Resolvin E1 attenuates doxorubicin-induced cardiac fibroblast senescence: A key role for IL-1ß.

Cardiac fibroblasts (CFs) undergo senescence in reaction to different stressors, leading to a poor prognosis of cardiac disease. Doxorubicin (Doxo) is an antineoplastic drug with strong cardiotoxic effects, which induces IL-1ß secretion and thus, triggers a potent pro-inflammatory response. Doxo induces CFs senescence; however, the mechanisms are not fully understood. Different pharmacological strategies have been used to eliminate senescent cells by inducing their apoptosis or modifying their secretome. However, Resolvin E1 (RvE1), a lipid derivative resolutive mediator with potent anti-inflammatory effects has not been used before to prevent CFs senescence. CFs were isolated from adult male C57BL/6J mice and subsequently stimulated with Doxo, in the presence or absence of RvE1. Senescence-associated ß-galactosidase activity (SA-ß-gal), γ-H2A.X, p53, p21, and senescence-associated secretory phenotype (SASP) were evaluated. The involvement of the NLRP3 inflammasome/interleukin-1 receptor (IL-1R) signaling pathway on CFs senescence was studied using an NLRP3 inhibitor (MCC950) and an endogenous IL-1R antagonist (IR1A). Doxo is able to trigger CFs senescence, as evidenced by an increase of γ-H2A.X, p53, p21, and SA-ß-gal, and changes in the SASP profile. These Doxo effects were prevented by RvE1. Doxo triggers IL-1ß secretion, which was dependent on NLRP3 activation. Doxo-induced CFs senescence was partially blocked by MCC950 and IR1A. In addition, IL-1ß also triggered CFs senescence, as evidenced by the increase of γ-H2A.X, p53, p21, SA-ß-gal activity, and SASP. All these effects were also prevented by RvE1 treatment. CONCLUSION: These data show the anti-senescent role of RvE1 in Doxo-induced CFs senescence, which could be mediated by reducing IL-1ß secretion.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation.

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10-100 nM) increased the number of SA-ß-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1ß. In fact, IL-1ß itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1ß by interfering with the increased expression of pP65, NLRP3, and pro-IL-1ß proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1ß axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

DPP4 Promotes Human Endothelial Cell Senescence and Dysfunction via the PAR2-COX-2-TP Axis and NLRP3 Inflammasome Activation.

BACKGROUND: Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. METHODS: Human endothelial cell senescence was assessed by senescence-associated ß-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. RESULTS: By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2-COX-2-TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. CONCLUSIONS: These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

Resolvin-D1 attenuation of angiotensin II-induced cardiac inflammation in mice is associated with prevention of cardiac remodeling and hypertension.

AIMS: Despite the broad pharmacological arsenal to treat hypertension, chronic patients may develop irreversible cardiac remodeling and fibrosis. Angiotensin II, the main peptide responsible for the Renin-Angiotensin-Aldosterone-System, has been closely linked to cardiac remodeling, hypertrophy, fibrosis, and hypertension, and some of these effects are induced by inflammatory mediators. Resolvin-D1 (RvD1) elicits potent anti-inflammatory and pro-resolving effects in various pathological models. In this study, we aimed to examine whether RvD1 ameliorates cardiac remodeling and hypertension triggered by angiotensin II. METHODS AND RESULTS: Alzet® osmotic mini-pumps filled with angiotensin II (1.5 mg/kg/day) were implanted in male C57BL/6 J mice for 7 or 14 days. RvD1 (3 µg/kg/day, i.p) was administered one day after the surgery and during the complete infusion period. Blood pressure and myocardial functional parameters were assessed by echocardiography. At the end of the experimental procedure, blood and heart tissue were harvested, and plasma and histological parameters were studied. After 7 and 14 days, RvD1 reduced the increase of neutrophil and macrophage infiltration triggered by angiotensin II, and also reduced ICAM-1 and VCAM-1 expression levels. RvD1 also reduced cytokine plasma levels (IL-1ß, TNF-α, IL-6, KC, MCP-1), cardiac hypertrophy, interstitial and perivascular fibrosis, and hypertension. CONCLUSIONS: This study unveils novel cardioprotective effects of RvD1 in angiotensin II-induced hypertension and cardiac remodeling by attenuating inflammation and provides insights into a potential clinical application.

Resolvin D1 and E1 promote resolution of inflammation in rat cardiac fibroblast in vitro.

Cardiac fibroblasts (CFs) have a key role in the inflammatory response after cardiac injury and are necessary for wound healing. Resolvins are potent agonists that control the duration and magnitude of inflammation. They decrease mediators of pro-inflammatory expression, reduce neutrophil migration to inflammation sites, promote the removal of microbes and apoptotic cells, and reduce exudate. However, whether resolvins can prevent pro-inflammatory-dependent effects in CFs is unknown. Thus, the present work was addressed to study whether resolvin D1 and E1 (RvD1 and RvE1) can prevent pro-inflammatory effects on CFs after lipopolysaccharide (LPS) challenge. For this, CFs were stimulated with LPS, in the presence or absence of RvD1 or RvE1, to analyze its effects on intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), monocyte adhesion and the cytokine levels of tumor necrosis factor alpha (TNF-α), interleukin-6(IL-6), interleukin-1beta (IL-1ß), monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10). Our results showed that CFs are expressing ALX/FPR2 and ChemR23, RvD1 and RvE1 receptors, respectively. RvD1 and RvE1 prevent the increase of ICAM-1 and VCAM-1 protein levels and the adhesion of spleen mononuclear cells to CFs induced by LPS. Finally, RvD1, but not RvE1, prevents the LPS-induced increase of IL-6, MCP-1, TNF-α, and IL-10. In conclusion, our findings provide evidence that in CFs, RvD1 and RvE1 might actively participate in the prevention of inflammatory response triggered by LPS.

Cardiovascular Damage in COVID-19: Therapeutic Approaches Targeting the Renin-Angiotensin-Aldosterone System.

Coronavirus disease 2019 (COVID-19) is usually more severe and associated with worst outcomes in individuals with pre-existing cardiovascular pathologies, including hypertension or atherothrombosis. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can differentially infect multiple tissues (i.e., lung, vessel, heart, liver) in different stages of disease, and in an age- and sex-dependent manner. In particular, cardiovascular (CV) cells (e.g., endothelial cells, cardiomyocytes) could be directly infected and indirectly disturbed by systemic alterations, leading to hyperinflammatory, apoptotic, thrombotic, and vasoconstrictive responses. Until now, hundreds of clinical trials are testing antivirals and immunomodulators to decrease SARS-CoV-2 infection or related systemic anomalies. However, new therapies targeting the CV system might reduce the severity and lethality of disease. In this line, activation of the non-canonical pathway of the renin-angiotensin-aldosterone system (RAAS) could improve CV homeostasis under COVID-19. In particular, treatments with angiotensin-converting enzyme inhibitors (ACEi) and angiotensin-receptor blockers (ARB) may help to reduce hyperinflammation and viral propagation, while infusion of soluble ACE2 may trap plasma viral particles and increase cardioprotective Ang-(1-9) and Ang-(1-7) peptides. The association of specific ACE2 polymorphisms with increased susceptibility of infection and related CV pathologies suggests potential genetic therapies. Moreover, specific agonists of Ang-(1-7) receptor could counter-regulate the hypertensive, hyperinflammatory, and hypercoagulable responses. Interestingly, sex hormones could also regulate all these RAAS components. Therefore, while waiting for an efficient vaccine, we suggest further investigations on the non-canonical RAAS pathway to reduce cardiovascular damage and mortality in COVID-19 patients.

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses.

Dual Effects of Resveratrol on Cell Death and Proliferation of Colon Cancer Cells.

Colorectal cancer remains a main cause of deaths worldwide, and novel agents are being searched to treat this disease. Polyphenols have emerged as promising therapeutic tools in cancer. Resveratrol (3,5,4'-trihydoxy-trans-stilbene) induces cell death in different tumor cell lines, and it also stimulates the proliferation of specific breast and prostate cancer cell lines. Here, we studied the impact of resveratrol over a 100-fold concentration range on cell death and proliferation of HT-29 colorectal adenocarcinoma cells. After 96 h of treatment, a biphasic pattern was observed. At lower concentrations (1 and 10 µmol/l), resveratrol increased the cell number, as did the polyphenol quercetin. At 50 or 100 µmol/l, resveratrol reduced the cell number and increased the percentage of apoptotic or necrotic cells, thus indicating cytotoxicity. On HCT116 colon cancer cells, however, no proliferative properties of resveratrol were observed. Resveratrol-induced cytotoxicity on HT-29 cells was associated with NADPH oxidase activation and increased levels of histone γH2AX, a marker of DNA damage, paralleled by enhanced sirtuin 6 levels, likely as a repair mechanism. Overall, resveratrol may be an effective tool in anti-tumor chemotherapy. However, since under some conditions it may favor tumor cell growth, appropriate local concentrations must be achieved to minimize unwanted effects of resveratrol.

Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation.

The Mas receptor is involved in the angiotensin (Ang)-(1-7) vasodilatory actions by increasing nitric oxide production (NO). We have previously demonstrated an increased production of Ang-(1-7) in human umbilical vein endothelial cells (HUVEC) exposed to estradiol (E2), suggesting a potential cross-talk between E2 and the Ang-(1-7)/Mas receptor axis. Here, we explored whether the vasoactive response and NO-related signalling exerted by E2 are influenced by Mas. HUVEC were exposed to 10nM E2 for 24h in the presence or absence of the selective Mas receptor antagonist A779, and the estrogen receptor (ER) antagonist ICI182780 (ICI). E2 increased Akt and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, measured by RT-PCR and Western blot, respectively. Furthermore, E2 increased Akt activity (determined by the levels of phospho-Ser473) and eNOS activity (by the enhanced phosphorylation of Ser1177, the activated form), resulting in increased NO production, which was measured by the fluorescence probe DAF-2-FM. These signalling events were dependent on ER and Mas receptor activation, since they were abolished in the presence of ICI or A779. In ex-vivo functional experiments performed with a small-vessel myograph in isolated mesenteric vessels from wild-type mice pre-contracted with noradrenaline, the relaxant response to physiological concentrations of E2 was blocked by ICI and A779, to the same extent to that obtained in the vessels isolated from Mas-deficient. In conclusion, E2 induces NO production and vasodilation through mechanisms that require Mas receptor activation.

Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway.

BACKGROUND: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1ß. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1ß. METHODS: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1ß in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-κB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined. RESULTS: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1ß. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1ß activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1ß by a mechanism which was abrogated by the inhibition of the PPP. CONCLUSIONS: A pro-inflammatory stimulus like IL1ß transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia.

Soluble dipeptidyl peptidase-4 induces microvascular endothelial dysfunction through proteinase-activated receptor-2 and thromboxane A2 release.

BACKGROUND: Dipeptidyl peptidase-4 (DPP4) is a key protein in glucose homeostasis and a pharmacological target in type 2 diabetes mellitus. This study explored whether the novel adipokine soluble DPP4 (sDPP4) can cause endothelial dysfunction, an early marker of impaired vascular reactivity. METHOD: Reactivity was studied in mesenteric arteries from 3-month-old female mice, using a small vessel myograph. Thromboxane A2 (TXA2) release was explored in cultured human coronary artery endothelial cells by enzyme immunoassay. RESULTS: Neither the contractility to noradrenaline nor the endothelium-independent relaxations induced by sodium nitroprusside were modified by sDPP4. However, sDPP4 impaired in a concentration-dependent manner the endothelium-dependent relaxation elicited by acetylcholine. The DPP4 inhibitors K579 and linagliptin prevented the defective relaxation induced by sDPP4, as did the protease-activated receptor 2 (PAR2) inhibitor GB83. Downstream of PAR2, the cyclooxygenase (COX) inhibitor indomethacin, the COX2 inhibitor celecoxib or the thromboxane receptors blocker SQ29548 prevented the deleterious effects of sDPP4. Accordingly, sDPP4 triggered the release of TXA2 by endothelial cells, whereas TXA2 release was prevented by inhibiting DPP4, PAR2 or COX. CONCLUSION: In summary, these findings reveal sDPP4 as a direct mediator of endothelial dysfunction, acting through PAR2 activation and the release of vasoconstrictor prostanoids. By interfering with these actions, DPP4 inhibitors might help preserving endothelial function in the context of cardiometabolic diseases.

The interleukin-1 receptor antagonist anakinra improves endothelial dysfunction in streptozotocin-induced diabetic rats.

BACKGROUND: Endothelial dysfunction is a crucial early phenomenon in vascular diseases linked to diabetes mellitus and associated to enhanced oxidative stress. There is increasing evidence about the role for pro-inflammatory cytokines, like interleukin-1ß (IL-1ß), in developing diabetic vasculopathy. We aimed to determine the possible involvement of this cytokine in the development of diabetic endothelial dysfunction, analysing whether anakinra, an antagonist of IL-1 receptors, could reduce this endothelial alteration by interfering with pro-oxidant and pro-inflammatory pathways into the vascular wall. RESULTS: In control and two weeks evolution streptozotocin-induced diabetic rats, either untreated or receiving anakinra, vascular reactivity and NADPH oxidase activity were measured, respectively, in isolated rings and homogenates from mesenteric microvessels, while nuclear factor (NF)-κB activation was determined in aortas. Plasma levels of IL-1ß and tumor necrosis factor (TNF)-α were measured by ELISA. In isolated mesenteric microvessels from control rats, two hours incubation with IL-1ß (1 to 10 ng/mL) produced a concentration-dependent impairment of endothelium-dependent relaxations, which were mediated by enhanced NADPH oxidase activity via IL-1 receptors. In diabetic rats treated with anakinra (100 or 160 mg/Kg/day for 3 or 7 days before sacrifice) a partial improvement of diabetic endothelial dysfunction occurred, together with a reduction of vascular NADPH oxidase and NF-κB activation. Endothelial dysfunction in diabetic animals was also associated to higher activities of the pro-inflammatory enzymes cyclooxygenase (COX) and the inducible isoform of nitric oxide synthase (iNOS), which were markedly reduced after anakinra treatment. Circulating IL-1ß and TNF-α levels did not change in diabetic rats, but they were lowered by anakinra treatment. CONCLUSIONS: In this short-term model of type 1 diabetes, endothelial dysfunction is associated to an IL-1 receptor-mediated activation of vascular NADPH oxidase and NF-κB, as well as to vascular inflammation. Moreover, endothelial dysfunction, vascular oxidative stress and inflammation were reduced after anakinra treatment. Whether this mechanism can be extrapolated to a chronic situation or whether it may apply to diabetic patients remain to be established. However, it may provide new insights to further investigate the therapeutic use of IL-1 receptor antagonists to obtain vascular benefits in patients with diabetes mellitus and/or atherosclerosis.

Soluble DPP4 induces inflammation and proliferation of human smooth muscle cells via protease-activated receptor 2.

DPP4 is an ubiquitously expressed cell-surface protease that is shedded to the circulation as soluble DPP4 (sDPP4). We recently identified sDPP4 as a novel adipokine potentially linking obesity to the metabolic syndrome. The aim of this study was to investigate direct effects of sDPP4 on human vascular smooth muscle cells (hVSMCs) and to identify responsible signaling pathways. Using physiological concentrations of sDPP4, we could observe a concentration-dependent activation of ERK1/2 (3-fold) after 6h, which remained stable for up to 24h. Additionally, sDPP4 treatment induced a 1.5-fold phosphorylation of the NF-κB subunit p65. In accordance with sDPP4-induced stress and inflammatory signaling, sDPP4 also stimulates hVSMC proliferation. Furthermore we could observe an increased expression and secretion of pro-inflammatory cytokines like interleukin (IL)-6, IL-8 and MCP-1 (2.5-, 2.4- and 1.5-fold, respectively) by the sDPP4 treatment. All direct effects of sDPP4 on signaling, proliferation and inflammation could completely be prevented by DPP4 inhibition. Bioinformatic analysis and signaling signature induced by sDPP4 suggest that sDPP4 might be an agonist for PAR2. After the silencing of PAR2, the sDPP4-induced ERK activation as well as the proliferation was totally abolished. Additionally, the sDPP4-induced upregulation of IL-6 and IL-8 could completely be prevented by the PAR2 silencing. In conclusion, we show for the first time that sDPP4 directly activates the MAPK and NF-κB signaling cascade involving PAR2 and resulting in the induction of inflammation and proliferation of hVSMC. Thus, our in vitro data might extend the current view of sDPP4 action and shed light on cardiovascular effects of DPP4-inhibitors.

Visfatin as a novel mediator released by inflamed human endothelial cells.

BACKGROUND: Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. METHODS AND RESULTS: Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1ß (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 µmol/L) markedly enhanced intracellular visfatin content. Using IL-1ß (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1ß modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1ß-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1ß promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. CONCLUSIONS: Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation.

Visfatin/Nampt: an adipokine with cardiovascular impact.

Adipose tissue is acknowledged as an endocrine organ that releases bioactive factors termed adipokines. Visfatin was initially identified as a novel adipokine with insulin-mimetic properties in mice. This adipokine was identical to two previously described molecules, namely, pre-B cell colony-enhancing factor (PBEF) and the enzyme nicotinamide phosphoribosyltransferase (Nampt). Enhanced circulating visfatin/Nampt levels have been reported in metabolic diseases, such as obesity and type 2 diabetes. Moreover, visfatin/Nampt circulating levels correlate with markers of systemic inflammation. In cardiovascular diseases, visfatin/Nampt was initially proposed as a clinical marker of atherosclerosis, endothelial dysfunction, and vascular damage, with a potential prognostic value. Nevertheless, beyond being a surrogate clinical marker, visfatin/Nampt is an active player promoting vascular inflammation, and atherosclerosis. Visfatin/Nampt effects on cytokine and chemokine secretion, macrophage survival, leukocyte recruitment by endothelial cells, vascular smooth muscle inflammation and plaque destabilization make of this adipokine an active factor in the development and progression of atherosclerosis. Further research is required to fully understand the mechanisms mediating the cellular actions of this adipokine and to better characterize the factors regulating visfatin/Nampt expression and release in all these pathologic scenarios. Only then, we will be able to conclude whether visfatin/Nampt is a therapeutical target in cardiometabolic diseases.

Complete blockade of the vasorelaxant effects of angiotensin-(1-7) and bradykinin in murine microvessels by antagonists of the receptor Mas.

The heptapeptide angiotensin-(1-7) is a biologically active metabolite of angiotensin II, the predominant peptide of the renin-angiotensin system. Recently, we have shown that the receptor Mas is associated with angiotensin-(1-7)-induced signalling and mediates, at least in part, the vasodilatory properties of angiotensin-(1-7). However, it remained controversial whether an additional receptor could account for angiotensin-(1-7)-induced vasorelaxation. Here, we used two different angiotensin-(1-7) antagonists, A779 and d-Pro-angiotensin-(1-7), to address this question and also to study their influence on the vasodilatation induced by bradykinin. Isolated mesenteric microvessels from both wild-type and Mas-deficient C57Bl/6 mice were precontracted with noradrenaline, and vascular reactivity to angiotensin-(1-7) and bradykinin was subsequently studied using a small-vessel myograph. Furthermore, mechanisms for Mas effects were investigated in primary human umbilical vein endothelial cells. Both angiotensin-(1-7) and bradykinin triggered a concentration-dependent vasodilatation in wild-type microvessels, which was absent in the presence of a nitric oxide synthase inhibitor. In these vessels, the pre-incubation with the Mas antagonists A779 or d-Pro-angiotensin-(1-7) totally abolished the vasodilatory capacity of both angiotensin-(1-7) and bradykinin, which was nitric oxide mediated. Accordingly, Mas-deficient microvessels lacked the capacity to relax in response to either angiotensin-(1-7) or bradykinin. Pre-incubation of human umbilical vein endothelial cells with A779 prevented bradykinin-mediated NO generation and NO synthase phosphorylation at serine 1177. The angiotensin-(1-7) antagonists A779 and d-Pro-angiotensin-(1-7) equally block Mas, which completely controls the angiotensin-(1-7)-induced vasodilatation in mesenteric microvessels. Importantly, Mas also appears to be a critical player in NO-mediated vasodilatation induced by renin-angiotensin system-independent agonists by altering phosphorylation of NO synthase.

Characterization of endothelium-dependent relaxations in the mesenteric vasculature: a comparative study with potential pathophysiological relevance.

BACKGROUND: Endothelium-dependent relaxations in human adult mesenteric microvessels involve 3 different main mechanisms: cyclooxygenase (COX)-derived prostanoids, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF), which elicits vascular smooth muscle hyperpolarization and relaxation. There are some pathological conditions with an abnormal balance between mesenteric vasoconstriction and vasodilatation inputs leading to endothelial dysfunction and tissue injury. PURPOSE: The purpose was to characterize the mechanisms mediating endothelium-dependent relaxation and differences in children and adult mesenteric microvessels. METHODS: Microvessels were dissected from omentum obtained from children (3-6 years old) and adults (25-41 years old) and mounted as ring preparations in a small vessel myograph. RESULTS: In microvessels precontracted with a thromboxane analogue, the endothelium-dependent relaxations to bradykinin (10 nmol/L to 30 µmol/L) mediated by EDHF, that is, nonsensitive to COX (10 µmol/L indomethacin) and NO synthase blockade (100 µmol/L N-nitro-L-arginine methyl ester), were higher in children than in adults. When EDHF was blunted by a depolarizing precontraction with KCl, the remaining COX- and NO-dependent relaxations were significantly lower in children. CONCLUSIONS: The EDHF's role in the endothelium-dependent relaxations is higher in children's vasculature. This suggests that endothelial dysfunction in mesenteric microvessels in children is likely more dependent on EDHF-related mechanisms rather than on NO- or COX-derived prostanoids.

Visfatin impairs endothelium-dependent relaxation in rat and human mesenteric microvessels through nicotinamide phosphoribosyltransferase activity.

Visfatin, also known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 µmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 µmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 µmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 µmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 µmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 µmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.

Pathways responsible for apoptosis resulting from amadori-induced oxidative and nitrosative stress in human mesothelial cells.

BACKGROUND: Apoptosis and inflammatory/oxidative stress have been associated with hyperglycemia in human peritoneal mesothelial cells (HPMCs) and other cell types. We and others have highlighted the role of early products of non-enzymatic protein glycation in inducing proinflammatory conditions and increasing apoptotic rates in HPMCs. Loss of HPMCs seems to be a hallmark of complications associated with peritoneal membrane dysfunction. The aim of this work is to elucidate the mechanisms by which Amadori adducts may act upon HPMC apoptosis. METHODS: HPMCs isolated from different patients were exposed to different Amadori adducts, i.e. highly glycated hemoglobin (10 nM) and glycated bovine serum albumin (250 µg/ml), to study cell death and several proapoptotic markers by different experimental approaches. RESULTS: Amadori adducts, but not their respective controls, impaired cell proliferation and cell viability by means of apoptosis in a time-dependent manner. They regulated the intrinsic mitochondrial cell death signaling pathway and modulated activation of caspases, Bax, iNOS, p53, NF-κB, and mitogen-activated protein kinases (p38 and JNK) through different reactive oxygen and nitrosative species. CONCLUSIONS: Our data strongly support the idea that long-term hyperglycemia could act as an inducer of apoptosis in HPMCs through Amadori adducts, involving different oxidative and nitrosative reactive species.

Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial proliferation, arterial relaxation, vascular permeability and angiogenesis by dobesilate.

Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis and vascular permeability which is associated with many pathological processes. 2,5-hydroxybenzene sulfonate (DHBS; dobesilate) is a small molecule with anti-angiogenic activity that has been described as an inhibitor of fibroblast growth factors (FGF). The aim of the present study was to evaluate the effects of DHBS on VEGF-induced actions. The effects of DHBS were evaluated on VEGF-induced proliferation in human umbilical vein endothelial cells (HUVEC) and rat aorta relaxation, as well as on in vivo VEGF-induced skin vascular permeability and neovascularization in rats. DHBS at 50 and 100 µM concentration significantly inhibited the proliferation of HUVEC induced by VEGF (10 ng/ml), without significantly affecting HUVEC proliferation in the absence of VEGF. Rapid VEGF-induced activation of Akt in HUVEC was also prevented by DHBS (100 µM). Additionally, DHBS (2 µM) specifically inhibited the relaxation of rat aorta induced by VEGF (0.1 to 30 ng/ml), but not endothelium-dependent relaxation to acetylcholine (1 nM to 10 µM). The in vivo enhancement of vascular permeability caused by VEGF injection (50 µl at 10 ng/ml) in rat skin was also inhibited by DHBS co-administration (200 µM) (74.8±3.8% inhibition of dye extravasation). Administration of DHBS (200 mg/kg/day; i.p.) also reduced VEGF-induced angiogenesis in vivo. DHBS inhibits main responses elicited in vitro and in vivo by VEGF. As a dual antagonist of VEGF and FGF activities, DHBS could be of therapeutic interest in the treatment of diseases related to VEGF/FGF overproduction and excessive angiogenesis.