Role In Vascular Dysfunction Research Articles

Vascular dysfunction in retinitis pigmentosa.

The relationship between ocular haemodynamics and retinitis pigmentosa (RP) has not been fully understood. Reductions in blood flow have been established in RP patients by a variety of studies; however, questions have yet to be answered regarding the role of vascular dysfunction in photoreceptors (PR) degeneration, the causes of vascular dysfunction in RP, as well as the diagnostic, prognostic and perhaps therapeutic potential of measuring ocular haemodynamics in RP patients. While significant evidence supports the theory that vascular dysfunction is associated with but not the cause of PR death in retinitis pigmentosa, evidence suggests that vascular abnormalities in the foveal and parafoveal regions may exacerbate cone cell loss. Additional evidence demonstrates that vascular dysfunction likely results from changes in metabolic demand due to death of PR cells in the retina. Detection and monitoring of ocular blood flow, retinal oxygen saturation, endothelin-1 levels and vascular structural abnormalities could provide diagnostic, prognostic and therapeutic potential for patients with RP.

Mitochondrial Deacetylase Sirt3 as a New Target in Cardiovascular Diseases

Multiple risk factors such as aging, vascular dysfunction, metabolic disorders and hypertension contribute to the pathogenesis of heart failure. The convergent mechanism that may underlie the interplay of these major risk factors is not clear but may provide novel strategies for treatment. Clinical studies show that Sirtuin 3 (Sirt3) expression declines by 40% by age 65 and sedentary lifestyle paralleling the increased incidence of cardiovascular diseases and heart failure while aerobic exercise increases Sirt3 expression and attenuates age‐dependent Sirt3 decline. Meanwhile, the pathophysiological role of Sirt3 depletion is not clear. Mitochondrial deacetylase Sirt3 is critical in regulation of mitochondrial metabolism and it activates a key antioxidant enzyme, superoxide dismutase 2 by deacetylation of specific lysine residues. We hypothesized that Sirt3 plays critical role in vascular dysfunction and hypertension. To test this hypothesis, we have developed new tamoxifen‐inducible endothelium specific Sirt3 knockout (EcSirt3KO), endothelial Sirt3 overexpressing (EcSirt3OX), smooth muscle Sirt3 knockout (SmcSirt3KO), smooth muscle Sirt3 overexpressing (SmcSirt3OX) and global Sirt3 overexpressing (Sirt3OX) mice on C57Bl/6J background, and examined the effect of Sirt3 expression on vascular dysfunction and angiotensin II‐induced hypertension. Sirt3 depletion in EcSirt3KO and SmcSirt3KO mice raises basal vascular permeability in the heart by 2‐fold, reduces endothelial NO, increases blood pressure and accelerates vascular aging compared with wild‐type mice. Angiotensin II infusion in EcSirt3KO and SmcSirt3KO mice caused vascular hyperpermeability, increased vascular hypertrophy, exacerbated endothelial dysfunction and hypertension. Meanwhile, Sirt3 overexpression attenuates vascular aging, normalizes vascular permeability, diminishes vascular inflammation, inhibits vascular oxidative stress, preserves endothelial‐dependent relaxation, reduces vascular hypertrophy and attenuates hypertension in Sham and angiotensin II‐infused EcSirt3OX and SmcSirt3OX mice compared with the wild‐type littermates. We suggest that Sirt3 is central in redox and metabolic regulations of smooth muscle and endothelial cells. As vascular permeability increases, the access of cytokines, inflammatory cells and LDL to tissue is greater contributing to cardiac inflammation, altered tissue remodeling, hypertrophy and dysfunction. Our data support a therapeutic potential of targeting Sirt3 in cardiovascular dysfunction and hypertension which can improve treatment of the heart failure.Support or Funding InformationThis work was supported by funding from National Institute of Health (NHLBI 1R01HL124116) and American Heart Association (16GRNT31230017).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beneficial Changes Promoted by L‐arginine and Aliskiren in Resistance Vessels in 2K1C hypertensive rats

Renovascular hypertension is characterized by increased angiotensin II and oxidative stress and by endothelial dysfunction. By acting on multiple targets and promoting diverse actions, angiotensin II (Ang II) plays a pivotal role in vascular dysfunction. The purpose of this study was to test whether the administration of aliskiren (ALSK) and L‐arginine (L‐arg) would restore endothelial dysfunction and reduce inflammation and oxidative stress in a rat renovascular hypertension model. Hypertension was induced by clipping the right renal artery, and the following five groups were divided: SHAM operated; 2‐kidney, 1‐clip (2K1C); 2K1C plus ALSK; 2K1C plus L‐ARG; and 2K1C plus ALSK+ L‐ARG. The vascular reactivity to norepinephrine (NE; 10(−9) to 2 × 10(−3) mol/L) and acetylcholine (ACh; 10(−10) to 10(−3) mol/L) was evaluated in the mesenteric arteriolar bed through concentration‐effect curves. The structure was assessed by histology and biomolecular analyzes were done via western blot, lipid peroxidation, DHE and DAF. For 4 weeks, blood pressure (BP) was monitored and endothelium‐dependent vasoconstriction and relaxation were assessed in mesenteric arteriolar. ALSK+L‐arg reduced BP and the contractile response to norepinephrine and improved acetylcholine relaxation. Incubation with N‐nitro‐L‐arginine methyl ester (L‐NAME) and L‐NAME+ Indometacin increased the response to norepinephrine in the 2K1C group and the effect was greater and partially improved, respectively, with the treatments. However, It was improved acetylcholine relaxation with the both of the treatments. The bioavailability of nitric oxide increased with the treatments, and the inflammation and oxidative stress was reduced with the treatments. L‐arginine and aliskiren attenuates the morphofunctional deleterious effects of Ang II on resistance vessels. The benefits of L‐arginine and aliskiren seem to occur through restoring the balance of ROS/NO. Therefore, this study opened new avenues for further clinical targeting of the treatment of cardiovascular diseases related to activation of the renin‐angiotensin system. In conclusion, combined ALSK+L‐arg treatment normalizes BP and prevents endothelial dysfunction in 2K1C hypertensive rats.Support or Funding InformationThis research was supported by a CNPq research grants. This study was funded by Fundação de Amparo a Pesquisa do Espirito Santo (FAPES‐Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq‐Brazil).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P53 plays a crucial role in endothelial dysfunction associated with hyperglycemia and ischemia

p53 is a guardian of the genome that protects against carcinogenesis. There is accumulating evidence that p53 is activated with aging. Such activation has been reported to contribute to various age-associated pathologies, but its role in vascular dysfunction is largely unknown. The aim of this study was to investigate whether activation of endothelial p53 has a pathological effect in relation to endothelial function. We established endothelial p53 loss-of-function and gain-of-function models by breeding endothelial-cell specific Cre mice with floxed Trp53 or floxed Mdm2/Mdm4 mice, respectively. Then we induced diabetes by injection of streptozotocin. In the diabetic state, endothelial p53 expression was markedly up-regulated and endothelium-dependent vasodilatation was significantly impaired. Impairment of vasodilatation was significantly ameliorated in endothelial p53 knockout (EC-p53 KO) mice, and deletion of endothelial p53 also significantly enhanced the induction of angiogenesis by ischemia. Conversely, activation of endothelial p53 by deleting Mdm2/Mdm4 reduced both endothelium-dependent vasodilatation and ischemia-induced angiogenesis. Introduction of p53 into human endothelial cells up-regulated the expression of phosphatase and tensin homolog (PTEN), thereby reducing phospho-eNOS levels. Consistent with these results, the beneficial impact of endothelial p53 deletion on endothelial function was attenuated in EC-p53 KO mice with an eNOS-deficient background. These results show that endothelial p53 negatively regulates endothelium-dependent vasodilatation and ischemia-induced angiogenesis, suggesting that inhibition of endothelial p53 could be a novel therapeutic target in patients with metabolic disorders.

Vascular Oxidative Stress: Impact and Therapeutic Approaches.

Oxidative stress has been defined as an imbalance between oxidants and antioxidants and more recently as a disruption of redox signaling and control. It is generally accepted that oxidative stress can lead to cell and tissue injury having a fundamental role in vascular dysfunction. Physiologically, reactive oxygen species (ROS) control vascular function by modulating various redox-sensitive signaling pathways. In vascular disorders, oxidative stress instigates endothelial dysfunction and inflammation, affecting several cells in the vascular wall. Vascular ROS are derived from multiple sources herein discussed, which are prime targets for therapeutic development. This review focuses on oxidative stress in vascular physiopathology and highlights different strategies to inhibit ROS production.

Resveratrol ameliorates endothelial dysfunction in diabetic and obese mice through sirtuin 1 and peroxisome proliferator-activated receptor δ.

Sirtuin 1 (SIRT1) activation reduces oxidative stress, inhibits inflammatory responses, and retards cellular senescence in endothelial cells in mouse models of diabetes. However, whether SIRT1 also plays a protective role in vascular dysfunction of diabetic and obese mice is not fully characterized. Previous work showed that peroxisome proliferator-activated receptor δ (PPARδ) is beneficial in diabetic vascular dysfunction. Whether PPARδ is involved in the beneficial effect of SIRT1 on vascular endothelial function is unknown. We used mice with overexpression of endothelial cell-specific human SIRT1 (SIRT1-Tg) and dominant-negative SIRT1 (SIRT1-mut) fed with normal chow and high fat diet to show that expression of functional SIRT1 in endothelium protects against vascular dysfunction in diet-induced obese mice. Endothelial-specific overexpression of SIRT1 improved endothelium-dependent dilation in aortas treated with risk factors including high glucose, angiotensin II, and lysophosphatidylcholine. Oral treatment with resveratrol treatment improves endothelial function in high fat diet fed wild type Ppard-wt but not in PPARδ knockout Ppard-mut mice. Experiments on isolated arteries also showed that the effect of resveratrol or SIRT1 activator CAY10602 was inhibited by PPARδ antagonist GSK0660. Resveratrol increased PPARδ transcriptional activity in endothelial cells. Results demonstrated here indicated that PPARδ contributes to the beneficial effect of SIRT1 to ameliorate endothelial dysfunction in diabetic and obese mice. These results help to understand SIRT1-based strategy for treating vascular and metabolic dysfunction in the context of obesity and insulin resistance.

6 - Resveratrol improves endothelial function in diabetic and obese mice through SIRT1-PPARδ

SIRT1 activation reduces vascular oxidative stress, inhibits inflammatory responses, and retards cellular senescence in mouse models of diabetes. However, whether SIRT1 also plays a protective role in vascular dysfunction of diabetic and obese mice is not fully characterized. Previous work showed that PPARδ is beneficial in diabetic vascular dysfunction. Whether PPARδ is involved in the effect of SIRT1 on vascular endothelial function is unknown. We used mice with overexpression of endothelial cell-specific human SIRT1 (SIRT1-Tg) and dominant negative SIRT1 (SIRT1-mut) fed with normal chow and high fat diet to show expression of functional SIRT1 in endothelium protects against vascular dysfunction in diet-induced obese mice. Endothelial overexpression of SIRT1 improved endothelium-dependent dilation in aorta treated with risk factors including high glucose, angiotensin II, and lysophosphatidylcholine. Wild type (Ppard-wt) and PPARδ knockout (Ppard-mut) mice on high fat diet were treated with resveratrol. Resveratrol treatment improves endothelial function in Ppard-wt but not Ppard-mut mice. Experiments on isolated arteries ex vivo also showed that the effect of SIRT1 activator resveratrol or CAY10602 can be inhibited by PPARδ antagonist GSK0660. Resveratrol increased PPARδ transcriptional activity in endothelial cells. Results demonstrated here indicated that PPARδ contributes to the beneficial effect of SIRT1 against endothelial dysfunction in diabetic and obese mice. These results help to understand SIRT1-based strategy for treating vascular and metabolic dysfunction in the context of obesity and insulin resistance.

Abstract 049: Endothelial-Derived Microparticles as Biomarkers and Mediators of Endothelial Cell Injury in VEGF Inhibitor-Treated Cancer Patients: Implications in Hypertension

Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, induce severe hypertension although the underlying molecular mechanisms are still unclear. Endothelial microparticles (MPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether MP status is altered in cancer patients treated with VEGFRi and whether they influence endothelial cell function associated with vascular dysfunction in hypertension. Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi. Human aortic endothelial cells (HAEC) were stimulated with human plasma-isolated MPs (10 6 MPs/mL). MP characterization was assessed by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Patients treated with VEGFRi had significantly increased in endothelial cell-derived MPs (EMP) (0.19±0.02 Pre vs. 0.31±0.04 Post-treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (4.10±0.97 vs. 10.57±3.54), corroborating the raise in ET-1 levels (μg/mL: 631.9±46.1 vs. 780.2±37.2) observed in HAEC stimulated with vatalanib (VEGFRi). Post-treatment MPs increased ROS generation in HAEC (100.0 vs. 5 min- 123.4±7.7, 30 min- 162.4±22.5, 60 min- 183.9±44.7), effects that were attenuated by ET A and ET B receptor blockers. VEGFRi post-treatment MPs increased phosphorylation of the inhibitory site of eNOS (Thr 495 ) (100.0 vs. 181.3±18.7) and decreased NO levels in HAEC (100.0 vs. 72.7±9.9) which was inhibited by ET B receptor blockade (eNOS: 109.8±14.9; NO: 142.1±25.9). Gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs (1.0 vs. TNF-α: 5.4±1.8, MCP-1: 2.1±0.3, iNOS: 3.1±0.8, COX2: 2.4±0.4, ICAM1: 2.7±0.6), effects inhibited by BQ123 and BQ788. In conclusion, our data identify EMPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated endothelial cell signalling. These molecular processes may play a role in vascular dysfunction associated with hypertension in VEGFRi-treated cancer patients.

Abstract 114: Increased Circulating Levels of Mitochondrial N-Formyl Peptides Leads to Vascular Dysfunction and High Blood Pressure in Spontaneously Hypertensive Rats

Aims: The most powerful signaling pathway that induces actin polymerization and movement in neutrophils is due to formyl peptide receptor (FPR) activation. FPR was originally identified by its ability to bind bacterial N-formyl peptides. Mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Consequently, mitochondrial N-formyl peptides (F-MITs) are recognized by FPR. Recently, we have observed that FPR is expressed in endothelial and vascular smooth muscle cells (VSMC). We hypothesized that increased plasma levels of F-MITs activates FPR and contributes to microvascular damage and hypertension. Methods: Male SHR (12-week old) were treated for 10 days with FPR antagonist cocktail: Cyclosporin H (CsH, 0.1 mg/kg/day i.p. ) and WRW4 (0.1 mg/kg/day i.p. ) or vehicle. Blood pressure (BP) was measured by telemetry. We also treated Wistar rats (12 week old) with F-MITs (formylated peptide corresponding to the NH2-terminus of mitochondria ND6; 0.02 mg/rat/day i.p. ) or non-formylated peptides (control) for 10 days. Blood was collected to measure F-MITs. Intrarenal arteries and mesenteric resistance arteries (MRA) were isolated and mounted in a wire myograph. Wistar VSMCs were treated with F-MITs (20 min, 10 μM) or control in the presence or absence of FPR antagonists to evaluate actin polymerization. T-test *p<0.05; n=5-6. Results and Conclusion: F-MITs plasma levels were increased in SHR (1.7-fold* vs. Wistar). FPR blockade decreased BP (SHR + vehicle: 151 ± 7.2 vs. SHR+CsH+WRW4: 138 ± 0.4* mmHg) and ameliorated acetylcholine-induced relaxation (Intrarenal; Emax: SHR+vehicle: 10 ± 5 vs. SHR+CsH+WRW4: 28 ± 6* %); (MRA; Emax: SHR+vehicle: 74±10 vs. SHR+CsH+WRW4: 99±0.6* %). F-MITs infusion increased BP in Wistar rats (Control: 133 ± 1.9 vs. F-MITs: 145 ± 4.7* mmHg) and decreased acetylcholine-induced relaxation (EC50; MRA: Control: -8.765 ± 0.05 vs. F-MIT: -8.348 ± 0.08*) (Emax; Intrarenal: Control: 63±4 vs. F-MIT: 46 ± 3* %). F-MITs induced VSMCs actin polymerization (1.8 fold* vs. control) and FPR blockers abolished this result. In conclusion, FMITs play a role in vascular dysfunction and hypertension via FPR activation.

Abstract 060: Mitochondrial Deacetylase Sirt3 in Endothelial and Smooth Muscle Cells Protects From Vascular Dysfunction and Attenuates Hypertension

Vascular dysfunction plays a key role in the development of hypertension and heart disease which causes one-third of deaths worldwide. Cardiovascular disease risk factors reduce mitochondrial deacetylase Sirt3 and Sirt3 declines with age paralleling the increased incidence of cardiovascular disease and hypertension; however, the role of Sirt3 was largely ignored. We hypothesized that Sirt3 plays critical role in vascular dysfunction and hypertension. To test this hypothesis we have developed novel tamoxifen-inducible endothelium specific Sirt3 knockout (Ec Sirt3KO ), endothelial Sirt3 overexpressing (Ec Sirt3OX ), smooth muscle Sirt3 knockout (Smc Sirt3KO ), smooth muscle Sirt3 overexpressing (Smc Sirt3OX ) and global Sirt3 overexpressing (Sirt3OX) mice on C57Bl/6J background, and examined the effect of Sirt3 expression on vascular dysfunction and angiotensin II-induced hypertension. It was found that cell-specific Sirt3 depletion in endothelial and smooth muscle in Ec Sirt3KO and Smc Sirt3KO mice increases vascular permeability by 2-fold, raises T cell and monocyte vascular accumulation, diminishes endothelial NO and increases blood pressure by 10 mm Hg compared with wild-type Sham mice. Angiotensin II infusion in Ec Sirt3KO and Smc Sirt3KO mice caused vascular hyperpermeability, increased vascular hypertrophy, exacerbated endothelial dysfunction and hypertension. Meanwhile, Sirt3 overexpression reduced vascular permeability and diminished vascular inflammation compared to Sham wild-type mice. Sirt3 overexpression in endothelial and smooth muscle prevents angiotensin II-induced vascular hyperpermeability, inhibits vascular oxidative stress, preserves endothelial-dependent relaxation, diminishes vascular hypertrophy and attenuates hypertension by 20 mm Hg in angiotensin II-infused Ec Sirt3OX and Smc Sirt3OX mice compared with the wild-type littermates. Interestingly, Sirt3 depletion in human aortic endothelial cells and human vascular smooth muscle cells reduces expression of differentiation markers. We suggest that Sirt3 is central in redox and metabolic regulations of smooth muscle and endothelial cells. Our data support a therapeutic potential of targeting Sirt3 in vascular dysfunction and hypertension.

Toll‐Like Receptor 9 (TLR9) Is Associated with Vascular Dysfunction in 2‐Kydney 1‐Clip (2K1C) Hipertensive Mice.

IntroductionRenovascular hypertension is the leading cause of secondary hypertension and commonly resistant to antihypertensive therapy. The involvement of toll‐like receptors 9 (TLR9) has already been demonstrated in spontaneously hypertensive rats (SHR) and their activation seems to contribute to the increase in arterial pressure (AP). Therefore, we hypothesized that TLR9 are activated in two‐kidney one‐ clip (2K1C) hypertension, eliciting inflammation and vascular dysfunction, playing a role in the increase of the AP.Methods and ResultsWild type C57BL (WT) and TLR9 knockout (TLR9_KO) mice were anesthetized with isoflurane and were implanted with a silver clip (0.12 mm) around the left renal artery (SHAM operated mice were not implanted with the clip). After 4 weeks, the mice were also anesthetized with isoflurane for direct AP measurement. Afterwards the third‐order mesenteric resistance arteries (MRA) were removed for vascular function evaluation. AP was higher in 2K1C WT mice (133 ± 2 vs 93 ± 4 mmHg in SHAM WT mice), while this increase was partially prevented by the absence of TLR9 (114 ± 5 mmHg in 2K1C TLR9_KO mice). Vascular dysfunction, characterized by increased contractile responses to phenylephrine (Emax: 167 ± 2.2 vs 126 ± 3.5 KCl % in SHAM WT mice) and reduced relaxation to acetylcholine (Emax: 69 ± 1.6 vs 88 ± 1.4 PE % in SHAM WT mice) and sodium nitroprusside (pD2: 7.31 ± 0.07 vs 7.85 ± 0.05 in SHAM WT mice) was found in 2K1C WT mice but not in 2K1C TLR9_KO mice (Emax: 128 ± 2.9 KCl %, 89 ± 2.0 PE % and pD2: 7.78 ± 0.06, when compared to SHAM WT mice, to phenylephrine, acetylcholine and sodium nitroprusside, respectively).ConclusionTLR9 play an important role in vascular dysfunction and AP increase in 2K1C hypertension.Support or Funding InformationFinancial support: FAPESP (Proc. 2015/21976‐1, 2013/20549‐7 and 2016/11988‐5).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gut Microbiota: An Integral Moderator in Health and Disease.

The gut microbiota, as the main member in gut microecology, is an essential mediator in health and disease. The gut microbiota interacts with various organs and systems in the body, including brain, lung, liver, bone, cardiovascular system, and others. Microbiota-derived metabolites such as the short chain fatty acid (SCFA) butyrate are primary signals, which link the gut microbiota and physiology. Recently, the gut microbiota has been identified as the origin of a number of diseases by influencing the related cell signaling pathways such as WNT/beta-catenin pathway in colorectal cancer and T cell receptor signaling in the central nervous system. Moreover, several microRNAs participate in signaling networks through the intervention of the gut microbiota. The interaction between the gut microbiota and miRNAs plays a crucial role in vascular dysfunction and hepatocellular carcinoma (HCC). In this review, we will report and discuss recent findings about the crosstalk between the gut microbiota and physical organs and how the gut microbiota and miRNAs regulate each other while influencing the host via genes, proteins, or metabolites.

Characterization of vascular dysregulation in meriones shawi after high-calorie diet feeding

ABSTRACTThe present study was initiated to characterize vascular dysregulations (contraction and relaxation) associated with metabolic defects in Merions shawi, a rodent from the gerbillidae family, submitted to 12 weeks high-calorie diet. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. In diabetic meriones, body weight increase was associated with hyperglycemia, increased insulinemia, and insulin resistance. Compared to lean meriones, diabetic meriones showed decreased aorta contraction to noradrenaline, which was normalized after NOS inhibition. Endothelium-dependent relaxation to carbachol was enhanced, while relaxing effects of the NO donor SNAP and of diazoxide were unchanged. Insulin-evoked relaxation was depressed in aorta from diabetic meriones, and L-arginine relaxed contracted arteries from diabetic meriones, but not from lean meriones. Urine NOX level and iNOS mRNA muscle expression were significantly higher in diabetic meriones compared to lean animals. These data strongly suggest that iNOS may have a pathogenic role in vascular dysfunction observed in diet-induced diabetic meriones.

Plasma microparticles in Alzheimer's disease: The role of vascular dysfunction.

Cerebrovascular lesions, a potent stimulus for endothelial cell activation, trigger cognitive and degenerative changes and contribute to pathology of Alzheimer's disease (AD). Circulating microparticles (MPs) are actively involved in the pathogenesis of AD and cerebrovascular diseases, which share common vascular risk factors. We examined the plasma changes of endothelial MPs (EMPs) and platelet MPs (PMPs) in AD patients with vascular risk factors. The plasma Annexin V+ CD 41a- CD144+ EMPs and Annexin V+ CD41a+ CD144- PMPs of 37 patients with AD, with or without vascular risk factors (hypertension, diabetes, dyslipidemia, stroke, coronary artery disease, and smoking), and 10 age-matched controls were quantified by flow cytometry. Pearson correlation analysis used to evaluate the linear relationship between variables. Significantly higher plasma levels of EMPs were observed in AD patients with vascular risk factors as compared to the patients without vascular risk factors [Mean Difference (MD): 2587.80, 95% confidence interval (CI) 770.30-4404.80], and control subjects (MD: 4990.60, 95% CI, 3054.40-6926.79). Significant correlations were found between circulating EMPs, total MPs, and PMPs. There were no significant correlations between plasma levels of EMPs/ PMPs, and cognitive decline indices. Circulating EMP levels are influenced by AD disease status, and plasma levels of MPs and PMPs are associated with vascular risk factors in patients with AD. EMP phenotyping, as cellular biomarkers of vascular injury/dysfunction, and their effects on cerebral perfusion, and cognitive decline should be further investigated. Graphical abstract Vascular endothelial cell activation results in release of endothelial-derived microparticles (EMPs), which contributing to vascular dysfunction and cognitive decline.

Abstract P217: Nox Compartmentalization, Protein Oxidation and ER Stress in Vascular Smooth Muscle Cells in Hypertension

In hypertension, activation of NADPH oxidases (Noxs) is associated with oxidative stress and vascular dysfunction. The exact role of each isoform in hypertension-associated vascular injury is still unclear. We investigated the compartmentalization of Noxs in VSMC from resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Expression of Nox1 and Nox4 was increased in SHR cells (96.6±28.7% and 48.2±21.2% vs WKY, p<0.05), as well as basal ROS levels measured by chemiluminescence (110.2±26.4% vs WKY, p<0.05) and amplex red (105.2±33.2% vs WKY, p<0.05). Phosphorylation of unfolded protein response activators, PERK and IRE1α, and expression of ER chaperone BiP were elevated in SHR cells (p<0.05 vs WKY), indicating activation of ER stress response. Immunoblotting after organelle fractionation demonstrated that Noxs are expressed in an organelle-specific manner, with Nox1, 2 and 4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHR cells, NoxA1ds (Nox1 inhibitor, 10μM) and GKT136901 (Nox1/4 inhibitor, 10μM) decreased AngII-induced ROS levels (p<0.001 vs Ctl). Additionally, mito-tempol (mitochondrial-targeted antioxidant, 50nM) and 4-PBA (ER stress inhibitor, 1mM) decreased basal ROS levels in SHR cells (p<0.05 vs Ctl). Furthermore, oxidation of the antioxidant enzymes Peroxiredoxins (Prx) was increased in SHRSP compared to WKY (2.51±0.14 vs 0.56±0.07, p<0.001). One-dimensional isoelectric focusing revealed that cytosolic Prx2 and mitochondrial Prx3 were more oxidized in SHRSP than WKY cells. Using a biotin-tagged dimedone-based probe (DCP-Bio) we identified oxidation of ER stress proteins BiP and IRE1. To investigate the effect of protein oxidation in vascular function, vascular reactivity was evaluated in isolated mesenteric arteries. Inhibition of general oxidation (DTT 1mM; Emax: 111.7±33.1) and peroxiredoxin (Conoidin A 10nM; Emax: 116.0±7.3) reduces vascular contraction in response to noradrenalin in WKY rats (Emax: 166.6±30.2; p<0.05). These findings suggest an important role for Nox1/4 in redox-dependent organelle dysfunction and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

181 Nox compartmentalization and protein oxidation in vascular smooth muscle cells – implications in vascular dysfunction in hypertension

NADPH oxidases (Noxs) are a major source of reactive oxygen species (ROS) in vascular cells. ROS are important signalling molecules with diverse actions. Mechanisms underlying differential ROS effects may relate,...

Restoration of perivascular adipose tissue function in diet-induced obese mice without changing bodyweight.

Background and PurposeWe have recently shown that a reduced function of endothelial nitric oxide synthase (eNOS) in the perivascular adipose tissue (PVAT) contributes crucially to obesity‐induced vascular dysfunction in mice. The current study was conducted to test the hypothesis that vascular dysfunction in obesity can be reversed by in vivo improvement of PVAT eNOS activity.Experimental ApproachMale C57BL/6J mice were fed a high‐fat diet (HFD) for 22 weeks to induce obesity. During the last 4 weeks of HFD feeding, the obese mice were treated p.o. with the standardized Crataegus extract WS® 1442, which has been shown previously to improve eNOS activity.Key ResultsDiet‐induced obesity in mice markedly reduced the vasodilator response of thoracic aorta to acetylcholine in wire myograph experiments. Strikingly, this vascular dysfunction was only evident in PVAT‐containing aorta but not in PVAT‐free aorta. In vivo treatment of obese mice with WS® 1442 had no effect on body weight or epididymal fat mass, but completely restored the vascular function of PVAT‐containing aorta. Feeding a HFD led to a reduced phosphorylation and an enhanced acetylation of PVAT eNOS, both effects were reversed by WS® 1442 treatment.Conclusion and ImplicationsPVAT plays a key role in vascular dysfunction in diet‐induced obese mice. Not obesity itself, but a PVAT dysfunction is responsible for obesity‐induced vascular disorders. Improving PVAT function by pharmacological means (e.g. with WS® 1442) can ameliorate vascular function even without reducing body weight or fat mass.Linked ArticlesThis article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue – Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc

Role of vascular reactive oxygen species in regulating cytochrome P450-4A enzyme expression in Dahl salt-sensitive rats.

The potential contribution of CYP4A enzymes to endothelial dysfunction in Dahl salt-sensitive rats was determined by comparison to SS-5BN consomic rats having chromosome 5 carrying CYP4A alleles from the BN rat introgressed into the SS genetic background. The following experiments were performed in cerebral arteries from HS-fed SS and SS-5BN rats ± the SOD inhibitor DETC and/or the superoxide scavenger Tempol: (i) endothelial function was determined via video microscopy ± acute addition of the CYP4A inhibitor DDMS or Tempol; (ii) vascular oxidative stress was assessed with DHE fluorescence ± acute addition of DDMS, l-NAME, or PEG-SOD; and (iii) CYP4A protein levels were compared by western blotting. In DETC-treated SS-5BN and HS-fed SS rats, (i) DDMS or Tempol ameliorated vascular dysfunction, (ii) DDMS reduced vascular oxidative stress to control levels, (iii) chronic Tempol treatment reduced vascular CYP4A protein expression, and (iv) combined treatment with Tempol and l-NAME prevented the reduction in CYP4A protein expression in MCA of HS-fed SS rats. The CYP4A pathway plays a role in vascular dysfunction in SS rats and there appears to be a direct role of reduced NO availability due to salt-induced oxidant stress in upregulating CYP4A enzyme expression.

Impaired vascular function contributes to exercise intolerance in chronic kidney disease.

Exercise intolerance is an important feature in patients with chronic kidney disease (CKD) and is prognostic for both increased morbidity and mortality. Little is known about the underlying mechanisms in predialysis CKD. This study aimed to gain more insight into the role of vascular dysfunction in the exercise intolerance of predialysis CKD. In addition, vascular-related microRNAs (miRNAs)-as epigenetic regulators of exercise capacity-were analysed. Sixty-three patients with CKD stages 1-5 and 18 healthy controls were included. Peak oxygen consumption (VO2peak) was determined by cardiopulmonary exercise testing, endothelial function by flow-mediated dilation (FMD) and arterial stiffness by carotid-femoral pulse wave velocity (PWV). Plasma miRNA levels (miR-21, miR-126, miR-146a, miR-150 and miR-210) were quantified by quantitative RT-PCR. VO2peak was already impaired in mild CKD (stages 1-3A) and significantly correlated with estimated glomerular filtration rate (eGFR; r = 0.525, P < 0.001). Likewise, both FMD and PWV were significantly correlated with eGFR (r = 0.319, P = 0.007 and r = -0.365, P = 0.001, respectively). In multiple regression analysis, PWV remained one of the strongest independent determinants of VO2peak (β = -0.301, P = 0.01). Of the studied miRNA, circulating levels of miR-146a and miR-150 correlated with eGFR, PWV and VO2peak, but the association with the latter was lost when correcting for PWV. Arterial stiffness contributes to the observed reduced aerobic capacity in predialysis CKD, independent of age, haemoglobin levels and endothelial function and represents a promising therapeutic target for improving exercise capacity in this population. Future work is required to elucidate why higher circulating levels of miR-146a and miR-150 are associated with impaired renal function and increased arterial stiffness.

4-Phenylbutyrate Benefits Traumatic Hemorrhagic Shock in Rats by Attenuating Oxidative Stress, Not by Attenuating Endoplasmic Reticulum Stress.

Vascular dysfunction such as vascular hyporeactivity following severe trauma and shock is a major cause of death in injured patients. Oxidative stress and endoplasmic reticulum stress play an important role in vascular dysfunction. The objective of the present study was to determine whether or not 4-phenylbutyrate can improve vascular dysfunction and elicit antishock effects by inhibiting oxidative and endoplasmic reticulum stress. Prospective, randomized, controlled laboratory experiment. State key laboratory of trauma, burns, and combined injury. Five hundred and fifty-two Sprague-Dawley rats. Rats were anesthetized, and a model of traumatic hemorrhagic shock was established by left femur fracture and hemorrhage. The effects of 4-phenylbutyrate (5, 20, 50, 100, 200, and 300 mg/kg) on vascular reactivity, animal survival, hemodynamics, and vital organ function in traumatic hemorrhagic shock rats and cultured vascular smooth muscle cells, and the relationship to oxidative stress and endoplasmic reticulum stress was observed. Lower doses of 4-phenylbutyrate significantly improved the vascular function, stabilized the hemodynamics, and increased the tissue blood flow and vital organ function in traumatic hemorrhagic shock rats, and markedly improved the survival outcomes. Among all dosages observed in the present study, 20 mg/kg of 4-phenylbutyrate had the best effect. Further results indicated that 4-phenylbutyrate significantly inhibited the oxidative stress, decreased shock-induced oxidative stress index such as the production of reactive oxygen species, increased the antioxidant enzyme levels such as superoxide dismutase, catalase, and glutathione, and improved the mitochondrial function by inhibiting the opening of the mitochondrial permeability transition pore in rat artery and vascular smooth muscle cells. In contrast, 4-phenylbutyrate did not affect the changes of endoplasmic reticulum stress markers following traumatic hemorrhagic shock. Furthermore, 4-phenylbutyrate increased the nuclear levels of nuclear factor-E2-related factor 2, and decreased the nuclear levels of nuclear factor κB in hypoxic vascular smooth muscle cells. 4-phenylbutyrate has beneficial effects for traumatic hemorrhagic shock including improving animal survival and protecting organ function. These beneficial effects of 4-phenylbutyrate in traumatic hemorrhagic shock result from its vascular function protection via attenuation of the oxidative stress and mitochondrial permeability transition pore opening. Nuclear factor-E2-related factor 2 and nuclear factor-κB may be involved in 4-phenylbutyrate-mediated inhibition of oxidative stress.