Vascular Dysfunction In Hypertension Research Articles

Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension.

Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

The angiotensin II type 1 receptor mediates the induction of oxidative stress, apoptosis, and autophagy in HUVECs induced by angiotensin II

Angiotensin (Ang) II, which is the central effector of the renin-angiotensin system (RAS), is one of the principal mediators of vascular dysfunction in hypertension and cardiovascular diseases. Proper vascular function is mediated by oxidative stress, cell apoptosis, and autophagy. However, the underlying signaling pathways and the major RAS components involved in this process are still not fully understood. In the present study, the effect of Ang II on reactive oxygen species (ROS) production, apoptosis induction, and autophagy in human umbilical vein endothelial cells (HUVECs) was investigated. An Annexin V kit was used for the apoptosis analysis, and caspase-3/7 activities were measured with the Caspase-Glo 3/7 Assay Kit. ROS production was measured using a 2,7-dichlorodihydrofluorescein diacetate probe whereas detection of autophagy was performed using acridine orange staining. We found that Ang II increases oxidative stress via ROS production, cell apoptosis via caspase 3/7, and the mitochondrial membrane potential (MMP). Interestingly, losartan, being an antagonist of the angiotensin II type 1 receptor (AT1R), has demonstrated the ability to restore autophagy levels to that of the control group subsequent to its induction by Ang II. The latter can thus induce endothelial cell damage, through excessive oxidative stress and defective autophagy-related apoptosis, which can be inhibited by losartan. These findings reinforce the pivotal role played by the Ang II/AT1R axis in the pathogenesis of vascular damage and bolster our knowledge of the role played by ROS/autophagy-related apoptosis via AT1R in the pathogenesis of hypertension and vascular diseases.

Calcium handling coupled to the endothelin ETA and ETB receptor-mediated vasoconstriction in resistance arteries: Differential regulation by PI3K, PKC and RhoK

Abnormal endothelin-1 (ET-1) activity is involved in the pathogenesis of vascular diseases such as essential and pulmonary arterial hypertension, coronary artery disease, and cerebrovascular disease, blockade of ET receptors having shown efficacy in clinical assays and experimental models of hypertension. Augmented Ca2+ influx and changes in Ca2+ sensitization associated with arterial vasoconstriction underlie increased systemic vascular resistance in hypertension. Since peripheral resistance arteries play a key role in blood pressure regulation, we aimed to determine here the specific Ca2+ signaling mechanisms linked to the ET receptor-mediated vasoconstriction in resistance arteries and their selective regulation by protein kinase C (PKC), Rho kinase (RhoK), the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK). ET-1-induced contraction was mediated by the endothelin ETA receptor with a minor contribution of vascular smooth muscle (VSM) endothelin ETB receptors. ET receptor activation elicited Ca2+ mobilization from intracellular stores, extracellular Ca2+ influx and Ca2+ sensitization associated with contraction in resistance arteries. Vasoconstriction induced by ET-1 was largely dependent on activation of canonical transient receptor potential channel 3 (TRPC3) and extracellular Ca2+ influx through nifedipine-sensitive voltage-dependent Ca2+ channels. PI3K inhibition reduced intracellular Ca2+ mobilization and Ca2+ entry without altering vasoconstriction elicited by ET-1, while PKC has dual opposite actions by enhancing Ca2+ influx associated with contraction, and by inhibiting Ca2+ release from intracellular stores. RhoK was a major determinant of the enhanced sensitivity of the contractile filaments underlying ET-1 vasoconstriction, with also a modulatory positive action on Ca2+ influx and intracellular Ca2+ release. Augmented RhoK and PKC activities are involved in vascular dysfunction in hypertension and vascular complications of insulin-resistant states, and these kinases are thus potential pharmacological targets in vascular diseases in which the ET pathway is impaired.

Protein disulfide isomerase regulation of Nox1 contributes to vascular dysfunction in hypertension

Protein disulfide isomerase regulation of Nox1 contributes to vascular dysfunction in hypertension

Abstract 020: Nox5 Regulates Vascular Smooth Muscle Cell De-differentiation In Human Hypertension

Nox5 is an important source of reactive oxygen species and aberrant redox-sensitive signalling in vascular smooth muscle cells (VSMC) in human hypertension. We aimed to characterize the VSMC proteomic profile and investigate the effects of Nox5-derived ROS on VSMC phenotype in human hypertension. VSMC from resistance arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Proteins were labelled with isobaric tandem mass tags and identified by liquid chromatography tandem mass spectrometry. The oxidative proteome was assessed using stable isotope-labelled iodoacetamide to target cysteine thiols. Nox5 silencing was performed by siRNA. Protein expression was detected by western blotting. Pro-inflammatory cytokines (IL-6, IL-8) and pro-collagen I was measured by ELISA in the culture media. Proteomic analysis identified 207 proteins upregulated in HT subjects (fold change>1.5, p<0.05). Gene ontology enrichment analysis showed proteins upregulated in HT were involved in extracellular matrix (ECM) organization, immune response and cell proliferation. ECM proteins (COL1A1, COL9A1, COL10A1, FBN1, FBLN1) and proteins related to interferon and IL-1β pathways (IFIT1, IFIT2, IFIT3, MX1, MX2, ABCA1, ABCA2, IL1RAP, CD36, ICAM1) were increased in cells from HT subjects. The VSMC oxidative proteome analysis identified 88 cysteine-containing peptides highly oxidized in HT (fold change>1.5, p<0.05), including COL11A1, COL16A1, FBLN1 and FBLN2. VSMCs from HT exhibit increased expression of the proliferation marker, PCNA (0.162±0.3 vs NT:0.051±0.04RFU, p<0.05) and pro-collagen I (23.6±2 vs NT:13.2±0.3ng/ml, p<0.05). Production of pro-inflammatory cytokines IL-6 (501.8±23.6 vs NT:121.7±6.4pg/mL) and IL-8 (373.6±34.1 vs NT:262.5±24.6pg/mL, p<0.05) were increased in HT. Nox5 silencing in VSMC from HT subjects reduced PCNA expression(43%), pro-collagen I release (8%), baseline and LPS-induced IL-6 (30% baseline, 43% LPS-induced) and IL-8 (21% baseline, 23% LPS-induced) release (p<0.05). We provide new insights into the proteomic changes related to vascular phenotype in hypertension and demonstrated that Nox5 plays an important role in VSMC phenotypic switching associated with vascular dysfunction in hypertension.

Antihypertensive effect of Mali-Nil surin rice bran hydrolysate and its mechanisms related to the EDHF-mediated vasorelaxation and L-type Ca2+ channel-mediated vasoconstriction in L-NAME hypertensive rats.

Mali-Nil Surin rice bran hydrolysate (MRH) contains highly nutritional proteins and beneficial phenolic compounds. This study investigated an antihypertensive effect of MRH and evaluated the mechanisms mediating this action in Nω-nitro-L-arginine-methyl ester (L-NAME)-induced hypertensive rats. Antihypertensive activity was determined in male rats orally administered with MRH (100 or 300mg/kg) or enalapril (15mg/kg) daily together with L-NAME (50mg/kg/day) in drinking water, for 21 days. Concurrent oral treatment with MRH lowered the high blood pressure in the L-NAME-induced hypertensive rats. MRH treatment improved endothelial function and increased the endothelium-derived hyperpolarizing factor-mediated vasorelaxation in L-NAME hypertensive rats. L-NAME rats treated with MRH had reduced adrenergic hypercontractility, which was associated with a decrease in L-type calcium channel-mediated vasoconstriction. In addition, MRH exhibited antioxidant activity in hypertensive rats, as indicated by suppression of vascular superoxide anion production and reduction of malondialdehyde levels, as well as magnification of superoxide dismutase and catalase activities in serum. This study demonstrated the nutraceutical potential of MRH to prevent oxidative stress-related vascular dysfunction in hypertension.

Abstract P090: Nox5 Induces Vascular Damage Through C-src Activation In Human Hypertension

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%) (p<0.05, vs NT). Activation of c-Src (101±26%), PKC (96±33%), MLC 20 (416±71%) and Ang II-induced Ca 2+ influx (574±44 vs NT:451±26) were also increased in HT (p<0.05, vs NT). Melittin reduced Ang II-induced ROS generation in both groups (p<0.05 vs Ctl). In contrast, p22phox silencing increased ROS in both groups, an effect blocked by melittin (p<0.05 vs Ctl). Nox5 inhibition reduced Ang II-induced c-Src phosphorylation and oxidation. In HT, p22phox silencing was associated with sustained Ang II-induced PKC (83±21% vs Ctl) and MLC 20 (89±22% vs Ctl) phosphorylation, effects blocked by melittin and PP2 (p<0.05 vs Ctl). Nox5 and c-Src inhibition reduced Ca 2+ 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.

Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress.

Hypertension represents a major risk factor for stroke, myocardial infarction, and heart failure and affects 30% of the adult population. Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear. The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level. We hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in hypertension, but increased Sirt3 protects vascular function and decreases hypertension. To test the therapeutic potential of targeting Sirt3 expression, we developed new transgenic mice with global Sirt3OX (Sirt3 overexpression), which protects from endothelial dysfunction, vascular oxidative stress, and hypertrophy and attenuates Ang II (angiotensin II) and deoxycorticosterone acetate-salt induced hypertension. Global Sirt3 depletion in Sirt3-/- mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM [vascular cell adhesion molecule-1], ICAM [intercellular adhesion molecule-1], and MCP1 [monocyte chemoattractant protein 1]), increases inflammatory cell infiltration in the kidney, reduces telomerase expression, and accelerates vascular senescence and age-dependent hypertension; conversely, increased Sirt3 expression in Sirt3OX mice prevents these deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with normotensive subjects. We suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.

Circulating Extracellular Vesicles in Normotension Restrain Vasodilation in Resistance Arteries.

Extracellular vesicles (EVs) have been described as novel biomarkers and bioactivators in vascular dysfunction in hypertension. However, the mechanism(s) by which EVs affect vascular function is unknown. To examine the effects of EVs on endothelial-dependent vasodilation (acetylcholine), we isolated circulating EVs from platelet-poor plasma using a low centrifugation speed (17 000g) and mesenteric resistance arteries from 12-week-old normotensive WKYs (Wistar-Kyoto rats) and SHRs (spontaneously hypertensive rats). Arteries were cannulated on a pressure myograph, and EVs were added to the vessel lumen and circulating bath. We found that circulating EVs from normotensive WKY reduced vasodilation of normotensive WKY arteries but had no effect on hypertensive SHR arteries. In contrast, EVs from hypertensive SHR failed to reduce vasodilation of arteries from both WKY and SHR. The restraining effect on vasodilation by EVs from normotensive WKY may be mediated by inhibition of eNOS (endothelial NO synthase), as addition of L-nitro-arginine methyl ester did not provide any additive effect. Moreover, circulating EVs from normotensive 6-week-old SHR-an age where SHRs have not yet developed hypertension-had similar restraining effect on vasodilation. In addition, delipidation of EVs did not alter the restraining effect of EVs from WKY but did restore the restraining effect of EVs from SHR. Finally, EVs from normotensive humans also restrained vasodilation of normotensive mouse arteries-an effect not observed in EVs from hypertensive humans. Taken together, our data support a vasoactive role of EVs that is altered in hypertension.

Relaxin reduces endothelium-derived vasoconstriction in hypertension: Revealing new therapeutic insights.

Endothelium-derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium-derived prostacyclin (PGI2 ) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium-derived vasoconstriction has never been investigated. We tested the hypothesis that short-term relaxin treatment mitigates endothelium-derived vasoconstriction in spontaneously hypertensive rats (SHR). Male Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20mmol·L-1 sodium acetate) or relaxin (13.3μg·kg-1 ·hr-1 ) using osmotic minipumps for 3days. Vascular reactivity to the endothelium-dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC-MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively. Relaxin treatment ameliorated hypertension-induced endothelial dysfunction by increasing NO-dependent relaxation and reducing endothelium-dependent contraction. Notably, short-term relaxin treatment up-regulated mesenteric PGI2 receptor (IP) expression, permitting PGI2 -IP-mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension-induced increase in prostanoid-producing enzymes and reduction in PGI2 -evoked contractions. Relaxin has region-dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium-derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium-derived vasoconstriction including hypertension.

Abstract 068: Interplay Between Nox5 and Endoplasmic Reticulum Stress Regulates Vascular Signalling in Human Hypertension

Nox5, a major ROS-generating oxidase in human vessels, regulates vascular contraction. We demonstrated an ER-perinuclear Nox5 localization and questioned the role of ER stress in Nox5 regulation. Vascular smooth muscle cells (VSMC) were isolated from small arteries from subcutaneous fat from normotensive (NT) and hypertensive (HT) subjects. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); peroxiredoxin/DJ-1 oxidation, activation of ER stress and contractile signalling (IRE1α, Src, PKC, MLC phosphorylation; immunoblotting) and actin cytoskeleton organization (phalloidin staining) were assessed. In hypertension, ROS levels (139±27% vs NT, p<0.05), oxidation of peroxiredoxin (870.4±188.7% vs NT, p<0.05) and DJ-1 (125±34% vs NT, p<0.05) were increased. IRE1α phosphorylation was increased in the HT group (58±21% vs NT, p<0.05). ER stress inhibition (4-PBA, 1mM) reduced ROS levels in HT subjects (20±6% vs NT, p<0.05), suggesting association between ER and oxidative stress. Nox5 expression was increased in the HT group (103±23% vs NT, p<0.05) in a compartment specific manner: Nox5 levels were reduced in plasma membrane (45±7% vs NT, p<0.05), but increased in the ER/nuclear fraction (46±13% vs NT, p<0.05). IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in the HT group (65±5% vs Ctl, p<0.05), while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p<0.05). To investigate the role of Nox5 on contractile signalling, cells were treated with mellitin (100nM), a Nox5 inhibitor. ROS generation and phosphorylation of c-Src, PKC and MLC 20 induced by Ang II were reduced by mellitin in both groups (p<0.05 vs Ctl). In contrast, silencing of p22phox increased ROS and activation of c-Src, PKC and MLC 20 in both groups, an effect blocked by mellitin (p<0.05 vs Ctl). VSMC from hypertensive subjects had increased number of stress fibres, an effect attenuated by mellitin. Our findings demonstrate that Nox5 is upregulated in a compartment specific manner and is regulated by ER stress in hypertension. Nox5 upregulation influences pro-contractile signalling and cytoskeleton reorganization in VSMC, processes that contribute to vascular dysfunction in hypertension.

Antioxidant and antihypertensive responses to oral nitrite involves activation of the Nrf2 pathway

Impaired redox balance contributes to the cardiovascular alterations of hypertension and activation of nuclear factor erythroid 2–related factor 2 (Nrf2) pathway may counteract these alterations. While nitrite recycles back to NO and exerts antioxidant and antihypertensive effects, the mechanisms involved in these responses are not fully understood. We hypothesized that nitrite treatment of two-kidney, one-clip (2K1C) hypertensive rats activates the Nrf2 pathway, promotes the transcription of antioxidant genes, and improves the vascular redox imbalance and dysfunction in this model. Two doses of oral nitrite were studied: 15 mg/kg and the sub-antihypertensive dose of 1 mg/kg. Nitrite 15 mg/kg (but not 1 mg/kg) decreased blood pressure and increased circulating plasma nitrite and nitrate. Both doses blunted hypertension-induced increases in mesenteric artery reactive oxygen species concentrations assessed by DHE technique and restored the impaired mesenteric artery responses to acetylcholine. While 2K1C hypertension decreased nuclear Nrf2 accumulation, both doses of nitrite increased nuclear Nrf2 accumulation and mRNA expression of Nrf2-regulated genes including superoxide dismutase-1 (SOD1), catalase (CAT), glutathione peroxidase (GPX), thioredoxin-1(TRDX-1) and -2 (TRDX-2). To further confirm nitrite-mediated antioxidant effects, we measured vascular SOD and GPX activity and we found that nitrite at 1 or 15 mg/kg increased the activity of both enzymes (P < 0.05). These results suggest that activation of the Nrf2 pathway promotes antioxidant effects of nitrite, which may improve the vascular dysfunction in hypertension, even when nitrite is given at a sub-antihypertensive dose. These findings may have many clinical implications, particularly in the therapy of hypertension and other cardiovascular diseases.

Role of Complement-Related Inflammation and Vascular Dysfunction in Hypertension.

Role of Complement-Related Inflammation and Vascular Dysfunction in Hypertension.

Extracellular vesicles from Wistar Kyoto and spontaneously hypertensive rats have differential vasodilatory responses in resistance arteries

Extracellular vesicles (EVs) have been described as novel bio‐markers and bio‐activators in vascular dysfunction in hypertension (HTN). However, the mechanism by which EVs affect vascular function is unknown. To examine the effects of EVs on acetylcholine (ACh)‐mediated vasodilation, we isolated 3rd order mesenteric arteries and EVs from 12‐wk old normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Arteries were cannulated on a pressure myograph and EVs (~6×107EV/mL) were added to the vessel lumen and circulating bath. No significant difference in ACh vasodilation was observed in control WKY and SHR arteries (no EVs). Interestingly, WKY arteries treated with WKY EVs reduced vasodilation compared to control arteries, an effect lost when treated with SHR EVs. ACh‐mediated vasodilation was not altered in SHR arteries when treated with either WKY or SHR EVs. WKY arteries treated with EVs isolated from 6‐wk old WKY and SHR, an age where SHR rats have not yet developed HTN, or with delipidated EVs from 12‐wk old WKY and SHR rats resulted in reduced vasodilation. Together, these data suggest that normotensive EVs reduce ACh‐mediated vasodilation and upon development of hypertension fail to reduced vasodilation. However, reduced vasodilation can be restored by delipidation of the SHR EVs or SHR EVs obtained prior to development of hypertension. Analysis of isolated EVs showed a larger number of CD45‐positive EVs in 12 week‐old SHR rats, indicating an increase in EVs from inflammatory cells following the development of hypertension. Using EVs isolated from human blood samples, a similar reduction in vasodilation was observed in arteries exposed to EVs isolated from normotensive humans. This effect was lost when arteries were treated with EVs isolated from hypertensive patients. This data supports the functional role of EVs in vascular regulation that is altered in HTN.Support or Funding InformationK99HL143165, K23HL12610, HL088554This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Influence of Age on Anticontractile Effect of Perivascular Adipose Tissue in Normotensive and Hypertensive Rats.

Perivascular adipose tissue (PVAT) and its vasomodulatory effects play an important role in the physiology and pathophysiology of blood vessels. Alterations in PVAT associated with reduction in its anticontractile influence are proven to contribute to vascular dysfunction in hypertension. The aim of this study was to examine whether the changes in PVAT properties could participate in progression of vascular abnormalities in developing spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) rats and SHR, both in 5th and in 12th week of age, were used. Systolic blood pressure was similar between WKY rats and SHR in 5th week of age; however, in 12th week, it was significantly increased in SHR comparing to WKY rats. The amount of retroperitoneal fat was higher in WKY rats in both age groups, whereas body weight was higher in WKY rats only in 12th week, when compared to age-matched SHR. From isolated superior mesenteric arteries, two ring preparations were prepared for isometric tension recording, one with PVAT intact and other with PVAT removed. In WKY rats as well as in SHR, arterial contractile responses to noradrenaline, applied cumulatively on rings, were significantly inhibited in the presence of intact PVAT. In both age groups, anticontractile effect of PVAT was higher in WKY rats than in SHR. Neurogenic contractions, induced by electrical stimulation of perivascular sympathoadrenergic nerves, were significantly attenuated in the presence of PVAT in WKY mesenteric arteries from both age groups; however, in arteries from SHR, intact PVAT had no influence on this type of contractile responses. The results suggest that in SHR impairment of anticontractile effect of PVAT precedes hypertension and might contribute to its development.

A10274 Interplay between Nox-regulated oxidative stress and ER stress response in experimental hypertension

Objectives: Nox-derived ROS and ER stress are implicated in vascular dysfunction in hypertension. Evidence suggests a cross-talk between these processes; however the mechanisms involved are still unknown. We investigated how oxidative stress impacts the oxidative proteome and the ER stress response in VSMC from SHR rats. Methods: Nox compartmentalization (cell fractionation), O2- (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase (PTP), peroxiredoxin (Prx) oxidation) and ER stress (PERK, IRE1α, phosphorylation/oxidation) were assessed in SHR VSMC. VSMC proliferation was measured by FACS and vascular reactivity by myography. Results: High levels of O2- (67%) and H2O2 (204%) were observed in SHR, p < 0.05 vs WKY. Reversible and irreversible protein oxidation were also increased in SHR (p < 0.05 vs WKY). ER stress was upregulated in SHR, as phosphorylation of PERK and IRE1α were increased (p < 0.05 vs WKY). Noxs were upregulated in a compartment-specific manner in SHR: Nox1 in plasma membrane (1.6 fold) and Nox4 in ER (1.3 fold, p < 0.05 vs WKY). Inhibition of Nox1 (NoxA1ds) and Nox1/4 (GKT137831) normalized vascular ROS generation and protein oxidation in SHR. NoxA1ds reduced PERK phosphorylation, while GKT137831 reduced IRE1α sulfenylation. Inhibition of ER stress (4-PBA) reduced ROS levels and expression of Nox4 in SHR. Increased VSMC proliferation in SHR (51%, p < 0.05 vs WKY) was normalized by GKT137831 and 4-PBA. Vascular hypercontractility, assessed in mesenteric arteries, was attenuated by 4-PBA. Conclusion: Our findings demonstrate that in hypertension oxidative stress is associated with ER stress through processes involving Nox decompartmentalization, where Nox1 mediated activation of PERK and Nox4 induced oxidation of IRE1α.

A9595 Crosstalk Between Vascular Redox and Calcium Signalling in Hypertension Involves Transient Receptor Potential Melastatin 2 Channel Activation

Objectives: ROS regulates Ca2+ homeostasis by mechanisms still not fully understood. TRPM2, a redox-sensitive channel permeable to Ca2+ and Na+, is activated by H2O2 via oxidation or ADPR production. We tested the hypothesis that TRPM2 is a molecular target of ROS leading to regulation of Ca2+ levels and vascular function in hypertension. Methods: VSMCs from normotensive and hypertensive subject were used. Ca2+ and Na+ influx was assessed by fluorescence microscopy. Results: O2- (1.65 fold) and H2O2 (1.88 fold) generation were enhanced in VSMCs from hypertensive subjects (p < 0.05). Ca2+ (AUC: 1.36 fold) and Na+ influx (AUC: 1.2 fold) were also increased in cells from hypertensive subjects (p < 0.05), an effect blocked by PEG-catalase and, 2-APB, 8-br or olaparib (TRPM2 inhibitors). Additionally, in the absence of Na+, [Ca2+]i was reduced to levels detected in cells from normotensive subjects. TRPM2-induced Na+ influx may influence the sodium-calcium exchanger (NCX), which in reverse mode, promotes Ca2+ influx. Benzamil, KB-R7943 and YM244769 (NCX inhibitors) also ameliorated the increased Ca2+ influx observed in hypertension. MLC20 activation, a marker of cell contraction, was increased in VSMCs from hypertensive subjects (1.72 fold), an effect reversed by TRPM2 and NCX inhibitors (p < 0.05). Arteries from hypertensive mice exhibit increased TRPM2 (1.5 fold) and NCX (1.9 fold) mRNA expression and increased U46619-induced vasoconstriction (Emax: 7.6 ± 0.2 vs 10.21 ± 0.3), effect reversed by TRPM2 and NCX inhibitors (p < 0.05). Conclusion: In conclusion, activation of redox-dependent TRPM2 channel contributes to increased calcium and sodium influx, partially through NCX activation. The results highlight a novel pathway linking ROS to Ca2+ and Na+ signalling through TRPM2/NCX, leading to vascular dysfunction in hypertension.

A15156 G protein-coupled receptor kinase 2 is a novel modulator of oxidative stress and prostanoids production in adipocytes in hypertension

Objectives:Oxidative stress and prostanoids released from adipose tissue participate in vascular dysfunction in hypertension and obesity. G protein-coupled receptor kinase 2 (GRK2) is increased in obesity and hypertension and regulates adiposity. We investigated the role of GRK2 in oxidative stress