ACh-induced Vasodilation Research Articles

Abstract 44: VEGF Inhibitor-induced Vascular Dysfunction Is Ameliorated By PARP/TRPM2 Inhibition

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.

Exercise training mitigates ER stress and UCP2 deficiency-associated coronary vascular dysfunction in atherosclerosis

Endoplasmic reticulum (ER) stress and uncoupling protein-2 (UCP2) activation are opposing modulators of endothelial dysfunction in atherosclerosis. Exercise reduces atherosclerosis plaques and enhances endothelial function. Our aim was to understand how exercise affects ER stress and UCP2 activation, and how that relates to endothelial dysfunction in an atherosclerotic murine model. Wild type (C57BL/6, WT) and apolipoprotein-E-knockout (ApoEtm1Unc, ApoE KO) mice underwent treadmill exercise training (EX) or remained sedentary for 12 weeks. Acetylcholine (ACh)-induced endothelium-dependent vasodilation was determined in the presence of an eNOS inhibitor (L-NAME), UCP2 inhibitor (genipin), and ER stress inducer (tunicamycin). UCP2, ER stress markers and NLRP3 inflammasome signaling were quantified by western blotting. p67phox and superoxide were visualized using immunofluorescence and DHE staining. Nitric oxide (NO) was measured by nitrate/nitrite assay. ACh-induced vasodilation was attenuated in coronary arterioles of ApoE KO mice but improved in ApoE KO-EX mice. Treatment of coronary arterioles with L-NAME, tunicamycin, and genipin significantly attenuated ACh-induced vasodilation in all mice except for ApoE KO mice. Exercise reduced expression of ER stress proteins, TXNIP/NLRP3 inflammasome signaling cascades, and Bax expression in the heart of ApoE KO-EX mice. Further, exercise diminished superoxide production and NADPH oxidase p67phox expression in coronary arterioles while simultaneously increasing UCP2 expression and nitric oxide (NO) production in the heart of ApoE KO-EX mice. Routine exercise alleviates endothelial dysfunction in atherosclerotic coronary arterioles in an eNOS, UCP2, and ER stress signaling specific manner, and resulting in reduced TXNIP/NLRP3 inflammasome activity and oxidative stress.

Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation.

Deletion of pannexin-1 (Panx-1) leads not only to a reduction in endothelium-derived hyperpolarization but also to an increase in NO-mediated vasodilation. Therefore, we evaluated the participation of Panx-1-formed channels in the control of membrane potential and [Ca2+]i of endothelial cells. Changes in NO-mediated vasodilation, membrane potential, superoxide anion (O2·–) formation, and endothelial cell [Ca2+]i were analyzed in rat isolated mesenteric arterial beds and primary cultures of mesenteric endothelial cells. Inhibition of Panx-1 channels with probenecid (1 mM) or the Panx-1 blocking peptide 10Panx (60 μM) evoked an increase in the ACh (100 nM)-induced vasodilation of KCl-contracted mesenteries and in the phosphorylation level of endothelial NO synthase (eNOS) at serine 1177 (P-eNOSS1177) and Akt at serine 473 (P-AktS473). In addition, probenecid or 10Panx application activated a rapid, tetrodotoxin (TTX, 300 nM)-sensitive, membrane potential depolarization and [Ca2+]i increase in endothelial cells. Interestingly, the endothelial cell depolarization was converted into a transient spike after removing Ca2+ ions from the buffer solution and in the presence of 100 μM mibefradil or 10 μM Ni2+. As expected, Ni2+ also abolished the increment in [Ca2+]i. Expression of Nav1.2, Nav1.6, and Cav3.2 isoforms of voltage-dependent Na+ and Ca2+ channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O2·– production. Treatment with 10 μM TEMPOL or 100 μM apocynin prevented the increase in O2·– formation, ACh-induced vasodilation, P-eNOSS1177, and P-AktS473 observed in response to Panx-1 inhibition. These findings indicate that the Panx-1 channel blockade triggers a novel complex signaling pathway initiated by the sequential activation of TTX-sensitive Nav channels and Cav3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOSS1177, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.

Systemic microvascular endothelial dysfunction is associated with reduction of left ventricular ejection fraction in chronic Chagas disease patients

Abstract Introduction Chagas disease (CD) affects millions of patients around the world, many of whom may develop cardiovascular complications, including heart failure and stroke. Notwithstanding, little is known about the effects of CD in the reactivity of the systemic microcirculation. Purpose The present study aims to compare the microvascular reactivity (MR) of a group of patients in the chronic phase of CD with healthy volunteers, matched for sex and age. In addition, we sought to evaluate the association between MR and left ventricular ejection fraction (LVEF) in CD patients. Methods Systemic microvascular flow and endothelium-dependent vasodilation were assessed using laser speckle contrast imaging, coupled with acetylcholine (Ach) iontophoresis in the forearm skin. Vascular reactivity data were expressed as area under the curve (AUC) of Ach-induced vasodilation. Anthropometry, medications, biochemistry, and echocardiographic data were obtained from medical records. Results Thirty-six patients were compared to 25 healthy individuals (controls). Ach-induced vasodilation was higher in controls, when compared to patients (maximum increase of microvascular flow from baseline of 106% and of 48% respectively, P<0.0001; Mann Whitney test). There was a significant association between LVEF, stratified into quartiles, and MR (P value for linear trend = 0.002; one way ANOVA, Figure 1). In addition, there was no difference in MR between CD patients with normal LVEF and the control group. In CD patients, MR was independent of the presence of arterial hypertension or diabetes. Conclusion We have shown that the reduction of MR is associated with a decrease of LVEF in a group of chronic CD patients. The results were not affected by comorbidities, such as hypertension or diabetes. The evaluation of systemic endothelial function may be useful to tailor therapeutic and preventive approaches, targeted at systolic left ventricular failure associated with chronic CD cardiomyopathy. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro

NO-induced vasodilation correlates directly with BP in smooth muscle-Na/Ca exchanger-1-engineered mice: elevated BP does not attenuate endothelial function.

Arterial smooth muscle Na+/Ca2+ exchanger-1 (SM-NCX1) promotes vasoconstriction or vasodilation by mediating, respectively, Ca2+ influx or efflux. In vivo, SM-NCX1 mediates net Ca2+ influx to help maintain myogenic tone (MT) and neuronally activated constriction. SM-NCX1-TG (overexpressing transgenic) mice have increased MT and mean blood pressure (MBP; +13.5 mmHg); SM-NCX1-KO (knockout) mice have reduced MT and MBP (-11.1 mmHg). Endothelium-dependent vasodilation (EDV) is often impaired in hypertension. We tested whether genetically engineered SM-NCX1 expression and consequent BP changes similarly alter EDV. Isolated, pressurized mesenteric resistance arteries with MT from SM-NCX1-TG and conditional SM-NCX1-KO mice, and femoral arteries in vivo from TG mice were studied. Acetylcholine (ACh)-dilated TG arteries with MT slightly more than control or KO arteries, implying that SM-NCX1 overexpression does not impair EDV. In preconstricted KO, but not TG mouse arteries, however, ACh- and bradykinin-triggered vasodilation was markedly attenuated. To circumvent the endothelium, phenylephrine-constricted resistance arteries were tested with Na-nitroprusside [SNP; nitric oxide (NO) donor] and cGMP. This endothelium-independent vasodilation was augmented in TG but attenuated in KO arteries that lack NCX1-mediated Ca2+ clearance. Baseline cytosolic Ca2+ ([Ca2+]cyt) was elevated in TG femoral arteries in vivo, supporting the high BP; furthermore, SNP-triggered [Ca2+]cyt decline and vasodilation were augmented as NO and cGMP promote myocyte polarization thereby enhancing NCX1-mediated Ca2+ efflux. The TG mouse data indicate that BP elevation does not attenuate endothelium-dependent vasodilation. Thus, in essential hypertension and many models the endothelial impairment that supports the hypertension apparently is not triggered by BP elevation but by extravascular mechanisms.NEW & NOTEWORTHY Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na+/Ca2+ exchangers (NCX1) are upregulated in many forms of hypertension. Surprisingly, mildly hypertensive smooth muscle-specific (SM)-NCX1 transgenic mice exhibited modestly enhanced EDV and augmented endothelium-independent vasodilation (EIV). Conversely, mildly hypotensive SM-NCX1-knockout mice had greatly attenuated EIV. These adaptations help compensate for NCX1 expression-induced alterations in cytosolic Ca2+ and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.

Exposure to maternal diabetes induces endothelial dysfunction and hypertension in adult male rat offspring

The adverse environment in early life can modulate adult phenotype, including blood pressure. Our previous study shows, in a rat streptozotocin (STZ)-induced maternal diabetes model, fetal exposure to maternal diabetes is characterized by established hypertension in the offspring. However, the exact mechanisms are not known. Our present study found, as compared with male control mother offspring (CMO), male diabetic mother offspring (DMO) had higher blood pressure with arterial dysfunction, i.e., decreased acetylcholine (Ach)-induced vasodilation. But there is no difference in blood pressure between female CMO and DMO. The decreased Ach-induced vasodilation was related to decreased nitric oxide (NO) production in the endothelium, not NO sensitivity in vascular smooth muscle because sodium nitroprusside (SNP)-mediated vasodilation was preserved; there was decreased NO production and lower eNOS phosphorylation in male DMO. The reactive oxygen species (ROS) level was increased in male DMO than CMO; normalized ROS levels with tempol increased NO production, normalized Ach-mediated vasodilation, and lowered blood pressure in male DMO rats. It indicates that diabetic programming hypertension is related to arterial dysfunction; normalizing ROS might be a potential strategy for the prevention of hypertension in the offspring.

Abstract P004: Endothelial Function In Rat Juxtamedullary Afferent Arterioles After Renal Ischemia-reperfusion Injury

Renal ischemia-reperfusion (IR) induced kidney injury exhibits reduced renal blood flow (RBF) and glomerular filtration rate (GFR). Both RBF and GFR are regulated by renal microvascular function. Our previous study showed that IR caused impaired afferent arteriole autoregulatory capability via excess ROS accumulation and inflammation in renal microvessels. Other mechanisms contributing to renal microvascular dysfunction in IR have not been fully defined. In this study, we conducted acetylcholine (ACh) concentration-response experiments (n=5-7/each group) to assess endothelial function using the in vitro blood-perfused juxtamedullary nephron preparation. IR was induced by 60 minutes of bilateral renal artery occlusion followed by 24 hours or 7 days of reperfusion. Renal microvessels (RMV) were collected at either 24 hours or 7 days post-surgery for mRNA analysis. Systolic blood pressure was normal in IR rats vs. sham ranging from 126-136 mmHg (>0.05) over 7 days. IR markedly increased plasma creatinine concentration (Cr) to 3.9±0.2 mg/dL at 24 hours post-IR ( P <0.05 vs. 0.9±0.1 mg/dL in sham). Plasma Cr remained elevated in IR rats by day 7 of reperfusion (1.5±0.2 vs. 1.0±0.0 mg/dL in sham, P <0.05). Baseline afferent arteriole diameter was smaller in IR rats 24 hours post-IR and averaged 11.1±0.7 μm (P<0.05 vs. 14.2±0.7 μm in sham). Superfusion of ACh (10 -8 to 10 -4 M) caused concentration-dependent vasodilation in sham rats, increasing diameter to 23.5±2.6 μm (169±14% of the control, P <0.05) at 10 -4 M ACh. The ACh-induced vasodilation was not altered by IR insult and was indistinguishable from the sham responses. IR diameter reached a maximum of 19.7±0.9 μm (179±8% of the control) at 10 -5 M ACh. The ACh responses were also similar between sham and IR rats on day 7 post-IR. mRNA expression of endothelial markers, iCAM and vCAM, were not statistically different in RMV collected at 24 hours post-surgery between two groups, but was markedly elevated in IR RMV on day 7 post-IR (4-fold in iCAM and 2-fold in vCAM, P<0.05 vs. sham). In conclusion, upregulation of iCAM and vCAM mRNA expression in RMV 7 days post-IR suggests renal microvascular inflammation, but rat juxtamedullary afferent arterioles still maintain normal endothelium-dependent reactivity to ACh.

The Protective Mechanism Of Exercise Training For Coronary Vascular Dysfunction In Atherosclerosis: Er Stress And Ucp-2

PURPOSE: Endoplasmic reticulum (ER) stress and uncoupling protein-2 (UCP-2) are a key modulator for endothelial dysfunction in atherosclerosis. We determined the effects of exercise on ER stress and UCP-2 in endothelial dysfunction in atherosclerosis. METHODS: We used 4 groups of mice; wild type (WT), WT with exercise training, running on the treadmill for 12 weeks (WT-EX), apolipoprotein E knockout (ApoE KO) and ApoE KO with exercise training (ApoE KO-EX). We measured endothelium-dependent acetylcholine (ACh)-induced vasodilation of isolated and pressurized coronary arterioles in a concentration-dependent manner. Also, ACh-induced vasodilation was elicited in the presence of an inhibitor of eNOS and UCP-2 (L-NAME and Genipin) and the ER stress inducer (Tunicamycin). Immunoblotting was performed to measure the protein expression of ER stress markers (GRP78, IRE1, eIF2α, and CHOP), NLRP3 inflammasome signaling (NLRP3, caspase-1, IL-1β), Bax, TXNIP, and UCP-2 in the heart. The expression of p67phox and superoxide were visualized using immunofluorescence and DHE staining in coronary arterioles. NO production was measured by nitrate/nitrite assay. RESULTS: ACh-induced endothelium-dependent vasodilation was attenuated in coronary arterioles of ApoE KO, but it was improved in ApoE KO-EX. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reduced the expressions of ER stress (GRP78, p/t-IRE1, p-eIF2α, and CHOP), NLRP3 inflammasome (TXNIP, caspase-1 p20, and IL-1β), Bax, superoxide production, and NADPH oxidase p67phox, but it increased NO production in ApoE KO-EX mice. CONCLUSION: Our findings suggest that exercise training alleviates endothelial dysfunction in atherosclerotic coronary arterioles through the NOS, UCP-2, and ER stress signaling pathways including TXNIP/NLRP3 inflammasome and oxidative stress.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy.

To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10-6 M), and subsequently dilated in acetylcholine (ACh, 10-6 M - 10-4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10-4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10-6 M) followed by ACh (10-5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. ACh (10-6 M - 10-4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10-4 M) significantly attenuated the vasodilatation and the intracellular NO release. This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Acute Systemic Inhibition of Inflammation Augments Endothelium‐dependent Dilation in Women with a History of Preeclamptic Pregnancy

Women who have had preeclampsia demonstrate microvascular endothelial‐dysfunction, mediated in part by reduced nitric oxide (NO)‐dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if systemic inhibition of inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of inflammation (oral salsalate; 1500mg/twice daily/4 days) would improve endothelium‐ and NO‐dependent vasodilation in women with a history of preeclampsia (PrEC) but not in women with a history of uncomplicated pregnancy (HC). Eleven HC (30±1yrs, 9±2 months post‐partum) and 8 PrEC (29±3yrs, 6±1 months post‐partum) participated in a double‐blind placebo‐controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (ACh, 10−7–102 mM) or ACh+15mM L‐NAME (NO synthase antagonist). Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). ACh‐induced (75±3 vs. 92±4%CVCmax; p=0.002) and NO‐dependent (18±8 vs. 32±4%; p=0.04) vasodilation were attenuated in PrEC compared to HC following placebo. Salsalate augmented ACh‐induced (92±3%CVCmax; p=0.001) and NO‐dependent (38±4%; p=0.04) dilation in PrEC compared to placebo but had no effect in HC (all p>0.05). Women who have had preeclampsia demonstrate attenuated microvascular endothelial function compared to women with a history of uncomplicated pregnancy. Salsalate treatment augmented endothelium‐dependent vasodilation via NO‐mediated pathways in these women, suggesting that inflammatory signaling plays a role in mediating persistent endothelial‐dysfunction in women with a history of preeclampsia. ClinicalTrials.gov registration # NCT03482440Support or Funding InformationNIH K99 HL138133 (Stanhewicz); NIH R01 HL093238 (Alexander); NIH T32 AG049676 (Serviente)

Dietary supplementation with copper nanoparticles influences the markers of oxidative stress and modulates vasodilation of thoracic arteries in young Wistar rats.

We aimed to study the physiological effects of diet supplemented with copper (Cu) nanoparticles (NPs). During the eight weeks of the experiment, young Wistar rats (at seven weeks of age, n = 9) were supplemented with 6.5 mg of Cu either as NPs or carbonate salt (Cu6.5). A diet that was not supplemented with Cu served as a negative control (Cu0). The impact of nano Cu supplementation on lipid (reflected as thiobarbituric acid reactive substances-TBARS) and protein peroxidation (thiol and carbonyl groups) in blood plasma as well as the influence on the vasodilatory mechanism(s) of isolated rat thoracic arteries were studied. Supplementation with Cu enhanced lipid peroxidation (TBARS) in NP6.5 (x2.4) and in Cu6.5 (x1.9) compared to the negative control. Significant increase in TBARS was also observed in NP6.5 (x1.3) compared to the Cu6.5 group. The level of thiol groups increased in NP6.5 (x1.6) compared to Cu6.5. Meanwhile, significant (x0.6) decrease was observed in the Cu6.5 group compared to the negative control. Another marker of protein oxidation, carbonyl groups increased in NP6.5 (x1.4) and Cu6.5 (x2.3) compared to the negative control. However significant difference (x0.6) was observed between NP6.5 and Cu6.5. Arteries from Cu supplemented rats exhibited an enhanced vasodilation to gasotransmitters: nitric oxide (NO) and carbon monoxide (CO). An enhanced vasodilation to NO was reflected in the increased response to acetylcholine (ACh) and calcium ionophore A23187. The observed responses to ACh and CO releasing molecule (CORM-2) were more pronounced in NP6.5. The activator of cGMP-dependent protein kinases (8-bromo-cGMP) induced similar vasodilation of thoracic arteries in NP6.5 and Cu0 groups, while an increased response was observed in the Cu6.5 group. Preincubation with the inducible nitric oxide (iNOS) synthase inhibitor- 1400W, decreased the ACh-induced vasodilation in NP6.5, exclusively. Meanwhile the eicosanoid metabolite of arachidonic acid (20-HETE) synthesis inhibitor-HET0016, enhanced vasodilation of arteries from Cu0 group. In conclusion, this study demonstrates that supplementation with nano Cu influences oxidative stress, which further has modified the vascular response.

Resveratrol modulates the blood plasma levels of Cu and Zn, the antioxidant status and the vascular response of thoracic arteries in copper deficient Wistar rats

Copper (Cu) deficiency plays an important role in the development of cardiovascular disorders. Resveratrol (RSV) possesses pleiotropic cardiovascular benefits; however, the mechanism(s) by which RSV exerts protective effects are not completely understood.Male Wistar rats at 6 weeks of age were fed for 8 weeks with a Cu deficient diet (no added Cu, Cu = 0). In addition, Cu deficient rats were supplemented with RSV (500 mg/kg of diet, n = 9). Blood and intestinal samples were taken for further analysis together with internal organs and thoracic arteries.RSV supplementation resulted in elevated blood plasma levels of Cu (x2.1) and Zn (x1.1), in an increased activity of superoxide dismutase (SOD, x1.5) and ferric reducing antioxidant power (FRAP, x1.2). Meanwhile, markers of lipid peroxidation expressed as malondialdehyde (MDA, x1.5) and lipid hydroperoxides (LOOH, x1.1) were also increased in a significant way. Food intake, body weight, blood glucose, catalase, ceruloplasmin, lipid profile and intestinal samples were not modified.RSV enhanced the vasoconstriction of isolated thoracic arteries to noradrenaline (x1.4), potentiated the vasodilation to acetylcholine (ACh, x1.4) and increased the sensitivity to sodium nitroprusside (SNP). In addition, preincubation with the cyclooxygenase (COX)-inhibitor, indomethacin, potentiated the ACh-induced vasodilation, which was more pronounced in animals not supplemented with RSV.The KATP channel opener, pinacidil, induced a similar response in both studied groups.In conclusion, this study demonstrates that RSV supplementation influences oxidative stress and the antioxidant status, which may modify the vascular response in Cu deficiency.

Controlled Feeding of an 8-d, High-Dairy Cheese Diet Prevents Sodium-Induced Endothelial Dysfunction in the Cutaneous Microcirculation of Healthy, Older Adults through Reductions in Superoxide

Background:While excess dietary sodium impairs vascular function by increasing oxidative stress, the dietary incorporation of dairy foods improves vascular health. We demonstrated that single-meal cheese consumption ameliorates acute, sodium-induced endothelial dysfunction. However, controlled feeding studies examining the inclusion of cheese, a dairy product that contains both bioactive constituents and sodium, are lacking. Objectives:We tested the hypothesis that microcirculatory endothelium-dependent dilation (EDD) would be impaired by a high-sodium diet, but a sodium-matched diet high in dairy cheese would preserve EDD through oxidant stress mechanisms. Methods:We gave 11 adults without salt-sensitive blood pressure (<10 mmHg Δ mean arterial pressure; 64 ± 2 y) 4 separate 8-d controlled dietary interventions in a randomized, crossover design: a low-sodium, no-dairy intervention (LNa; 1500 mg/d sodium); a low-sodium, high-cheese intervention (LNaC; 1500 mg/d sodium, 170 g/d cheese); a high-sodium, no-dairy intervention (HNa; 5500 mg/d sodium); and a high-sodium, high-cheese intervention (HNaC; 5500 mg/d sodium, 170 g/d cheese). On Day 8 of each diet, EDD was assessed through a localized infusion (intradermal microdialysis) of acetylcholine (ACh), both alone and during coinfusion of NG-nitro-L-arginine methyl ester (NO synthase inhibitor), L-ascorbate (nonspecific antioxidant), apocynin [NAD(P)H oxidase inhibitor], or tempol (superoxide scavenger). Results:Compared with LNa, microvascular responsiveness to ACh was attenuated during HNa (LNa: –4.82 ± 0.20 versus HNa: –3.21 ± 0.55 M logEC50; P = 0.03) but not LNaC (–5.44 ± 0.20 M logEC50) or HNaC (–4.46 ± 0.50 M logEC50). Further, ascorbate, apocynin, and tempol administration each increased ACh-induced vasodilation during HNa only (Ringer's: 38.9 ± 2.4; ascorbate: 48.0 ± 2.5; tempol: 45.3 ± 2.7; apocynin: 48.5 ± 2.6% maximum cutaneous vascular conductance; all Pvalues < 0.01). Conclusions:These results demonstrate that incorporating dairy cheese into a high-sodium diet preserves EDD by decreasing the concentration of superoxide radicals. Consuming sodium in cheese, rather than in nondairy sources of sodium, may be an effective strategy to reduce cardiovascular disease risk in salt-insensitive, older adults. This trial was registered at clinicaltrials.govas NCT03376555.

Effects of Angiotensin-(1-7) and Angiotensin II on Acetylcholine-Induced Vascular Relaxation in Spontaneously Hypertensive Rats.

Endothelial dysfunction of small arteries occurs in patients with hypertension and in various hypertensive models. Endothelial function is usually evaluated by the degree of acetylcholine- (ACh-) induced vascular relaxation. Our previous study has found that compared to Wistar-Kyoto rats (WKY), ACh-induced vasodilatation was attenuated significantly in the mesenteric artery (MA), coronary artery (CA), and pulmonary artery (PA) of spontaneously hypertensive rats (SHR). This study investigated the influence of angiotensin- (Ang-) (1-7) and Ang II on blood pressure and ACh-induced vascular relaxation, as well as their interactive roles and downstream signal pathways in SHR and WKY. Intravenous injection of Ang II significantly increased, while Ang-(1-7) decreased the mean arterial pressure (MAP) in SHR. Ang-(1-7) improved ACh-induced relaxation in the MA, CA, and PA of SHR, while Ang II further attenuated it, which were inhibited by pretreatment with Mas receptor antagonist A-779 or AT1 receptor antagonist losartan, respectively. Ang-(1-7) decreased the basal arterial tension, and Ang II induced great vasoconstriction in SHR. Pretreatment with Ang-(1-7) inhibited the Ang II-induced pressor response, vasoconstriction, and the effects on ACh-induced relaxation in SHR. AT1 receptor expression was higher, while nitric oxide (NO), cGMP, and protein kinase G (PKG) levels of arteries were lower in SHR than in WKY. Ang II decreased, while Ang-(1-7) increased, the levels of NO, cGMP, and PKG of arteries. In addition, pretreatment with Ang-(1-7) inhibited the Ang II-induced reduction of NO, cGMP, and PKG in SHR. These results indicate that the activation of the Mas receptor by Ang-(1-7) can improve endothelial function and decrease MAP in SHR and inhibit the deteriorative effect of Ang II on endothelial function through the NO-cGMP-PKG pathway.

The interaction between resveratrol and two forms of copper as carbonate and nanoparticles on antioxidant mechanisms and vascular function in Wistar rats.

Experimental studies have emphasized that cardiovascular alterations can be improved by the long-term use of resveratrol (trans-3,5,4'-trihydroxystilbene; RSV) as well as dietary copper (Cu) intake. Male Wistar rats were supplemented for 8 weeks with Cu (6.5 mg/kg diet) as either nanoparticles (40 nm, CuNPs) or carbonate (CuCO3). Half of the studied animals were supplemented with RSV (500 mg/kg diet). Vascular function and blood plasma antioxidant status, expressed as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lipid hydroperoxides (LOOH) and malondialdehyde (MDA) were analyzed. The activity of ceruloplasmin (Cp), lipid profile, fasting glucose, and concentrations of Cu and zinc (Zn) were analyzed. RSV supplementation resulted in the elevated activity of SOD and decreased CAT, GPx and LDL-cholesterol in both groups. RSV supplementation on CuNPs increased the participation of vasoconstrictor prostanoids and decreased ACh-induced vasodilation, while the participation of hyperpolarizing mechanism(s) was restored by activating KATP channels. Blood plasma glucose was decreased. RSV supplementation on CuCO3 enhanced ACh- and SNP-induced vasodilation and decreased NA-induced vasoconstriction. The lipid profile was improved, as well as Zn concentration. Meanwhile, Cu and Cp, and the markers of lipid peroxidation, reflected as LOOH and MDA, were decreased. The use of RSV during CuCO3 intake improves vascular responses, the lipid profile and the antioxidant mechanism(s). The beneficial role of RSV was not observed in the CuNP group and decreased ACh-induced vasodilation and increased participation of vasoconstrictor prostanoids in the vascular regulation were noticed.

Betulinic acid attenuates lipopolysaccharide-induced vascular hyporeactivity in the rat aorta by modulating Nrf2 antioxidative function

Betulinic acid (BA), a pentacyclic triterpenoid, has been reported to inhibit cardiovascular dysfunction under sepsis-induced oxidative stress. Nuclear factor erythroid-2 related factor-2 (Nrf2) is regarded as a key transcription factor regulating expression of endogenous antioxidative genes. To explore the preventive effects of BA against vascular hyporeactivity and the related antioxidative mechanism in sepsis, contraction and relaxation in aortas isolated from lipopolysaccharide (LPS)-challenged rats were performed. Male Sprague-Dawley rats were pretreated with brusatol (Bru, 0.4mg/kg/2days, i.p.), an inhibitor of Nrf2, and BA (10, 25, 50mg/kg/day, i.g.) for 3days and injected with LPS (10mg/kg, i.p.) at the 4th day. Rats were anesthetized and killed by cervical dislocation after they were treated with LPS for 4h. Thoracic aortas were immediately dissected out to determine contraction and relaxation using the organ bath system. Pro-inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and oxidative stress were measured in aortic tissues and plasma. mRNA expression of Nrf2-regulated antioxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), in rat aortas was determined. Increases of IL-1β, TNF-α, nitric oxide, and malondialdehyde and the decrease of glutathione induced by LPS were significantly attenuated by pretreatment with different doses of BA in plasma and aortas (p < 0.05 versus LPS), all of which were blocked by Bru (p < 0.01). Inhibition of phenylephrine (PE)- and KCl-induced contractions and acetylcholine (ACh)-induced vasodilatation in aortas from LPS-challenged rats was dose-dependently reduced by BA (p < 0.05; percentage improvements by BA in PE-induced contraction were 55.38%, 96.41%, and 104.33%; those in KCl-induced contraction were 15.11%, 23.96%, and 22.96%; and those in ACh-induced vasodilatation were 16.08%, 42.99%, and 47.97%), all of which were reversed by Bru (p < 0.01). Improvements of SOD, GPx, and HO-1 mRNA expression conferred by BA in LPS-challenged rat aortas were inhibited by Bru (p < 0.01; 145.45% versus 17.42%, 160.69% versus 22.76%, and 166.88% versus 23.57%). These findings suggest that BA attenuates impairments of aortic contraction and relaxation in LPS-challenged rats by activating Nrf2-regulated antioxidative pathways.

High Sodium Intake Impairs Small Artery Vasoreactivity in vivo in Dahl Salt-Sensitive Rats

The effects of high sodium intake on the functionality of resistance arteries have been repeatedly studied in vitro, but no study has focused on salt-sensitive hypertension in vivo. We studied the in vivo reactivity of mesenteric small arteries (MSAs) to vasoactive agents in Dahl salt-sensitive (DS) rats with various sodium diets. Twenty-four male DS rats were randomized into 3 groups: LS (0.3% NaCl diet), NS (0.6% NaCl diet), and HS (8% NaCl diet). After a 12-week intervention, the diameter changes of the MSAs after noradrenaline (NA) and acetylcholine (ACh) exposure were detected by a microscope, and changes in blood perfusion through the MSAs were measured by full-field laser perfusion imaging. HS enhanced the constrictive response of the MSAs to NA and attenuated the relaxing response to ACh. Low sodium intake reduced the response of the MSAs to NA and promoted ACh-induced vasodilatation. HS also aggravated NA-induced blood perfusion reduction and impaired ACh-induced hyperperfusion of the MSAs. Pathologically, HS was associated with arteriolar structural damage and fibrosis of the MSAs. We conclude that sodium intake affects the responsiveness of the MSAs to vasoactive agents in DS rats and might play important roles in modulating blood pressure in hypertensive individuals.

Cutaneous Microvascular Function in Hypertensive Adults is not Altered Following 16‐weeks of Pharmacological Antihypertensive Treatment

Hypertension is characterized by pervasive microvascular endothelial dysfunction, resulting in a shift toward a proconstrictor vascular profile and contributing to increased cardiovascular morbidity and mortality. Cross‐sectional data suggest that pharmacologically lowering blood pressure (BP) is associated with improvements in microvascular function via endothelium‐dependent mechanisms. Well controlled longitudinal studies directly assessing mechanisms underlying microvascular dysfunction in hypertensive adults before and after a pharmacological antihypertensive intervention have not been performed. We tested the hypothesis that endothelium‐dependent dilation would be improved and adrenergic‐mediated vasoconstriction would be attenuated following 16‐weeks of pharmacological treatment to lower BP. Microvascular function was assessed before and after 16‐weeks of antihypertensive treatment (diuretic or angiotensin converting enzyme inhibitor) in eleven stage II hypertensive adults (9 men, 54±3 yrs, 29±1 kg/m2, resting BP 143±4/93±3 mmHg). Red blood cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis perfusion of the endothelium‐dependent agonist acetylcholine (ACh; 10−10–10−1 M) and the adrenergic receptor agonist norepinephrine (NE, 10−12–10−2 M). Cutaneous vascular conductance (CVC; flux/mmHg) was normalized to and expressed as a percentage of either baseline or maximal conductance (28 mM sodium nitroprusside and heat to 43°C). Resting BP was reduced following treatment (143±4/93±3 pre vs. 105±4/68±2 mmHg post; p=0.003). Neither baseline (0.19±0.03 pre vs. 0.19±0.03 post flux/mmHg; p=0.97) nor maximal CVC (2.16±0.48 pre vs. 1.44±0.24 post flux/mmHg; p=0.24) were different following treatment. There was no difference in ACh‐induced vasodilation following antihypertensive treatment (logEC50: −4.28±0.48 pre vs. −3.15±4.68 post; p=0.83). Further, NE‐induced vasoconstriction was not different after treatment (logEC50: −4.85±1.23 pre vs. −6.28±0.68 post; p=0.65). Contrary to our hypothesis, these preliminary data suggest that a 16‐week pharmacological antihypertensive treatment to reduce BP does not improve cutaneous microvascular function in middle‐aged hypertensive adults.Support or Funding InformationNIH R01HL093238This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preserved cardiovascular homeostasis despite blunted acetylcholine-induced dilation in mice with endothelial muscarinic M3 receptor deletion.

Muscarinic acetylcholine receptors (AChMR1-5) are fundamental for cellular responses upon release of the neurotransmitter acetylcholine (ACh) from parasympathetic nerve fibers. ACh is the prototypical agonist stimulating endothelium-dependent dilation, but most blood vessels lack parasympathetic innervation, raising the question as to the physiologic function of endothelial AChMR in vivo. Global deletion of AChM3R revealed a role in ACh-induced vasodilation in vitro and food uptake, but overall cardiovascular homeostasis has not been examined thoroughly. To characterize the function of endothelial AChM3R in vivo, we deleted AChM3R specifically in endothelial cells with an inducible or a non-inducible Cre-loxP system, driven by the endothelium-specific promoters VE-cadherin (indEC-M3R-/- ) or TIE2 (tek2; EC-M3R-/- ) and examined arteriolar dilation in the cremaster microcirculation, arterial pressure and cardiac function in these mice in vivo. In both EC-M3R-/- , ACh-induced dilation was strongly impaired in arterioles in vivo, while responses to other dilators were mostly preserved. However, arterial pressure (indEC-M3R-/- ) and arteriolar tone as a surrogate for peripheral vascular resistance did not differ between EC-M3R-/- and control mice. Aged EC-M3R-/- mice (74-78weeks) did not differ in body weight, heart weight, cardiac structure or contractile function from controls. We conclude that AChM3R elicits the endothelium-dependent dilation upon ACh also in arterioles in vivo. Despite this prominent role, the endothelial deletion of AChM3R does not affect overall cardiovascular homeostasis. Thus, their physiologic function in endothelial cells remains obscure.

Abstract 515: Bariatric Surgery Reverses Vascular Pathology in Mice With Morbid Obesity and Type 2 Diabetes

Objective: The mechanism of improved insulin sensitivity and vascular function following bariatric surgery remains unclear. We assessed the role of bariatric surgery in coronary arteriolar function in murine type 2 diabetic mice. Approach and Results: The animals were randomly allocated into normal control (Lean) sham, Lean surgery, db/db sham, and db/db surgery (at 5, 10, 20 and 30 days post-operatively). Impaired vasodilation to acetylcholine (ACh) in db/db mice was partially restored by bariatric surgery. Ghrelin administration restored endothelium-dependent dilation in db/db mice, but surgery-induced restoration was disappeared by ghrelin antagonist. Additionally, the incubation of coronary arterioles with IL-6 prevented the restoration of ACh-induced vasodilation. Bariatric surgery attenuated plasma concentration and protein expression of IL-6 in db/db. Bariatric surgery increased protein expression of eNOS and SOD2 in db/db mice. Conclusions: Decreased IL-6 expression after bariatric surgery induce up-regulation of eNOS and SOD2, and down-regulation of iNOS and O 2 .− , leading to restoration of endothelial function in coronary arterioles in type 2 diabetes.