Lysyl oxidase expression in smooth muscle cells determines the level of intima calcification in hypercholesterolemia-induced atherosclerosis. / La expresión de la lisil oxidasa en las células musculares lisas determina el nivel de calcificación de la íntima en la aterosclerosis inducida por hipercolesterolemia.

INTRODUCTION: Cardiovascular calcification is an important public health issue with an unmeet therapeutic need. We had previously shown that lysyl oxidase (LOX) activity critically influences vascular wall smooth muscle cells (VSMCs) and valvular interstitial cells (VICs) calcification by affecting extracellular matrix remodeling. We have delved into the participation of LOX in atherosclerosis and vascular calcification, as well as in the mineralization of the aortic valve. METHODS: Immunohistochemical and expression studies were carried out in human atherosclerotic lesions and experimental models, valves from patients with aortic stenosis, VICs, and in a genetically modified mouse model that overexpresses LOX in CMLV (TgLOXCMLV). Hyperlipemia and atherosclerosis was induced in mice through the administration of adeno-associated viruses encoding a PCSK9 mutated form (AAV-PCSK9D374Y) combined with an atherogenic diet. RESULTS: LOX expression is increased in the neointimal layer of atherosclerotic lesions from human coronary arteries and in VSMC-rich regions of atheromas developed both in the brachiocephalic artery of control (C57BL/6J) animals transduced with PCSK9D374Y and in the aortic root of ApoE-/- mice. In TgLOXCMLV mice, PCSK9D374Y transduction did not significantly alter the enhanced aortic expression of genes involved in matrix remodeling, inflammation, oxidative stress and osteoblastic differentiation. Likewise, LOX transgenesis did not alter the size or lipid content of atherosclerotic lesions in the aortic arch, brachiocephalic artery and aortic root, but exacerbated calcification. Among lysyl oxidase isoenzymes, LOX is the most expressed member of this family in highly calcified human valves, colocalizing with RUNX2 in VICs. The lower calcium deposition and decreased RUNX2 levels triggered by the overexpression of the nuclear receptor NOR-1 in VICs was associated with a reduction in LOX. CONCLUSIONS: Our results show that LOX expression is increased in atherosclerotic lesions, and that overexpression of this enzyme in VSMC does not affect the size of the atheroma or its lipid content, but it does affect its degree of calcification. Further, these data suggest that the decrease in calcification driven by NOR-1 in VICs would involve a reduction in LOX. These evidences support the interest of LOX as a therapeutic target in cardiovascular calcification.

Upregulation of NOR-1 in calcified human vascular tissues: impact on osteogenic differentiation and calcification.

Cardiovascular calcification is a significant public health issue whose pathophysiology is not fully understood. NOR-1 regulates critical processes in cardiovascular remodeling, but its contribution to ectopic calcification is unknown. NOR-1 was overexpressed in human calcific aortic valves and calcified atherosclerotic lesions colocalizing with RUNX2, a factor essential for osteochondrogenic differentiation and calcification. NOR-1 and osteogenic markers were upregulated in calcifying human valvular interstitial cells (VICs) and human vascular smooth muscle cells (VSMCs). Gain- and loss-of-function approaches demonstrated that NOR-1 negatively modulates the expression of osteogenic genes relevant for the osteogenic transdifferentiation (RUNX2, IL-6, BMP2, and ALPL) and calcification of VICs. VSMCs from transgenic mice overexpressing NOR-1 in these cells (TgNOR-1VSMC) expressed lower basal levels of osteogenic genes (IL-6, BMP2, ALPL, OPN) than cells from WT littermates, and their upregulation by a high-phosphate osteogenic medium (OM) was completely prevented by NOR-1 transgenesis. Consistently, this was associated with a dramatic reduction in the calcification of both transgenic VSMCs and aortic rings from TgNOR-1VSMC mice exposed to OM. Atherosclerosis and calcification were induce in mice by the administration of AAV-PCSK9D374Y and a high-fat/high-cholesterol diet. Challenged-TgNOR-1VSMC mice exhibited decreased vascular expression of osteogenic markers, and both less atherosclerotic burden (assessed in whole aorta and lesion size in aortic arch and brachiocephalic artery) and less vascular calcification (assessed either by near-infrared fluorescence imaging or histological analysis) than WT mice. Our data indicate that NOR-1 negatively modulates the expression of genes critically involved in the osteogenic differentiation of VICs and VSMCs, thereby restraining ectopic cardiovascular calcification.

Rolipram impacts on redox homeostasis and cellular signaling in an experimental model of abdominal aortic aneurysm. / Impacto del tratamiento con rolipram sobre la homeostasis rédox y la señalización celular en un modelo experimental de aneurisma de aorta abdominal.

INTRODUCTION: Cyclic nucleotide phosphodiesterases (PDEs) of the PDE4 subfamily are responsible for the hydrolysis and subcellular compartmentalization of cAMP, a second messenger that modulates vascular functionality. We had shown that PDE4B is induced in abdominal aortic aneurysms (AAA) and that PDE4 inhibition by rolipram limits experimental aneurysms. In this study we have delved into the mechanisms underlying the beneficial effect of rolipram on AAA. METHODS: AAA were induced in ApoE-/- mice by angiotensin II (Ang II) infusion. Aneurysm formation was evaluated by ultrasonography. The expression of enzymes involved in rédox homeostasis was analyzed by real-time RT-PCR and the activation of signaling pathways by Western blot. RESULTS: Induction of PDE4B in human AAA has been confirmed in a second cohort of patients. In Ang II-infused ApoE-/- mice, rolipram increased the percentage of animals free of aneurysms without affecting the percentage of aortic ruptures. Quantitative analyses determined that this drug significantly attenuated aortic collagen deposition. Additionally, rolipram reduced the increased Nox2 expression triggered by Ang II, exacerbated Sod1 induction, and normalized Sod3 expression. Likewise, PDE4 inhibition decreased the activation of both ERK1/2 and the canonical Wnt pathway, while AKT activity was not altered. CONCLUSIONS: The inhibition of PDE4 activity modulates the expression of enzymes involved in rédox homeostasis and affects cell signaling pathways involved in the development of AAA.

PDE4 Phosphodiesterases in Cardiovascular Diseases: Key Pathophysiological Players and Potential Therapeutic Targets.

3',5'-cyclic adenosine monophosphate (cAMP) is a second messenger critically involved in the control of a myriad of processes with significant implications for vascular and cardiac cell function. The temporal and spatial compartmentalization of cAMP is governed by the activity of phosphodiesterases (PDEs), a superfamily of enzymes responsible for the hydrolysis of cyclic nucleotides. Through the fine-tuning of cAMP signaling, PDE4 enzymes could play an important role in cardiac hypertrophy and arrhythmogenesis, while it decisively influences vascular homeostasis through the control of vascular smooth muscle cell proliferation, migration, differentiation and contraction, as well as regulating endothelial permeability, angiogenesis, monocyte/macrophage activation and cardiomyocyte function. This review summarizes the current knowledge and recent advances in understanding the contribution of the PDE4 subfamily to cardiovascular function and underscores the intricate challenges associated with targeting PDE4 enzymes as a therapeutic strategy for the management of cardiovascular diseases.

Lysyl oxidase-dependent extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease.

Extracellular matrix (ECM) is an active player in cardiovascular calcification (CVC), a major public health issue with an unmet need for effective therapies. Lysyl oxidase (LOX) conditions ECM biomechanical properties; thus, we hypothesized that LOX might impact on mineral deposition in calcific aortic valve disease (CAVD) and atherosclerosis. LOX was upregulated in calcified valves from two cohorts of CAVD patients. Strong LOX immunostaining was detected surrounding calcified foci in calcified human valves and atherosclerotic lesions colocalizing with RUNX2 on valvular interstitial cells (VICs) or vascular smooth muscle cells (VSMCs). Both LOX secretion and organized collagen deposition were enhanced in calcifying VICs exposed to osteogenic media. ß-aminopropionitrile (BAPN), an inhibitor of LOX, attenuated collagen deposition and calcification. VICs seeded onto decellularized matrices from BAPN-treated VICs calcified less than cells cultured onto control scaffolds; instead, VICs exposed to conditioned media from cells over-expressing LOX or cultured onto LOX-crosslinked matrices calcified more. Atherosclerosis was induced in WT and transgenic mice that overexpress LOX in VSMC (TgLOXVSMC) by AAV-PCSK9D374Y injection and high-fat feeding. In atherosclerosis-challenged TgLOXVSMC mice both atherosclerosis burden and calcification assessed by near-infrared fluorescence (NIRF) imaging were higher than in WT mice. These animals also exhibited larger calcified areas in atherosclerotic lesions from aortic arches and brachiocephalic arteries. Moreover, LOX transgenesis exacerbated plaque inflammation, and increased VSMC cellularity, the rate of RUNX2-positive cells and both connective tissue content and collagen cross-linking. Our findings highlight the relevance of LOX in CVC and postulate this enzyme as a potential therapeutic target for CVC.

Safety and efficacy of avelumab plus carboplatin in patients with metastatic castration-resistant prostate cancer in an open-label Phase Ib study.

BACKGROUND: Single-agent PD-1/PD-L1 inhibitors have shown limited efficacy in unselected mCRPC. The evidence of a survival benefit with sipuleucel-T and ipilimumab, provides a rationale to study further increasing immunogenicity in mCRPC through combinations. METHODS: Safety and efficacy avelumab plus carboplatin was investigated in a single-arm Phase Ib study in mCRPC, progressing to at least one taxane and one androgen-receptor inhibitor. The primary endpoint was safety. Secondary endpoints included PSA/radiographic responses, progression-free survival (PFS) and overall survival (OS). Germline/somatic mutation analysis was performed. RESULTS: In total, 26 patients were included. Patients were heavily pretreated: 76.9% received ≥3 and 42.3% ≥4 prior lines. A DNA damage repair (DDR) alteration was found in three patients (11.5%). The safety profile was acceptable with 73% Grade 3-4 treatment-related adverse events. PSA response rate ≥50% was seen in 7.7% of patients. The objective response rate was 17.6%, including one complete response (5.9%). Two of these responders had a known DDR alteration (one BRCA2, one ATM). The median response duration was 6 months. Median radiographic PFS was 6.6 months (95% CI 4.28-9.01), and median OS 10.6 months (95% CI 6.68-NR). CONCLUSIONS: Avelumab plus carboplatin has an acceptable safety profile and was associated with a prolonged OS given the heavily pretreated population.

CD69 expression on regulatory T cells protects from immune damage after myocardial infarction.

Increasing evidence has pointed to the important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed significant CD69 overexpression on Tregs after MI. Our results in mice showed that CD69 expression on Tregs increased survival after left anterior descending (LAD) coronary artery ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Tregs, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Tregs into Cd69-/- mice after LAD ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from 2 independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of rehospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex, and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Tregs as a potential prognostic factor and a therapeutic option to prevent HF after MI.

El receptor nuclear NOR-1 (Neuron-derived Orphan Receptor-1) en el remodelado vascular patológico / Nuclear receptor NOR-1 (Neuron-derived Orphan Receptor-1) in pathological vascular remodelling and vascular remodelling

En el remodelado vascular patológico juegan un papel clave las células vasculares y su interacción con las células inflamatorias y del sistema inmune. En este proceso intervienen una gran cantidad de genes y proteínas regulados de forma coordinada por un reducido número de factores de transcripción. En los últimos años las investigaciones sobre una pequeña subfamilia de factores de transcripción, la subfamilia NR4A, han tenido un gran impacto sobre nuestra comprensión de la biología vascular. Los receptores NR4A1 (Nur77), NR4A2 (Nurr1) y NR4A3 (NOR-1) son productos de genes de respuesta temprana cuya expresión es inducida por múltiples estímulos fisiopatológicos y físicos. Su amplia distribución en los diferentes tejidos y células los sitúan en el control de numerosos procesos como la diferenciación, la proliferación, la supervivencia y la apoptosis celular, así como la inflamación y el metabolismo de lípidos y carbohidratos. Esta revisión analiza el papel de estos receptores, particularmente de NOR-1, en el remodelado vascular patológico asociado a la aterosclerosis, el aneurisma de aorta abdominal y la hipertensión arterial pulmonar. (AU)

Vascular cells and their interaction with inflammatory cells and the immune system play a key role in pathological vascular remodeling. A large number of genes and proteins regulated in a coordinated manner by a small number of transcription factors are involved in this process. In recent years, research on a small subfamily of transcription factors, the NR4A subfamily, has had a major impact on our understanding of vascular biology. The NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (NOR-1) receptors are products of early response genes whose expression is induced by multiple pathophysiological and physical stimuli. Their wide distribution in different tissues and cells places them in the control of numerous processes such as cell differentiation, proliferation, survival and apoptosis, as well as inflammation and the metabolism of lipids and carbohydrates. This review analyzes the role of these receptors, particularly NOR-1, in pathological vascular remodeling associated with atherosclerosis, abdominal aortic aneurysm and pulmonary arterial hypertension. (AU)

Nuclear receptor NOR-1 (Neuron-derived Orphan Receptor-1) in pathological vascular remodelling and vascular remodelling. / El receptor nuclear NOR-1 (Neuron-derived Orphan Receptor-1) en el remodelado vascular patológico.

Vascular cells and their interaction with inflammatory cells and the immune system play a key role in pathological vascular remodeling. A large number of genes and proteins regulated in a coordinated manner by a small number of transcription factors are involved in this process. In recent years, research on a small subfamily of transcription factors, the NR4A subfamily, has had a major impact on our understanding of vascular biology. The NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (NOR-1) receptors are products of early response genes whose expression is induced by multiple pathophysiological and physical stimuli. Their wide distribution in different tissues and cells places them in the control of numerous processes such as cell differentiation, proliferation, survival and apoptosis, as well as inflammation and the metabolism of lipids and carbohydrates. This review analyzes the role of these receptors, particularly NOR-1, in pathological vascular remodeling associated with atherosclerosis, abdominal aortic aneurysm and pulmonary arterial hypertension.

NR4A3: A Key Nuclear Receptor in Vascular Biology, Cardiovascular Remodeling, and Beyond.

The mechanisms committed in the activation and response of vascular and inflammatory immune cells play a major role in tissue remodeling in cardiovascular diseases (CVDs) such as atherosclerosis, pulmonary arterial hypertension, and abdominal aortic aneurysm. Cardiovascular remodeling entails interrelated cellular processes (proliferation, survival/apoptosis, inflammation, extracellular matrix (ECM) synthesis/degradation, redox homeostasis, etc.) coordinately regulated by a reduced number of transcription factors. Nuclear receptors of the subfamily 4 group A (NR4A) have recently emerged as key master genes in multiple cellular processes and vital functions of different organs, and have been involved in a variety of high-incidence human pathologies including atherosclerosis and other CVDs. This paper reviews the major findings involving NR4A3 (Neuron-derived Orphan Receptor 1, NOR-1) in the cardiovascular remodeling operating in these diseases.

Impacto del confinamiento por Covid-19 en la salud mental y emocional en la población gestante / Impact of Covid-19 confinement on mental and emotional health in the pregnant population

Objetivo principal: analizar el impacto del confinamiento por Covid-19 en la salud mental de las gestantes y describir diversas variables que pudieran influir en ello. Metodología: estudio transversal exploratorio, en una muestra de 362 gestantes en el Hospital Universitario La Paz de Madrid. Resultados principales: un 51,9 % estaba de baja por enfermedad/maternidad, 3,9 % trabajaba fuera de casa. El 36,7 % vivía en piso sin posibilidad de estar al aire libre. La puntuación del Test STAI Rasgo aumentó de 16,4 ± 8,3 a 22,3 ± 12,7 en el STAI Estado en el momento del confinamiento. El 61,6 % no eran capaces de expresar sentimientos de forma adecuada. Conclusión principal: El confinamiento ha tenido un efecto negativo sobre la salud mental de las embarazadas, se ve influenciado por variables habitacionales y ocupacionales. La inteligencia emocional es una habilidad que protege frente al estrés y ansiedad en las embarazadas. (AU)

Objective: Analyses the impact of Covid-19 confinement on the mental health of the pregnant and describe various variables that could influence it. Methods: The analysis was through descriptive statistics. At last no sample of 362 pregnant women undergoing follow-up in the area of the La Paz University Hospital. Results: 51.9 % of pregnant women were on sick / maternity leave, 3.9 % worked outside the home. 36.7 % lived in an apartment without the possibility of being outdoors. The STAI Trait Test score was 16.4 ± 8.3 and increased to 22.3 ± 12.7 when assessing stress / anxiety through the STAI State Test at the time of confinement. 61.6 % were not able to express feelings adequately. Conclusions: Confinement due to SARS-Cov2 has had a negative effect on anxiety and depression in pregnant women. Adequate emotional intelligence is a skill that protects pregnant women against stress and anxiety. (AU)

Targeting Tyrosine Hydroxylase for Abdominal Aortic Aneurysm: Impact on Inflammation, Oxidative Stress, and Vascular Remodeling.

[Figure: see text].

High NOR-1 (Neuron-Derived Orphan Receptor 1) Expression Strengthens the Vascular Wall Response to Angiotensin II Leading to Aneurysm Formation in Mice.

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-1ß, 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.

Neuron-derived orphan receptor-1 modulates cardiac gene expression and exacerbates angiotensin II-induced cardiac hypertrophy.

Hypertensive cardiac hypertrophy (HCH) is a common cause of heart failure (HF), a major public health problem worldwide. However, the molecular bases of HCH have not been completely elucidated. Neuron-derived orphan receptor-1 (NOR-1) is a nuclear receptor whose role in cardiac remodelling is poorly understood. The aim of the present study was to generate a transgenic mouse over-expressing NOR-1 in the heart (TgNOR-1) and assess the impact of this gain-of-function on HCH. The CAG promoter-driven transgenesis led to viable animals that over-expressed NOR-1 in the heart, mainly in cardiomyocytes and also in cardiofibroblasts. Cardiomyocytes from TgNOR-1 exhibited an enhanced cell surface area and myosin heavy chain 7 (Myh7)/Myh6 expression ratio, and increased cell shortening elicited by electric field stimulation. TgNOR-1 cardiofibroblasts expressed higher levels of myofibroblast markers than wild-type (WT) cells (α 1 skeletal muscle actin (Acta1), transgelin (Sm22α)) and were more prone to synthesise collagen and migrate. TgNOR-1 mice experienced an age-associated remodelling of the left ventricle (LV). Angiotensin II (AngII) induced the cardiac expression of NOR-1, and NOR-1 transgenesis exacerbated AngII-induced cardiac hypertrophy and fibrosis. This effect was associated with the up-regulation of hypertrophic (brain natriuretic peptide (Bnp), Acta1 and Myh7) and fibrotic markers (collagen type I α 1 chain (Col1a1), Pai-1 and lysyl oxidase-like 2 (Loxl2)). NOR-1 transgenesis up-regulated two key genes involved in cardiac hypertrophy (Myh7, encoding for ß-myosin heavy chain (ß-MHC)) and fibrosis (Loxl2, encoding for the extracellular matrix (ECM) modifying enzyme, Loxl2). Interestigly, in transient transfection assays, NOR-1 drove the transcription of Myh7 and Loxl2 promoters. Our findings suggest that NOR-1 is involved in the transcriptional programme leading to HCH.

The nuclear receptor NOR-1 modulates redox homeostasis in human vascular smooth muscle cells.

The nuclear receptor NOR-1 (Neuron-derived Orphan Receptor-1) has recently been involved in vascular remodeling and coronary artery disease, however, to date, only a few NOR-1 target genes have been described. We aimed to identify genes regulated by NOR-1 in human vascular smooth muscle cells (VSMC). Lentiviral overexpression of NOR-1 increases reactive oxygen species (ROS) in human VSMC. In accordance, NOR-1 strongly increased NADPH oxidase NOX1 mRNA and protein levels, while NOR-1 silencing significantly reduced NOX1 expression. Luciferase reporter, site-directed mutagenesis and EMSA studies identified two nerve growth factor-induced clone B (NGFI-B)-response elements (NBREs) in NOX1 promoter as essential elements for NOR-1 responsiveness. NOR-1 and NOX1 were co-expressed by VSMC in human atherosclerotic lesions, and NOX1 knockdown counteracted the increased ROS production and cell migration induced by NOR-1 overexpression. NOR-1 also modulated the expression of other enzymes involved in cellular redox status, in particular, upregulated superoxide dismutase-1 (SOD1) and SOD3 while downregulated SOD2 and NOX4. NOR-1 induced SOD1 and SOD3 transcriptional activity and participated in the modulation of SOD3 by inflammatory stimuli. By contrast, NOR-1 impaired SOD2 transcription antagonizing NFκB signaling. These results indicate that NOR-1 induces NOX1 in human VSMC and participates in the complex gene networks regulating oxidative stress and redox homeostasis in the vasculature.

The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells.

The nuclear receptor NOR-1 (NR4A3) has recently been involved in the regulation of extracellular matrix (ECM) proteins associated with neointimal thickening and the vascular control of hemostasis. We sought to find as-yet unidentified NOR-1 target genes in human vascular smooth muscle cells (VSMCs). An in silico analysis identified putative NOR-1 response elements in the proximal promoter region of several genes encoding for ECM proteins, including vitronectin (VTN). Lentiviral overexpression of NOR-1 strongly increased VTN mRNA and protein levels, whereas NOR-1 silencing significantly reduced VTN expression. Deletion and site-directed mutagenesis studies, as well as EMSA and chromatin immunoprecipitation, identified the NBRE(-202/-195) site in the VTN promoter as an essential element for NOR-1 responsiveness. Furthermore, NOR-1 and VTN colocalized in VSMCs in human atherosclerotic lesions. VTN levels were increased in cell supernatants from VSMCs that overexpress NOR-1. Cell supernatants from VSMCs overexpressing NOR-1 induced cell migration to a greater extent than supernatants from control cells, and this effect was attenuated when cell supernatants were preincubated with anti-VTN blocking antibodies or VTN was silenced in supernatant-generating cells. These results indicate that VTN is a target of NOR-1 and suggest that this multifunctional glycoprotein may participate in vascular responses mediated by this nuclear receptor.-Martí-Pàmies, I., Cañes, L., Alonso, J., Rodríguez, C., Martínez-González, J. The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells.

NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress.

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.

The nuclear receptor NOR-1 regulates the small muscle protein, X-linked (SMPX) and myotube differentiation.

Recent works have highlighted the role of NOR-1 in both smooth and skeletal muscle, and have proposed this nuclear receptor as a nexus that coordinates muscle performance and metabolic capacity. However, no muscle specific genes regulated by NOR-1 have been identified so far. To identify NOR-1 target genes, we over-expressed NOR-1 in human vascular smooth muscle cells (VSMC). These cells subjected to sustained over-expression of supraphysiological levels of NOR-1 experienced marked phenotypic changes and up-regulated the skeletal muscle protein X-linked (SMPX), a protein typically expressed in striated muscle and associated to cell shape. By transcriptional studies and DNA-protein binding assays, we identified a non-consensus NBRE site in human SMPX promoter, critical for NOR-1 responsiveness. The expression of SMPX was higher in human skeletal muscle myoblasts (HSMM) than in human VSMC, and further increased in HSMM differentiated to myotubes. NOR-1 silencing prevented SMPX expression in HSMM, as well as their differentiation to myotubes, but the up-regulation of SMPX was dispensable for HSMM differentiation. Our results indicate that NOR-1 regulate SMPX in human muscle cells and acts as a muscle regulatory factor, but further studies are required to unravel its role in muscle differentiation and hypertrophy.

NR4A receptors up-regulate the antiproteinase alpha-2 macroglobulin (A2M) and modulate MMP-2 and MMP-9 in vascular smooth muscle cells.

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.