NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration.

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

In utero exposure to maternal diabetes exacerbates dietary sodium intake-induced endothelial dysfunction by activating cyclooxygenase 2-derived prostanoids.

Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2+/C96Y mice (Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E2 (PGE2) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE2. This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood.NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E2, and inflammation.

Endothelin-1 down-regulates nuclear factor erythroid 2-related factor-2 and contributes to perivascular adipose tissue dysfunction in obesity.

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.

Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via downregulation of the Nrf2 transcriptional factor.

The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target-organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target for cytokines. Considering that cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum from individuals with severe COVID-19 decreases endothelial cells' main antioxidant defense, i.e., the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from patients with severe COVID-19 at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from individuals with COVID-19 increased oxidant species, as indicated by higher DHE (dihydroethydine) oxidation, increased protein carbonylation, and induced mitochondrial reactive oxygen species (ROS) generation and dysfunction. Serum from patients with COVID-19, but not serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were decreased in endothelial cells exposed to serum from individuals with COVID-19. In addition, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-CoV-2 infection is linked to decreased endothelial antioxidant defense via IL-6-dependent mechanisms. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.NEW & NOTEWORTHY We demonstrate that endothelial cell dysfunction in SARS-CoV-2-infected individuals is linked to decreased activity of the major antioxidant system regulator, the Nrf2 transcription factor. We provide evidence that this phenomenon relies on IL-6, an important cytokine involved in the pathophysiology of COVID-19. Our data support the view that Nrf2 activation is a potential therapeutical strategy to prevent oxidative stress and vascular inflammation in severe cases of COVID-19.

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

Melatonin reverses the loss of the anticontractile effect of perivascular adipose tissue in obese rats.

Perivascular adipose tissue (PVAT) undergoes functional changes in obesity. Increased oxidative stress is a central mechanism whereby obesity induces loss of the anticontractile effect of PVAT. Melatonin is an antioxidant that displays vasoprotective action in cardiovascular disease. Here, we sought to investigate whether melatonin would restore the anticontractile effect of periaortic PVAT in obesity. Male Wistar Hannover rats were treated for 10 weeks with a high-calorie diet. Melatonin (5 mg/kg/d, p.o., gavage) was administered for 2 weeks. Functional findings showed that obesity-induced loss of the anticontractile effect of PVAT and treatment with melatonin reversed this response. Tiron [a scavenger of superoxide anion (O2 - )] restored the anticontractile effect of PVAT in aortas from obese rats, suggesting a role for reactive oxygen species (ROS) in such response. Decreased superoxide dismutase (SOD) activity and augmented levels of ROS were detected in periaortic PVAT from obese rats. These responses were accompanied by decreased levels of nitric oxide (NO) in PVAT. Treatment with melatonin restored SOD activity, decreased ROS levels, and increased NO bioavailability in PVAT from obese rats. Here, we first reported the beneficial effects of melatonin in periaortic PVAT in obesity. Melatonin reversed the adverse effects of obesity in PVAT that included overproduction of ROS, reduced SOD activity, and decreased bioavailability of NO. Therefore, PVAT may constitute an important target for the treatment of obesity-induced vascular dysfunction and melatonin emerges as a potential tool in the management of the vascular complications induced by obesity.

Inhibition of inducible nitric oxide synthase protects against the deleterious effects of sub-lethal sepsis and ethanol in the cardiorenal system.

We tested the hypothesis that ethanol would aggravate the deleterious effects of sub-lethal cecal ligation and puncture (SL-CLP) sepsis in the cardiorenal system and that inhibition of inducible nitric oxide synthase (iNOS) would prevent such response. Male C57BL/6 mice were treated with ethanol for 12 weeks. One hour before SL-CLP surgery, mice were treated with N6-(1-iminoethyl)-lysine (L-NIL, 5 mg/kg, i.p.), a selective inhibitor of iNOS. A second dose of L-NIL was administered 24 h after SL-CLP surgery. Mice were killed 48 h post surgery and the blood, the renal cortex, and the left ventricle (LV) were collected for biochemical analysis. L-NIL attenuated the increase in serum creatinine levels induced by ethanol, but not by SL-CLP. Ethanol, but not SL-CLP, increased creatine kinase (CK)-MB activity and L-NIL did not prevent this response. In the renal cortex, L-NIL prevented the redox imbalance induced by ethanol and SL-CLP. Inhibition of iNOS also decreased lipoperoxidation induced by ethanol and SL-CLP in the LV. L-NIL prevented the increase of pro-inflammatory cytokines and reactive oxygen species induced by ethanol and (or) SL-CLP in the cardiorenal system, suggesting that iNOS modulated some of the molecular mechanisms that underlie the deleterious effects of both conditions in the cardiorenal system.

Aldosterone Negatively Regulates Nrf2 Activity: An Additional Mechanism Contributing to Oxidative Stress and Vascular Dysfunction by Aldosterone.

High levels of aldosterone (Aldo) trigger oxidative stress and vascular dysfunction independent of effects on blood pressure. We sought to determine whether Aldo disrupts Nrf2 signaling, the main transcriptional factor involved in antioxidant responses that aggravate cell injury. Thoracic aorta from male C57Bl/6J mice and cultured human endothelial cells (EA.hy926) were stimulated with Aldo (100 nM) in the presence of tiron [reactive oxygen species (ROS) scavenger, eplerenone [mineralocorticoid receptor (MR) antagonist], and L-sulforaphane (SFN; Nrf2 activator). Thoracic aortas were also isolated from mice infused with Aldo (600 µg/kg per day) for 14 days. Aldo decreased endothelium-dependent vasorelaxation and increased ROS generation, effects prevented by tiron and MR blockade. Pharmacological activation of Nrf2 with SFN abrogated Aldo-induced vascular dysfunction and ROS generation. In EA.hy926 cells, Aldo increased ROS generation, which was prevented by eplerenone, tiron, and SFN. At short times, Aldo-induced ROS generation was linked to increased Nrf2 activation. However, after three hours, Aldo decreased the nuclear accumulation of Nrf2. Increased Keap1 protein expression, but not activation of p38 MAPK, was linked to Aldo-induced reduced Nrf2 activity. Arteries from Aldo-infused mice also exhibited decreased nuclear Nrf2 and increased Keap1 expression. Our findings suggest that Aldo reduces vascular Nrf2 transcriptional activity by Keap1-dependent mechanisms, contributing to mineralocorticoid-induced vascular dysfunction.

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors.

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Enhanced proteolytic clearance of plasma Aß by peripherally administered neprilysin does not result in reduced levels of brain Aß in mice.

Accumulation of ß-amyloid (Aß) in the brain is believed to contribute to the pathology of Alzheimer's Disease (AD). Aß levels are controlled by the production of Aß from amyloid precursor protein, degradation by proteases, and peripheral clearance. In this study we sought to determine whether enhancing clearance of plasma Aß with a peripherally administered Aß-degrading protease would reduce brain Aß levels through a peripheral sink. Neprilysin (NEP) is a zinc-dependent metalloprotease that is one of the key Aß-degrading enzymes in the brain. We developed a NEP fusion protein with in vitro degradation of Aß and a 10 day plasma half-life in mouse. Intravenous administration of NEP to wild-type and APP23 transgenic mice resulted in dose-dependent clearance of plasma Aß. However, this did not correspond to reduced levels of soluble brain Aß with treatment up to 5 weeks in WT mice or formic acid-extractable brain Aß with 3 month treatment in aged APP23. In contrast, intracranial injection of NEP resulted in an acute decrease in soluble brain Aß. We found no change in amyloid precursor protein gene expression in mice treated with intravenous NEP, suggesting that the lack of effects in the brain following this route of administration was not caused by compensatory upregulation of Aß production. Taken together, these results suggest a lack of a robust peripheral Aß efflux sink through which brain amyloid burdens can be therapeutically reduced.

Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins.

Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins. HiBiT is a 1.3 kDa reporter peptide capable of generating bioluminescence through complementation with LgBiT, an 18 kDa subunit derived from NanoLuc. Using HiBiT CRISPR/Cas9-modified cell lines in combination with fluorescent antibodies, we developed a HiBiT-BRET immunoassay (a.k.a. Immuno-BRET). This is a homogeneous immunoassay capable of monitoring post-translational modifications on diverse protein targets. Its usefulness was demonstrated for the detection of phosphorylation of multiple signaling pathway targets (EGFR, STAT3, MAPK8 and c-MET), as well as chromatin containing histone H3 acetylation on lysine 9 and 27. These results demonstrate the ability to efficiently monitor endogenous biological processes modulated by post-translational modifications using a small bioluminescent peptide tag and fluorescent antibodies, providing sensitive quantitation of the response dynamics to multiple stimuli.

Pharmacological activation of nuclear factor erythroid 2-related factor-2 prevents hyperglycemia-induced renal oxidative damage: Possible involvement of O-GlcNAcylation.

Hyperglycemia is a major risk factor for kidney diseases. Oxidative stress, caused by reactive oxygen species, is a key factor in the development of kidney abnormalities related to hyperglycemia. The nuclear factor erythroid 2-related factor-2 (Nrf2) plays a crucial role in defending cells against oxidative stress by activating genes that produce antioxidants. L-sulforaphane (SFN), a drug that activates Nrf2, reduces damage caused by hyperglycemia. Hyperglycemic Wistar rats and HEK 293 cells maintained in hyperglycemic medium exhibited decreased Nrf2 nuclear translocation and reduced expression and activity of antioxidant enzymes. SFN treatment increased Nrf2 activity and reversed decreased renal function, oxidative stress and cell death associated with hyperglycemia. To investigate mechanisms involved in hyperglycemia-induced reduced Nrf2 activity, we addressed whether Nrf2 is modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a post-translational modification that is fueled in hyperglycemic conditions. In vivo, hyperglycemia increased O-GlcNAc-modified Nrf2 expression. Increased O-GlcNAc levels, induced by pharmacological inhibition of OGA, decreased Nrf2 activity in HEK 293 cells. In conclusion, hyperglycemia reduces Nrf2 activity, promoting oxidative stress, cell apoptosis and structural and functional renal damage. Pharmacological treatment with SFN attenuates renal injury. O-GlcNAcylation negatively modulates Nrf2 activity and represents a potential mechanism leading to oxidative stress and renal damage in hyperglycemic conditions.

Role of the CCL5 and Its Receptor, CCR5, in the Genesis of Aldosterone-Induced Hypertension, Vascular Dysfunction, and End-Organ Damage.

BACKGROUND: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown. METHODS: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction. RESULTS: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5. CONCLUSIONS: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.

Progranulin Maintains Blood Pressure and Vascular Tone Dependent on EphrinA2 and Sortilin1 Receptors and Endothelial Nitric Oxide Synthase Activation.

Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin+/+) and B6(Cg)-Grntm1.1Aidi/J (progranulin-/-) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin-/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin+/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.

Adjusted vascular contractility relies on integrity of progranulin pathway: Insights into mitochondrial function.

Objective: Cardiovascular disease (CVD) is a global health crisis and a leading cause of mortality. The intricate interplay between vascular contractility and mitochondrial function is central to CVD pathogenesis. The progranulin gene (GRN) encodes glycoprotein progranulin (PGRN), a ubiquitous molecule with known anti-inflammatory property. However, the role of PGRN in CVD remains enigmatic. In this study, we sought to dissect the significance of PGRN in the regulation vascular contractility and investigate the interface between PGRN and mitochondrial quality. Method: Our investigation utilized aortae from male and female C57BL6/J wild-type (PGRN+/+) and B6(Cg)-Grntm1.1Aidi/J (PGRN-/-) mice, encompassing wire myograph assays to assess vascular contractility and primary aortic vascular smooth muscle cells (VSMCs) for mechanistic insights. Results: Our results showed suppression of contractile activity in PGRN-/- VSMCs and aorta, followed by reduced α-smooth muscle actin expression. Mechanistically, PGRN deficiency impaired mitochondrial oxygen consumption rate (OCR), complex I activity, mitochondrial turnover, and mitochondrial redox signaling, while restoration of PGRN levels in aortae from PGRN-/- mice via lentivirus delivery ameliorated contractility and boosted OCR. In addition, VSMC overexpressing PGRN displayed higher mitochondrial respiration and complex I activity accompanied by cellular hypercontractility. Furthermore, increased PGRN triggered lysosome biogenesis by regulating transcription factor EB and accelerated mitophagy flux in VSMC, while treatment with spermidine, an autophagy inducer, improved mitochondrial phenotype and enhanced vascular contractility. Finally, angiotensin II failed to induce vascular contractility in PGRN-/- suggesting a key role of PGRN to maintain the vascular tone. Conclusion: Our findings suggest that PGRN preserves the vascular contractility via regulating mitophagy flux, mitochondrial complex I activity, and redox signaling. Therefore, loss of PGRN function appears as a pivotal risk factor in CVD development.

Vascular injury associated with ethanol intake is driven by AT1 receptor and mitochondrial dysfunction.

BACKGROUND: Renin-angiotensin (Ang II)-aldosterone system (RAAS) is crucial for the cardiovascular risk associated with excessive ethanol consumption. Disturbs in mitochondria have been implicated in multiple cardiovascular diseases. However, if mitochondria dysfunction contributes to ethanol-induced vascular dysfunction is still unknown. We investigated whether ethanol leads to vascular dysfunction via RAAS activation, mitochondria dysfunction, and mitochondrial reactive oxygen species (mtROS). METHODS: Male C57/BL6J or mt-keima mice (6-8-weeks old) were treated with ethanol (20% vol./vol.) for 12 weeks with or without Losartan (10 mg/kg/day). RESULTS: Ethanol induced aortic hypercontractility in an endothelium-dependent manner. PGC1α (a marker of biogenesis), Mfn2, (an essential protein for mitochondria fusion), as well as Pink-1 and Parkin (markers of mitophagy), were reduced in aortas from ethanol-treated mice. Disturb in mitophagy flux was further confirmed in arteries from mt-keima mice. Additionally, ethanol increased mtROS and reduced SOD2 expression. Strikingly, losartan prevented vascular hypercontractility, mitochondrial dysfunction, mtROS, and restored SOD2 expression. Both MnTMPyP (SOD2 mimetic) and CCCP (a mitochondrial uncoupler) reverted ethanol-induced vascular dysfunction. Moreover, L-NAME (NOS inhibitor) and EUK 134 (superoxide dismutase/catalase mimetic) did not affect vascular response in ethanol group, suggesting that ethanol reduces aortic nitric oxide (NO) and H2O2 bioavailability. These responses were prevented by losartan. CONCLUSION: AT1 receptor modulates ethanol-induced vascular hypercontractility by promoting mitochondrial dysfunction, mtROS, and reduction of NO and H2O2 bioavailability. Our findings shed a new light in our understanding of ethanol-induced vascular toxicity and open perspectives of new therapeutic approaches for patients with disorder associated with abusive ethanol drinking.

Suppressed vascular Rho-kinase activation is a protective cardiovascular mechanism in obese female mice.

BACKGROUND: Obesity is the number one cardiovascular risk factor for both men and women and is a complex condition. Although a sex dimorphism on vascular function has already been noted, the underlying processes remain unclear. The Rho-kinase pathway has a unique role in controlling vascular tone, and in obese male mice, hyperactivation of this system results in worsened vascular constriction. We investigated whether female mice exhibit decreased Rho-kinase activation as a protective mechanism in obesity. METHODS: We exposed male and female mice to a high-fat diet (HFD) for 14 weeks. At the end, energy expenditure, glucose tolerance, adipose tissue inflammation, and vascular function were investigated. RESULTS: Male mice were more sensitive to HFD-induced body weight gain, glucose tolerance, and inflammation than female mice. After establishing obesity, female mice demonstrated increase in energy expenditure, characterized by an increase in heat, whereas male mice did not. Interestingly, obese female mice, but not male, displayed attenuated vascular contractility to different agonists, such difference was blunted by inhibition of Rho-kinase, which was accompanied by a suppressed Rho-kinase activation, measured by Western blot. Finally, aortae from obese male mice displayed an exacerbated inflammation, whereas obese female demonstrated a mild vascular inflammation. CONCLUSION: In obesity, female mice demonstrate a vascular protective mechanism-suppression of vascular Rho-kinase-to minimize the cardiovascular risk associated with obesity, whereas male mice do not generate any adaptive response. Future investigations can help to understand how Rho-kinase becomes suppressed in female during obesity.

Role Of The C-C Motif Chemokine Ligand 5 (CCL5) And Its Receptor, C-C Motif Chemokine Receptor 5 (CCR5) In The Genesis Of Aldosterone-induced Hypertension, Vascular Dysfunction, And End-organ Damage.

Background: Aldosterone, a mineralocorticoid steroid hormone, has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of C-C Motif Chemokine Ligand 5 (CCL5) and its receptor, C-C Motif Chemokine Receptor 5 (CCR5), are well known in infectious diseases, but their roles in the genesis of aldosterone-induced vascular injury and hypertension are unknown. Methods: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg/day for 14 days) while receiving 1% saline to drink. Results: Here, we show that CCR5 plays a central role in aldosterone-induced vascular injury, hypertension, and renal damage. Long-term infusion of aldosterone in CCR5+/+ mice resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression. Aldosterone treatment also triggered vascular injury, characterized by endothelial dysfunction and inflammation, hypertension, and renal damage. Mice lacking CCR5 were protected from aldosterone-induced vascular damage, hypertension, and renal injury. Mechanistically, we demonstrated that CCL5 increased NADPH oxidase 1 (Nox1) expression, reactive oxygen species (ROS) formation, NFκB activation, and inflammation and reduced nitric oxide production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aortae incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking Nox1, NFκB, or with Maraviroc treatment. Conclusions: Our data demonstrate that CCL5/CCR5, through activation of NFkB and Nox1, is critically involved in aldosterone-induced vascular and renal damage and hypertension. Our data place CCL5 and CCR5 as potential targets for therapeutic interventions in conditions with aldosterone excess.