We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

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Objective-Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). Methods and Results-In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5Ϯ3.2 versus 9.1Ϯ1.3 pg/mL; PϽ0.01). LDL induced the expression of CCL20 in VSMC in a dose-and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-B. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-B site (Ϫ80/Ϫ71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. A therosclerosis is essentially an inflammatory chronic disease. [1][2][3] Inflammation is a necessary response to injury and infection. Virtually all cardiovascular risk factors are capable of promoting an inflammatory response; among them, however, elevated levels of plasma cholesterol, in particular LDL, are recognized as one of the most important risk factors for atherosclerosis. 4,5 The inflammatory response involves the coordinated regulation of cell adhesion and migration and the establishment of a chemotactic gradient that guides inflammatory cells to damaged tissues. Key elements in this communication network are cytokines and chemokines, which orchestrate the recruitment, survival, expansion, and effector function of inflammatory cells. 6 -8 Chemokines are a superfamily of structurally related small chemotactic cytokines that control leukocyte function through interactions with their cognate 7-transmembranedomain G protein-coupled receptors. Monocytes/macrophages and T lymphocytes are the most abundant inflammatory cells found in atherosclerotic plaques, 9,10 but also B cells, dendritic cells, and neutrophils contribute to the pathogenesis of atherosclerosis. 9,11,12 Native and modified LDL modulate the expression of key genes involved in the recruitment and trafficking of inflammatory cells including cellular adhesion molecules and chemokines such as monocyte chemotactic protein 1. 4,5,[13][14][15] Recent studies have implicated other chemokines in atherosclerosis and have extended the knowledge about the regulation of chemokines/chemokine receptors on vascular cells, 6 -8 but the complete picture of these molecules involved in atherogenesis is not completely understood. Conclusion-ThisIncreasing data involving innate and adapti...

No drug therapy has shown to limit abdominal aortic aneurysm (AAA) growth or rupture, and the understanding of the disease biology is incomplete; whereby, one challenge of vascular medicine is the development of good animal models and therapies for this life-threatening condition. The nuclear receptor NOR-1 (neuron-derived orphan receptor 1) controls biological processes involved in AAA; however, whether it plays a role in this pathology is unknown. Through a gain-of-function approach we assessed the impact of NOR-1 expression on the vascular response to Ang II (angiotensin II). We used 2 mouse models that overexpress human NOR-1 in the vasculature, one of them specifically in vascular smooth muscle cells. NOR-1 transgenesis amplifies the response to Ang II enhancing vascular inflammation (production of proinflammatory cytokines, chemokines, and reactive oxygen species), increasing MMP (matrix metalloproteinase) activity and disturbing elastin integrity, thereby broking the resistance of C57BL/6 mice to Ang II-induced AAA. Genes encoding for proteins critically involved in AAA formation ( Il [interleukin]-6 , Il-1β , Cxcl2 , [C-X-C motif chemokine ligand 2], Mcp-1 [monocyte chemoattractant protein 1] , and Mmp2 ) were upregulated in aneurysmal tissues. Both animal models show a similar incidence and severity of AAA, suggesting that high expression of NOR-1 in vascular smooth muscle cell is a sufficient condition to strengthen the response to Ang II. These alterations, including AAA formation, were prevented by the MMP inhibitor doxycycline. Microarray analysis identified gene sets that could explain the susceptibility of transgenic animals to Ang II-induced aneurysms, including those related with extracellular matrix remodeling, inflammatory/immune response, sympathetic activity, and vascular smooth muscle cell differentiation. These results involve NOR-1 in AAA and validate mice overexpressing this receptor as useful experimental models.

Matrix metalloproteinases (MMPs) are associated with tissue remodelling and repair. In non-vascular tissues, NR4A receptors have been involved in the regulation of MMPs by transcriptional repression mechanisms. Here, we analyse alternative mechanisms involving NR4A receptors in the modulation of MMP activity in vascular smooth muscle cells (VSMC). Lentiviral overexpression of NR4A receptors (NOR-1, Nurr1 and Nur77) in human VSMC strongly decreased MMP-2 and MMP-9 activities (analysed by zymography and DQ-gelatin assays) and protein levels. NR4A receptors also down-regulated MMP-2 mRNA levels. Real-time PCR analysis evidenced that alpha-2-macroglobulin (A2M), but not other MMP inhibitors (TIMP-1 and TIMP-2) were up-regulated in NR4A-transduced cells. Interestingly, A2M was expressed in human vascular tissues including the smooth muscle media layer. While NR4A receptors increased A2M expression and secretion in VSMC, NR4A knockdown significantly reduced basal A2M expression in these cells. The direct transcriptional regulation of the human A2M promoter by NR4A receptors was characterised in luciferase reporter assays, electrophoretic mobility shift assays and by chromatin immunoprecipitation, identifying a NGFI-B response element (NBRE-71/-64) essential for the NR4A-mediated induction. The blockade of A2M partially prevented the reduction of MMPs activity observed in NR4A-transduced cells. Although mouse A2M promoter was unresponsive to NR4A receptors, vascular MMP expression was attenuated in transgenic mice over-expressing human NOR-1 in VSMC challenged with lipopolysaccharide. Our results show that the pan-proteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M.

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Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells.

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: