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.

β-catenin/TCF signaling regulates a varied set of cellular functions including development and remodeling. Fibronectin is a TCF-regulated gene that is highly expressed in arterial endothelium during atherosclerosis development and contributes to the pathophysiology of the disease. However, the activation of endothelial β-catenin/TCF signaling and its role in fibronectin expression in atherosclerosis are not currently known. Objective To assess the activity of β-catenin/TCF signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. Methods and Results Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta prior to and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of β-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required β-catenin, TCF and the transcriptional co-activator CBP. Finally, we identified PECAM-1as a critical regulator of constitutive β-catenin and GSK-3β activities. Conclusions This data uncovers novel constitutive activation of the endothelial β-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.

The effects of energy balance on hormonal secretion patterns and the structure of recovered oocytes were evaluated in 20 lactating Holstein cows during two trial periods. Cows were randomly assigned to one of two dietary treatments formulated so that dry matter consumption was 3.6% of body weight (high energy; 1.78 Mcal/kg) or 3.2% of body weight (low energy; 1.52 Mcal/kg). Ovum recovery procedures were conducted twice weekly between d 30 and 100 of lactation. Follicle size and number were recorded. Follicular fluid aspirated from the largest follicle and serum samples were collected for hormone assay. Milk yield averaged 41.6 +/- 0.3 kg/d (mean +/- SE) for high energy fed cows and 32.8 +/- 0.3 kg/d for low energy fed cows. Oocyte numbers increased linearly from d 30 to 100 postpartum. Cows fed high energy diets produced more good (+) oocytes than did cows fed low energy diets.

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 ␣2␤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. 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...

Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center toward the leading edge of migrating cells in a Cdc42-dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to 4 days, but not shear stress-induced morphology. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor-free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and induced an increase in the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant-negative Par6, a major downstream effector of Cdc42-induced polarity. Shear stress-induced directed migratory polarity is modulated by exogenous growth factors and dependent on Par6 activity and shear stress direction.

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Prolonged dietary energy restriction has been shown to influence the secretion of ovarian hormones and follicular development. Energy balance affects the ability of the cow to support a functional reproductive system during early lactation. Follicular aspiration is a tool that can be used to assess the impact of dietary energy and stage of lactation on follicle development in dairy cattle. Follicular aspiration was conducted twice weekly from d 30 to 100 postpartum in cows fed either 104 or 80% of NRC energy requirements. Oocyte numbers recovered increased linearly from d 30 to 100 postpartum. Cows on high energy diets produced more high quality oocytes, but also produced more poor quality oocytes than cows fed the lower energy diet. Follicles < 5 mm predominated throughout. Cows in early (d 28) and mid (d 117) lactation were used to assess stage of lactation effects on serum and follicular fluid hormones, and quality of oocytes recovered by follicular aspiration procedures for 10 wk. Serum IGF-1 decreased in early lactation cows, but increased in midlactation cows. Follicular fluid progesterone in early lactation cows decreased from 275 to 235 ng/ml, whereas in midlactation cows it increased from 65 to 550 ng/ml at aspiration wk 10. The percentage of high quality ova was higher in early lactation cows (11%) than midlactation cows (6%), but fell to 1% by aspiration wk 7 and increased to 10% by wk 10, whereas percentage of high quality ova in midlactation cows steadily declined to 2% by wk 10. Milk yield and energy balance affect patterns of folliculogenesis in lactating dairy cows.