Ryan E. Feaver

Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice.

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.

Endothelial Cell PECAM-1 Promotes Atherosclerotic Lesions in Areas of Disturbed Flow in ApoE-Deficient Mice

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-&kgr;B 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-&kgr;B 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-&kgr;B–mediated gene expression.

GRP78 Upregulation by Atheroprone Shear Stress Via p38-, α2β1-Dependent Mechanism in Endothelial Cells

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 &agr;2&bgr;1. 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.

Atheroprone Hemodynamics Regulate Fibronectin Deposition to Create Positive Feedback that Sustains Endothelial Inflammation

Rationale: The extracellular matrix protein fibronectin (FN) is focally deposited in regions of atherosclerosis, where it contributes to inflammatory signaling. Objective: To elucidate the mechanism by which FN deposition is regulated by local shear stress patterns, its dependence on platelet-endothelial cell adhesion molecule (PECAM)-1 mechanotransduction and the role this pathway plays in sustaining an atheroprone/proinflammatory phenotype. Methods and Results: Human endothelial cells were exposed in vitro to atheroprone or atheroprotective shear stress patterns derived from human carotid arteries. Onset of atheroprotective flow induced a transient increase in FN deposition, whereas atheroprone flow caused a steady increase in FN expression and integrin activation over time, leading to a significant and sustained increase in FN deposition relative to atheroprotective conditions. Comparing FN staining in ApoE−/− and ApoE−/−PECAM−/− mice showed that PECAM-1 was essential for FN accumulation in atheroprone regions of the aortic arch. In vitro, small interfering RNA against PECAM-1 blocked the induction of FN and the activation of nuclear factor (NF)-&kgr;B by atheroprone flow, which was rescued by the addition of exogenous FN. Additionally, blocking NF-&kgr;B activation attenuated the flow-induced FN expression. Small interfering RNA against FN significantly reduced NF-&kgr;B activity, which was rescued by the addition of exogenous FN. Conclusions: These results indicate that FN gene expression and assembly into matrix fibrils is induced by atheroprone fluid shear stress. This effect is mediated at least in part by the transcription factor NF-&kgr;B. Additionally, because FN promotes activation of NF-&kgr;B, atheroprone shear stress creates a positive feedback to maintain inflammation.

Human haemodynamic frequency harmonics regulate the inflammatory phenotype of vascular endothelial cells

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 0 th 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 endotheliums exquisite sensitivity to complex shear stress frequency harmonics and provide a mechanism for the focal development of vascular inflammation.

Human IL-8 Regulates Smooth Muscle Cell VCAM-1 Expression in Response to Endothelial Cells Exposed to Atheroprone Flow

Objective—Interleukin-8 (IL-8) is a soluble human-specific chemokine implicated in the development of the chronic inflammatory disease atherosclerosis. Recently, we showed that atheroprone hemodynamics induced IL-8 secretion from endothelial cells (ECs) concurrent with increased EC/smooth muscle cell (SMC) VCAM-1 expression in a human hemodynamic coculture model. Despite an IL-8 association with inflammation, we show here that blocking IL-8 activity during atheroprone flow resulted in increased levels of EC/SMC VCAM-1 expression. We tested the hypothesis that IL-8 limits SMC VCAM-1 expression in response to inflammatory stimuli, either atheroprone flow or cytokine interleukin-1β (IL-1β) addition. Methods and Results—Atheroprone flow increased monocyte adhesion in both EC/SMCs, concurrent with the induction of VCAM-1 protein. VCAM-1 antisera attenuated this response. IL-1β upregulated VCAM-1 in SMCs by 3-fold, a response inhibited by the addition of IL-8 at 24 hours. Neither IL-1β nor IL-8 induced proliferation or migration. Neutralization of the IL-8 receptor, CXCR2, further induced VCAM-1 in the presence of IL-1β, and phospho-p38 was required for NF-&kgr;B activation and VCAM-1 expression. Additionally, IL-8 reduced p38 activation and NF-&kgr;B activity induced by IL-1β alone. Conclusions—Together, these findings provide evidence for a novel role whereby IL-8 limits the inflammatory response in ECs/SMCs via VCAM-1 modulation.

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis.

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation

Objectives—The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. Approach and Results—We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. Conclusions—The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Non-alcoholic fatty liver disease (NAFLD) models in drug discovery

ABSTRACT Introduction: The progressive disease spectrum of non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), is a rapidly emerging public health crisis with no approved therapy. The diversity of various therapies under development highlights the lack of consensus around the most effective target, underscoring the need for better translatable preclinical models to study the complex progressive disease and effective therapies. Areas covered: This article reviews published literature of various mouse models of NASH used in preclinical studies, as well as complex organotypic in vitro and ex vivo liver models being developed. It discusses translational challenges associated with both kinds of models, and describes some of the studies that validate their application in NAFLD. Expert opinion: Animal models offer advantages of understanding drug distribution and effects in a whole body context, but are limited by important species differences. Human organotypic in vitro and ex vivo models with physiological relevance and translatability need to be used in a tiered manner with simpler screens. Leveraging newer technologies, like metabolomics, proteomics, and transcriptomics, and the future development of validated disease biomarkers will allow us to fully utilize the value of these models to understand disease and evaluate novel drugs in isolation or combination.

Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western world, affecting about 1/3 of the US general population and remaining as a significant cause of morbidity and mortality. The hallmark of the disease is the excessive accumulation of fat within the liver cells (hepatocytes), which eventually paves the way to cellular stress, injury and apoptosis. NAFLD is strongly associated with components of the metabolic syndrome and is fast emerging as a leading cause of liver transplant in the USA. Based on clinico-pathologic classification, NAFLD may present as isolated lipid collection (steatosis) within the hepatocytes (referred to as non-alcoholic fatty liver; NAFL); or as the more aggressive phenotype (known as non-alcoholic steatohepatitis; NASH). There are currently no regulatory agency- approved medication for NAFLD, despite the enormous work and resources that have gone into the study of this condition. Therefore, there remains a huge unmet need in developing and utilizing pre-clinical models that will recapitulate the disease condition in humans. In line with progress being made in developing appropriate disease models, this review highlights the cutting-edge preclinical in vitro and animal models that try to recapitulate the human disease pathophysiology and/or clinical manifestations.