A clear relationship between diabetes and cardiovascular disease has been established for decades. Despite this, the mechanisms by which diabetes contributes to plaque formation remain in question. Some of this confusion derives from studies in type 2 diabetics where multiple components of metabolic syndrome show proatherosclerotic effects independent of underlying diabetes. However, the hyperglycemia that defines the diabetic condition independently affects atherogenesis in cell culture systems, animal models, and human patients. Endothelial cell biology plays a central role in atherosclerotic plaque formation regulating vessel permeability, inflammation, and thrombosis. The current paper highlights the mechanisms by which hyperglycemia affects endothelial cell biology to promote plaque formation.

Objective Endothelial cell activation results in altered cell-cell interactions with adjacent endothelial cells and with infiltrating leukocytes. Eph receptors and their ephrin ligands regulate cell-cell interactions during tissue remodeling, and multiple proinflammatory mediators induce endothelial EphA receptor and ephrinA ligand expression. Therefore, we sought to elucidate the role of EphA receptors and ephrinA ligands in endothelial cell activation and atherosclerosis. Methods and Results qRT-PCR screening for EphA/ephrinA expression in atherosclerosis-prone macrovascular endothelium identified EphA2, EphA4, and ephrinA1 as the dominant isoforms. Endothelial activation with oxidized LDL (oxLDL) and proinflammatory cytokines induced EphA2 and ephrinA1 expression and sustained EphA2 activation, whereas EphA4 expression was unaffected. Atherosclerotic plaques from mice and humans show enhanced EphA2 and ephrinA1 expression colocalizing in the endothelial cell layer. EphA2 activation with recombinant Fc-ephrinA1 induces proinflammatory gene expression (ex. VCAM-1, E-Selectin) and stimulates monocyte adhesion, while inhibiting EphA2 (siRNA, pharmacological inhibitors) abrogated both ephrinA1-induced and oxLDL-induced VCAM-1 expression. Conclusions The current data suggest that enhanced EphA2 signaling during endothelial cell activation perpetuates proinflammatory gene expression. Coupled with EphA2 expression in mouse and human atherosclerotic plaques, these data implicate EphA2 as a novel proinflammatory mediator and potential regulator of atherosclerotic plaque development.

Background Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. Methods Here, we assessed EphA2's role in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. Following 8-16 weeks Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. Results Despite enhanced weight gain and plasma lipid levels compared to Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. While plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2-/-Apoe-/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression following smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. Conclusions Together these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting monocyte firm adhesion, whereas smooth muscle EphA2 expression may regulate the progression to advanced atherosclerosis by regulating smooth muscle proliferation and extracellular matrix deposition.

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5β2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases.

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Key Words: shear stress Ⅲ extracellular matrix Ⅲ protein kinase A Ⅲ p21-activated kinase Ⅲ NF-B A therosclerosis, a chronic inflammatory disease of artery walls, originates as regions of local endothelial cell dysfunction characterized by enhanced permeability, inflammatory gene expression, and turnover. 1 Although classic risk factors for atherosclerosis, including hypercholesterolemia, hyperglycemia, and smoking occur throughout the vasculature, atherosclerotic plaques preferentially form at vessel curvatures, branch points, and bifurcations, where blood flow is of lower magnitude and exhibits complex features including turbulence, oscillations, separation, and reattachment, which we term disturbed flow. 2 Thus, flow patterns critically regulate the local susceptibility to atherosclerosis.In vitro, laminar flow inhibits endothelial activation and turnover, whereas disturbed flow induces inflammatory signaling, enhanced turnover, and other features of atherosclerosis-susceptible regions of arteries. 3 Interestingly, laminar and disturbed flow both initially activate nuclear factor (NF)-B 4 and JNK (c-Jun N-terminal kinase), 5 intercellular adhesion molecule (ICAM)-1 expression, 6 and enhanced permeability. 7 However, in laminar flow, these events decrease at later times as the cells align in the direction of flow, whereas in disturbed flow, these events remain elevated. 3 Thus, the inability of cells to adapt to disturbed flow may mediate the differential cellular responses to these 2 flow patterns.Integrins mediate an important subset of the endothelial response to flow. Shear stress stimulates integrin activation, 8,9 conversion from a low affinity to a high affinity state, which triggers new integrin-matrix binding; integrins thereby regulate both flow-induced endothelial cell alignment and proinflammatory gene expression. 10 Individual integrin heterodimers differ in both their ligand preferences and signaling Original received September 30, 2009; revision received February 22, 2010; accepted February 25, 2010 7 and PAK mediates matrix-specific activation of the proinflammatory transcription factor NF-B, 13,14 inflammatory gene expression, 13 and increased endothelial permeability 7 by flow. PAK is activated in atheroprone regions in vivo, which correlates with areas of fibronectin deposition and inflammatory gene expression. 7 However, the mechanisms mediating matrix-specific PAK activation remain unknown.PAK is activated by the small GTPases Rac and Cdc42, 12 and suppressed by PAK inhibitory proteins (eg, nischarin, hPIP1), 15,16 by dephosphorylation by phosphatases (PP2A, POPX1/2), 17,18 and by phosphorylation by protein kinase (PK)A. 19 We therefore set out to elucidate the mechanism of differential PAK activation on different matrix proteins. Our results identify PKA as the critical mediator of matrixspecific PAK activation and hence proinflammatory signaling through NF-B. MethodsBriefly, bovine aortic endothelial cells (BAECs) and human aortic endothelial cells (HAECs) on glass slides coated with s...

PAK2 mediates shear stress–induced NF-κB activation. Basement membrane proteins limit the proinflammatory response to shear by blocking the interaction of PAK2 with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain.

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin 2 (), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a mouse model of autosomal recessive Alport syndrome and increased proteinuria in females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen 345(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.