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.

Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/βII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.

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...

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and the underlying cause of cardiovascular disease, a major cause of mortality worldwide. The over-accumulation of modified cholesterol-containing low-density lipoproteins (e.g. oxLDL) in the artery wall and the subsequent recruitment and activation of macrophages contributes to the development of atherosclerosis. The excessive uptake of modified-LDL by macrophages leads to a lipid-laden “foamy” phenotype and pro-inflammatory cytokine production. Modified-LDLs promote foam cell formation in part by stimulating de novo lipid biosynthesis. However, it is unknown if lipid biosynthesis directly regulates foam cell pro-inflammatory mediator production. Lipin-1, a phosphatidate phosphohydrolase required for the generation of diacylglycerol during glycerolipid synthesis has recently been demonstrated to contribute to bacterial-induced pro-inflammatory responses by macrophages. In this study we present evidence demonstrating the presence of lipin-1 within macrophages in human atherosclerotic plaques. Additionally, reducing lipin-1 levels in macrophages significantly inhibits both modified-LDL-induced foam cell formation in vitro, as observed by smaller/fewer intracellular lipid inclusions, and ablates modified-LDL-elicited production of the pro-atherogenic mediators tumor necrosis factor-α, interleukin-6, and prostaglandin E2. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to modified-LDL. These data begin to link the processes of foam cell formation and pro-inflammatory cytokine production within macrophages.

Background: Mitochondrial reactive oxygen species (ROS) contribute to inflammation and vascular remodeling during atherosclerotic plaque formation. C57BL/6N (6N) and C57BL/6J (6J) mice display distinct mitochondrial redox balance due to the absence of nicotinamide nucleotide transhydrogenase (NNT) in 6J mice. We hypothesize that differential NNT expression between these animals alters plaque development. Methods: 6N and 6J mice were treated with AAV8-PCSK9 (adeno-associated virus serotype 8/proprotein convertase subtilisin/kexin type 9) virus leading to hypercholesterolemia, increased low-density lipoprotein, and atherosclerosis in mice fed a high-fat diet (HFD). Mice were co-treated with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO to assess the contribution of mitochondrial ROS to atherosclerosis. Results: Baseline and HFD-induced vascular superoxide is increased in 6J compared to 6N mice. MitoTEMPO diminished superoxide in both groups demonstrating differential production of mitochondrial ROS among these strains. PCSK9 treatment and HFD led to similar increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of plaque formation. MitoTEMPO reduced plasma lipids but did not affect plaque formation in 6N mice. In contrast, MitoTEMPO surprisingly increased plaque formation in 6J mice. Conclusion: These data indicate that loss of NNT increases vascular ROS production and exacerbates atherosclerotic plaque development.

Hemodynamic shear stress critically regulates endothelial activation and atherogenesis by affecting cytoskeletal dynamics and endothelial gene expression. The Nck adaptor proteins (Nck1 and Nck2) couple tyrosine kinase signaling to induction of cytoskeletal remodeling pathways, and we previously demonstrated that a peptide corresponding to a Nck-binding sequence in p21 activate kinase (PAK) blunts shear stress-induced proinflammatory signaling (PAK, NF-κB) and permeability in vitro and in vivo . However, the specific roles of Nck1 and Nck2 in flow-induced endothelial activation remain to be elucidated. Here, we demonstrate that Nck1, but not Nck2, critically regulates shear stress-induced endothelial activation. We show that Nck1 deficiency (siRNA knockdown, genetic knockout) decreases basal and shear stress-induced proinflammatory signaling (PAK2, NF-κB) and proinflammatory gene expression (ICAM-1, VCAM-1). In addition, only Nck1, but not Nck2, knockdown/knockout limited shear-induced endothelial permeability. However, other shear stress-dependent pathways, such as Akt activation, proved Nck1-independent. By contrast, selective Nck2 depletion paradoxically increased basal PAK2 activation without significant effects on NF-κB signaling or permeability. Using the partial carotid ligation model of disturbed flow, we found that Nck1 knockout mice showed significantly reduced proinflammatory gene expression that was not further diminished upon Nck2 deletion. However, Nck2 was not inconsequential to the flow response, as Nck2 knockdown or knockout reduced actin cytoskeletal alignment with laminar flow. Taken together, our data suggests that Nck1 plays a dominant role in flow-induced endothelial activation in response to shear stress, whereas Nck2 may critically regulate flow-induced cytoskeletal remodeling and endothelial alignment.

Modified low density lipoproteins (modLDL) elicit macrophage generation into foam cells that release pro-inflammatory mediators driving atherosclerotic lesion progression causing cardiovascular disease. The molecular mechanisms that elicit foam cell inflammatory responses have not yet been fully elucidated. The lipid-laden phenotype that is characteristic of macrophage foam cells is due to lipid droplet biogenesis in response to excess cholesterol. Lipid droplet biogenesis is a process that is thought to be symptomatic of, but not drive atherosclerosis. Lipid droplet biogenesis requires glycerolipid synthesis, during which, lipin-1 converts phosphatidate into diglyceride as the penultimate step of lipid droplet generation. We had previously demonstrated lipin-1 is also required for modLDL-elicited pro-inflammatory response from macrophages. We hypothesized that modLDL elicits chronic diglyceride generation, via lipin-1 enzymatic activity, that activates signaling cascades responsible for foam cell pro-inflammatory responses. To test our hypotheses we stimulated wild type and lipin-1 depleted bone marrow-derived macrophages (BMDMs) with oxidized LDLs (oxLDLs). Stimulation of wild type BMDMs resulted in chronic activation of the signaling kinases PKCα/βII, ERK1/2 and the AP-1 transcription factor subunit cJun (up to 48 hours after stimulation). This pathway was not observed to be active in BMDMs depleted of lipin-1 either genetically or with siRNA. The pharmacological inhibition of lipin-1, PKCα/βII, ERK1/2 strongly suggest lipin-1- PKCα/βII-ERK1/2-cJun represents a signaling axis. Finally, each of these proteins were required for oxLDL-elicited pro-inflammatory responses by macrophages. These results suggest that augmented glycerolipid synthesis in macrophages due to modLDL stimulation is not just symptomatic of atherosclerosis but promote inflammatory responses that drive lesion progression

Rationale: Sigma 1 receptor (Sigmar1) is a highly expressed mitochondrion-associated ER membrane resident protein in different cell lines. We recently reported that Sigmar1 is highly expressed in cardiomyocytes but it’s molecular functions and role in the stress response still remains unknown. Objective: We investigated the functional role of Sigmar1 in mediating mitochondrial autophagy, mitochondrial fission and effects on stress resistance in the heart. Methods and Results: Subcellular fractionation and biochemical experiments confirmed Sigmar1 expression in the mitochondria, where it resides as an integral mitochondrial outer membrane protein. Sigmar1 overexpression induced mitochondrial fission, increased autophagosome formation and autophagic flux in cardiomyocytes. Similarly, cardiac specific Sigmar1 transgenic (Tg) mice showed increased levels of mitochondrial fission and mitochondrial autophagy without adverse effects. Conversely, Sigmar1 knockdown induced both mitochondrial elongation and accumulation of damaged mitochondria, whereas autophagosome formation and autophagic flux were reduced at baseline and in response to glucose deprivation in cardiomyocytes. Parallel studies using Sigmar1 knockout mice showed increased accumulation of abnormal mitochondria and significantly altered cardiac contractility. To define the functional significance of Sigmar1 in the cardiac stress response, we subjected the mice to ischemia/reperfusion (I/R) injury. Sigmar1 Tg mouse showed reduced infarct size, protected from I/R-injury induced adverse cardiac remodeling, and improved cardiac function associated with enhanced mitochondrial autophagy even 12 weeks after reperfusion injury. In contrary, knockdown of Sigmar1 evoked mitochondrial dysfunction, accumulation of abnormal mitochondria, enhanced adverse cardiac remodeling, aggravated cardiac dysfunction and increased susceptibility to I/R-injury. Conclusions: Our findings suggested that Sigmar1 is an integral mitochondrial outer membrane protein dispensable for constitutive mitochondrial quality control in normal hearts. Sigmar1 regulates mitochondrial autophagy to protect the heart against I/R injury-induced cardiac remodeling and dysfunction.