Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

OBJECTIVE: Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. APPROACH AND RESULTS: Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. CONCLUSIONS: These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease.

RAGE regulates the metabolic and inflammatory response to high-fat feeding in mice.

In mammals, changes in the metabolic state, including obesity, fasting, cold challenge, and high-fat diets (HFDs), activate complex immune responses. In many strains of rodents, HFDs induce a rapid systemic inflammatory response and lead to obesity. Little is known about the molecular signals required for HFD-induced phenotypes. We studied the function of the receptor for advanced glycation end products (RAGE) in the development of phenotypes associated with high-fat feeding in mice. RAGE is highly expressed on immune cells, including macrophages. We found that high-fat feeding induced expression of RAGE ligand HMGB1 and carboxymethyllysine-advanced glycation end product epitopes in liver and adipose tissue. Genetic deficiency of RAGE prevented the effects of HFD on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. RAGE deficiency had no effect on genetic forms of obesity caused by impaired melanocortin signaling. Hematopoietic deficiency of RAGE or treatment with soluble RAGE partially protected against peripheral HFD-induced inflammation and weight gain. These findings demonstrate that high-fat feeding induces peripheral inflammation and weight gain in a RAGE-dependent manner, providing a foothold in the pathways that regulate diet-induced obesity and offering the potential for therapeutic intervention.

Aldose reductase drives hyperacetylation of Egr-1 in hyperglycemia and consequent upregulation of proinflammatory and prothrombotic signals.

Sustained increases in glucose flux via the aldose reductase (AR) pathway have been linked to diabetic vascular complications. Previous studies revealed that glucose flux via AR mediates endothelial dysfunction and leads to lesional hemorrhage in diabetic human AR (hAR) expressing mice in an apoE(-/-) background. Our studies revealed sustained activation of Egr-1 with subsequent induction of its downstream target genes tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in diabetic apoE(-/-)hAR mice aortas and in high glucose-treated primary murine aortic endothelial cells expressing hAR. Furthermore, we observed that flux via AR impaired NAD(+) homeostasis and reduced activity of NAD(+)-dependent deacetylase Sirt-1 leading to acetylation and prolonged expression of Egr-1 in hyperglycemic conditions. In conclusion, our data demonstrate a novel mechanism by which glucose flux via AR triggers activation, acetylation, and prolonged expression of Egr-1 leading to proinflammatory and prothrombotic responses in diabetic atherosclerosis.

PKCß promotes vascular inflammation and acceleration of atherosclerosis in diabetic ApoE null mice.

OBJECTIVE: Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)ß, it was logical to investigate the role of PKCß in modulation of atherosclerosis in diabetes mellitus. APPROACH AND RESULTS: ApoE(-/-) and PKCß(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCß(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCß reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCß and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1ß in U937 macrophages. CONCLUSIONS: These data link enhanced activation of PKCß to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCß in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCß activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Formin mDia1 mediates vascular remodeling via integration of oxidative and signal transduction pathways.

RATIONALE: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. OBJECTIVE: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. METHODS AND RESULTS: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3ß, and consequent smooth muscle cell migration. CONCLUSIONS: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.

RAGE gene deletion inhibits the development and progression of ductal neoplasia and prolongs survival in a murine model of pancreatic cancer.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is implicated in pancreatic tumorigenesis. Activating Kras mutations and p16 inactivation are genetic abnormalities most commonly detected as pancreatic ductal epithelium progresses from intraepithelial neoplasia (PanIN) to adenocarcinoma (PDAC). OBJECTIVE: The aim of this study was to evaluate the effect of RAGE (or AGER) deletion on the development of PanIN and PDAC in conditional Kras ( G12D ) mice. MATERIALS AND METHODS: Pdx1-Cre; LSL-Kras ( G12D/+) mice were crossed with RAGE (-/-) mice to generate Pdx1-Cre; LSL-Kras ( G12D/+) ; RAGE (-/-) mice. Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-) mice were crossed with RAGE (-/-) mice to generate Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice. Pancreatic ducts were scored and compared to the relevant RAGE (+/+) controls. RESULTS: At 16 weeks of age, Pdx1-Cre; LSL-Kras ( G12D/+); RAGE (-/-) mice had significantly fewer high-grade PanIN lesions than Pdx1-Cre; LSL-Kras ( G12D/+); RAGE (+/+) controls. At 12 weeks of age, none of the Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice had PDAC compared to a 45.5% incidence of PDAC in Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (+/+) controls. Finally, Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice also displayed markedly longer median survival. CONCLUSION: Loss of RAGE function inhibited the development of PanIN and progression to PDAC and significantly prolonged survival in these mouse models. Further work is needed to target the ligand-RAGE axis for possible early intervention and prophylaxis in patients at risk for developing pancreatic cancer.

Reduction of PKCbetaII activity in smooth muscle cells attenuates acute arterial injury.

OBJECTIVE: The ubiquitous enzyme protein kinase C (PKC) has been linked to the pathogenesis of vascular injury, but the cell-specific and discrete functions of the betaII isoform have yet to be discovered in this setting. Our previous findings demonstrated significantly increased PKCbetaII in the membrane fraction of injured femoral arteries in wild type (WT) mice and revealed reduction of neointimal expansion in PKCbeta(-/-) mice after acute vascular injury. As PKCbeta(-/-) mice are globally devoid of PKCbeta, we established novel transgenic (Tg) mice to test the hypothesis that the action of PKCbetaII specifically in smooth muscle cells (SMCs) mediates the formation of neointimal lesions in response to arterial injury. METHODS: Tg mice expressing SM22alpha promoter-targeted mouse carboxyl-terminal deletion mutant PKCbetaII were produced using standard techniques, subjected to femoral artery injury and compared with littermate controls. Smooth muscle cells (SMCs) were isolated from wild type (WT) and Tg mice and exposed to a prototypic stimulus, tumor necrosis factor (TNF)-alpha. Multiple strategies were employed in vivo and in vitro to examine the molecular mechanisms underlying the specific effects of SMC PKCbetaII in neointimal expansion. RESULTS: In vivo and in vitro analyses demonstrated that PKCbetaII activity in SMCs was critical for neointimal expansion in response to arterial injury, at least in part via regulation of ERK1/2, Egr-1 and induction of MMP-9. CONCLUSIONS: These data identify the SMC-specific regulatory role of PKCbetaII in neointimal expansion in response to acute arterial injury, and suggest that targeted inactivation of PKCbetaII may be beneficial in limiting restenosis via suppression of the neointima-mediating effects of Egr-1 and MMP-9.

Mice deficient in PKCbeta and apolipoprotein E display decreased atherosclerosis.

Endothelial activation is a central initiating event in atheroma formation. Evidence from our laboratory and others has demonstrated links between activation of early growth response-1 (Egr-1) and atherosclerosis and also has demonstrated that activated protein kinase C (PKC) betaII is a critical upstream regulator of Egr-1 in response to vascular stress. We tested the role of PKCbeta in regulating key events linked to atherosclerosis and show that the aortas of apoE(-/-) mice display an age-dependent increase in PKCbetaII antigen in membranous fractions vs. C57BL/6 animals with a approximately 2-fold increase at age 6 wk and a approximately 4.5-fold increase at age 24 wk. Consistent with important roles for PKCbeta in atherosclerosis, a significant decrease in atherosclerotic lesion area was evident in PKCbeta(-/-)/apoE(-/-) vs. apoE(-/-) mice by approximately 5-fold, in parallel with significantly reduced vascular transcripts for Egr-1 and matrix metalloproteinase (MMP)-2 antigen and activity vs. apoE(-/-) mice. Significant reduction in atherosclerosis of approximately 2-fold was observed in apoE(-/-) mice fed ruboxistaurin chow (PKCbeta inhibitor) vs. vehicle. In primary murine and human aortic endothelial cells, the PKCbeta-JNK mitogen-activated protein kinase pathway importantly contributes to oxLDL-mediated induction of MMP2 expression. Blockade of PKCbeta may be beneficial in mitigating endothelial perturbation and atherosclerosis.

Inflammatory stress in primary venous and aortic endothelial cells of type 1 diabetic mice.

OBJECTIVE: The progression of diabetes is associated with profound endothelial dysfunction. We tested the hypothesis that cellular stress would be detectable in ECs retrieved from arterial and venous vessels of diabetic mice. METHOD: We describe a method for direct isolation of well-characterised aortic and venous ECs from mice in which cells are not subjected to propagation in culture. RESULTS: Gene expression profiling, confirmed by real-time PCR, revealed a progressive increase in markers of injury within two main gene families, EC activation and EC apoptosis, in aortic and venous ECs recovered from diabetic versus non-diabetic mice. In short-term diabetes, Il1b mRNA transcripts were higher in aortic and venous ECs of diabetic mice versus controls. In long-term diabetes, casp-1 mRNA transcripts were higher in aortic and venous ECs of diabetic mice versus controls. CONCLUSION: These data suggest that diabetes imparts diffuse endothelial perturbation in the arterial and venous endothelium.

Oxygen deprivation triggers upregulation of early growth response-1 by the receptor for advanced glycation end products.

Myocardial infarction, stroke, and venous thromboembolism are characterized by oxygen deprivation. In hypoxia, biological responses are activated that evoke tissue damage. Rapid activation of early growth response-1 in hypoxia upregulates fundamental inflammatory and prothrombotic stress genes. We probed the mechanisms mediating regulation of early growth response-1 and demonstrate that hypoxia stimulates brisk generation of advanced glycation end products (AGEs) by endothelial cells. Via AGE interaction with their chief signaling receptor, RAGE, membrane translocation of protein kinase C-betaII occurs, provoking phosphorylation of c-Jun NH(2)-terminal kinase and increased transcription of early growth response-1 and its downstream target genes. These findings identify RAGE as a master regulator of tissue stress elicited by hypoxia and highlight this receptor as a central therapeutic target to suppress the tissue injury-provoking effects of oxygen deprivation.

PKCbeta modulates ischemia-reperfusion injury in the heart.

Protein kinase C-betaII (PKCbetaII) is an important modulator of cellular stress responses. To test the hypothesis that PKCbetaII modulates the response to myocardial ischemia-reperfusion (I/R) injury, we subjected mice to occlusion and reperfusion of the left anterior descending coronary artery. Homozygous PKCbeta-null (PKCbeta(-/-)) and wild-type mice fed the PKCbeta inhibitor ruboxistaurin displayed significantly decreased infarct size and enhanced recovery of left ventricular (LV) function and reduced markers of cellular necrosis and serum creatine phosphokinase and lactate dehydrogenase levels compared with wild-type or vehicle-treated animals after 30 min of ischemia followed by 48 h of reperfusion. Our studies revealed that membrane translocation of PKCbetaII in LV tissue was sustained after I/R and that gene deletion or pharmacological blockade of PKCbeta protected ischemic myocardium. Homozygous deletion of PKCbeta significantly diminished phosphorylation of c-Jun NH(2)-terminal mitogen-activated protein kinase and expression of activated caspase-3 in LV tissue of mice subjected to I/R. These data implicate PKCbeta in I/R-mediated myocardial injury, at least in part via phosphorylation of JNK, and suggest that blockade of PKCbeta may represent a potent strategy to protect the vulnerable myocardium.

Receptor for advanced-glycation end products: key modulator of myocardial ischemic injury.

BACKGROUND: The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury. METHODS AND RESULTS: In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine. CONCLUSIONS: These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R.

Central role of PKCbeta in neointimal expansion triggered by acute arterial injury.

We tested the hypothesis that PKCbeta contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKCbeta null (-/-) mice or wild-type mice fed the PKCbeta inhibitor, ruboxistaurin, displayed significantly decreased neointimal expansion in response to acute femoral artery endothelial denudation injury compared with controls. In vivo and in vitro analyses demonstrated that PKCbetaII is critically linked to SMC activation, at least in part via regulation of ERK1/2 MAP kinase and early growth response-1. These data highlight novel roles for PKCbeta in the SMC response to acute arterial injury and suggest that blockade of PKCbeta may represent a therapeutic strategy to limit restenosis.

PKCbeta regulates ischemia/reperfusion injury in the lung.

Activation of PKCbetaII is associated with the response to ischemia/reperfusion (I/R), though its role, either pathogenic or protective, has not been determined. In a murine model of single-lung I/R, evidence linking PKCbeta to maladaptive responses is shown in the following studies. Homozygous PKCbeta-null mice and WT mice fed the PKCbeta inhibitor ruboxistaurin subjected to I/R displayed increased survival compared with controls. In PKCbeta-null mice, phosphorylation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), JNK, and p38 MAPK was suppressed in I/R. Expression of the immediate early gene, early growth response-1 (Egr-1), and its downstream target genes was significantly increased in WT mice in I/R, particularly in mononuclear phagocytes (MPs), whereas this expression was attenuated in PKCbeta-null mice or WT mice fed ruboxistaurin. In vitro, hypoxia/reoxygenation-mediated induction of Egr-1 in MPs was suppressed by inhibition of PKCbeta, ERK1/2, and JNK, but not by inhibition of p38 MAPK. These findings elucidate key roles for PKCbetaII activation in I/R by coordinated activation of MAPKs (ERK1/2, JNK) and Egr-1.

Early growth response-1 promotes atherogenesis: mice deficient in early growth response-1 and apolipoprotein E display decreased atherosclerosis and vascular inflammation.

Early growth response-1 (Egr-1) regulates expression of proinflammatory and procoagulant genes in acute cell stress. Experimental evidence suggested that Egr-1 transcripts were upregulated in human atherosclerotic plaques versus adjacent unaffected tissue. To test the impact of Egr-1 in chronic vascular stress, we examined its role in a murine model of atherosclerosis. Real-time PCR analysis of aortae retrieved from apoE-/- mice demonstrated increased Egr-1 transcripts in an age-dependent manner, compared with aortae retrieved from C57BL/6 control animals. Therefore, homozygous Egr-1-/- mice were bred into the apoE-/- background. Homozygous double-knockout mice (Egr-1-/-/apoE-/-) in the C57BL/6 background were maintained on normal chow diet. At age 14 and 24 weeks, atherosclerotic lesion area and complexity at the aortic root were strikingly decreased in mice deficient in both Egr-1 and apoE compared with mice deficient in apoE alone. In parallel, transcripts for genes regulating the inflammatory/prothrombotic response were diminished in Egr-1-/-/apoE-/- aortae versus apoE-/-. In vitro, oxidized low-density lipoprotein (OxLDL), a key factor inciting atherogenic mechanisms in the vasculature, upregulated Egr-1 expression in monocytes via the MEK-ERK1/2 pathway. We conclude that Egr-1 broadly regulates expression of molecules critically linked to atherogenesis and lesion progression.