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BACKGROUND: Obesity-induced hypogonadism (OIH) is a prevalent, but often neglected condition in men, which aggravates the metabolic complications of overweight. While hypothalamic suppression of Kiss1-encoded kisspeptin has been suggested to contribute to OIH, the molecular mechanisms for such repression in obesity, and the therapeutic implications thereof, remain unknown. METHODS: A combination of bioinformatic, expression and functional analyses was implemented, assessing the role of the evolutionary-conserved miRNAs, miR-137 and miR-325, in mediating obesity-induced suppression of hypothalamic kisspeptin, as putative mechanism of central hypogonadism and metabolic comorbidities. The implications of such miR-137/325-kisspeptin interplay for therapeutic intervention in obesity were also explored using preclinical OIH models. RESULTS: MiR-137/325 repressed human KISS1 3'-UTR in-vitro and inhibited hypothalamic kisspeptin content in male rats, while miR-137/325 expression was up-regulated, and Kiss1/kisspeptin decreased, in the medio-basal hypothalamus of obese rats. Selective over-expression of miR-137 in Kiss1 neurons reduced Kiss1/ kisspeptin and partially replicated reproductive and metabolic alterations of OIH in lean mice. Conversely, interference of the repressive actions of miR-137/325 selectively on Kiss1 3'-UTR in vivo, using target-site blockers (TSB), enhanced kisspeptin content and reversed central hypogonadism in obese rats, together with improvement of glucose intolerance, insulin resistance and cardiovascular and inflammatory markers, despite persistent exposure to obesogenic diet. Reversal of OIH by TSB miR-137/325 was more effective than chronic kisspeptin or testosterone treatments in obese rats. CONCLUSIONS: Our data disclose that the miR-137/325-Kisspeptin repressive interaction is a major player in the pathogenesis of obesity-induced hypogonadism and a putative druggable target for improved management of this condition and its metabolic comorbidities in men suffering obesity. SIGNIFICANCE STATEMENT: Up to half of the men suffering obesity display also central hypogonadism, an often neglected complication of overweight that can aggravate the clinical course of obesity and its complications. The mechanisms for such obesity-induced hypogonadism remain poorly defined. We show here that the evolutionary conserved miR137/miR325 tandem centrally mediates obesity-induced hypogonadism via repression of the reproductive-stimulatory signal, kisspeptin; this may represent an amenable druggable target for improved management of hypogonadism and other metabolic complications of obesity.
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Hipogonadismo , Hipotálamo , Kisspeptinas , MicroARNs , Obesidad , MicroARNs/genética , MicroARNs/metabolismo , Hipogonadismo/genética , Hipogonadismo/metabolismo , Hipogonadismo/complicaciones , Kisspeptinas/genética , Kisspeptinas/metabolismo , Animales , Obesidad/metabolismo , Obesidad/complicaciones , Obesidad/genética , Masculino , Ratas , Hipotálamo/metabolismo , Humanos , Ratones , Ratas Wistar , ComorbilidadRESUMEN
Lysyl oxidase (LOX)-mediated extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease; however, this enzyme also induces oxidative stress. We addressed the contribution of LOX-dependent oxidative stress to cardiovascular calcification. LOX is upregulated in human-calcified atherosclerotic lesions and atheromas from atherosclerosis-challenged LOX transgenic mice (TgLOXVSMC) and colocalized with a marker of oxidative stress (8-oxo-deoxyguanosine) in vascular smooth muscle cells (VSMCs). Similarly, in calcific aortic valves, high LOX expression was detected in valvular interstitial cells (VICs) positive for 8-oxo-deoxyguanosine, while LOX and LOXL2 expression correlated with osteogenic markers (SPP1 and RUNX2) and NOX2. In human VICs, mito-TEMPO and TEMPOL attenuated the increase in superoxide anion levels and the mineralization induced by osteogenic media (OM). Likewise, in OM-exposed VICs, ß-aminopropionitrile (a LOX inhibitor) ameliorated both oxidative stress and calcification. Gain- and loss-of-function approaches in VICs demonstrated that while LOX silencing negatively modulates oxidative stress and calcification induced by OM, lentiviral LOX overexpression exacerbated oxidative stress and VIC calcification, effects that were prevented by mito-TEMPO, TEMPOL, and ß-aminopropionitrile. Our data indicate that LOX-induced oxidative stress participates in the procalcifying effects of LOX activity in ectopic cardiovascular calcification, and highlight the multifaceted role played by LOX isoenzymes in cardiovascular diseases.
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During resolution of inflammation, specialized proresolving mediators (SPMs), including resolvins, are produced to restore tissue homeostasis. We hypothesized that there might be a dysregulation of SPMs pathways in pathological vascular remodeling and that resolvin D2 (RvD2) might prevent vascular remodeling and contractile and endothelial dysfunction in a model of obesity and hypertension. In aortic samples of patients with or without abdominal aortic aneurysms (AAA), we evaluated gene expression of enzymes involved in SPMs synthesis (ALOXs), SPMs receptors and pro-inflammatory genes. In an experimental model of aortic dilation induced by high fat diet (HFD, 60%, eighteen weeks) and angiotensin II (AngII) infusion (four weeks), we studied the effect of RvD2 administration in aorta and small mesenteric arteries structure and function and markers of inflammation. In human macrophages we evaluated the effects of AngII and RvD2 in macrophages function and SPMs profile. In patients, we found positive correlations between AAA and obesity, and between AAA and expression of ALOX15, RvD2 receptor GPR18, and pro-inflammatory genes. There was an inverse correlation between the expression of aortic ALOX15 and AAA growth rate. In the mice model, RvD2 partially prevented the HFD plus AngII-induced obesity and adipose tissue inflammation, hypertension, aortic and mesenteric arteries remodeling, hypercontratility and endothelial dysfunction, and the expression of vascular proinflammatory markers and cell apoptosis. In human macrophages, RvD2 prevented AngII-induced impaired efferocytosis and switched SPMs profile. RvD2 might represent a novel protective strategy in preventing vascular damage associated to hypertension and obesity likely through effects in vascular and immune cells.
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Ácidos Docosahexaenoicos , Hipertensión , Ratones Endogámicos C57BL , Obesidad , Remodelación Vascular , Animales , Masculino , Humanos , Ácidos Docosahexaenoicos/farmacología , Hipertensión/metabolismo , Hipertensión/tratamiento farmacológico , Obesidad/complicaciones , Obesidad/metabolismo , Remodelación Vascular/efectos de los fármacos , Ratones , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Dieta Alta en Grasa/efectos adversos , Angiotensina II , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Ratones Obesos , Vasoconstricción/efectos de los fármacos , Inflamación/patología , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Modelos Animales de EnfermedadRESUMEN
The angiotensin (Ang)-(1-12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1-12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2). Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1-12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1-12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1-12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only. In conclusion, the chymase-independent vascular Ang-(1-12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.
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Insuficiencia Cardíaca , Enfermedades Vasculares , Animales , Ratones , Angiotensina I , Angiotensina II/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina , Quimasas , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Ratones EndogámicosRESUMEN
Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor-associated macrophages (TAM) have a string of immunosuppressive functions and they are considered a therapeutic target in cancer. This study aimed to analyze the effects of trabectedin, an antitumor agent, on the tumor-microenvironment through the characterization of the electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but their treatment (16 h) with sub-cytotoxic concentrations of trabectedin increased their KV current due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv) exhibited an M2-like phenotype. TAMiv generated a small KV current and express high levels of M2 markers. K+ current from TAMs isolated from tumors generated in mice is a mixture of KV and KCa, and in TAM isolated from tumors generated in trabectedin-treated mice, the current is mostly driven by KCa. We conclude that the antitumor capacity of trabectedin is not only due to its effects on tumor cells, but also to the modulation of the tumor microenvironment, due, at least in part, to the modulation of the expression of different macrophage ion channels.
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Macrófagos , Microambiente Tumoral , Ratones , Animales , Trabectedina/farmacología , Macrófagos/metabolismo , Activación de Macrófagos , Fenómenos ElectrofisiológicosRESUMEN
BACKGROUND: Resolution of inflammation is orchestrated by specialized proresolving lipid mediators (SPMs), and this would be impaired in some cardiovascular diseases. Among SPMs, resolvins (Rv) have beneficial effects in cardiovascular pathologies, but little is known about their effect on cardiovascular damage in hypertension. METHODS: Aorta, small mesenteric arteries, heart, and peritoneal macrophages were taken from C57BL/6J mice, infused or not with angiotensin II (AngII; 1.44 mg/kg/day, 14 days) in presence or absence of resolvin D2 (RvD2) (100 ng/mice, every second day) starting 1 day before or 7 days after AngII infusion. RESULTS: Enzymes and receptors involved in SPMs biosynthesis and signaling were increased in aorta or heart from AngII-infused mice. We also observed a differential regulation of SPMs in heart from these mice. Preventive treatment with RvD2 partially avoided AngII-induced hypertension and protected the heart and large and small vessels against functional and structural alterations induced by AngII. RvD2 increased the availability of vasoprotective factors, modified SPMs profile, decreased cardiovascular fibrosis, and increased the infiltration of pro-resolving macrophages. When administered in hypertensive animals with established cardiovascular damage, RvD2 partially improved cardiovascular function and structure, decreased fibrosis, reduced the infiltration of neutrophils, and shifted macrophage phenotype toward a pro-resolving phenotype. CONCLUSIONS: There is a disbalance between proinflammatory and resolution mediators in hypertension. RvD2 protects cardiovascular function and structure when administered before and after the development of hypertension by modulating vascular factors, fibrosis and inflammation. Activating resolution mechanisms by treatment with RvD2 may represent a novel therapeutic strategy for the treatment of hypertensive cardiovascular disease.
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Angiotensina II , Hipertensión , Ratones , Animales , Ratones Endogámicos C57BL , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , FibrosisRESUMEN
In recent years, gut dysbiosis has been related to some peripheral vascular alterations linked to hypertension. In this work, we explore whether gut dysbiosis is related to vascular innervation dysfunction and altered nitric oxide (NO) production in the superior mesenteric artery, one of the main vascular beds involved in peripheral vascular resistance. For this purpose, we used spontaneously hypertensive rats, either treated or not with the commercial synbiotic formulation Prodefen® (108 colony forming units/day, 4 weeks). Prodefen® diminished systolic blood pressure and serum endotoxin, as well as the vasoconstriction elicited by electrical field stimulation (EFS), and enhanced acetic and butyric acid in fecal samples, and the vasodilation induced by the exogenous NO donor DEA-NO. Unspecific nitric oxide synthase (NOS) inhibitor L-NAME increased EFS-induced vasoconstriction more markedly in rats supplemented with Prodefen®. Both neuronal NO release and neuronal NOS activity were enhanced by Prodefen®, through a hyperactivation of protein kinase (PK)A, PKC and phosphatidylinositol 3 kinase-AKT signaling pathways. The superoxide anion scavenger tempol increased both NO release and DEA-NO vasodilation only in control animals. Prodefen® caused an increase in both nuclear erythroid related factor 2 and superoxide dismutase activities, consequently reducing both superoxide anion and peroxynitrite releases. In summary, Prodefen® could be an interesting non-pharmacological approach to ameliorate hypertension.
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Vascular remodeling is a typical feature of vascular diseases, such as atherosclerosis, aneurysms or restenosis. Excessive inflammation is a key mechanism underlying vascular remodeling via the modulation of vascular fibrosis, phenotype and function. Recent evidence suggests that not only augmented inflammation but unresolved inflammation might also contribute to different aspects of vascular diseases. Resolution of inflammation is mediated by a family of specialized pro-resolving mediators (SPMs) that limit immune cell infiltration and initiate tissue repair mechanisms. SPMs (lipoxins, resolvins, protectins, maresins) are generated from essential polyunsaturated fatty acids. Synthases and receptors for SPMs were initially described in immune cells, but they are also present in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), where they regulate processes important for vascular physiology, such as EC activation and VSMC phenotype. Evidence from genetic models targeting SPM pathways and pharmacological supplementation with SPMs have demonstrated that these mediators may play a protective role against the development of vascular remodeling in atherosclerosis, aneurysms and restenosis. This review focuses on the latest advances in understanding the role of SPMs in vascular cells and their therapeutic effects in the vascular remodeling associated with different cardiovascular diseases.
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Aterosclerosis , Mediadores de Inflamación , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/uso terapéutico , Eicosanoides/farmacología , Células Endoteliales/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Remodelación VascularRESUMEN
AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.
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Aneurisma de la Aorta Abdominal , Hipertensión , Ratones , Humanos , Animales , Elastina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Angiotensina II/metabolismo , Interferones/metabolismo , Leucocitos Mononucleares/metabolismo , Grosor Intima-Media Carotídeo , Estrés Oxidativo , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertensión/metabolismo , Oxidación-Reducción , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/prevención & control , Inflamación , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND PURPOSE: KV 1.3 channels are expressed in vascular smooth muscle cells (VSMCs), where they contribute to proliferation rather than contraction and participate in vascular remodelling. KV 1.3 channels are also expressed in macrophages, where they assemble with KV 1.5 channels (KV 1.3/KV 1.5), whose activation generates a KV current. In macrophages, the KV 1.3/KV 1.5 ratio is increased by classical activation (M1). Whether these channels are involved in angiotensin II (AngII)-induced vascular remodelling, and whether they can modulate the macrophage phenotype in hypertension, remains unknown. We characterized the role of KV 1.3 channels in vascular damage in hypertension. EXPERIMENTAL APPROACH: We used AngII-infused mice treated with two selective KV 1.3 channel inhibitors (HsTX[R14A] and [EWSS]ShK). Vascular function and structure were measured using wire and pressure myography, respectively. VSMC and macrophage electrophysiology were studied using the patch-clamp technique; gene expression was analysed using RT-PCR. KEY RESULTS: AngII increased KV 1.3 channel expression in mice aorta and peritoneal macrophages which was abolished by HsTX[R14A] treatment. KV 1.3 inhibition did not prevent hypertension, vascular remodelling, or stiffness but corrected AngII-induced macrophage infiltration and endothelial dysfunction in the small mesenteric arteries and/or aorta, via a mechanism independent of electrophysiological changes in VSMCs. AngII modified the electrophysiological properties of peritoneal macrophages, indicating an M1-like activated state, with enhanced expression of proinflammatory cytokines that induced endothelial dysfunction. These effects were prevented by KV 1.3 blockade. CONCLUSIONS AND IMPLICATIONS: We unravelled a new role for KV 1.3 channels in the macrophage-dependent endothelial dysfunction induced by AngII in mice which might be due to modulation of macrophage phenotype.
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Angiotensina II , Hipertensión , Angiotensina II/toxicidad , Animales , Hipertensión/inducido químicamente , Macrófagos , Ratones , Miocitos del Músculo Liso , Remodelación VascularRESUMEN
The acute-on-chronic liver failure (ACLF) is a syndrome characterized by liver decompensation, hepatic encephalopathy (HE) and high mortality. We aimed to determine the mechanisms implicated in the development of HE-associated cerebral vasculopathy in a microsurgical liver cholestasis (MHC) model of ACLF. Microsurgical liver cholestasis was induced by ligating and extracting the common bile duct and four bile ducts. Sham-operated and MHC rats were maintained for eight postoperative weeks Bradykinin-induced vasodilation was greater in middle cerebral arteries from MHC rats. Both Nω-Nitro-L-arginine methyl ester and indomethacin diminished bradykinin-induced vasodilation largely in arteries from MHC rats. Nitrite and prostaglandin (PG) F1α releases were increased, whereas thromboxane (TX) B2 was not modified in arteries from MHC. Expressions of endothelial nitric oxide synthase (eNOS), inducible NOS, and cyclooxygenase (COX) 2 were augmented, and neuronal NOS (nNOS), COX-1, PGI2 synthase, and TXA2S were unmodified. Phosphorylation was augmented for eNOS and unmodified for nNOS. Altogether, these endothelial alterations might collaborate to increase brain blood flow in HE.
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An important link exists between hypertension and inflammation. Hypertensive patients present elevated circulating levels of proinflammatory cytokines, including interleukin-17A (IL-17A). This cytokine participates in host defense, autoimmune and chronic inflammatory pathologies, and cardiovascular diseases, mainly through the regulation of proinflammatory factors. Emerging evidence also suggests that IL-17A could play a role in regulating blood pressure and end-organ damage. Here, our preclinical studies in a murine model of systemic IL-17A administration showed that increased levels of circulating IL-17A raised blood pressure induced inward remodeling of small mesenteric arteries (SMAs) and arterial stiffness. In IL-17A-infused mice, treatment with hydralazine and hydrochlorothiazide diminished blood pressure elevation, without modifying mechanical and structural properties of SMA, suggesting a direct vascular effect of IL-17A. The mechanisms of IL-17A seem to involve an induction of vascular smooth muscle cell (VSMC) hypertrophy and phenotype changes, in the absence of extracellular matrix (ECM) proteins accumulation. Accordingly, treatment with an IL-17A neutralizing antibody diminished SMA remodeling in a model of angiotensin II (Ang II) infusion. Moreover, in vitro studies in VSMCs reported here, provide further evidence of the direct effects of IL-17A on cell growth responses. Our experimental data suggest that IL-17A is a key mediator of vascular remodeling of the small arteries, which might contribute, at least in part, to blood pressure elevation.
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Presión Sanguínea/efectos de los fármacos , Interleucina-17/farmacología , Arterias Mesentéricas/efectos de los fármacos , Remodelación Vascular/efectos de los fármacos , Angiotensina II/administración & dosificación , Angiotensina II/farmacología , Animales , Forma de la Célula/efectos de los fármacos , Humanos , Hipertensión/fisiopatología , Interleucina-17/administración & dosificación , Masculino , Arterias Mesentéricas/fisiología , Ratones Endogámicos C57BL , Músculo Liso Vascular , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Vasoconstrictores/administración & dosificación , Vasoconstrictores/farmacologíaRESUMEN
Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.
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Aletas de Animales/fisiología , Corazón/fisiología , Macrófagos/metabolismo , Regeneración , Proteínas WT1/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Macrófagos/citología , Proteínas WT1/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
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.
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Homeostasis/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Oxidación-Reducción , Análisis de Varianza , Movimiento Celular/fisiología , Células Cultivadas , Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/patología , Redes Reguladoras de Genes , Homeostasis/genética , Humanos , Isoenzimas/metabolismo , NADPH Oxidasa 1/genética , NADPH Oxidasa 1/metabolismo , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Estadísticas no Paramétricas , Superóxido Dismutasa/metabolismo , Activación TranscripcionalRESUMEN
mPGES-1 (microsomal prostaglandin E synthase-1), the downstream enzyme responsible for PGE2 (prostaglandin E2) synthesis in inflammatory conditions and oxidative stress are increased in vessels from hypertensive animals. We evaluated the role of mPGES-1-derived PGE2 in the vascular dysfunction and remodeling in hypertension and the possible contribution of oxidative stress. We used human peripheral blood mononuclear cells from asymptomatic patients, arteries from untreated and Ang II (angiotensin II)-infused mPGES-1-/- and mPGES-1+/+ mice, and vascular smooth muscle cells exposed to PGE2 In human cells, we found a positive correlation between mPGES-1 mRNA and carotid intima-media thickness (r=0.637; P<0.001) and with NADPH oxidase-dependent superoxide production (r=0.417; P<0.001). In Ang II-infused mice, mPGES-1 deletion prevented all of the following: (1) the augmented wall:lumen ratio, vascular stiffness, and altered elastin structure; (2) the increased gene expression of profibrotic and proinflammatory markers; (3) the increased vasoconstrictor responses and endothelial dysfunction; (4) the increased NADPH oxidase activity and the diminished mitochondrial membrane potential; and (5) the increased reactive oxygen species generation and reduced NO bioavailability. In vascular smooth muscle cells or aortic segments, PGE2 increased NADPH oxidase expression and activity and reduced mitochondrial membrane potential, effects that were abolished by antagonists of the PGE2 receptors (EP), EP1 and EP3, and by JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular-signal-regulated kinases 1/2) inhibition. Deletion of mPGES-1 augmented vascular production of PGI2 suggesting rediversion of the accumulated PGH2 substrate. In conclusion, mPGES-1-derived PGE2 is involved in vascular remodeling, stiffness, and endothelial dysfunction in hypertension likely through an increase of oxidative stress produced by NADPH oxidase and mitochondria.
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Arterias Carótidas/fisiopatología , Regulación de la Expresión Génica , Hipertensión/genética , Músculo Liso Vascular/metabolismo , Estrés Oxidativo , Prostaglandina-E Sintasas/genética , Rigidez Vascular , Animales , Arterias Carótidas/metabolismo , Modelos Animales de Enfermedad , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/fisiología , Prostaglandina-E Sintasas/biosíntesis , ARN/genéticaRESUMEN
Cyclooxygenase-2 (COX-2) derived-prostanoids participate in the altered vascular function and mechanical properties in cardiovascular diseases. We investigated whether regulator of calcineurin 1 (Rcan1) participates in vascular contractility and stiffness through the regulation of COX-2. For this, wild type (Rcan1+/+) and Rcan1-deficient (Rcan1-/-) mice untreated or treated with the COX-2 inhibitor rofecoxib were used. Vascular function and structure were analysed by myography. COX-2 and phospo-p65 expression were studied by western blotting and immunohistochemistry and TXA2 production by ELISA. We found that Rcan1 deficiency increases COX-2 and IL-6 expression and NF-κB activation in arteries and vascular smooth muscle cells (VSMC). Adenoviral-mediated re-expression of Rcan1.4 in Rcan1-/- VSMC normalized COX-2 expression. Phenylephrine-induced vasoconstrictor responses were greater in aorta from Rcan1-/- compared to Rcan1+/+ mice. This increased response were diminished by etoricoxib, furegrelate, SQ 29548, cyclosporine A and parthenolide, inhibitors of COX-2, TXA2 synthase, TP receptors, calcineurin and NF-κB, respectively. Endothelial removal and NOS inhibition increased phenylephrine responses only in Rcan1+/+ mice. TXA2 levels were greater in Rcan1-/- mice. In small mesenteric arteries, vascular function and structure were similar in both groups of mice; however, vessels from Rcan1-/- mice displayed an increase in vascular stiffness that was diminished by rofecoxib. In conclusion, our results suggest that Rcan1 might act as endogenous negative modulator of COX-2 expression and activity by inhibiting calcineurin and NF-kB pathways to maintain normal contractility and vascular stiffness in aorta and small mesenteric arteries, respectively. Our results uncover a new role for Rcan1 in vascular contractility and mechanical properties.
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Aorta Torácica/fisiología , Ciclooxigenasa 2/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Arterias Mesentéricas/fisiología , Proteínas Musculares/fisiología , Músculo Liso Vascular/fisiología , Animales , Proteínas de Unión al Calcio , Células Cultivadas , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiologíaRESUMEN
INTRODUCTION: Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed. METHODS: Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H2O2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy. RESULTS: LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H2O2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H2O2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis. CONCLUSIONS: LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodelling.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Estrés Oxidativo/fisiología , Proteína-Lisina 6-Oxidasa/genética , Animales , Colágeno/metabolismo , Elastina/metabolismo , Matriz Extracelular/genética , Regulación de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Remodelación Vascular/genética , Remodelación Vascular/fisiologíaRESUMEN
AIMS: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. RESULTS: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with ß-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2.- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. INNOVATION: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. CONCLUSION: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379-397.
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Proteínas de la Matriz Extracelular/metabolismo , Hipertensión/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Transducción de Señal , Rigidez Vascular , Animales , Modelos Animales de Enfermedad , Elastina/química , Proteínas de la Matriz Extracelular/genética , Hipertensión/genética , Masculino , Ratones , Estrés Oxidativo , Proteína-Lisina 6-Oxidasa/genética , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Reactive oxygen species (ROS) are key signaling molecules that regulate vascular function and structure in physiological conditions. A misbalance between the production and detoxification of ROS increases oxidative stress that is involved in the vascular remodeling associated with cardiovascular diseases such as hypertension by affecting inflammation, hypertrophy, migration, growth/apoptosis and extracellular matrix protein turnover. The major and more specific source of ROS in the cardiovascular system is the NADPH oxidase (NOX) family of enzymes composed of seven members (NOX1-5, DUOX 1/2). Vascular cells express several NOXs being NOX-1 and NOX-4 the most abundant NOXs present in vascular smooth muscle cells. This review focuses on specific aspects of NOX-1 and NOX-4 isoforms including information on regulation, function and their role in vascular remodeling. In order to obtain a more integrated view about the role of the different NOX isoforms in different types of vascular remodeling, we discuss the available literature not only on hypertension but also in atherosclerosis, restenosis and aortic dilation.
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Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/patología , NADPH Oxidasas/metabolismo , Remodelación Vascular , Animales , Enfermedades Cardiovasculares/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , NADPH Oxidasa 1/análisis , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 4/análisis , NADPH Oxidasa 4/metabolismo , NADPH Oxidasas/análisis , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Fatty acids cause endothelial dysfunction involving increased ROS (reactive oxygen species) and reduced NO (nitric oxide) bioavailability. We show that in MAECs (mouse aortic endothelial cells), the PPARß/δ (peroxisome- proliferator-activated receptor ß/δ) agonist GW0742 prevented the decreased A23187-stimulated NO production, phosphorylation of eNOS (endothelial nitric oxide synthase) at Ser1177 and increased intracellular ROS levels caused by exposure to palmitate in vitro. The impaired endothelium-dependent relaxation to acetylcholine in mouse aorta induced by palmitate was restored by GW0742. In vivo, GW0742 treatment prevented the reduced aortic relaxation, phosphorylation of eNOS at Ser1177, and increased ROS production and NADPH oxidase in mice fed on a high-fat diet. The PPARß/δ antagonist GSK0660 abolished all of these protective effects induced by GW0742. This agonist enhanced the expression of CPT (carnitine palmitoyltransferase)-1. The effects of GW0742 on acetylcholine- induced relaxation in aorta and on NO and ROS production in MAECs exposed to palmitate were abolished by the CPT-1 inhibitor etomoxir or by siRNA targeting CPT-1. GW0742 also inhibited the increase in DAG (diacylglycerol), PKCα/ßII (protein kinase Cα/ßII) activation, and phosphorylation of eNOS at Thr495 induced by palmitate in MAECs, which were abolished by etomoxir. In conclusion, PPARß/δ activation restored the lipid-induced endothelial dysfunction by up-regulation of CPT-1, thus reducing DAG accumulation and the subsequent PKC-mediated ROS production and eNOS inhibition.