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A subset of interleukin (IL)-17A-producing γδ T cells called γδT17 cells may contribute to progression of hypertension. γδT17 cell development is in part dependent upon IL-23 receptor (IL-23R) stimulation. We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in Il23r knock-in (Il23r

Introduction: The emergence of the global COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus, SARS-CoV-2, has created a substantial burden on healthcare systems worldwide. The systemic impacts of COVID-19 infection are severe and broad in their implications, and the cardiovascular system is no exception. Discussion: Patients with a history of cardiovascular disease are at an increased risk for hospitalization and mortality, and COVID-19 infection has now been demonstrated to initiate acute, but serious, episodes of cardiovascular events such as stroke. Considering the rapid spread of COVID-19 across the globe and the inability of healthcare systems to address and adequately respond to the pandemic, therein lies an increased need for understanding the interplay between COVID-19 infection and cardiovascular disease. SARS-CoV-2 relies on binding the angiotensin-converting enzyme-2 (ACE2) receptor to infect host cells, with ACE2 representing a critical regulator of blood pressure homeostasis and proper cardiovascular functioning. Conclusion: Identifying the exact role of ACE2 in COVID-19 infection will have major implications for understanding the disease; therefore, here we have reviewed ACE2’s involvement in the pathogenesis of COVID-19 infection and the resulting end-organ damage. In addition, we have summarized how COVID-19 affects cardiovascular physiology, and how COVID-19 infection can manifest in acute cardiovascular events. Finally, we examine why patients with cardiovascular disease are at an increased risk of succumbing to COVID-19 and what the long-term cardiovascular implications of COVID-19 infection could mean. Relevance: This paper discusses the cardiovascular consequences of the global COVID-19 pandemic.

Aims NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing human ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe-/-) mice enhanced high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We tested the hypothesis that ET-1 overexpression in the endothelium would worsen atherosclerosis in type-1 diabetes through a mechanism involving NOX1 but not NOX4. Methods and results Six-week-old male Apoe-/- and eET-1/Apoe-/- mice with or without Nox1 (Nox1-/y) or Nox4 knockout (Nox4-/-) were injected intraperitoneally with either vehicle or streptozotocin (55 mg/kg/day) for 5 days to induce type-1 diabetes and were studied 14 weeks later. ET-1 overexpression increased 2.5-fold and 5-fold the atherosclerotic lesion area in the aortic sinus and arch of diabetic Apoe-/- mice, respectively. Deletion of Nox1 reduced aortic arch plaque size by 60%; by contrast, Nox4 knockout increased lesion size by 1.5-fold. ET-1 overexpression decreased aortic sinus and arch plaque alpha smooth muscle cell content by ∼35% and ∼50%, respectively, which was blunted by Nox1 but not Nox4 knockout. Reactive oxygen species production was increased 2-fold in aortic arch perivascular fat of diabetic eET-1/Apoe-/- and eET-1/Apoe-/- / Nox4-/- mice but not eET-1/Apoe-/- / Nox1y/- mice. ET-1 overexpression enhanced monocyte/macrophage and CD3+ T cell infiltration ∼2.7-fold in the aortic arch perivascular fat of diabetic Apoe-/- mice. Both Nox1 and Nox4 knockout blunted CD3+ T cell infiltration whereas only Nox1 knockout prevented the monocyte/macrophage infiltration in diabetic eET-1/Apoe-/- mice. Conclusions Endothelium ET-1 overexpression enhances the progression of atherosclerosis in type-1 diabetes, perivascular oxidative stress and inflammation through NOX1. Translational perspective We demonstrate that endothelial cell-restricted human ET-1 overexpression worsens atherosclerosis in diabetes and causes perivascular oxidative stress and inflammation, extending our previous findings to a model of type-1 diabetes mellitus. We also show that these effects are mediated through NOX1 and that atherosclerosis is worsened by loss of NOX4, providing evidence that NADPH oxidase isoforms play differential roles in plaque progression. These results offer new approaches to prevent the progression of atherosclerosis in diabetes, which is associated with significantly increased risk of cardiovascular events.

We evaluated the effects of long-term (48 h) electrical stimulation of the carotid sinus (CS) in hypertensive rats. L-NAME-treated (10 days) Wistar rats were implanted with a catheter in the femoral artery and a miniaturized electrical stimulator attached to electrodes positioned around the left CS, encompassing the CS nerve. One day after implantation, arterial pressure (AP) was directly recorded in conscious animals for 60 min. Square pulses (1 ms, 3 V, 30 Hz) were applied intermittently (20/20 s ON/OFF) to the CS for 48 h. After the end of stimulation, AP was recorded again. Nonstimulated rats (control group) and rats without electrodes around the CS (sham-operated) were also studied. Next, the animals were decapitated, and segments of mesenteric resistance arteries were removed to study vascular function. After the stimulation period, AP was 16 ± 5 mmHg lower in the stimulated group, whereas sham-operated and control rats showed similar AP between the first and second recording periods. Heart rate variability (HRV) evaluated using time and frequency domain tools and a nonlinear approach (symbolic analysis) suggested that hypertensive rats with electrodes around the CS, stimulated or not, exhibited a shift in cardiac sympathovagal balance towards parasympathetic tone. The relaxation response to acetylcholine in endothelium-intact mesenteric arteries was enhanced in rats that underwent CS stimulation for 48 h. In conclusion, long-term CS stimulation is effective in reducing AP levels, improving HRV and increasing mesenteric vascular relaxation in L-NAME hypertensive rats. Moreover, only the presence of electrodes around the CS is effective in eliciting changes in HRV similar to those observed in stimulated rats.

Aldosterone is a mineralocorticoid hormone that controls body fluid and electrolyte balance. Excess aldosterone is associated with cardiovascular and metabolic diseases. Inflammation plays a critical role on vascular damage promoted by aldosterone and aggravates vascular abnormalities, including endothelial dysfunction, vascular remodeling, fibrosis and oxidative stress, and other manifestations of end-organ damage that are associated with hypertension, other forms of cardiovascular disease, and diabetes mellitus and the metabolic syndrome. Over the past few years, many studies have consistently shown that aldosterone activates cells of the innate and adaptive immune systems. Macrophages and T cells accumulate in the kidneys, heart, and vasculature in response to aldosterone, and infiltration of immune cells contributes to end-organ damage in cardiovascular and metabolic diseases. Aldosterone activates various subsets of innate immune cells such as dendritic cells and monocytes/macrophages, as well as adaptive immune cells such as T lymphocytes, and, by activation of mineralocorticoid receptors stimulates proinflammatory transcription factors and the production of adhesion molecules and inflammatory cytokines and chemokines. This review will briefly highlight some of the studies on the involvement of aldosterone in activation of innate and adaptive immune cells and its impact on the cardiovascular system. Since aldosterone plays a key role in many cardiovascular and metabolic diseases, these data will open up promising perspectives for the identification of novel biomarkers and therapeutic targets for prevention and treatment of diseases associated with increased levels of aldosterone, such as arterial hypertension, obesity, the metabolic syndrome, and heart failure., Graphical Abstract Graphical Abstract

Baroreflex activation by electric stimulation of the carotid sinus (CS) effectively lowers blood pressure. However, the degree to which differences between stimulation protocols impinge on cardiovascular outcomes has not been defined. To address this, we examined the effects of short- and long-duration (SD and LD) CS stimulation on hemodynamic and vascular function in spontaneously hypertensive rats (SHRs). We fit animals with miniature electrical stimulators coupled to electrodes positioned around the left CS nerve that delivered intermittent 5/25 s ON/OFF (SD) or 20/20 s ON/OFF (LD) square pulses (1 ms, 3 V, 30 Hz) continuously applied for 48 h in conscious animals. A sham-operated control group was also studied. We measured mean arterial pressure (MAP), systolic blood pressure variability (SBPV), heart rate (HR), and heart rate variability (HRV) for 60 min before stimulation, 24 h into the protocol, and 60 min after stimulation had stopped. SD stimulation reversibly lowered MAP and HR during stimulation. LD stimulation evoked a decrease in MAP that was sustained even after stimulation was stopped. Neither SD nor LD had any effect on SBPV or HRV when recorded after stimulation, indicating no adaptation in autonomic activity. Both the contractile response to phenylephrine and the relaxation response to acetylcholine were increased in mesenteric resistance vessels isolated from LD-stimulated rats only. In conclusion, the ability of baroreflex activation to modulate hemodynamics and induce lasting vascular adaptation is critically dependent on the electrical parameters and duration of CS stimulation.

Baroreflex activation by electric stimulation of the carotid sinus (CS) effectively lowers blood pressure. However, the degree to which differences between stimulation protocols impinge on cardiovascular outcomes has not been defined. To address this, we examined the effects of short- and long-duration (SD and LD) CS stimulation on hemodynamic and vascular function in spontaneously hypertensive rats (SHRs). We fit animals with miniature electrical stimulators coupled to electrodes positioned around the left CS nerve that delivered intermittent 5/25 s ON/OFF (SD) or 20/20 s ON/OFF (LD) square pulses (1 ms, 3 V, 30 Hz) continuously applied for 48 h in conscious animals. A sham-operated control group was also studied. We measured mean arterial pressure (MAP), systolic blood pressure variability (SBPV), heart rate (HR), and heart rate variability (HRV) for 60 min before stimulation, 24 h into the protocol, and 60 min after stimulation had stopped. SD stimulation reversibly lowered MAP and HR during stimulation. LD stimulation evoked a decrease in MAP that was sustained even after stimulation was stopped. Neither SD nor LD had any effect on SBPV or HRV when recorded after stimulation, indicating no adaptation in autonomic activity. Both the contractile response to phenylephrine and the relaxation response to acetylcholine were increased in mesenteric resistance vessels isolated from LD-stimulated rats only. In conclusion, the ability of baroreflex activation to modulate hemodynamics and induce lasting vascular adaptation is critically dependent on the electrical parameters and duration of CS stimulation.

Beyond the regulation of cardiovascular function, baroreceptor afferents play polymodal roles in health and disease. Sepsis is a life-threatening condition characterized by systemic inflammation (SI) and hemodynamic dysfunction. We hypothesized that baroreceptor denervation worsens lipopolysaccharide (LPS) induced-hemodynamic collapse and SI in conscious rats. We combined: (a) hemodynamic and thermoregulatory recordings after LPS administration at a septic-like non-lethal dose (b) analysis of the cardiovascular complexity, (c) evaluation of vascular function in mesenteric resistance vessels, and (d) measurements of inflammatory cytokines (plasma and spleen). LPS-induced drop in blood pressure was higher in sino-aortic denervated (SAD) rats. LPS-induced hemodynamic collapse was associated with SAD-dependent autonomic disbalance. LPS-induced vascular dysfunction was not affected by SAD. Surprisingly, SAD blunted LPS-induced surges of plasma and spleen cytokines. These data indicate that baroreceptor afferents are key to alleviate LPS-induced hemodynamic collapse, affecting the autonomic control of cardiovascular function, without affecting resistance blood vessels. Moreover, baroreflex modulation of the LPS-induced SI and hemodynamic collapse are not dependent of each other given that baroreceptor denervation worsened hypotension and reduced SI.

Beyond the regulation of cardiovascular function, baroreceptor afferents play polymodal roles in health and disease. Sepsis is a life-threatening condition characterized by systemic inflammation (SI) and hemodynamic dysfunction. We hypothesized that baroreceptor denervation worsens lipopolysaccharide (LPS) induced-hemodynamic collapse and SI in conscious rats. We combined: (a) hemodynamic and thermoregulatory recordings after LPS administration at a septic-like non-lethal dose (b) analysis of the cardiovascular complexity, (c) evaluation of vascular function in mesenteric resistance vessels, and (d) measurements of inflammatory cytokines (plasma and spleen). LPS-induced drop in blood pressure was higher in sino-aortic denervated (SAD) rats. LPS-induced hemodynamic collapse was associated with SAD-dependent autonomic disbalance. LPS-induced vascular dysfunction was not affected by SAD. Surprisingly, SAD blunted LPS-induced surges of plasma and spleen cytokines. These data indicate that baroreceptor afferents are key to alleviate LPS-induced hemodynamic collapse, affecting the autonomic control of cardiovascular function, without affecting resistance blood vessels. Moreover, baroreflex modulation of the LPS-induced SI and hemodynamic collapse are not dependent of each other given that baroreceptor denervation worsened hypotension and reduced SI.

Sepsis is a systemic inflammatory response syndrome (SIRS) resulting from a severe infection that is characterized by immune dysregulation, cardiovascular derangements, and end-organ dysfunction. The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) influences many of the key processes that are altered during sepsis, including the production of inflammatory mediators and vascular contractility. Here, we investigated whether O-GlcNAc affects the inflammatory response and cardiovascular dysfunction associated with sepsis. Mice received an intraperitoneal injection of lipopolysaccharide (LPS, 20 mg/Kg) to induce endotoxic shock and systemic inflammation, resembling sepsis-induced SIRS. The effects of an acute increase in O-GlcNAcylation, by treatment of mice with glucosamine (GlcN, 300 mg/Kg, i.v.) or thiamet-G (ThG, 150 μg/Kg, i.v.), on LPS-associated mortality, production and release of cytokines by macrophages and vascular cells, vascular responsiveness to constrictors and blood pressure were then determined. Mice under LPS-induced SIRS exhibited a systemic and local inflammatory response with increased levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), as well as severe hypotension and vascular hyporesponsiveness, characterized by reduced vasoconstriction to phenylephrine. In addition, LPS increased neutrophil infiltration in lungs and produced significant lethality. Treatment with GlcN and ThG reduced systemic inflammation and attenuated hypotension and the vascular refractoriness to phenylephrine, improving survival. GlcN and ThG also decreased LPS-induced production of inflammatory cytokines by bone marrow-derived macrophages and nuclear transcription factor-kappa B (NF-κB) activation in RAW 264.7 NF-κB promoter macrophages. Treatment of mice with ThG increased O-glycosylation of NF-κB p65 subunit in mesenteric arteries, which was associated with reduced Ser536 phosphorylation of NF-κB p65. Finally, GlcN also increased survival rates in mice submitted to cecal ligation and puncture (CLP), a sepsis model. In conclusion, increased O-GlcNAc reduces systemic inflammation and cardiovascular disfunction in experimental sepsis models, pointing this pathway as a potential target for therapeutic intervention.

Background: NLRP3 inflammasome activation in response to several signals, including mitochondrial DNA (mDNA), regulates inflammatory responses by caspase-1 activation and interleukin-1β (IL-1β) release. Circulating mDNA is linked to micro and macrovascular complications in diabetes. However, a role for mDNA in endothelial dysfunction is not clear. We tested the hypothesis that mDNA contributes to diabetes-associated endothelial dysfunction and vascular inflammation via NLRP3 activation. Methods: Vascular reactivity, reactive oxygen species (ROS) generation, calcium (Ca2+) influx and caspase-1 and IL-1β activation were determined in mesenteric resistance arteries from normoglicemic and streptozotocin-induced diabetic C57BL/6 and NLRP3 knockout (Nlrp3-/- ) mice. Endothelial cells and mesenteric arteries were stimulated with mDNA from control (cmDNA) and diabetic (dmDNA) mice. Results: Diabetes reduced endothelium-dependent vasodilation and increased vascular ROS generation and caspase-1 and IL-1β activation in C57BL/6, but not in Nlrp3-/- mice. Diabetes increased pancreatic cytosolic mDNA. dmDNA decreased endothelium-dependent vasodilation. In endothelial cells, dmDNA activated NLRP3 via mitochondrial ROS and Ca2+ influx. Patients with type 1 diabetes exhibited increased circulating mDNA as well as caspase-1 and IL-1β activation. Conclusion: dmDNA activates endothelial NLRP3 inflammasome by mechanisms that involve Ca2+ influx and mitochondrial ROS generation. NLRP3 deficiency prevents diabetes-associated vascular inflammatory damage and endothelial dysfunction. Our study highlights the importance of NLRP3 inflammasome in diabetes-associated vascular dysfunction, which is key to diabetic complications.

We evaluated the effects of long-term (48 h) electrical stimulation of the carotid sinus (CS) in hypertensive rats. L-NAME-treated (10 days) Wistar rats were implanted with a catheter in the femoral artery and a miniaturized electrical stimulator attached to electrodes positioned around the left CS, encompassing the CS nerve. One day after implantation, arterial pressure (AP) was directly recorded in conscious animals for 60 min. Square pulses (1 ms, 3 V, 30 Hz) were applied intermittently (20/20 s ON/OFF) to the CS for 48 h. After the end of stimulation, AP was recorded again. Nonstimulated rats (control group) and rats without electrodes around the CS (sham-operated) were also studied. Next, the animals were decapitated, and segments of mesenteric resistance arteries were removed to study vascular function. After the stimulation period, AP was 16 ± 5 mmHg lower in the stimulated group, whereas sham-operated and control rats showed similar AP between the first and second recording periods. Heart rate variability (HRV) evaluated using time and frequency domain tools and a nonlinear approach (symbolic analysis) suggested that hypertensive rats with electrodes around the CS, stimulated or not, exhibited a shift in cardiac sympathovagal balance towards parasympathetic tone. The relaxation response to acetylcholine in endothelium-intact mesenteric arteries was enhanced in rats that underwent CS stimulation for 48 h. In conclusion, long-term CS stimulation is effective in reducing AP levels, improving HRV and increasing mesenteric vascular relaxation in L-NAME hypertensive rats. Moreover, only the presence of electrodes around the CS is effective in eliciting changes in HRV similar to those observed in stimulated rats.

Beyond the regulation of cardiovascular function, baroreceptor afferents play polymodal roles. We hypothesized that baroreceptor denervation affects lipopolysaccharide (LPS)-induced systemic inflammation (SI) and hemodynamic collapse in conscious rats, and that these parameters are interconnected. We combine: a) hemodynamic and thermoregulatory recordings after LPS administration at a septic-like dose b) analysis of the cardiovascular complexity, c) evaluation of vascular function in mesenteric resistance vessels, and d) measurements of inflammatory cytokines (plasma and spleen). LPS-induced drop in blood pressure was higher in sino-aortic denervated (SAD) rats. LPS-induced hemodynamic collapse was associated with SAD-dependent autonomic disbalance. LPS-induced vascular dysfunction was not affected by SAD. Surprisingly, SAD blunted LPS-induced surges of plasma and spleen cytokines. These data indicate that sino-aortic afferents are key to alleviate LPS-induced cardiovascular collapse, affecting the autonomic cardiovascular control, without affecting resistance blood vessels. Moreover, baroreflex modulation of the LPS-induced SI and hemodynamic collapse seem not to be interconnected.

Aldosterone excess aggravates endothelial dysfunction in diabetes and hypertension by promoting the increased generation of reactive oxygen species, inflammation, and insulin resistance. Aldosterone activates the molecular platform inflammasome in immune system cells and contributes to vascular dysfunction induced by the mineralocorticoid hormone. It is unclear as to whether the NLRP3 inflammasome associated with the mineralocorticoid receptor contributes to vascular dysfunction in diabetic conditions. Here, we tested the hypothesis that an excess of aldosterone induces vascular dysfunction in type 2 diabetes, via the activation of mineralocorticoid receptors (MR) and assembly of the NLRP3 inflammasome. Mesenteric resistance arteries from control (db/m) and diabetic (db/db) mice treated with vehicle, spironolactone (MR antagonist) or an NLRP3 selective inhibitor (MCC950) were used to determine whether NLRP3 contributes to diabetes-associated vascular dysfunction. Db/db mice exhibited increased vascular expression/activation of caspase-1 and IL-1&beta, increased plasma IL-1&beta, levels, active caspase-1 in peritoneal macrophages, and reduced acetylcholine (ACh) vasodilation, compared to db/m mice. Treatment of db/db mice with spironolactone and MCC950 decreased plasma IL-1&beta, and partly restored ACh vasodilation. Spironolactone also reduced active caspase-1-positive macrophages in db/db mice, events that contribute to diabetes-associated vascular changes. These data clearly indicate that MR and NLRP3 activation contribute to diabetes-associated vascular dysfunction and pro-inflammatory phenotype.

Background: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. Methods: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout ( NLRP3 −/− ), caspase-1 knockout ( Casp-1 −/− ), and interleukin-1 receptor knockout ( IL-1R −/− ) mice treated with vehicle or aldosterone (600 µg·kg −1 ·d −1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Results: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1β levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1β secretion by bone marrow–derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1β in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Conclusions: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation via ETA receptors. ET-1 has been shown to stimulate the release of aldosterone from the adrenal cortex. Whether aldosterone plays a role in ET-1 endothelium overexpression-induced BP elevation is still unknown. Methods and Results: Nine to 12-week-old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreER T2 ) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and studied 3 months later. Plasma aldosterone level measured by ELISA was higher in ieET-1 compared with ieCre mice (1.21±0.14 vs. 0.70±0.09 ng/mL, P Scnn1a ), TSC22 domain family member 3 ( Rsc22d3 ), serum- and glucocorticoid-induced kinase 1 ( Sgk1 ), and ET type A and B receptors in the kidney or adrenal glands. The mRNA expression of aldosterone synthase ( Cyp11b2 , fold change: 0.52±0.06 vs 1.00±0.16, P Hsd3b1) and 6 (Hsd3b6) or steroidogenic acute regulatory protein ( Star ), were decreased in the adrenal cortex of ieET-1 compared with ieCre mice. CYP11B2 and HSD3B1 protein levels measured by Western Blotting were unchanged. Treatment of ieET-1 mice with MR antagonist eplerenone (100 mg/kg per day) during the last 2 weeks decreased systolic BP more during the day (128±2 vs. 134±3 mm Hg) than during the night (137±2 vs. 140±2 mm Hg) compared to untreated ieET-1 mice. Conclusions: These results showed that aldosterone contributes at least in part to the BP elevation caused by endothelial human ET-1 overexpression.

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

NLRP3 Inflammasome is a platform that regulates inflammatory responses by caspase-1 activation and processing of pro-IL-1β and pro-IL-18 to mature cytokines. NLRP3 is activated by several mechanisms including mitochondrial DNA (mitDNA). Circulating mitDNA is increased in diabetes, a condition associated with NLRP3 activation. We tested the hypothesis that mitDNA release is increased in type 1 diabetes (T1D) leading to NLRP3 activation and contributing to vascular inflammatory and oxidative processes. Wild type (WT) and NLRP3-deficient (NLRP3 -/- ) mice were treated with vehicle or streptozotocin (40 mg/kg), i.p. for 5 days. Vascular reactivity was determined in mesenteric resistance arteries (MA). Cultured vascular smooth muscle cells (VSMC) were stimulated with mitDNA of T1D (dmDNA) and control (cmDNA) mice. Caspase-1 and IL-1β activation was evaluated by western blot analysis and reactive oxygen species (ROS) by fluorescence to DHE. DNA was extracted, purified and amplified by real-time-PCR. Data are presented as mean ± standard error of mean, Veh vs. T1D. NLRP3 -/- T1D mice exhibited attenuated hyperglycemia vs. WT T1D mice [mg/dL, 241.0±27.7 vs. 337.6±18.1, pvs. Veh [E max , 46.6±4.0 vs . 91.5±2.8, p-/- T1D mice. Diabetes increased vascular caspase-1 [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1, pvs. 0.8±0.5 p -/- T1D. T1D mice exhibited increased NLRP3 activation and mitDNA release in pancreatic cells and increased circulating mitDNA. dmDNA, but not cmDNA, increased NLRP3 activation in VSMC (i.e. activated caspase-1 and increased IL-1β levels) [a.u., 4.2±0.1 vs. 1.9±0.1; 2.3±0.1 vs. 0.7±0.1, p-/- VMSC, but not in WT VSMC incubated with a TLR-9 antagonist. Increased ROS generation was observed in response to dmDNA, which was prevented by a mitochondrial uncoupler. Our data show that T1D increases mitDNA release, which promotes vascular NLRP3 activation via mitochondrial superoxide production, contributing to T1D-associated vascular dysfunction. Financial Support: FAPESP, CNPq.

Diabetic patients and animal models of type 2 diabetes (DM2) display increased plasma aldosterone (aldo) levels. Aldo induces vascular inflammation and endothelial dysfunction. NOD-like receptors, which are pattern recognition receptors involved in a variety of host innate immune responses, promote vascular inflammation. We hypothesized that aldo via mineralocorticoid receptors (MR) activates the inflammasome platform in the vasculature of DM2 mice. Control (db/+) and diabetic (db/db) mice were treated with vehicle or spironolactone (spiro - MR antagonist, 50 mg/Kg/day). Mesenteric resistance arteries (MA) from db/db mice exhibited reduced acetylcholine (ACh) dilation, which was reversed by spiro [Emax (% of relaxation): db/+: 78.5±4.1; db/db: 40.5±6.4; db/+spiro: 77.0±3.8; db/db+spiro: 62.8±5.9 n=3-6 pIn vitro, aldo increased mature IL-1β in vascular smooth muscle cells (VSMC) (cont: 0.9±0.01 ; LPS+Nigericine: 6.1±2.1 ; Aldo 4h: 9.7±2.6; LPS+Aldo 4h: 12.8±1.9 n=3-5, pin vivo directly activates NLRP3/inflammasome in the vasculature and whether NLRP3 activation contributes to aldo-induced vascular injury, aldo was infused (600 ug/Kg/day for 14 days) in wild type (WT) and NLRP3 knockout mice ( NLRP3-/- ) after bone marrow transplantation from WT donor. The groups were constituted: WT->WT, WT->WT+aldo and WT-> NLRP3 -/-+aldo. NLRP3 -/- mice were protected against aldo-induced endothelial dysfunction [Emax: WT: 89.3±2.9; WT+aldo: 39.8±1.8; NLRP3-/- +aldo: 87.7±4.2, pWT mice, but WT-> NLRP3 -/- mice were protected from aldo-induced endothelial dysfunction [Emax: WT->WT: 95.1±3.1; WT->WT+aldo: 57.1±4.7; WT->NLRP3-/-+aldo: 85.3±3.1 p

Aldosterone promotes non-genomic effects in endothelial and vascular smooth muscle cells via activation of mineralocorticoid receptors (MR) and G protein-coupled estrogen receptors (GPER). GPER activation is associated with beneficial/protective effects in the vasculature. Considering that vascular dysfunction plays a major role in diabetes-associated complications, we hypothesized that the beneficial effects mediated by vascular GPER activation, in response to aldosterone, are decreased in diabetes. Mesenteric resistance arteries from female, 14–16 weeks-old, control and diabetic (db/db) mice were used. Phenylephrine (PhE)-induced contractions were greater in arteries from db/db vs. control mice. Aldosterone (10 nM) increased maximal contractile responses to PhE in arteries from control mice, an effect elicited via activation of GPER. Although aldosterone did not increase PhE responses in arteries from db/db mice, blockade of GPER, and MR decreased PhE-induced contractile responses in db/db mesenteric arteries. Aldosterone also reduced the potency of acetylcholine (ACh)-induced relaxation in arteries from both control and db/db mice via MR-dependent mechanisms. GPER antagonism further decreased ACh-induced relaxation in the control group, but did not affect ACh responses in the diabetic group. Aldosterone increased extracellular signal-regulated kinase 1/2 phosphorylation in arteries from control and db/db mice by a GPER-dependent mechanism. GPER, but not MR, gene, and protein expression, determined by RT-PCR and immunoblotting/immunofluorescence assays, respectively, were increased in arteries from db/db mice vs. control arteries. These findings indicate that aldosterone activates both vascular MR and GPER and that the beneficial effects of GPER activation are decreased in arteries from diabetic animals. Our results further elucidate the mechanisms by which aldosterone influences vascular function and contributes to vascular dysfunction in diabetes. Financial Support: FAPESP, CNPq, and CAPES, Brazil.

Aldosterone (Aldo) excess aggravates endothelial dysfunction in diabetes. Aldo exerts its effects via activation of both mineralocorticoid receptors (MR) and G protein-coupled estrogen receptors (GPER). Considering that GPER activation has beneficial effects in the vasculature, we hypothesized that GPER-mediated vascular effects of aldosterone are decreased/abrogated in diabetes. Second-order mesenteric arteries from control (B6BKS-Leprdb/+) and diabetic (db/db) female mice were incubated with 10 nM Aldo, in the presence of either vehicle (veh), the MR antagonist eplerenone (Eple, 10 μM) or the GPER antagonist G15 (1μM), and the effects on phenylephrine (Phe) vascular reactivity were determined. Aldo increased Phe maximal response (Emax, % of KCl contraction) in arteries from control (veh: 112.5±3.2 vs. Aldo: 129.1±2.8 p0.05). In control vessels, Eple did not alter Phe Emax either in the presence of Aldo or veh (p>0.05), whereas G15 abrogated Aldo-induced increase in Phe Emax (Aldo: 129.1±2.8 vs. G15+Aldo: 110.3±3.6 p

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) has properties that go beyond its antidepressant effects and alters mechanisms involved in the regulation of vasomotor tone. While there are many studies demonstrating the acute effects of fluoxetine in the vasculature, studies on the chronic effects of this SSRI are still limited. Here we postulated that chronic treatment with fluoxetine enhances vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling. The effects of chronic treatment with fluoxetine on vascular reactivity were determined in resistance mesenteric arteries from Wistar rats, which were treated with (I) vehicle (water for 21 days) or (II) fluoxetine (10 mg/kg/day for 21 days in the drinking water). Fluoxetine treatment increased endothelium-dependent (pEC50, Veh = -7.08±0.07; Fluox = -7.4±0.11, p0.05). In conclusion, chronic treatment with fluoxetine increases endothelium-dependent and -independent relaxation response in resistance mesenteric arteries by mechanisms that involve increased NOS activity, NO generation and KATP channels activation. These effects may contribute to the cardiovascular side effects associated with fluoxetine treatment.