Specialized Pro-Resolving Lipid Mediators: New Therapeutic Approaches for Vascular Remodeling.

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.

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

OBJECTIVE: microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE-/- mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE-/- mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk. CONCLUSIONS: Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.

A Blunted Sympathetic Function and an Enhanced Nitrergic Activity Contribute to Reduce Mesenteric Resistance in Hyperthyroidism.

We aimed to determine whether an experimental model of hyperthyroidism could alter the function of sympathetic and nitrergic components of mesenteric innervation. For this purpose, male Wistar rats were divided into (1) control rats (CT) and (2) rats infused with L-Thyroxine (HT). Body weight gain and adipose tissue accumulation were lower in HT rats, while systolic blood pressure and citrate synthase activity in the soleus muscle were increased by HT. In segments from the superior mesenteric artery, the application of an electrical field stimulation (EFS) induced a vasoconstrictor response, which was lower in arteries from HT animals. The alpha-adrenoceptor antagonist phentolamine diminished EFS-induced vasoconstriction to a lower extent in HT arteries, while the purinergic receptor antagonist suramin reduced contractile response to EFS only in segments from CT. In line with this, noradrenaline release, tyrosine hydroxylase expression and activation and dopamine ß hydroxylase expression were diminished in HT. The unspecific nitric oxide synthase (NOS) inhibitor L-NAME increased EFS-induced vasoconstriction more markedly in segments from HT rats. NO release was enhanced in HT, probably due to an enhancement in neuronal NOS activity, in which a hyperactivation of both PKC and PI3K-AKT signaling pathways might play a relevant role. In conclusion, perivascular mesenteric innervation might contribute to reduce the vascular resistance observed in hyperthyroidism.

Interleukin-17A induces vascular remodeling of small arteries and blood pressure elevation.

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.

Branched-chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation.

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro-inflammatory responses through the transcription factor NF-κB that resulted in the release of intracellular adhesion molecule-1 and E-selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA-induced pro-oxidant and pro-inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre-contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.

Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids.

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.

Mercury-induced vascular dysfunction is mediated by angiotensin II AT-1 receptor upregulation.

Low doses of mercury (Hg) promote deleterious effects on cardiovascular system, but the mechanisms implicated remain unclear. This study analyzed whether angiotensin II AT-1 receptors are involved in the vascular dysfunction caused by chronic exposure to low HgCl2 doses. For this, rats were divided into four groups and untreated (saline by im injections and tap water by gavage) or treated for 30 days as follows: Mercury (HgCl2im, first dose of 4.6 µg kg-1 and subsequent doses of 0.07 µg kg-1 day-1, and tap water by gavage); Losartan (saline im and losartan, 15 mg kg-1 day-1, by gavage); Losartan-Mercury (HgCl2im and Losartan by gavage). Systolic blood pressure was measured by tail plethysmography, vascular reactivity in aorta by isolated organ bath, oxidative stress by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and antioxidant capacity (FRAP) and protein expression of AT-1 receptors by Western Blot. As results, co-treatment with losartan prevented the increased aortic vasoconstrictor responses to phenylephrine (Phe), the involvement of ROS and prostanoids on the response to Phe and the reduced negative endothelial modulation by nitric oxide on these responses. Moreover, this co-treatment avoided the increase in plasmatic and vascular oxidative stress and AT-1 protein expression in aorta. In conclusion, these results suggest that AT-1 receptors upregulation might play a key role in the vascular damage induced by Hg exposure by increasing oxidative stress and probably by reducing NO bioavailability.

Deficiency of MMP17/MT4-MMP proteolytic activity predisposes to aortic aneurysm in mice.

RATIONALE: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.

The cessation of the long-term exposure to low doses of mercury ameliorates the increase in systolic blood pressure and vascular damage in rats.

This study aimed to verify whether a prolonged exposure to low-level mercury promotes haemodynamic disorders and studied the reversibility of this vascular damage. Rats were divided into seven groups: three control groups received saline solution (im) for 30, 60 or 90 days; two groups received HgCl2 (im, first dose, 4.6µg/kg, subsequent doses 0.07µg/kg/day) for 30 or 60 days; two groups received HgCl2 for 30 or 60 days (im, same doses) followed by a 30-day washout period. Systolic blood pressure (SBP) was measured, along with analysis of vascular response to acetylcholine (ACh) and phenylephrine (Phe) in the absence and presence of endothelium, a nitric oxide (NO) synthase inhibitor, an NADPH oxidase inhibitor, superoxide dismutase, a non-selective cyclooxygenase (COX) inhibitor and an AT1 receptor blocker. Reactive oxygen species (ROS) levels and antioxidant power were measured in plasma. HgCl2 exposure for 30 and 60 days: a) reduced the endothelium-dependent relaxation; b) increased the Phe-induced contraction and the contribution of ROS, COX-derived vasoconstrictor prostanoids and angiotensin II acting on AT1 receptors to this response while the NO participation was reduced; c) increased the oxidative stress in plasma; d) increased the SBP only after 60 days of exposure. After the cessation of HgCl2 exposure, SBP, endothelium-dependent relaxation, Phe-induced contraction and the oxidative stress were normalised, despite the persistence of the increased COX-derived prostanoids. These results demonstrated that long-term HgCl2 exposure increases SBP as a consequence of vascular dysfunction; however, after HgCl2 removal from the environment the vascular function ameliorates.

Reproductive dysfunction after mercury exposure at low levels: evidence for a role of glutathione peroxidase (GPx) 1 and GPx4 in male rats.

Mercury is a ubiquitous environmental pollutant and mercury contamination and toxicity are serious hazards to human health. Some studies have shown that mercury impairs male reproductive function, but less is known about its effects following exposure at low doses and the possible mechanisms underlying its toxicity. Herein we show that exposure of rats to mercury chloride for 30 days (first dose 4.6µgkg-1, subsequent doses 0.07µgkg-1day-1) resulted in mean (±s.e.m.) blood mercury concentrations of 6.8±0.3ngmL-1, similar to that found in human blood after occupational exposure or released from removal of amalgam fillings. Even at these low concentrations, mercury was deposited in reproductive organs (testis, epididymis and prostate), impaired sperm membrane integrity, reduced the number of mature spermatozoa and, in the testes, promoted disorganisation, empty spaces and loss of germinal epithelium. Mercury increased levels of reactive oxygen species and the expression of glutathione peroxidase (GPx) 1 and GPx4. These results suggest that the toxic effects of mercury on the male reproductive system are due to its accumulation in reproductive organs and that the glutathione system is its potential target. The data also suggest, for the first time, a possible role of the selenoproteins GPx1 and GPx4 in the reproductive toxicity of mercury chloride.