Evis Harja

Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE–/– mice

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.

Identification, classification, and expression of RAGE gene splice variants

The receptor for advanced glycation end‐products (RAGE) is a single‐transmembrane, mul tiligand receptor of the immunoglobulin superfamily. RAGE up‐regulation is implicated in numerous patho logical states including vascular disease, diabetes, can cer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease patho genesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand‐binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals~50% of identified variants are targeted to the nonsense‐mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the under standing of the gene in the normal and pathological state.—Hudson, B. I., Carter, A. M., Harja, E., Kalea, A. Z., Arriero, M., Yang, H., Grant, P. J., Schmidt, A. M. Identification, classification, and expression of RAGE gene splice variants. FASEB J. 22, 1572–1580 (2008)

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42.

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as “bait” in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.

Early Growth Response-1 Promotes Atherogenesis Mice Deficient in Early Growth Response-1 and Apolipoprotein E Display Decreased Atherosclerosis and Vascular Inflammation

Abstract— 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.

Mice deficient in PKCβ 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) βII is a critical upstream regulator of Egr‐1 in response to vascular stress. We tested the role of PKCβ in regulating key events linked to atherosclerosis and show that the aortas of apoE–/– mice display an age‐dependent increase in PKCβll antigen in membranous fractions vs. C57BL/6 animals with a ~2‐fold increase at age 6 wk and a ~4.5‐fold increase at age 24 wk. Consistent with important roles for PKCβ in atherosclerosis, a significant decrease in atherosclerotic lesion area was evident in PKCβ–/– apoE–/– vs. apoE–/– mice by ~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 ~2‐fold was observed in apoE–/– mice fed ruboxistaurin chow (PKCβ inhibitor) vs. vehicle. In primary murine and human aortic endothelial cells, the PKCβ‐JNK mitogen‐activated protein kinase pathway importantly contributes to oxLDL‐mediated induction of MMP2 expression. Blockade of PKCβ may be beneficial in mitigating endothelial perturbation and atherosclerosis.—Harja, E., Chang, J. S., Lu, Y., Leitges, M., Zou, Y. S., Schmidt, A. M., Yan, S.‐F. Mice deficient in PKC β and apolipoprotein E display decreased atherosclerosis. FASEB J. 23, 1081–1091 (2009)

Alternatively Spliced RAGEv1 Inhibits Tumorigenesis through Suppression of JNK Signaling

Receptor for advanced glycation end products (RAGE) and its ligands are overexpressed in multiple cancers. RAGE has been implicated in tumorigenesis and metastasis, but little is known of the mechanisms involved. In this study, we define a specific functional role for an alternate splice variant termed RAGE splice variant 1 (RAGEv1), which encodes a soluble endogenous form of the receptor that inhibits tumorigenesis. RAGEv1 was downregulated in lung, prostate, and brain tumors relative to control matched tissues. Overexpressing RAGEv1 in tumor cells altered RAGE ligand stimulation of several novel classes of genes that are critical in tumorigenesis and metastasis. Additionally, RAGEv1 inhibited tumor formation, cell invasion, and angiogenesis induced by RAGE ligand signaling. Analysis of signal transduction pathways underlying these effects revealed marked suppression of c-jun-NH(2)-kinase (JNK) pathway signaling, and JNK inhibition suppressed signaling through the RAGE pathway. Tumors expressing RAGEv1 were significantly smaller than wild-type tumors and displayed prominently reduced activation of JNK. Our results identify RAGEv1 as a novel suppressor, the study of which may offer new cancer therapeutic directions.

Central Role of PKCβ in Neointimal Expansion Triggered by Acute Arterial Injury

We tested the hypothesis that PKC&bgr; contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKC&bgr; null (−/−) mice or wild-type mice fed the PKC&bgr; 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 PKC&bgr;II 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 PKC&bgr; in the SMC response to acute arterial injury and suggest that blockade of PKC&bgr; may represent a therapeutic strategy to limit restenosis.