John Y.-J. Shyy

Objective—: Patients with rheumatoid arthritis are prone to atherosclerosis. We explored the role of elevated level of monocyte chemotactic protein-1 (MCP-1)–induced protein (MCPIP) in endothelial dysfunction associated with rheumatoid arthritis. Approach and Results—: The level of MCP-1 was elevated in sera from mice with collagen-induced arthritis (CIA) and was negatively correlated with endothelium-dependent vessel dilation. Aortas from CIA mice showed increased expression of MCPIP but decreased bioavailability of endothelial NO synthase–derived NO. Administering MCP-1 neutralizing antibody to CIA mice decreased the MCPIP level in aortas and alleviated endothelial dysfunction. In vitro, treating cultured vascular endothelial cells with MCP-1 or sera from CIA mice or rheumatoid arthritis patients increased the expression of MCPIP but inhibited endothelial NO synthase phosphorylation. These detrimental effects were reproduced in endothelial cells overexpressing MCPIP, with elevated redox stress. Small interfering RNA knockdown of MCPIP restored the endothelial NO synthase–derived NO bioavailability. Administering simvastatin to CIA mice ameliorated the endothelial dysfunction, with attendant decreased aortic level of MCPIP. The beneficial effect of the statin was mediated by inhibiting nuclear factor κB binding to the MCPIP gene enhancer. Conclusions—: Increased MCPIP is found in rheumatoid arthritis leading to endothelial dysfunction. Statin treatment or MCP-1 neutralizing antibody administration antagonizes MCPIP expression, thereby attenuating the endothelial dysfunction.

Despite a general repression of translation under hypoxia, cells selectively upregulate a set of hypoxia-inducible genes. Results from deep sequencing revealed that Let-7 and miR-103/107 are hypoxia-responsive microRNAs (HRMs) that are strongly induced in vascular endothelial cells. In silico bioinformatics and in vitro validation showed that these HRMs are induced by HIF1α and target argonaute 1 (AGO1), which anchors the microRNA-induced silencing complex (miRISC). HRM targeting of AGO1 resulted in the translational desuppression of VEGF mRNA. Inhibition of HRM or overexpression of AGO1 without the 3′ untranslated region decreased hypoxia-induced angiogenesis. Conversely, AGO1 knockdown increased angiogenesis under normoxia in vivo. In addition, data from tumor xenografts and human cancer specimens indicate that AGO1-mediated translational desuppression of VEGF may be associated with tumor angiogenesis and poor prognosis. These findings provide evidence for an angiogenic pathway involving HRMs that target AGO1 and suggest that this pathway may be a suitable target for anti- or proangiogenesis strategies.

B-cell lymphoma–6 protein (Bcl-6) is a corepressor for inflammatory mediators such as vascular cell adhesion molecule–1 and monocyte chemotactic protein–1 and –3, which function to recruit monocytes to vascular endothelial cells upon inflammation. Poly [ADP ribose] polymerase 1 (PARP-1) is proinflammatory, in part through its binding at theBcl-6intron 1 to suppress Bcl-6 expression. We investigated the mechanisms by which PARP-1 dissociates from theBcl-6intron 1, ultimately leading to attenuation of endothelial inflammation. Analysis of the PARP-1 primary sequence suggested that phosphorylation of PARP-1 Serine 177 (Ser-177) by AMP-activated protein kinase (AMPK) is responsible for the induction of Bcl-6. Our results show that AMPK activation with treatment of 5-aminoimidazole-4-carboxamide ribonucleotide, metformin, or pulsatile shear stress induces PARP-1 dissociation from theBcl-6intron 1, increases Bcl-6 expression, and inhibits expression of inflammatory mediators. Conversely, AMPKα suppression or knockdown produces the opposite effects. The results demonstrate an anti-infamatory pathway linking AMPK, PARP-1, and Bcl-6 in endothelial cells.

Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser⁷⁹, an AMPK-specific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca²⁺/calmodulin-dependent protein kinase kinase-β knockout (CaMKKβ^−/−) mice and cultured adipocytes, we further show that glucagon activates the CaMKKβ/AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKKβ^+/+but not CaMKKβ^−/−mice. These results indicate that CaMKKβ/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.

Background—: Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow–regulated KLF2. Methods and Results—: Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3′ untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow. Conclusions—: Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

Rationale:: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of γ/δ T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective:: To investigate whether shear stress regulates membrane translocation of ATP synthase β chain (ATPSβ) in ECs, leading to the increased γ/δ T-lymphocyte adhesion and the related functions. Method and Results:: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPSβ (ecto-ATPSβ) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPSβ and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with β-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPSβ translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of γ/δ T cells binding to ECs, which was blocked by anti-ATPSβ antibody. Furthermore, the incubation of γ/δ T cells with ECs increased tumor necrosis fact α and interferon-γ secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, γ/δ T-cell adhesion and ATPSβ membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE ^−/− mice fed a high-fat diet. Conclusions:: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote γ/δ T-lymphocyte activation by the membrane translocation of ATPSβ in ECs and in vivo in mice, which is a novel mechanism of endothelial activation.

Background: Proteinase-activated-receptor-2 (PAR₂) is a seven transmembrane receptor that can activate two separate signaling arms: one through Gαq and Ca²⁺ mobilization, and a second through recruitment of β-arrestin scaffolds. In some cases downstream targets of the Gαq/Ca²⁺ signaling arm are directly inhibited by β-arrestins, while in other cases the two pathways are synergistic; thus β-arrestins act as molecular switches capable of modifying the signal generated by the receptor. Results: Here we demonstrate that PAR₂ can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca²⁺-dependent Kinase Kinase β (CAMKKβ), while inhibiting AMPK through interaction with β-arrestins. The ultimate outcome of PAR₂ activation depended on the cell type studied; in cultured fibroblasts with low endogenous β-arrestins, PAR₂ activated AMPK; however, in primary fat and liver, PAR₂ only activated AMPK in β-arrestin-2^-/- mice. β-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKKβ under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR₂ activation. Addition of recombinant β-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKKβ on Thr172. Conclusions: Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for β-arrestin in the inhibition of AMPK signaling, raising the possibility that β-arrestin-dependent PAR₂ signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one.

Shear stress imposed by blood flow is crucial for maintaining vascular homeostasis. We examined the role of shear stress in regulating SIRT1, an NAD ⁺ -dependent deacetylase, and its functional relevance in vitro and in vivo. The application of laminar flow increased SIRT1 level and activity, mitochondrial biogenesis, and expression of SIRT1-regulated genes in cultured endothelial cells (ECs). When the effects of different flow patterns were compared in vitro, SIRT1 level was significantly higher in ECs exposed to physiologically relevant pulsatile flow than pathophysiologically relevant oscillatory flow. These results are in concert with the finding that SIRT1 level was higher in the mouse thoracic aorta exposed to atheroprotective flow than in the aortic arch under atheroprone flow. Because laminar shear stress activates AMP-activated protein kinase (AMPK), with subsequent phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser-633 and Ser-1177, we studied the interplay of AMPK and SIRT1 on eNOS. Laminar flow increased SIRT1-eNOS association and eNOS deacetylation. By using the AMPK inhibitor and eNOS Ser-633 and -1177 mutants, we demonstrated that AMPK phosphorylation of eNOS is needed to prime SIRT1-induced deacetylation of eNOS to enhance NO production. To verify this finding in vivo, we compared the acetylation status of eNOS in thoracic aortas from AMPKα2 ^−/− mice and their AMPKα2 ^+/+ littermates. Our finding that AMPKα2 ^−/− mice had a higher eNOS acetylation indicates that AMPK phosphorylation of eNOS is required for the SIRT1 deacetylation of eNOS. These results suggest that atheroprotective flow, via AMPK and SIRT1, increases NO bioavailability in endothelium.