Publications by authors named "Marta Saura"

33 Publications

Ivabradine Induces Cardiac Protection against Myocardial Infarction by Preventing Cyclophilin-A Secretion in Pigs under Coronary Ischemia/Reperfusion.

Int J Mol Sci 2021 Mar 12;22(6). Epub 2021 Mar 12.

Hospital Ramón y Cajal Research Unit (IRYCIS), Cardiology Department, Universidad Francisco de Vitoria, 28223 Madrid, Spain.

In response to cardiac ischemia/reperfusion, proteolysis mediated by extracellular matrix metalloproteinase inducer (EMMPRIN) and its secreted ligand cyclophilin-A (CyPA) significantly contributes to cardiac injury and necrosis. Here, we aimed to investigate if, in addition to the effect on the funny current (I(f)), Ivabradine may also play a role against cardiac necrosis by reducing EMMPRIN/CyPA-mediated cardiac inflammation. In a porcine model of cardiac ischemia/reperfusion (IR), we found that administration of 0.3 mg/kg Ivabradine significantly improved cardiac function and reduced cardiac necrosis by day 7 after IR, detecting a significant increase in cardiac CyPA in the necrotic compared to the risk areas, which was inversely correlated with the levels of circulating CyPA detected in plasma samples from the same subjects. In testing whether Ivabradine may regulate the levels of CyPA, no changes in tissue CyPA were found in healthy pigs treated with 0.3 mg/kg Ivabradine, but interestingly, when analyzing the complex EMMPRIN/CyPA, rather high glycosylated EMMPRIN, which is required for EMMPRIN-mediated matrix metalloproteinase (MMP) activation and increased CyPA bonding to low-glycosylated forms of EMMPRIN were detected by day 7 after IR in pigs treated with Ivabradine. To study the mechanism by which Ivabradine may prevent secretion of CyPA, we first found that Ivabradine was time-dependent in inhibiting co-localization of CyPA with the granule exocytosis marker vesicle-associated membrane protein 1 (VAMP1). However, Ivabradine had no effect on mRNA expression nor in the proteasome and lysosome degradation of CyPA. In conclusion, our results point toward CyPA, its ligand EMMPRIN, and the complex CyPA/EMMPRIN as important targets of Ivabradine in cardiac protection against IR.
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http://dx.doi.org/10.3390/ijms22062902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001911PMC
March 2021

miR-16 induces endoplasmic reticulum stress in ischemic dilated cardiomyopathy.

Rev Esp Cardiol (Engl Ed) 2021 Mar 26. Epub 2021 Mar 26.

Unidad de Fisiología, Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain; Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CIBERCV), Spain. Electronic address:

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http://dx.doi.org/10.1016/j.rec.2021.03.003DOI Listing
March 2021

Ivabradine induces cardiac protection by preventing cardiogenic shock-induced extracellular matrix degradation.

Rev Esp Cardiol (Engl Ed) 2020 Oct 29. Epub 2020 Oct 29.

Unidad de Investigación Cardiovascular, Departamento de Cardiología, Universidad Francisco de Vitoria, Hospital Ramón y Cajal (IRYCIS), Madrid, Spain; Centro de Investigación en Red de Enfermedades Cardiovasculares (CIBERCV), Spain. Electronic address:

Introduction And Objectives: Ivabradine reduces heart rate by blocking the I(f) current and preserves blood pressure and stroke volume through unknown mechanisms. Caveolin-3 protects the heart by forming protein complexes with several proteins, including extracellular matrix (ECM)-metalloproteinase-inducer (EMMPRIN) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HN4), a target of ivabradine. We hypothesized that ivabradine might also exert cardioprotective effects through inhibition of ECM degradation.

Methods: In a porcine model of cardiogenic shock, we studied the effects of ivabradine on heart integrity, the levels of MMP-9 and EMMPRIN, and the stability of caveolin-3/HCN4 protein complexes with EMMPRIN.

Results: Administration of 0.3 mg/kg ivabradine significantly reduced cardiogenic shock-induced ventricular necrosis and expression of MMP-9 without affecting EMMPRIN mRNA, protein, or protein glycosylation (required for MMP activation). However, ivabradine increased the levels of the caveolin-3/LG-EMMPRIN (low-glycosylated EMMPRIN) and caveolin-3/HCN4 protein complexes and decreased that of a new complex between HCN4 and high-glycosylated EMMPRIN formed in response to cardiogenic shock. We next tested whether caveolin-3 can bind to HCN4 and EMMPRIN and found that the HCN4/EMMPRIN complex was preserved when we silenced caveolin-3 expression, indicating a direct interaction between these 2 proteins. Similarly, EMMPRIN-silenced cells showed a significant reduction in the binding of caveolin-3/HCN4, which regulates the I(f) current, suggesting that, rather than a direct interaction, both proteins bind to EMMPRIN.

Conclusions: In addition to inhibition of the I(f) current, ivabradine may induce cardiac protection by inhibiting ECM degradation through preservation of the caveolin-3/LG-EMMPRIN complex and control heart rate by stabilizing the caveolin-3/HCN4 complex.
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http://dx.doi.org/10.1016/j.rec.2020.09.012DOI Listing
October 2020

Cardiac microvasculature and adverse remodeling after acute myocardial infarction. New evidence on the use of VEGF as a therapeutic target.

Rev Esp Cardiol (Engl Ed) 2021 02 17;74(2):124-125. Epub 2020 Oct 17.

Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CIBERCV), Spain; Unidad de Investigación Cardiovascular, Universidad Francisco de Vitoria/Servicio de Cardiología, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain.

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http://dx.doi.org/10.1016/j.rec.2020.07.019DOI Listing
February 2021

Bisphenol A impaired cell adhesion by altering the expression of adhesion and cytoskeleton proteins on human podocytes.

Sci Rep 2020 10 6;10(1):16638. Epub 2020 Oct 6.

Laboratory of Renal Physiology and Experimental Nephrology, Department of System Biology/Physiology Unit, University of Alcalá, Alcalá de Henares, Spain.

Bisphenol A (BPA), a chemical -xenoestrogen- used in food containers is present in the urine of almost the entire population. Recently, several extensive population studies have proven a significant association between urinary excretion of BPA and albuminuria. The alteration of glomerular podocytes or "podocytopathy" is a common event in chronic albuminuric conditions. Since many podocytes recovered from patients' urine are viable, we hypothesized that BPA could impair podocyte adhesion capabilities. Using an in vitro adhesion assay, we observed that BPA impaired podocyte adhesion, an effect that was abrogated by Tamoxifen (an estrogen receptor blocker). Genomic and proteomic analyses revealed that BPA affected the expression of several podocyte cytoskeleton and adhesion proteins. Western blot and immunocytochemistry confirmed the alteration in the protein expression of tubulin, vimentin, podocin, cofilin-1, vinculin, E-cadherin, nephrin, VCAM-1, tenascin-C, and β-catenin. Moreover, we also found that BPA, while decreased podocyte nitric oxide production, it lead to overproduction of ion superoxide. In conclusion, our data show that BPA induced a novel type of podocytopathy characterizes by an impairment of podocyte adhesion, by altering the expression of adhesion and cytoskeleton proteins. Moreover, BPA diminished production of podocyte nitric oxide and induced the overproduction of oxygen-free metabolites. These data provide a mechanism by which BPA could participate in the pathogenesis and progression of renal diseases.
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http://dx.doi.org/10.1038/s41598-020-73636-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538920PMC
October 2020

Ivabradine-Stimulated Microvesicle Release Induces Cardiac Protection against Acute Myocardial Infarction.

Int J Mol Sci 2020 Sep 8;21(18). Epub 2020 Sep 8.

Cardiology Department, Universidad Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), 28223 Madrid, Spain.

Ivabradine can reduce heart rate through inhibition of the current I() by still unexplored mechanisms. In a porcine model of ischemia reperfusion (IR), we found that treatment with 0.3 mg/kg Ivabradine increased plasma release of microvesicles (MVs) over Placebo, as detected by flow cytometry of plasma isolated from pigs 7 days after IR, in which a tenfold increase of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) containing (both high and low-glycosylated) MVs, was detected in response to Ivabradine. The source of MVs was investigated, finding a 37% decrease of CD31+ endothelial cell derived MVs, while CD41+ platelet MVs remained unchanged. By contrast, Ivabradine induced the release of HCN4+ (mostly cardiac) MVs. While no differences respect to EMMPRIN as a cargo component were found in endothelial and platelet derived MVs, Ivabradine induced a significant release of EMMPRIN+/HCN4+ MVs by day 7 after IR. To test the role of EMMPRIN+ cardiac MVs (EMCMV), H9c2 cell monolayers were incubated for 24 h with 10 EMCMVs, reducing apoptosis, and increasing 2 times cell proliferation and 1.5 times cell migration. The in vivo contribution of Ivabradine-induced plasma MVs was also tested, in which 10 MVs isolated from the plasma of pigs treated with Ivabradine or Placebo 7 days after IR, were injected in pigs under IR, finding a significant cardiac protection by increasing left ventricle ejection fraction and a significant reduction of the necrotic area. In conclusion ivabradine induces cardiac protection by increasing at least the release of EMMPRIN containing cardiac microvesicles.
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http://dx.doi.org/10.3390/ijms21186566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555962PMC
September 2020

Differential expression of circulating miRNAs as a novel tool to assess BAG3-associated familial dilated cardiomyopathy.

Biosci Rep 2019 03 15;39(3). Epub 2019 Mar 15.

Univ Puerta del Mar, School of Medicine, Cadiz, Spain

A new familial dilated cardiomyopathy (FDCM) was found related to mutations in gene. MicroRNAs (miRNAs) represent new targets of FDCM, although no studies have assessed clinical association between Bcl2-associated athanogene 3 (BAG3)-related DCM and miRNAs. Here, we studied whether a clinical association between BAG3-related FDCM and circulating miRNAs may have diagnostic and prognostic value in a small cohort of familial related individuals carrying a BAG3 mutation (BAG3+) and/or diagnosed of dilated cardiomyopathy (DCM) (DCM+). The analysis of 1759 circulating miRNAs showed significant differences between BAG3+ and BAG3- individuals for miRNAs mir-3191-3p, 6769b-3p, 1249-ep, 154-5p, 6855-5p, and 182-5p, while comparisons between BAG3+/DCM+ versus BAG3+/DCM- were restricted to miRNAs mir-154-5p, 6885-5p, and 182-5p, showing significant correlation with systolic and diastolic blood pressure, A wave, left atrium length, and left atrium area. Additionally, when stratified by gender and age, miRNAs were statistically correlated with critical parameters, including left ventricle ejection fraction (LVEF) and ventricular diameter, in women and young men. Likewise, 56% of BAG3+/DCM+, significantly co-expressed mir-154-5p and mir-182-5p, and a slight 4% did not express such combination, suggesting that co-expression of mir-154-5p and mir-182-5p may potentially show diagnostic value. Further studies will require long-term follow-up, and validation in larger populations.
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http://dx.doi.org/10.1042/BSR20180934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418398PMC
March 2019

Non-Invasive Detection of Extracellular Matrix Metalloproteinase Inducer EMMPRIN, a New Therapeutic Target against Atherosclerosis, Inhibited by Endothelial Nitric Oxide.

Int J Mol Sci 2018 Oct 19;19(10). Epub 2018 Oct 19.

Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), CIBERCV, 28223 Madrid, Spain.

Lack of endothelial nitric oxide causes endothelial dysfunction and circulating monocyte infiltration, contributing to systemic atheroma plaque formation in arterial territories. Among the different inflammatory products, macrophage-derived foam cells and smooth muscle cells synthesize matrix metalloproteinases (MMPs), playing a pivotal role in early plaque formation and enlargement. We found increased levels of MMP-9 and MMP-13 in human endarterectomies with advanced atherosclerosis, together with significant amounts of extracellular matrix (ECM) metalloproteinase inducer EMMPRIN. To test whether the absence of NO may aggravate atherosclerosis through EMMPRIN activation, double NOS3/apoE knockout (KO) mice expressed high levels of EMMPRIN in carotid plaques, suggesting that targeting extracellular matrix degradation may represent a new mechanism by which endothelial NO prevents atherosclerosis. Based on our previous experience, by using gadolinium-enriched paramagnetic fluorescence micellar nanoparticles conjugated with AP9 (NAP9), an EMMPRIN-specific binding peptide, magnetic resonance sequences allowed non-invasive visualization of carotid EMMPRIN in NOS3/apoE over apoE control mice, in which atheroma plaques were significantly reduced. Taken together, these results point to EMMPRIN as a new therapeutic target of NO-mediated protection against atherosclerosis, and NAP9 as a non-invasive molecular tool to target atherosclerosis.
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http://dx.doi.org/10.3390/ijms19103248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214015PMC
October 2018

Nanotechnology Applied to Preserve Extracelular Matrix as Teranostic Tool in Acute Myocardial Infarction.

Rev Esp Cardiol (Engl Ed) 2019 Feb 9;72(2):171-174. Epub 2018 Apr 9.

Servicio de Cardiología, Unidad de Investigación del Hospital Ramón y Cajal (IRYCIS)-Universidad Francisco de Vitoria, Madrid, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.rec.2017.12.023DOI Listing
February 2019

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium.

Arterioscler Thromb Vasc Biol 2017 07 25;37(7):1272-1281. Epub 2017 May 25.

From the Biology Systems Department, Physiology, School of Medicine and Health Sciences, Universidad Alcalá (IRYCIS), Madrid, Spain (P.R., M.A., S.M., M.S.); Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), Madrid, Spain (C.Z.); and Cardiology Department, School of Medicine, Cádiz University, Spain (R.T.).

Objective: ILK (integrin-linked kinase) plays a key role in controlling vasomotor tone and is decreased in atherosclerosis. The objective of this study is to test whether nitric oxide (NO) regulates ILK in vascular remodeling.

Approach And Results: We found a striking correlation between increased levels of inducible nitric oxide and decreased ILK levels in human atherosclerosis and in a mouse model of vascular remodeling (carotid artery ligation) comparing with iNOS (inducible NO synthase) knockout mice. iNOS induction produced the same result in mouse aortic endothelial cells, and these effects were mimicked by an NO donor in a time-dependent manner. We found that NO decreased ILK protein stability by promoting the dissociation of the complex ILK/Hsp90 (heat shock protein 90)/eNOS (endothelial NO synthase), leading to eNOS uncoupling. NO also destabilized ILK signaling platform and lead to decreased levels of paxillin and α-parvin. ILK phosphorylation of its downstream target GSK3-β (glycogen synthase kinase 3 beta) was decreased by NO. Mechanistically, NO increased ILK ubiquitination mediated by the E3 ubiquitin ligase CHIP (C terminus of HSC70-interacting protein), but ILK ubiquitination was not followed by proteasome degradation. Alternatively, NO drove ILK to degradation through the endocytic-lysosomal pathway. ILK colocalized with the lysosome marker LAMP-1 (lysosomal-associated membrane protein 1) in endothelial cells, and inhibition of lysosome activity with chloroquine reversed the effect of NO. Likewise, ILK colocalized with the early endosome marker EEA1 (early endosome antigen 1). ILK endocytosis proceeded via dynamin because a specific inhibitor of dynamin (Dyngo 4a) was able to reverse ILK endocytosis and its lysosome degradation.

Conclusions: Endocytosis regulates ILK signaling in vascular remodeling where there is an overload of inducible NO, and thus its inhibition may represent a novel target to fight atherosclerotic disease.
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http://dx.doi.org/10.1161/ATVBAHA.117.309560DOI Listing
July 2017

Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

PLoS One 2016 20;11(9):e0162912. Epub 2016 Sep 20.

Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), Ctra. Colmenar Viejo, km. 9100, 28034, Madrid, Spain.

Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029905PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162912PLOS
August 2017

Preclinical models of atherosclerosis. The future of Hybrid PET/MR technology for the early detection of vulnerable plaque.

Expert Rev Mol Med 2016 Apr 8;18:e6. Epub 2016 Apr 8.

Cardiology Department,University Francisco de Vitoria/Hospital Ramón y Cajal,Ctra. Colmenar Viejo,km 9,100,28034 Madrid,Spain.

Cardiovascular diseases are the leading cause of death in developed countries. The aetiology is currently multifactorial, thus making them very difficult to prevent. Preclinical models of atherothrombotic diseases, including vulnerable plaque-associated complications, are now providing significant insights into pathologies like atherosclerosis, and in combination with the most recent advances in new non-invasive imaging technologies, they have become essential tools to evaluate new therapeutic strategies, with which can forecast and prevent plaque rupture. Positron emission tomography (PET)/computed tomography imaging is currently used for plaque visualisation in clinical and pre-clinical cardiovascular research, albeit with significant limitations. However, the combination of PET and magnetic resonance imaging (MRI) technologies is still the best option available today, as combined PET/MRI scans provide simultaneous data acquisition together with high quality anatomical information, sensitivity and lower radiation exposure for the patient. The coming years may represent a new era for the implementation of PET/MRI in clinical practice, but first, clinically efficient attenuation correction algorithms and research towards multimodal reagents and safety issues should be validated at the preclinical level.
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http://dx.doi.org/10.1017/erm.2016.5DOI Listing
April 2016

EMMPRIN-Targeted Magnetic Nanoparticles for In Vivo Visualization and Regression of Acute Myocardial Infarction.

Theranostics 2016 15;6(4):545-57. Epub 2016 Feb 15.

1. Cardiology Department. University Francisco de Vitoria/Hospital Ramón y Cajal. Ctra. Colmenar Viejo, km. 9100. 28034 Madrid, Spain.

Unlabelled: Inhibition of extracellular matrix (ECM) degradation may represent a mechanism for cardiac protection against ischemia. Extracellular matrix metalloproteinase inducer (EMMPRIN) is highly expressed in response to acute myocardial infarction (AMI), and induces activation of several matrix metalloproteinases (MMPs), including gelatinases MMP-2 and MMP-9. We targeted EMMPRIN with paramagnetic/fluorescent micellar nanoparticles conjugated with the EMMPRIN binding peptide AP-9 (NAP9), or an AP-9 scrambled peptide as a negative control (NAPSC). We found that NAP9 binds to endogenous EMMPRIN in cultured HL1 myocytes and in mouse hearts subjected to ischemia/reperfusion (IR). Injection of NAP9 at the time of or one day after IR, was enough to reduce progression of myocardial cell death when compared to CONTROL and NAPSC injected mice (infarct size in NAP9 injected mice: 32%±6.59 vs

Control: 46%±9.04 or NAPSC injected mice: 48%±7.64). In the same way, cardiac parameters were recovered to almost healthy levels (LVEF NAP9 63% ± 7.24 vs CONTROL 42% ± 4.74 or NAPSC 39% ± 6.44), whereas ECM degradation was also reduced as shown by inhibition of MMP-2 and MMP-9 activation. Cardiac magnetic resonance (CMR) scans have shown a signal enhancement in the left ventricle of NAP9 injected mice with respect to non-injected, and to mice injected with NAPSC. A positive correlation between CMR enhancement and Evans-Blue/TTC staining of infarct size was calculated (R:0.65). Taken together, these results point to EMMPRIN targeted nanoparticles as a new approach to the mitigation of ischemic/reperfusion injury.
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http://dx.doi.org/10.7150/thno.13352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775864PMC
December 2016

Bisphenol A: An environmental factor implicated in renal vascular damage.

Nefrologia 2016 10;36(1):5-9. Epub 2015 Nov 10.

Laboratorio de Fisiopatología de la Pared Vascular, Unidad de Fisiología, Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, España.

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http://dx.doi.org/10.1016/j.nefro.2015.08.007DOI Listing
September 2017

Oral administration of bisphenol A induces high blood pressure through angiotensin II/CaMKII-dependent uncoupling of eNOS.

FASEB J 2014 Nov 7;28(11):4719-28. Epub 2014 Aug 7.

Physiology Unit, Department of Systems Biology, Laboratory of Renal Physiology and Experimental Nephrology.

Bisphenol A (BPA) is found in human urine and fat tissue. Higher urinary BPA concentrations are associated with arterial hypertension. To shed light on the underlying mechanism, we orally administered BPA (4 nM to 400 μM in drinking water) to 8-wk-old CD11 mice over 30 d. Mice developed dosage-dependent high blood pressure (systolic 130 ± 12 vs. 170 ± 12 mmHg; EC50 0.4 μM), impairment of acetylcholine (AcH)-induced carotid relaxation (0.66 ± 0.08 vs. 0.44 ± 0.1 mm), a 1.7-fold increase in arterial angiotensin II (AngII), an 8.7-fold increase in eNOS mRNA and protein, and significant eNOS-dependent superoxide and peroxynitrite accumulation. AngII inhibition with 0.5 mg/ml losartan reduced oxidative stress and normalized blood pressure and endothelium-dependent relaxation, which suggests that AngII uncouples eNOS and contributes to the BPA-induced endothelial dysfunction by promoting oxidative and nitrosative stress. Microarray analysis of mouse aortic endothelial cells revealed a 2.5-fold increase in expression of calcium/calmodulin-dependent protein kinase II-α (CaMKII-α) in response to 10 nM BPA, with increased expression of phosphorylated-CaMKII-α in carotid rings of BPA-exposed mice, whereas CaMKII-α inhibition with 100 nM autocamptide-2-related inhibitor peptide (AIP) reduced BPA-mediated increase of superoxide. Administration of CaMKII-α inhibitor KN 93 reduced BPA-induced blood pressure and carotid blood velocity in mice, and reverted BPA-mediated carotid constriction in response to treatment with AcH. Given that CaMKII-α inhibition prevents BPA-mediated high blood pressure, our data suggest that BPA regulates blood pressure by inducing AngII/CaMKII-α uncoupling of eNOS.
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http://dx.doi.org/10.1096/fj.14-252460DOI Listing
November 2014

Nitric oxide prevents aortic neointimal hyperplasia by controlling macrophage polarization.

Arterioscler Thromb Vasc Biol 2014 Aug 12;34(8):1739-46. Epub 2014 Jun 12.

From the Fundación Centro Nacional de Investigaciones Cardiovasculares CNIC Melchor Fernandez Almagro 3, Madrid, Spain (B.L., M.G., O.M.P., B.C., C.Z.); Departmento de Fisiología, Facultad de Medicina, Universidad de Alcala, Ctra Madrid-Barcelona, Alcala de Henares, Spain (M.S.); and Facultad de Medicina, Universidad Francisco de Vitoria, Pozuelo de Alarcon, Madrid, Spain (M.J.P., C.Z.).

Objective: Nitric oxide synthase 3 (NOS3) prevents neointima hyperplasia by still unknown mechanisms. To demonstrate the significance of endothelial nitric oxide in the polarization of infiltrated macrophages through the expression of matrix metalloproteinase (MMP)-13 in neointima formation.

Approach And Results: After aortic endothelial denudation, NOS3 null mice show elevated neointima formation, detecting increased mobilization of LSK (lineage-negative [Lin]-stem-cell antigen 1 [SCA1]+KIT+) progenitor cells, and high ratios of M1 (proinflammatory) to M2 (resolving) macrophages, accompanied by high expression of interleukin-5, interleukin-6, MCP-1 (monocyte chemoattractant protein), VEGF (vascular endothelial growth factor), GM-CSF (granulocyte-macrophage colony stimulating factor), interleukin-1β, and interferon-γ. In conditional c-Myc knockout mice, in which M2 polarization is defective, denuded aortas showed extensive wall thickening as well. Conditioned medium from NOS3-deficient endothelium induced extensive repolarization of M2 macrophages to an M1 phenotype, and vascular smooth muscle cells proliferated and migrated faster in conditioned medium from M1 macrophages. Among the different proteins participating in cell migration, MMP-13 was preferentially expressed by M1 macrophages. M1-mediated vascular smooth muscle cell migration was inhibited when macrophages were isolated from MMP-13-deficient mice, whereas exogenous administration of MMP-13 to vascular smooth muscle cell fully restored migration. Excess vessel wall thickening in mice lacking NOS3 was partially reversed by simultaneous deletion of MMP-13, indicating that NOS3 prevents neointimal hyperplasia by preventing MMP-13 activity. An excess of M1-polarized macrophages that coexpress MMP-13 was also detected in human carotid samples from endarterectomized patients.

Conclusions: These findings indicate that at least M1 macrophage-mediated expression of MMP-13 in NOS3 null mice induces neointima formation after vascular injury, suggesting that MMP-13 may represent a new promising target in vascular disease.
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http://dx.doi.org/10.1161/ATVBAHA.114.303866DOI Listing
August 2014

Endothelial mechanosensors of shear stress as regulators of atherogenesis.

Curr Opin Lipidol 2012 Oct;23(5):446-52

National Center for Cardiovascular Research, University of Alcalá, Alcalá de Henares, Madrid, Spain.

Purpose Of Review: Differences in local blood flow patterns along the endothelium may trigger abnormal vascular responses which can have profound pathophysiological consequences. While endothelial cells exposed to laminar blood flow (high shear stress) are protected from atherosclerosis formation, turbulent or disturbed blood flow, which occurs at bends and bifurcations of blood vessels, facilitates atherosclerosis formation. Here, we will highlight the endothelial cell mechanisms involved in detecting shear stress and their translation into downstream biochemical signals.

Recent Findings: Prior evidence supports a role for integrins as mechanotransducers in the endothelium by promoting phosphorylation of different targets through the activation of focal adhesion kinase. Our recent findings show that integrins contact integrin-linked kinase and regulate vasomotor responses by an endothelial nitric oxide synthase-dependent mechanism, which stabilizes the production of vasoactive factor nitric oxide. In addition, different structures of endothelial cells, mainly primary cilia, are investigated, as they can explain the differential responses to laminar versus disturbed flow.

Summary: The discovery of a connection between endothelial cell structures such as cilia, integrin, extracellular matrix, and signaling events opens today a new chapter in our understanding of the molecular mechanisms regulating vascular responses to the changes in flow.
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http://dx.doi.org/10.1097/MOL.0b013e328357e837DOI Listing
October 2012

Integrin-linked kinase regulates vasomotor function by preventing endothelial nitric oxide synthase uncoupling: role in atherosclerosis.

Circ Res 2012 Feb 22;110(3):439-49. Epub 2011 Dec 22.

National Center for Cardiovascular Research, Madrid, Spain.

Rationale: Atherosclerotic lesions develop in regions of disturbed flow, whereas laminar flow protects from atherogenesis; however, the mechanisms involved are not completely elucidated. Integrins are mechanosensors of shear stress in endothelial cells, and integrin-linked kinase (ILK) is important for blood vessel integrity and cardiovascular development.

Objectives: To explore the role of ILK in vascular function by studying conditionally ILK-deficient (cKO) mice and human atherosclerotic arteries.

Results: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK-shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner.

Conclusions: Our results identify ILK as a regulatory partner of eNOS in vivo that prevents eNOS uncoupling, and suggest ILK as a therapeutic target for prevention of endothelial dysfunction related to shear stress-induced vascular diseases.
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http://dx.doi.org/10.1161/CIRCRESAHA.111.253948DOI Listing
February 2012

The extracellular matrix metalloproteinase inducer EMMPRIN is a target of nitric oxide in myocardial ischemia/reperfusion.

Free Radic Biol Med 2011 Jul 16;51(2):387-95. Epub 2011 Apr 16.

Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid 28029, Spain.

Nitric oxide (NO) is an important defense against myocardial ischemia/reperfusion (I/R) injury. Although matrix metalloproteinase (MMP)-mediated necrosis of cardiac myocytes is well characterized, the role of inducible NO synthase (iNOS)-derived NO in this process is poorly understood. I/R injury was increased in iNOS-deficient mice and in mice treated with 1400 W (a pharmacological iNOS inhibitor) and was associated with significantly increased expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and EMMPRIN-associated MMPs. Transcriptional activity of an EMMPRIN luciferase promoter reporter expressed in cardiac myocytes was inhibited by NO in a cGMP-dependent manner, and this transcriptional inhibition was abolished by mutation of a putative E2F site. Consistent with these findings, EMMPRIN null mice, in which iNOS is normally induced, are partially protected against I/R injury. Pharmacological inhibition of iNOS in EMMPRIN null mice had no additional protective effect, suggesting that EMMPRIN is a downstream target of NO. Administration of anti-EMMPRIN neutralizing antibodies partly reduced the excess heart damage and MMP-9 expression induced by I/R in iNOS null mice, indicating that regulation of EMMPRIN is an important mechanism of NO-mediated cardioprotection.
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http://dx.doi.org/10.1016/j.freeradbiomed.2011.04.021DOI Listing
July 2011

H2O2 regulation of vascular function through sGC mRNA stabilization by HuR.

Arterioscler Thromb Vasc Biol 2011 Mar 16;31(3):567-73. Epub 2010 Dec 16.

Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA.

Objective: Hydrogen peroxide (H(2)O(2)) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H(2)O(2) on sGC expression and function and to explore the molecular mechanism involved.

Methods And Results: H(2)O(2) increased sGCβ1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time- and dose-dependent manner, and this effect was blocked by catalase. sGCα2 expression increased along with β1 subunit, whereas α1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H(2)O(2), and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H(2)O(2) was greatly increased after sodium nitroprusside treatment. The H(2)O(2)-dependent sGCβ1 upregulation was attributable to sGCβ1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGCβ1 3' untranslated region. HuR silencing reversed the effects of H(2)O(2) on sGCβ1 levels and cGMP synthesis.

Conclusions: Our results identify H(2)O(2) as an endogenous mediator contributing to the regulation of vascular tone and point to a key role of HuR in sGCβ1 mRNA stabilization.
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http://dx.doi.org/10.1161/ATVBAHA.110.219725DOI Listing
March 2011

Fibronectin upregulates cGMP-dependent protein kinase type Iβ through C/EBP transcription factor activation in contractile cells.

Am J Physiol Cell Physiol 2011 Mar 15;300(3):C683-91. Epub 2010 Dec 15.

Dept. of Physiology, Facultad de Medicina, Universidad de Alcalá, Campus Universitario s/n, Alcalá de Henares, 28871 Madrid, Spain.

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.
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http://dx.doi.org/10.1152/ajpcell.00251.2010DOI Listing
March 2011

Recent insights into the implication of nitric oxide in osteoblast differentiation and proliferation during bone development.

ScientificWorldJournal 2010 Apr 13;10:624-32. Epub 2010 Apr 13.

Departamento de Fisiologia, Facultad de Medicina, Universidad de Alcala, Madrid, Spain.

Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively. During bone development, extracellular matrix remodeling is required for osteoblast differentiation and the process is largely mediated by the proteolytic activity of extracellular matrix metalloproteinases (MMPs), which play a fundamental role in osteoblast migration, unmineralized matrix degradation, and cell invasion. The recent advances towards investigation in osteogenesis have provided significant information about the transcriptional regulation of several genes, including MMPs, by the expression of crucial transcription factors like NFAT, ATF4, osterix, TAZ, and Cbfa-1-responsive elements. Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by nitric oxide (NO), since mice deficient in endothelial nitric oxide synthase (eNOS) and mice deficient in the eNOS downstream effector (cGMP)-dependent protein kinase (PKG) show bone abnormalities, while inducible NOS (iNOS) null mice also show imbalances in bone osteogenesis and abnormalities in bone healing. Recently, in vitro data showed that Cbfa-1 and the MAPK pathways were crucial for osteoblastic cell differentiation, and NO was found to play a significant role. This article sheds light on some of the mechanisms that may influence NO-mediated actions in bone development.
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http://dx.doi.org/10.1100/tsw.2010.58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763674PMC
April 2010

Nitric oxide elicits functional MMP-13 protein-tyrosine nitration during wound repair.

FASEB J 2008 Sep 21;22(9):3207-15. Epub 2008 May 21.

Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.

Nitric oxide (NO) plays a critical role in wound healing, in part by promoting angiogenesis. However, the precise repair pathways affected by NO are not well defined. We now show that NO regulates matrix metalloproteinase-13 (MMP-13) release during wound repair. We find that normally MMP-13 is kept inside endothelial cells by an association with caveolin-1. However, nitration of MMP-13 on tyrosine residue Y338 causes it to dissociate from caveolin-1 and be released from endothelial cells. We next explored the functional significance of MMP-13 nitration in vivo. Skin injury increases nitration of MMP-13 in mice. Skin wounds in inducible nitric oxide synthase knockout mice release less MMP-13 and heal more slowly than skin wounds in wild-type mice. Conversely, skin wounds in caveolin-1 knockout mice have increased NO production, increased MMP-13 nitration, and accelerated wound healing. Collectively, our data reveal a new pathway through which NO modulates wound repair: nitration of MMP-13 promotes its release from endothelial cells, where it accelerates angiogenesis and wound healing.
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http://dx.doi.org/10.1096/fj.07-103804DOI Listing
September 2008

Inhibitor of NF kappa B alpha is a host sensor of coxsackievirus infection.

Cell Cycle 2007 Mar 25;6(5):503-6. Epub 2007 Mar 25.

Departamento de Fisiologia, Facultad de Medicina, Universidad de Alcala, Alcala de Henares, Spain.

Apoptosis is a host response to viral infection: programmed cell death can limit viral replication. Therefore, the knowledge of pathways by which cells detect viral infection and activate apoptosis may be of considerable interest when developing strategies against viral pathogens. We have shown that cells activate apoptosis in response to Coxsackievirus B3 (CVB3) infection. In an effort to discover how cells detect viral infections, we found that the viral protease 3C(pro) cleaves IkappaBalpha. Truncated IkappaBalpha forms a stable complex with NFkappaB, translocates to the nucleus and inhibits NFkappaB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells in which IkappaBalpha expression is reduced are more susceptible to viral infection, showing less apoptosis and more viral replication. IkBalpha thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection.
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http://dx.doi.org/10.4161/cc.6.5.3918DOI Listing
March 2007

Viral protease cleavage of inhibitor of kappaBalpha triggers host cell apoptosis.

Proc Natl Acad Sci U S A 2006 Dec 30;103(50):19051-6. Epub 2006 Nov 30.

Fundación Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernandez Almagro 3, 28029 Madrid, Spain.

Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3C(pro) cleaves inhibitor of kappaBalpha (IkappaBalpha). A proteolytic fragment of IkappaBalpha then forms a stable complex with NF-kappaB, translocates to the nucleus, and inhibits NF-kappaB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IkappaBalpha expression are more susceptible to viral infection, with less apoptosis and more viral replication. IkappaBalpha thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection.
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http://dx.doi.org/10.1073/pnas.0606019103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1748175PMC
December 2006

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2-dependent TGF-beta signaling.

J Cell Physiol 2007 Feb;210(2):456-68

Centro de Investigaciones Biologicas, CSIC, Madrid, Spain.

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium-derived nitric oxide in endoglin(+/-) (Eng(+/-)) mice and cultured endothelial cells. Here, we find that TGF-beta1 leads to an increased vasodilatation in Eng(+/+) mice that is severely impaired in Eng(+/-) mice, suggesting the involvement of endoglin in the TGF-beta regulated vascular homeostasis. The endoglin-dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF-beta receptor ALK5 and its downstream substrate Smad2. In addition, Smad2-specific signaling is upregulated in endoglin-induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin-dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin-inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome-dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF-beta signaling, and leading to an increased eNOS expression in endothelial cells.
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http://dx.doi.org/10.1002/jcp.20878DOI Listing
February 2007

Stat3 mediates interleukin-6 [correction of interelukin-6] inhibition of human endothelial nitric-oxide synthase expression.

J Biol Chem 2006 Oct 3;281(40):30057-62. Epub 2006 Aug 3.

Department of Physiology, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

Chronic activation of the acute phase response (APR) is associated with atherosclerosis. Elevated levels of interleukin-6, the major inducer of the APR, are associated with an increased risk of cardiovascular events. One of the clinical hallmarks of atherogenesis is endothelial dysfunction, characterized by a decrease in endothelial production of nitric oxide (NO). We hypothesized that interleukin-6 (IL-6) decreases endothelial NO synthase (eNOS) expression. We now show that IL-6 treatment of human aortic endothelial cells (HAEC) decreases steady-state levels of human eNOS mRNA and protein. This decrease in eNOS expression is caused in part by IL-6 inhibition of transactivation of the human eNOS promoter. To explore the mechanism by which IL-6 affects eNOS expression, we examined activation of signal transducer and transactivator-3 (Stat3). The IL-6 receptor (IL-6R) is expressed in HAEC, and Stat3 is phosphorylated in response to IL-6 stimulation of the IL-6R. We identified four consensus sequences for Stat3 binding (SIE) in the eNOS promoter at positions -1520, -1024, -840, and -540. Transfection of eNOS promoter mutants revealed that the SIE at -1024 mediates Stat3 inhibition of eNOS promoter activity. Gel-shift analysis of nuclear extracts from HAEC treated with IL-6 confirms that Stat3 binds to a complex containing the SIE at -1024. RNA silencing of STAT3 blocks the inhibitory effect of IL-6 on eNOS expression. Our data show that IL-6 has direct effects upon endothelial cells, inhibiting eNOS expression in part through Stat3. Decreased levels of eNOS may be an important component of the pro-atherogenic effect of the APR.
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http://dx.doi.org/10.1074/jbc.M606279200DOI Listing
October 2006

Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts.

J Cell Sci 2006 May;119(Pt 9):1896-902

Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain.

During bone development, osteoblast differentiation requires remodeling of the extracellular matrix. Although underlying mechanisms have not been elucidated, evidence points to the participation of the nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) system. Here, we detected increased matrix metalloproteinase (MMP)-13 mRNA, protein and activity, as well as increased inducible NO synthase (iNOS) and NO production during the differentiation of MC3T3-E1 osteoblasts. Transcriptional activity of the MMP-13 promoter was augmented by NO, 8-bromo-cGMP (8-Br-cGMP), and by a dominant-positive form of protein kinase G (PKG1-alpha). The stimulatory effect on the MMP-13 promoter was partially inhibited by mutation of the osteoblast-specific element 2 (OSE-2) binding site. Core binding factor-1 (Cbfa-1) expression peaked at 7 days of differentiation, and was phosphorylated by PKG in vitro. Cbfa-1 was localized to cell nuclei, and its translocation was inhibited by the iNOS inhibitor 1400W. Immunohistological examination revealed that MMP-13 and Cbfa-1 expression levels are both reduced in 17-day-old embryos of iNOS-deficient mice. Silencing of Cbfa-1 mRNA blocked MMP-13 expression without interfering with endogenous NO production, confirming its role in NO-induced MMP-13 expression by MC3T3-E1 cells. The results described here suggest a mechanism by which NO regulates osteogenesis.
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http://dx.doi.org/10.1242/jcs.02895DOI Listing
May 2006

Arg-Gly-Asp (RGD)-containing peptides increase soluble guanylate cyclase in contractile cells.

Cardiovasc Res 2006 Feb 19;69(2):359-69. Epub 2005 Dec 19.

Department of Physiology, Universidad de Alcalá, Alcalá de Henares, 28871-Madrid, Spain.

Objectives: Alterations in NO/cGMP signaling have been associated with vascular dysfunction. Here, we tested whether peptides containing arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins, could increase the expression and function of soluble guanylate cyclase (sGC) in human mesangial cell (HMC), and human aortic smooth muscle (HASMC) cells.

Methods And Results: Arginine-glycine-aspartic acid-serine (RGDS) promoted an up-regulation in the sGC beta1 subunit steady-state level, both in HMC and HASMC, in a time- and dose-dependent manner. The cellular effects of RGDS-stimulation of sGC expression was an enhanced cellular response to sodium nitroprusside, resulting in elevated cGMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in both kinds of cells, and an increased NO relaxing effect on cells precontracted with H(2)O(2) or Angiotensin II. Moreover, RGDS was able to restore the sGC levels that had been previously decreased by long term exposure to NO donors. RGDS effects on sGC regulation were due to the specific interaction with alpha(5)beta(1) integrin. To investigate the intracellular mechanisms activated after RGDS cell treatment, pharmacological kinase inhibitors were used. The effect of RGDS on sGC protein content was completely abolished by treating the cells with c-Jun N-terminal kinase (JNK) inhibitors. In addition, c-fos and c-jun were found in the cell nuclei after RGDS treatment, suggesting that the RGDS effect could be mediated by the AP-1 transcription factor.

Conclusion: Results provide evidence of a mechanism able to increase the sGC protein content linked to increased activity in contractile cells, not only in basal conditions, but also after the down-regulation of the receptor by its own substrate. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.
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http://dx.doi.org/10.1016/j.cardiores.2005.10.011DOI Listing
February 2006

Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells.

Circ Res 2005 Nov 20;97(11):1115-23. Epub 2005 Oct 20.

Departmento Fisiología, Universidad de Alcalá, Alcalá de Henares, Spain.

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-beta1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-beta-initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-beta-dependent gene expression. We show that NO inhibits TGF-beta/Smad-regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1alpha suppressed NO/cGMP inhibition of TGF-beta-induced gene expression. Inversely, overexpression of PKG-1alpha catalytic subunit blocked TGF-beta-induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas NG-nitro-L-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-beta. Aortas from endothelial NO synthase-deficient mice showed enhanced basal TGF-beta1 and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-beta signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-beta/Smad2 signaling by directing the proteasomal degradation of activated Smad.
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http://dx.doi.org/10.1161/01.RES.0000191538.76771.66DOI Listing
November 2005