifurcations) and correlate with regional development of inflammation and atherosclerosis. However, the complex frequency spectrum characteristics from these haemodynamics have never been exploited to test whether frequency variations are critical determinants of endothelial inflammatory phenotype. Here we utilize an experimental Fourier transform analysis to systematically manipulate individual frequency harmonics from human carotid shear stress waveforms applied in vitro to human endothelial cells. The frequency spectrum, specifically the 0th and 1st harmonics, is a significant regulator of inflammation, including NF-κB activity and downstream inflammatory phenotype. Further, a harmonic-based regression-model predicts eccentric NF-κB activity observed in the human internal carotid artery. Finally, short interfering RNA-knockdown of the mechanosensor PECAM-1 reverses frequency-dependent regulation of NF-κB activity. Thus, PECAM-1 may have a critical role in the endothelium’s exquisite sensitivity to complex shear stress frequency harmonics and provide a mechanism for the focal development of vascular inflammation.
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