Marta Saura

Nitric oxide inhibition of coxsackievirus replication in vitro.

Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism.

Nitric Oxide Regulates Transforming Growth Factor-β Signaling in Endothelial Cells

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-&bgr;1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-&bgr;–initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-&bgr;–dependent gene expression. We show that NO inhibits TGF-&bgr;/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-1&agr; suppressed NO/cGMP inhibition of TGF-&bgr;–induced gene expression. Inversely, overexpression of PKG-1&agr; catalytic subunit blocked TGF-&bgr;–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-&bgr;. Aortas from endothelial NO synthase–deficient mice showed enhanced basal TGF-&bgr;1 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-&bgr; 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-&bgr;/Smad2 signaling by directing the proteasomal degradation of activated Smad.

Stat3 Mediates Interelukin-6 Inhibition of Human Endothelial Nitric-oxide Synthase Expression

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.

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

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-α). 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.

Viral protease cleavage of inhibitor of κBα triggers host cell apoptosis

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 3Cpro cleaves inhibitor of κBα (IκBα). A proteolytic fragment of IκBα then forms a stable complex with NF-κB, translocates to the nucleus, and inhibits NF-κB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IκBα expression are more susceptible to viral infection, with less apoptosis and more viral replication. IκBα 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.

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

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.

Endothelial mechanosensors of shear stress as regulators of atherogenesis.

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.

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

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 caveo‐lin‐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.—Lizarbe, T. R., García‐Ram, C., Tarín, C., Saura, M., Calvo, E., López, J. A., López‐Otín, C., Folgueras, A. R., Lamas, S., Zaragoza, C. Nitric oxide elicits functional MMP‐13 protein‐tyrosine nitration during wound repair. FASEB J. 22, 3207–3215 (2008)

Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling Role in Atherosclerosis

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.

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

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.—Saura, M., Marquez, S., Reventun, P., Olea‐Herrero, N., Arenas, M.I., Moreno‐Gómez‐Toledano, R., Gómez‐Parrizas, M., Muñóz‐Moreno, C., González‐Santander, M., Zaragoza, C., Bosch, R.J. Oral administration of bisphenol A induces high blood pressure through angiotensin II/CaMKII‐dependent uncoupling of eNOS. FASEB J. 28, 4719–4728 (2014). www.fasebj.org