Andrew W. Pryor

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.

Hemodynamic Activation of β-Catenin and T-Cell-Specific Transcription Factor Signaling in Vascular Endothelium Regulates Fibronectin Expression

Objective—The goal of this study was to assess the activity of &bgr;-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. Methods and Results—Histological staining identified preferential nuclear localization of &bgr;-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with &bgr;-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of &bgr;-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required &bgr;-catenin, TCF, and the transcriptional coactivator CRBP-binding protein. Finally, we identified platelet endothelial cell adhesion molecule-1 as a critical regulator of constitutive &bgr;-catenin and glycogen synthase kinase-3&bgr; activities. Conclusion—These data reveal novel constitutive activation of the endothelial &bgr;-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.

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.

FIRST DEMONSTRATION THAT HEPATIC APOB100 AND INTESTINAL APOB48 CO-LOCALIZE WITH MACROPHAGES IN HUMAN CAROTID ATHEROSCLEROTIC PLAQUES

We previously demonstrated the presence and localization of hepatic apoB100 and intestinal apoB48 in human carotid atherosclerotic plaques by immunoperoxidase, immunofluorescence (IF), immunoelectron microscopy, and western blot; however, co-localization of apoB48 and apoB100 with macrophages has