Search

Background In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise. Methods and results Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P Conclusion Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar–Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Notch3 mutations cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), which predisposes to stroke and dementia. CADASIL is characterised by vascular dysfunction and granular osmiophilic material (GOM) accumulation in cerebral small vessels. Systemic vessels may also be impacted by Notch3 mutations. However vascular characteristics and pathophysiological processes remain elusive. We investigated mechanisms underlying the peripheral vasculopathy mediated by CADASIL-causing Notch3 gain-of-function mutation. We studied: (i) small arteries and vascular smooth muscle cells (VSMCs) from TgNotch3R169C mice (CADASIL model), (ii) VSMCs from peripheral arteries from CADASIL patients, and (iii) post-mortem brains from CADASIL individuals. TgNotch3R169C vessels exhibited GOM deposits, increased vasoreactivity and impaired vasorelaxation. Hypercontractile responses were normalised by fasudil (Rho kinase inhibitor) and 4-phenylbutyrate (4-PBA; endoplasmic-reticulum (ER) stress inhibitor). Ca2+ transients and Ca2+ channel expression were increased in CADASIL VSMCs, with increased expression of Rho guanine nucleotide-exchange factors (GEFs) and ER stress proteins. Vasorelaxation mechanisms were impaired in CADASIL, evidenced by decreased endothelial nitric oxide synthase (eNOS) phosphorylation and reduced cyclic guanosine 3′,5′-monophosphate (cGMP) levels, with associated increased soluble guanylate cyclase (sGC) oxidation, decreased sGC activity and reduced levels of the vasodilator hydrogen peroxide (H2O2). In VSMCs from CADASIL patients, sGC oxidation was increased and cGMP levels decreased, effects normalised by fasudil and 4-PBA. Cerebral vessels in CADASIL patients exhibited significant oxidative damage. In conclusion, peripheral vascular dysfunction in CADASIL is associated with altered Ca2+ homoeostasis, oxidative stress and blunted eNOS/sGC/cGMP signaling, processes involving Rho kinase and ER stress. We identify novel pathways underlying the peripheral arteriopathy induced by Notch3 gain-of-function mutation, phenomena that may also be important in cerebral vessels.

Over the past two decades, the treatment of cancer has been revolutionised by the highly successful introduction of novel molecular targeted therapies and immunotherapies, including small-molecule kinase inhibitors and monoclonal antibodies that target angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling pathways. Despite their anti-angiogenic and anti-cancer benefits, the use of VEGF inhibitors (VEGFi) and other tyrosine kinase inhibitors (TKIs) has been hampered by potent vascular toxicities especially hypertension and thromboembolism. Molecular processes underlying VEGFi-induced vascular toxicities still remain unclear but inhibition of endothelial NO synthase (eNOS), reduced nitric oxide (NO) production, oxidative stress, activation of the endothelin system, and rarefaction have been implicated. However, the pathophysiological mechanisms still remain elusive and there is an urgent need to better understand exactly how anti-angiogenic drugs cause hypertension and other cardiovascular diseases (CVDs). This is especially important because VEGFi are increasingly being used in combination with other anti-cancer dugs, such as immunotherapies (immune checkpoint inhibitors (ICIs)), other TKIs, drugs that inhibit epigenetic processes (histone deacetylase (HDAC) inhibitor) and poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, which may themselves induce cardiovascular injury. Here, we discuss vascular toxicities associated with TKIs, especially VEGFi, and provide an up-to-date overview on molecular mechanisms underlying VEGFi-induced vascular toxicity and cardiovascular sequelae. We also review the vascular effects of VEGFi when used in combination with other modern anti-cancer drugs.

Background Cisplatin-based chemotherapy increases the risk of cardiovascular and renal disease. Objectives We aimed to define the time course, pathophysiology, and approaches to prevent cardiovascular disease associated with cisplatin-based chemotherapy. Methods Two cohorts of patients with a history of testicular cancer (n = 53) were recruited. Cohort 1 consisted of 27 men undergoing treatment with: 1) surveillance; 2) 1 to 2 cycles of bleomycin, etoposide, and cisplatin (BEP) chemotherapy (low-intensity cisplatin); or 3) 3 to 4 cycles of BEP (high-intensity cisplatin). Endothelial function (percentage flow-mediated dilatation) and cardiovascular biomarkers were assessed at 6 visits over 9 months. Cohort 2 consisted of 26 men previously treated 1 to 7 years ago with surveillance or 3 to 4 cycles BEP. Vasomotor and fibrinolytic responses to bradykinin, acetylcholine, and sodium nitroprusside were evaluated using forearm venous occlusion plethysmography. Results In cohort 1, the percentage flow-mediated dilatation decreased 24 h after the first cisplatin dose in patients managed with 3 to 4 cycles BEP (10.9 ± 0.9 vs. 16.7 ± 1.6; p < 0.01) but was unchanged from baseline thereafter. Six weeks after starting 3 to 4 cycles BEP, there were increased serum cholesterol levels (7.2 ± 0.5 mmol/l vs. 5.5 ± 0.2 mmol/l; p = 0.01), hemoglobin A1c (41.8 ± 2.0 mmol/l vs. 35.5 ± 1.2 mmol/l; p < 0.001), von Willebrand factor antigen (62.4 ± 5.4 mmol/l vs. 45.2 ± 2.8 mmol/l; p = 0.048) and cystatin C (0.91 ± 0.07 mmol/l vs. 0.65 ± 0.09 mmol/l; p < 0.01). In cohort 2, intra-arterial bradykinin, acetylcholine, and sodium nitroprusside caused dose-dependent vasodilation (p < 0.0001). Vasomotor responses, endogenous fibrinolytic factor release, and cardiovascular biomarkers were not different in patients managed with 3 to 4 cycles of BEP versus surveillance. Conclusions Cisplatin-based chemotherapy induces acute and transient endothelial dysfunction, dyslipidemia, hyperglycemia, and nephrotoxicity in the early phases of treatment. Cardiovascular and renal protective strategies should target the early perichemotherapy period. (Clinical Characterisation of the Vascular Effects of Cis-platinum Based Chemotherapy in Patients With Testicular Cancer [VECTOR], NCT03557177; Intermediate and Long Term Vascular Effects of Cisplatin in Patients With Testicular Cancer [INTELLECT], NCT03557164), Central Illustration

Objective: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR–TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. Conclusions: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.

AimsAlthough effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria.Methods and resultsMale Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan.ConclusionsOur results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.

Increased generation of reactive oxygen species (ROS) and altered Ca 2+ handling cause vascular damage in hypertension. Mechanisms linking these systems are unclear, but TRPM2 (transient receptor potential melastatin 2) could be important because TRPM2 is a ROS sensor and a regulator of Ca 2+ and Na + transport. We hypothesized that TRPM2 is a point of cross-talk between redox and Ca 2+ signaling in vascular smooth muscle cells (VSMC) and that in hypertension ROS mediated-TRPM2 activation increases [Ca 2+ ] i through processes involving NCX (Na + /Ca 2+ exchanger). VSMCs from hypertensive and normotensive individuals and isolated arteries from wild type and hypertensive mice (LinA3) were studied. Generation of superoxide anion and hydrogen peroxide (H 2 O 2 ) was increased in hypertensive VSMCs, effects associated with activation of redox-sensitive PARP1 (poly [ADP-ribose] polymerase 1), a TRPM2 regulator. Ang II (angiotensin II) increased Ca 2+ and Na + influx with exaggerated responses in hypertension. These effects were attenuated by catalase−polyethylene glycol -catalase and TRPM2 inhibitors (2-APB, 8-Br-cADPR olaparib). TRPM2 siRNA decreased Ca 2+ in hypertensive VSMCs. NCX inhibitors (Benzamil, KB-R7943, YM244769) normalized Ca 2+ hyper-responsiveness and MLC20 phosphorylation in hypertensive VSMCs. In arteries from LinA3 mice, exaggerated agonist (U46619, Ang II, phenylephrine)-induced vasoconstriction was decreased by TRPM2 and NCX inhibitors. In conclusion, activation of ROS-dependent PARP1-regulated TRPM2 contributes to vascular Ca 2+ and Na + influx in part through NCX. We identify a novel pathway linking ROS to Ca 2+ signaling through TRPM2/NCX in human VSMCs and suggest that oxidative stress-induced upregulation of this pathway may be a new player in hypertension-associated vascular dysfunction.

AIMS NOX-derived reactive oxygen species (ROS) are mediators of signaling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signaling molecules, actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were downregulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs NOX5 was upregulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1) and hyperphosphorylation (PKC, ERK1/2, MLC20) of signaling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSIONS We define NOX5/ROS/c-Src as a novel feedforward signaling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury. TRANSLATIONAL PERSPECTIVE Oxidative stress is a major factor contributing to vascular damage in hypertension. We corroborate experimental evidence that NOX-derived ROS generation is increased in human vascular smooth muscle cells (VSMC) and demonstrate that in human hypertension NOX5 upregulation is a major trigger of VSMC dysfunction. We uncover new regulatory molecular mechanisms of NOX5 and define NOX5/ROS/c-Src as a novel signaling pathway in human VSMCs. This system is augmented in hypertension contributing to abnormal VSMC redox signaling, cytoskeletal disorganization and vascular dysfunction. Modulating the NOX5/ROS/c-Src pathway may have therapeutic potential by targeting redox signaling pathways involved in vascular dysfunction associated with hypertension.

Notch signalling is critically involved in vascular morphogenesis and function. Four Notch isoforms (Notch1–4) regulating diverse cellular processes have been identified. Of these, Notch3 is expressed almost exclusively in vascular smooth muscle cells (VSMCs), where it is critically involved in vascular development and differentiation. Under pathological conditions, Notch3 regulates VSMC switching between the contractile and synthetic phenotypes. Abnormal Notch3 signalling plays an important role in vascular remodelling, a hallmark of several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Because of the importance of Notch3 in VSMC (de)differentiation, Notch3 has been implicated in the pathophysiology of pulmonary vascular remodelling in PAH. Here we review the current literature on the role of Notch in VSMC function with a focus on Notch3 signalling in pulmonary artery VSMCs, and discuss potential implications in pulmonary artery remodelling in PAH.

Hypertension, vascular dysfunction and downregulation of the renin angiotensin system as sequelae of COVID-19 The long-term CV consequences of COVID are unknown however the potential for ongoing cardiac and vascular inflammation with RAAS alteration may increase the risk of developing hypertension and CV disease. Non-hypertensive patients hospitalised in April-May 2020 with either confirmed COVID19 (cases) or non-COVID (controls) diagnosis were recruited ≥12 weeks post-discharge. All underwent detailed BP and vascular/immune and RAAS phenotyping. The primary outcome was ABPM 24-hr SBP. Paired t-tests and multivariable regression models used to assess differences. Thirty cases and eighteen controls completed the study. Cases were older (51±7 vs 45±9 years) with lower discharge SBP (121±10 vs 128±15 mmHg; p0.01). ABPM at study visit was higher in the cases compared to controls (24-hour SBP (OR[95%CI]: 8.6[0.9-16.3]; p0.03), day-time SBP (8.6[1.5-17.3]; p0.02), day-time DBP (4.6[0.1-9.1]; p

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), pvs. C: 1011±268 RFU, p

High salt diet (HSD) has deleterious effects on the vasculature by mechanisms not fully elucidated. We demonstrated tight coupling between Na + and Ca 2+ levels in VSMCs, where PARP-regulated TRPM2, a redox-sensitive Ca 2+ channel, plays an important role. Increased [Ca 2+ ]i also contributes to inflammasome assembly, which dysregulates vascular function. We hypothesized that HSD induces a pro-oxidant environment that contributes to PARP-induced TRPM2-activation, Ca 2+ influx and inflammasome assembly, leading to vascular damage. WKY rats were treated with 1% HSD (3 weeks). Blood pressure was assessed by tail-cuff methodology, vascular reactivity was assessed in mesenteric arteries and calcium influx, ROS generation, inflammasome and pro-contractile marker in vascular smooth muscle cells (VSMCs) in presence and absence of HS medium (HSM-140mM). HSD did not increase blood pressure (BP), but vascular contractility was exaggerated (Emax(mN): WT 10.20 ± 0.70 vs 15.17 ± 1.74), effect reversed by PARP (Emax: 11.14 ± 1.24) and TRPM2 (8-br) (Emax: 11.45 ± 1.11) inhibitors. In VSMCs, HSM behaves as a pro-oxidant agent (ROS-AU: Control 77.51 ± 2.80 vs 130.04 ± 13.89 HSM), leading to increased [Ca 2+ ]i (AUC: Control 25562.45 ± 880.48 vs 30924.8 ± 1263.85 HSM) and activation of myosin light chain (pMLC-AU: Control 99.27 ± 1.01 vs 626.87 ±71.28 HSM), by mechanisms dependent on ROS and PARP/TRPM2 activation. HSM also increased expression of inflammasome components NLRP3 (2 ΔΔCt : Control 1.20 ± 0.01 vs 1.85 ± 0.19 HSM), ASC (2 ΔΔCt : Control 1.02 ± 0.01 vs 1.49 ± 0.17 HSM) and Caspase 1 (2 ΔΔCt : Control 1.06 ± 0.03 vs 2.35 ± 0.46 HSM), which was prevented by ROS scavenger Tiron and PARP inhibitor. In conclusion, HSD-induced vascular hypercontractility involves ROS activation of PARP/TRPM2 signalling. Activation of PARP/TRPM2 was associated with increased [Ca 2+ ]i and activation of pro-contractile signaling and inflammasome assembly. Normal BP in HSD-fed rats in the presence of vascular damage suggests that ROS/PARP/TRPM2 signaling induced by salt influences vascular function independently of BP elevation. We identify a novel pathway that underlies salt-induced vascular damage and suggest a potential therapeutic role for PARP/TRPM2 inhibitors in vascular dysfunction.

Hypertension is a common unwanted effect of VEGF inhibitors (VEGFi), which are used as anti-angiogenic drugs in cancer treatment. Clinical observations suggest that the combination of VEGFi with another anti-cancer drug, olaparib (PARP inhibitor [PARPi]), may attenuate the development of hypertension. However putative vascular mechanisms are unknown. PARP plays a major role in the activation of TRPM2, a redox-sensitive Ca 2+ channel, which is associated with hypertension-induced vascular dysfunction. We hypothesized that PARPi attenuates VEGFi-induced vascular injury through TRPM2/Ca 2+ -dependent pathways. Human vascular smooth muscle cells (hVSMC), human aortic endothelial cells (HAEC), and mouse mesenteric arteries were studied. Cells/arteries were exposed to axitinib (VEGFi) alone (3μM) or in combination with olaparib (1μM). Wire myography was used to assess vascular function. Axitinib reduced ACh-induced vasodilation (% relaxation: 70.5 [Ct] vs. 34.8 [Axi]), an effect blocked by olaparib. U46619- and ET-1-induced vasoconstriction were increased by axitinib (% KCl- U4 : 101.2 [Ct] vs. 141.4 [Axi]; ET-1 : 122.6 [Ct] vs. 152.5 [Axi]), an effect not observed with axitinib plus olaparib. TRPM2 channel blocker (8-Br-cADPR; 1μM) attenuated the hypercontractile effects and endothelial dysfunction induced by axitinib. Axitinib increased ROS production in hVSMC (RUL: 0.8±0.2 [Ct] vs. 1.1±0.09 [Axi]) and HAEC (0.7±0.4 [Ct] vs. 1.2±0.1 [Axi]), stimulated phosphorylation of the inhibitory site of eNOS (a.u.: 0.99±0.35 [Ct] vs. 1.35±0.10 [Axi]) and induced exaggerated Ca 2+ influx (AUC: 17541±4708 [Ct] vs. 22249±1438 [Axi]) in hVSMC. These effects were blocked by olaparib and 8-Br-cADPR. Axitinib also induced phosphorylation of MLC20 in hVSMC (a.u.: 0.028±0.02 [Ct] vs. 0.04±0.01 [Axi]) and aorta (a.u.: 0.3±0.01 [Ct] vs. 0.5±0.001 [Axi]). Our data indicate that PARP/TRPM2 inhibition attenuates axitinib-mediated vascular dysfunction and normalizes impaired hVSMC and HAEC signalling induced by VEGFi. We define a putative vasoprotective effect of olaparib that may ameliorate vascular injury and hypertension induced by VEGFi in cancer treatment.

Supplemental Digital Content is available in the text., Combined inhibition of NEP (neutral endopeptidase) and ACE (angiotensin-converting enzyme), without unwanted effects, remains an attractive therapeutic strategy in cardiovascular medicine. Omapatrilat, a dual NEP inhibitor–ACE inhibitor, was a promising antihypertensive drug but failed in trials due to angioedema, an effect possibly caused by inhibition of both the N- and C-domains of ACE. Here, we aimed to determine whether lisinopril-tryptophan (lisW-S), a C-domain specific ACE inhibitor that preserves the N-domain catalytic activity, together with sacubitril (NEP inhibitor), differentially influences cardiovascular function and vascular permeability in hypertension compared with omapatrilat and lisinopril+sacubitril which inhibits both the ACE C- and N-domains. Ang II (angiotensin II)–dependent hypertensive mice (transgenic mice expressing active human renin in the liver [also known as LinA3]) received vehicle, sacubitril, lisW-S, lisinopril, lisinopril+sacubitril, or lisW-S+sacubitril for 4 weeks. Systolic blood pressure was increased in LinA3 mice, along with cardiac hypertrophy/dysfunction, impaired endothelium-dependent vasorelaxation, hypercontractile responses, vascular remodeling, and renal inflammation. LisW-S+sacubitril, lisinopril+sacubitril, and omapatrilat reduced systolic blood pressure and normalized cardiovascular remodeling and vascular hypercontractile responses in LinA3 mice. Although lisinopril+sacubitril and omapatrilat improved Ach-induced vasorelaxation, lisW-S+sacubitril had no effect. Endothelial permeability (Evans Blue assessment) was increased in omapatrilat but not in LisW-S+sacubitril–treated mice. In conclusion, lisW-S combined with sacubitril reduced systolic blood pressure and improved cardiac dysfunction in LinA3 mice, similar to omapatrilat but without effects on endothelium-dependent vasorelaxation. Moreover, increased vascular leakage (plasma extravasation) induced by omapatrilat was not evident in mice treated with lisW-S+sacubitril. Targeting ACE C-domain and NEP as a combination therapy may be as effective as omapatrilat in lowering systolic blood pressure, but without inducing vascular permeability and endothelial injury.

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.

Introduction COVID‐19 is primarily a respiratory disease associated with cardiovascular risk. SARS‐CoV‐2, the virus causing COVID‐19, uses ACE2, an important enzyme in the cardiovascular system that regulates the conversion of Ang II (deleterious/pro‐hypertensive) to Ang 1‐7 (protective/anti‐hypertensive), as a receptor for host cell entry and infection. Considering the relationship between the viral S1‐protein and the host's ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether it also contributes to cardiovascular damage associated with COVID‐19. We hypothesisedthat SARS‐Cov‐2‐ACE2 interaction induces activation of vascular cell inflammatory responses that are influenced by ACE2 dependent and/or independent enzymatic Ang‐(1‐7) production. Methods Human microvascular endothelial cells (MEC) were used and stimulated with SARS‐CoV‐2 recombinant S1 protein (rS1p) (0.66 μg/mL) at 10/30 min (acute) and 5/24h (chronic). Activation of pro‐inflammatory signaling pathways (immunoblotting, real‐time PCR), microparticle (MP) generation (NanoSight), and cytokine production (ELISA) were assessed. In some experiments, cells were pre‐incubated with an ACE2 activator (DIZE – 190 nM) and inhibitor (MLN‐4760 – 440 pM). Results rS1P increased NFκB activation (Control ©=0.99±0.06 vs. 1.38±0.19 AU; p

The development of a wide range of novel antineoplastic therapies has improved the prognosis for patients with a wide range of malignancies, which has increased the number of cancer survivors substantially. Despite the oncological benefit, cancer survivors are exposed to short- and long-term adverse cardiovascular toxicities associated with anticancer therapies. Systemic hypertension, the most common comorbidity among cancer patients, is a major contributor to the increased risk for developing these adverse cardiovascular events. Cancer and hypertension have common risk factors, have overlapping pathophysiological mechanisms and hypertension may also be a risk factor for some tumor types. Many cancer therapies have prohypertensive effects. Although some of the mechanisms by which these antineoplastic agents lead to hypertension have been characterized, further preclinical and clinical studies are required to investigate the exact pathophysiology and the optimal management of hypertension associated with anticancer therapy. In this way, monitoring and management of hypertension before, during, and after cancer treatment can be improved to minimize cardiovascular risks. This is vital to optimize cardiovascular health in patients with cancer and survivors, and to ensure that advances in terms of cancer survivorship do not come at the expense of increased cardiovascular toxicities.

Epidermal growth factor (EGF), signals throught the EGF receptor (EGFR) and plays an important role in the pathogenesis of vascular remodeling. Transient receptor potential melastatin 7 (TRPM7) is a channel bound to a kinase domain important for Mg 2+ , Zn 2+ and Ca 2+ homeostasis. Cancer patients treated with EGFR inhibitors develop hypomagnesemia, suggesting a relationship between EGFR and TRPM7. Here we investigated the role of TRPM7 in EGF signaling in vascular smooth muscle cell (VSMC) from humans (hVSMC) and rats (rVSMC). VSMCs were stimulated with EGF (50ng/ml) for 5min and 24h with/without pretreatment of gefitinib (1μM), PP2 (10μM), 2APB (30μM) and NS8593 (40μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aortas were isolated from wild type (WT), TRPM7-deficient (TRPM7 +/Δkinase ) and kinase-dead (TRPM7 R/R ) mice. Protein expression was assessed by immunoblotting. Ca 2+ and Mg 2+ were assessed using Cal-520 and Mg-green probes respectively. EGFR/TRPM7 interaction was investigated by proximity ligation assay (PLA), immunoprecipitation and confocal microscopy. VSMC migration and proliferation were examined by wound healing and CFSE proliferation assays. In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), (p2+ and Ca 2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg 2+ ) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) through c-Src, EGFR and TRPM7, p+/Δkinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7 R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7 +/Δkinase showed reduced wall thickness (35%). Our findings demonstrate that interaction between EGFR/TRPM7 is a key process underlying EGF-induced VSMC migration and growth. This novel EGF-c-Src-EGFR-TRPM7 pathway may play an important role in vascular remodeling.

Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, convincing evidence suggests that fetuin-A is also involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel FetA vascular effects and associated molecular mechanisms, focusing on oxidative stress and toll-like receptor 4 (TLR4). Vascular function studies were performed in mesenteric resistance arteries from WKY rats, wild-type, Nox1 KO, Nox4 KO and Ang II-dependent hypertensive mice (LinA3) and rat aortic endothelial cells (RAEC). ROS production (chemiluminescence, Amplex Red, ELISA) and pro-inflammatory markers expression (RT-PCR) were measured in VSMCs from WKY rats and RAEC. FetA impaired endothelium-dependent (LogEC50 7.320±0.08 M vs control 8.025±0.06) and endothelium-independent vasorelaxation (LogEC50 6.48±0.19 M vs control 7.38±0.12), p

TRPM7 has cation channel and kinase properties, is permeable to Mg 2+ , Ca 2+ , and Zn 2+ and is protective in the cardiovascular system. Hyperaldosteronism, which induces hypertension and cardiovascular fibrosis, is associated with Mg 2+ wasting. Here we questioned whether TRPM7 plays a role in aldosterone- induced hypertension and fibrosis and whether it influences cation regulation. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo-salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel structure was assessed by pressure myography. Molecular mechanisms were investigated in cardiac fibroblasts (CF) from WT and M7+/Δ mice. Protein expression was assessed by western-blot and histology. M7+/Δ mice exhibited reduced TRPM7 expression (30%) and phosphorylation (62%), levels that were recapitulated in WT aldo-salt mice. M7+/Δ exhibited increased BP by aldo, salt and aldo-salt (135-140mmHg) vs M7+/Δ-veh (117mmHg) (p2+ -dependent phosphatase, was reduced (3-fold) only in M7+/Δ mice. M7+/Δ CF showed reduced proliferation (30%) and PPM1A (4-fold) and increased expression of TGFβ, IL-11 and IL-6 (2-3-fold), activation of Stat1 (2-fold), Smad3 (9-fold) and ERK1/2 (8-fold) compared with WT. Mg 2+ supplementation normalized cell proliferation and reduced protein phosphorylation in M7+/Δ CF (p2+ -dependent mechanisms.

CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3 R169C ) and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of Notch3 (WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p2 7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr 495 ) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (pR169C mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H 2 O 2 levels were decreased in TgNotch3 R169C mice (1.9±0.2 vs. 1.1±1.9; pR169C mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p

NOX4-induced H 2 O 2 production promotes vasodilation and is cardiovascular protective. H 2 O 2 also regulates TRPM2, a redox-sensitive channel that regulates Ca 2 influx. eNOS is a Ca 2+ -dependent enzyme, and hypertension-associated endothelial dysfunction involves eNOS inactivation. As NOX4-derived H 2 O 2 regulates TRPM2 and consequently Ca 2+ influx, we questioned whether downregulation of the H 2 O 2 -TRPM2-Ca 2+ axis in endothelial cells may contributes to impaired vascular relaxation in hypertension. WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was studied in mesenteric resistance arteries by wire myography. Ca 2+ influx was assessed by fluorescence microscopy in aortic endothelial cells, eNOS activation and TRPM2 expression were assessed by immunoblotting and immunohistochemistry, respectively. Blood pressure in TTRhRen (130.3±7.0 mmHg) and TTRhRen/NOX4 KO mice (141.3±18 mmHg) was significantly increased compared to control mice (98.1±8.0 mmHg). Endothelium-dependent relaxation was impaired in TTRhRen mice (Emax: WT 83.5±4.03 vs TTRhRen 59.1±3.5), effects worsened by NOX4 KO (37.9±5.4), p2 O 2 incubation (74.2±15.4), p2 O 2 generation (% of control: 138.23±9.04) followed by Ca 2+ influx (AUC - Ca 2+ : 19401.25±1940.21), an important regulator of eNOS. These processes were reduced by TRPM2 inhibition (AUC - Ca 2+ : 8-br-cADPR 15232.2±1052.0; Olaparib 14260±843.2 and 2-APB 13095.2±277.4, p2+ : Ang II 107357±1940.2 vs GKT 15067.5±255.6, p2 O 2 due to Nox4 downregulation is a major driver of this process. We identify a new axis in endothelial cells involving Nox4-H 2 O 2 -mediated activation of TRPM2-Ca 2+ -eNOS signalling which is vasoprotective.

Nox5 is the major ROS-generating Nox isoform in human vascular smooth muscle cells (VSMC). The role of Nox5 in oxidative stress and redox signaling underlying vascular dysfunction in hypertension is unclear. We examined molecular processes that regulate VSMC Nox5-induced ROS generation, focusing on c-Src. VSMC isolated from small arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 expression and phosphorylation (immunoblotting, immunoprecipitation); ROS generation (chemiluminescence); activation of contractile signaling pathways (immunoblotting), Ca 2+ influx (Cal-520AM fluorescence), reversible protein oxidation (cysteine sulfenic acid probe BCN-E-BCN), actin polymerization (phalloidin staining) and migration (wound healing assay) were assessed in absence/presence of Nox5 (melittin) and Src (PP2) inhibitors. To study Nox5-specific effects, we used p22phox-silenced VSMCs (siRNA). Vascular function in VSMC-specific Nox5 transgenic mice was studied by wire myography. In HT, ROS levels (139±27%), Nox5 expression (103±23%) and phosphorylation were increased (77±17.93%) (p20 (416±71%) and Ang II-induced Ca 2+ influx (574±44 vs NT:451±26) were also increased in HT (p20 (89±22% vs Ctl) phosphorylation, effects blocked by melittin and PP2 (p2+ influx, actin polymerization and migration in HT. Hypercontractility observed in Nox5 mice was abolished by melittin and PP2. Our findings demonstrate that Nox5 is upregulated in human hypertension. This is associated with activation of c-Src, increased redox signaling and VSMC cytoskeletal reorganization, migration and vascular contraction. We define a novel Nox5-ROS-c-Src signaling pathway that may play a role in vascular remodeling/dysfunction in hypertension.

Our understanding of Na+ homeostasis has recently been reshaped by the notion of skin as a depot for Na+ accumulation in multiple cardiovascular diseases and risk factors. The proposed water-independent nature of tissue Na+ could induce local pathogenic changes, but lacks firm demonstration. Here, we show that tissue Na+ excess upon high Na+ intake is a systemic, rather than skin-specific, phenomenon reflecting architectural changes, i.e. a shift in the extracellular-to-intracellular compartments, due to a reduction of the intracellular or accumulation of water-paralleled Na+ in the extracellular space. We also demonstrate that this accumulation is unlikely to justify the observed development of experimental hypertension if it were water-independent. Finally, we show that this isotonic skin Na+ excess, reflecting subclinical oedema, occurs in hypertensive patients and in association with aging. The implications of our findings, questioning previous assumptions but also reinforcing the importance of tissue Na+ excess, are both mechanistic and clinical., Na+ has been suggested to accumulate in tissues, particularly skin, in a hypertonic manner and to exert local pathogenic effects. Here, we reappraise this phenomenon which is systemic in nature and reflects isotonic changes in the relative extracellular volume in tissues, e.g. subclinical oedema; as such, it occurs in human hypertension and aging.

The etiology of hypertension involves complex interactions among genetic, environmental, and pathophysiologic factors that influence many regulatory systems. Hypertension is characteristically associated with vascular dysfunction, cardiovascular remodelling, renal dysfunction, and stimulation of the sympathetic nervous system. Emerging evidence indicates that the immune system is also important and that activated immune cells migrate and accumulate in tissues promoting inflammation, fibrosis, and target-organ damage. Common to these processes is oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the oxidants that leads to a disruption of oxidation-reduction (redox) signalling and control and molecular damage. Physiologically, reactive oxygen species (ROS) act as signalling molecules and influence cell function through highly regulated redox-sensitive signal transduction. In hypertension, oxidative stress promotes posttranslational modification (oxidation and phosphorylation) of proteins and aberrant signalling with consequent cell and tissue damage. Many enzymatic systems generate ROS, but NADPH oxidases (Nox) are the major sources in cells of the heart, vessels, kidneys, and immune system. Expression and activity of Nox are increased in hypertension and are the major systems responsible for oxidative stress in cardiovascular disease. Here we provide a unifying concept where oxidative stress is a common mediator underlying pathophysiologic processes in hypertension. We focus on some novel concepts whereby ROS influence vascular function, aldosterone/mineralocorticoid actions, and immunoinflammation, all important processes contributing to the development of hypertension.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation–associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL., ER stress and Rho kinase are potentially druggable targets in CADASIL, the most aggressive form of small vessel disease and dementia.

Overproduction of superoxide anion (•O2−) and O-linked β-N-acetylglucosamine (O-GlcNAc) modification in the vascular system are contributors to endothelial dysfunction. This study tested the hypothesis that increased levels of O-GlcNAc-modified proteins contribute to •O2− production via activation of NADPH oxidase, resulting in impaired vasodilation. Rat aortic segments and vascular smooth muscle cells (VSMCs) were incubated with vehicle (methanol) or O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc) (100 μM). PUGNAc produced a time-dependent increase in O-GlcNAc levels in VSMC and decreased endothelium-dependent relaxation, which was prevented by apocynin and tiron, suggesting that •O2− contributes to endothelial dysfunction under augmented O-GlcNAc levels. Aortic segments incubated with PUGNAc also exhibited increased levels of reactive oxygen species, assessed by dihydroethidium fluorescence, and augmented •O2− production, determined by lucigenin-enhanced chemiluminescence. Additionally, PUGNAc treatment increased Nox-1 and Nox-4 protein expression in aortas and VSMCs. Translocation of the p47phox subunit from the cytosol to the membrane was greater in aortas incubated with PUGNAc. VSMCs displayed increased p22phox protein expression after PUGNAc incubation, suggesting that NADPH oxidase is activated in conditions where O-GlcNAc protein levels are increased. In conclusion, O-GlcNAc levels reduce endothelium-dependent relaxation by overproduction of •O2− via activation of NADPH oxidase. This may represent an additional mechanism by which augmented O-GlcNAc levels impair vascular function.

Chemerin is an adipokine that regulates adipogenesis and metabolic functions of mature adipocytes mainly through the activation of chemokine-like receptor 1 (CMKLR1). Elevated levels of chemerin have been found in individuals with obesity, type 2 diabetes, and osteoporosis. This adipokine was identified as an inflammatory and metabolic syndrome marker. Considering that the association between metabolic syndrome and bone health remains unclear, the present study aimed to clarify the role of chemerin in the pathophysiology of bone loss induced by dyslipidemia, particularly modulating osteoclastogenesis. In vitro analyses showed a downregulation of CMKLR1 at the early stage of differentiation and a gradual increase at late stages. Strikingly, chemerin did not modify osteoclast differentiation markers or osteoclast formation; however, it increased the actin-ring formation and bone resorption activity in mature osteoclasts. The increased bone resorption activity induced by chemerin was effectively inhibited by CMKLR1 antagonist (CCX832). Chemerin boosting mature osteoclast activity involves ERK5 phosphorylation. Moreover, two models of dyslipidemia (high-fat diet [HFD]-treated C57/BL6 and db/db mice) exhibited significantly increased level of chemerin in the serum and gingival tissue. Morphometric analysis showed that HFD-treated and db/db mice exhibited increased alveolar bone loss compared to respective control mice, which was associated with an up-regulation of chemerin, CMKLR1 and cathepsin K mRNA expression in the gingival tissue. The treatment of db/db mice with CCX832 effectively inhibited bone loss. Antagonism of chemerin receptor also inhibited the expression of cathepsin K in the gingival tissue. Our results show that chemerin not only increases osteoclasts activity in vitro, but also that increased level of chemerin in dyslipidemic mice plays a critical role in bone homeostasis. © 2016 American Society for Bone and Mineral Research.

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.

CADASIL, a monogenic condition due to Notch3 mutations is a small vessel disease of the brain resulting in premature vascular dementia and stroke. CADASIL patients have cerebral vascular narrowing, progressive SMCs loss and granular osmiophilic material (GOM) deposition, however it is unclear whether peripheral arteries also exhibit dysfunction. We studied mice harbouring the CADASIL-causing Notch3 mutation (TgNotch3 R169C ), which recapitulates human CADASIL, to evaluate whether peripheral small arteries exhibit vascular dysfunction. Mesenteric arteries (MA) from male TgNotch3 R169C and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. Vascular structure and function were assessed by myography. No changes were observed in blood pressure and cardiac function in CADASIL mice, measured by tail cuff and echocardiography respectively. Expression of Notch3 and target genes was augmented in MA from CADASIL mice ( Notch3 : 2.0±0.5 vs. 6.0±1.3; HeyL: 1.1±0.4 vs. 2.9±0.6, p2 7.8±0.1 vs 6.8±0.3) relaxation and increased reactivity in response to AngII (Emax 33.7±6.8 vs. 72.8±4.4), phenylephrine (Emax: 70.6±7.2 vs. 92.1±4.2), and U46619 (Emax: 123.4±4.4 vs. 215.1±24.4) (pWT ); effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (pR169C ). MA from CADASIL mice also had hypotrophic remodelling. U46619-induced calcium influx was increased in VSMCs (AUC: 1.3 fold increase) from TgNotch3 R169C and gene expression of Rho GEFs (LARG: 1.1±0.1 vs. 2.1±0.3; PDZ: 0.9±0.1 vs. 2.9±0.4) was increased in MA from TgNotch3 R169C vs. TgNotch3 WT mice (p495 ) and BiP expression (ER stress marker) were increased in vessels from CADASIL mice (eNOS: 1.8 fold increase; Bip: 1.7 fold increase; pWT ). In conclusion, our data demonstrated that the vasculopathy associated with Notch3 mutation is also present in peripheral small vessels where the interplay between Notch3, Rho-kinase and ER stress may be important. We identify potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.

Patients with coronary microvascular dysfunction (CMD), a potential cause of heart ischemia, have systemic vascular dysfunction, characterized by increased vascular contraction to ET-1 and a thromboxane A2 analogue (U46619). Nox5 regulates vascular contraction and is involved in cardiovascular diseases. In our study, we questioned whether Nox5 plays a role in systemic vascular dysfunction in heart ischemia. As Nox5 expression has been described in the cardiovascular system of rabbits, a model of ischaemic cardiomyopathy (IC) was used. Coronary artery ligation was performed in Male New Zealand White rabbits. After 8 weeks, skin and mesenteric arteries were isolated and vascular function assessed by wire myography. Vascular contraction to NA (EMax %KCl: 122±4 vs sham 97±3.7) and U46619 (EMax %KCl: 82±3 vs sham 67±4) were exacerbated in skin arteries from IC (p2+ channels, but an increase in RyR was observed (2^-ddCT: 1.67±0.15 vs sham 0.98±0.08) in VSMCs isolated from IC animals. Peroxiredoxin (Prdx), antioxidant, mRNA was increased in IC (2^-ddCT: 1.95±0.4 vs sham 0.88±0.1, p11 /mL: 4.8±0.6 vs control 1.75±0.2), where Nox5 expression was also increased (AU: 0.11±0.02 vs control MP 0.03±0.006) (p

TRPM7 is a chanzyme that influences cellular Mg 2+ homeostasis and vascular signaling. We demonstrated that aldosterone mediates cellular effects through TRPM7-dependent signaling pathways. Since hyperaldosteronism causes hypertension and Mg 2+ wasting, we questioned whether TRPM7 plays a role in aldosterone-induced hypertension. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo/salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel function was investigated in mesenteric arteries by wire and pressure myography. Protein expression was assessed by western-blot and histology. Cardiac fibroblasts (CF) were isolated from WT and M7+/Δ. M7+/Δ exhibited increased BP by aldo (140mmHg), salt (135mmHg) and aldo/salt (137mmHg) vs M7+/Δ-veh (117mmHg) (p