Pressurized Mesenteric Arteries Research Articles

Abstract 15590: Smooth Muscle Specific Deletion of P90 Ribosomal S6 Kinase Alters the Vasomotor Response of Resistance Arteries

Background: Smooth muscle (SM) contraction is triggered by Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain of myosin (RLC 20 ). However, additional regulatory mechanisms exist. We found that global Rsk2 KO mice had more dilated mesenteric arteries (MAs) with reduced myogenic tone and RLC 20 phosphorylation, and significantly lower blood pressure. Furthermore, RSK2-dependent activation of the Na + /H + exchanger resulted in an increase in pH i and increased Ca 2+ i transients. However, it is unclear whether our observations were due to developmental abnormalities observed in Rsk2 global KO mice. Hypothesis: RSK2 signaling in SM is a significant regulator of myogenic tone, arterial stiffness, and Ca 2+ signaling. Methods: To study the role of RSK2 signaling in vascular tone, we generated inducible SM-specific RSK2 KO mice. Detection of total and phospho proteins were determined by western blot. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order MAs isolated from MYH11-Cre + Rsk2 fl/fl (RSK2 SM -/- ) and MYH11-Cre - Rsk2 fl/fl (RSK2 SM +/+ ) mice. We examined SM-specific Ca 2+ events in pressurized MAs by confocal fluorescent microscopy. Results: Western blot confirms knock down of RSK2 expression (~70%) in MAs and aorta. Intraluminal pressure-induced constriction of MAs is attenuated in RSK2 SM -/- . RSK2 SM -/- MAs display a delay in development of myogenic tone at 40 mmHg and decreased vascular stiffness, compared to RSK2 SM +/+ . Vasodilatory responses to Spermine NONOate (1-100 μM) in preconstricted (L-NNA; 10 μM) RSK2 SM -/- vessels were enhanced. RSK2 SM -/- MAs display fewer Ca 2+ events (97±19.6; n = 4) than RSK2 SM +/+ (400±24.5; n = 4) at 80 mmHg ( p = 0.0001). Additionally, Ca 2+ events are slower and longer-lasting in the RSK2 SM -/- . Decreased levels of p-RLC 20 are observed at 40 mmHg, but not 80 mmHg in RSK2 SM -/- vs. RSK2 SM +/+ mice, consistent with impeding tone development. Conclusion: RSK2 signaling contributes to myogenic tone and vessel stiffness by regulating sensitivity to NO, increasing Ca 2+ events, and RLC 20 activity. RSK2 signaling pathway is a potential therapeutic target for hypertension.

In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p>0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p>0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p>0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.

Loss of SR‐PM Contact Sites May Contribute to The Development of Hypertension in Spontaneously Hypertensive Rats

Sarcoplasmic reticulum (SR)‐plasma membrane (PM) contact sites in vascular smooth muscle (VSM) cells bring the SR Ca2+ release channels into proximity of plasma membrane large‐conductance Ca2+‐activated K+ (BKca) channels and regulate intracellular Ca2+ through BKca‐mediated membrane hyperpolarization. Loss of BKca channel activity leads to increased intracellular Ca2+ concentration, vascular hypercontractility, and activation of hypertrophic genes. We hypothesized that the loss of close contact between the SR and the PM contributes to the development of vascular hypercontractility and hypertrophy in the Spontaneously Hypertensive Rat (SHR). To evaluate this hypothesis, we compared BKca channel activity in response to the activation of inositol 1,4,5‐trisphosphate receptor (IP3R), an SR Ca2+ channel between SHR and normotensive Sprague Dawley (SD) rats. We found that in endothelium‐denuded pressurized mesenteric arteries, paxilline (1μM), a selective BKca channel blocker, significantly increased norepinephrine (NE, 0.5μM)‐induced vasoconstriction in SD arteries (n=5) by ~15% (% initial diameter from 78.58±3.93 to 66.77±3.07). However, paxilline failed to increase NE‐induced vasoconstriction in SHR arteries (Figure 1). Paxilline treatment significantly augmented the hypertrophic effect of angiotensin II (ANG II) in SD, where the total cell area of paxilline treated ANG II group was ~11% (from 3471.48±121.35 to 3854.86±129.99μm2) greater compared to the non‐paxilline‐treated ANG II group. In contrast, the paxilline‐induced increase in hypertrophic response was absent in SHR. Similarly, in WST‐1 cell proliferation assay, paxilline treatment significantly increased ANG II‐induced proliferation in SD by ~15% (Absorbance(A440nm‐A630nm) from 0.15±0.01 to 0.17±0.01) while having no effect on cell proliferation in SHR. Next, we compared the effect of 5‐HT on BKca channel activity between SHR and SD rat mesenteric VSM cells using patch‐clamp whole‐cell mode. 5‐HT activates IP3Rs by stimulating the production of IP3 in VSM cells. At +60mV, 5‐HT (0.3μM) significantly increased BKca current density at +60mV in SD VSM cells from 50.78±2.19 to 69.40±5.50 pA/pF (n=6 cells, P<0.05) but the increase in current density was non‐significant in SHR (from 36.77±3.11 to 47.45±8.83 pA/pF, n=6 cells, Figure 2). We also examined the Ca2+‐ and voltage‐sensitivity of BKca channels using inside‐out patch‐clamp. BKca channels in VSM cells isolated from mesenteric arteries of SHR and SD rats displayed identical Ca2+‐ and voltage‐sensitivity. In conclusion, our data suggest a lack of BKca activation in response to IP3R activation in VSM cells ofSHR, which may be due to the loss of close contact between the SR and the PM.

Dehydration results in a reduction in the expression and function of AT 1 R and V 1 R in resistance‐sized arteries

In the body, the state of dehydration occurs where there is a deficit of water compared to that which is required to function properly. Water transportation is crucial and carried out via aquaporins (AQPs) located throughout the body and as a response to dehydration events, hormones such as angiotensin II (Ang II) and arginine-vasopressin (AVP) are secreted to induce various normal physiological functions via a hormone-specific receptor and associated-receptor activation found in vascular smooth muscle cells (VSMCs). Consequently, dehydration results with overall blood vessel constriction and thickening of the blood, ultimately leading to heart strain and increased blood pressure. Currently, the effect of dehydration on both the angiotensin II receptor type 1 (AT1R) and vasopressin receptor (V1R) expressed in resistance-sized artery VSMCs is unclear. C57BL/6J male mice were deprived of water for 48 hours before analyzing AT1R and V1R expression and function in freshly isolated mesenteric arteries, compared to controls. Dehydration for 48 hours results in a reduction in AT1R and V1R message and protein expression as well as a reduction in angiotensin- and arginine vasopressin (AVP)-induced vasoconstriction in pressurized mesenteric arteries. These data suggest that AT1R and V1R function is reduced in resistance-sized mesenteric arteries as a response to an elevation in plasma vasopressin and angiotensin II that occurs after dehydration.

Dichotomous effects of smooth muscle TRPV4 channels on vascular contractility

Ca2+ signaling mechanisms in smooth muscle cells (SMCs) determine vascular contractility. Transient receptor potential vanilloid 4 (TRPV4) ion channels have been described as a crucial Ca2+ influx pathway in SMCs from small, resistance-sized arteries. However, the impact of SMC TRPV4 (TRPV4SMC) channel activity on blood pressure remains unknown, mostly due to the lack of studies in SMC-specific knockout mice. We hypothesized that TRPV4SMC channels control the resting blood pressure. Radiotelemetric blood pressure recordings showed reduced daytime and nighttime blood pressures in the inducible, SMC-specific TRPV4 knockout (TRPV4SMC-/-) mice compared to the control mice. The effect of TRPV4SMC-/- on vascular contractility was assessed using two endogenous vasoconstrictor mechanisms- 1) SMC α1 adrenergic receptor (α1AR) stimulation and 2) increased intraluminal pressure. Phenylephrine (α1AR agonist)-induced constriction of pressurized mesenteric arteries (MAs, 80 mm Hg, 1 nM-10 mM phenylephrine) and blood pressure elevation (10 mg/kg phenylephrine, i.p.) were blunted in TRPV4SMC-/- mice, identifying a central role of TRPV4SMC channels in α1AR-induced vasoconstriction and blood pressure elevation. Individual Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) were recorded using spinning disk confocal imaging in fluo-4-loaded MAs pressurized to 80 mm Hg. Phenylephrine (1 mM) activated TRPV4SMC sparklets, an effect abolished by protein kinase C (PKC) inhibitor (Go6976, 1 mM). These results indicated that PKC-TRPV4SMC channel signaling is a major contributor to α1AR-induced vasoconstriction. In direct contrast with PE-induced constriction, intraluminal pressure-induced constriction was higher in MAs from TRPV4SMC-/- mice, suggesting that SMC TRPV4 channels limit pressure-induced vasoconstriction. Previous studies have shown that TRPV4SMC channels can activate large-conductance, Ca2+-sensitive K+ (BK) channels, thereby decreasing pressure-induced vasoconstriction. Consistent with these findings, vasoconstriction in response to BK channel inhibitor (paxilline, 1 mM) was reduced in MAs from TRPV4SMC-/- mice, supporting a dilatory TRPV4SMC channel-BK channel signaling under resting conditions. In situ proximity ligation assay revealed co-localization of TRPV4SMC channels with both α1ARs and BK channels. Overall, TRPV4SMC channels increase the resting blood pressure but have divergent effects on vascular contractility. TRPV4SMC channels contribute to α1AR-induced vasoconstriction but limit pressure-induced vasoconstriction. Therefore, selective vasoactive stimulus-TRPV4SMC channel coupling may determine the impact of TRPV4SMC channels on vascular resistance.

Abstract P1109: Cholesterol Regulation of Hydrogen Sulfide-Mediated Vasodilation

Hydrogen sulfide (H 2 S) is produced within the vascular wall by cystathionine γ-lyase (CSE). CSE is expressed throughout the vascular tree however, the sensitivity to H 2 S-induced dilation in small resistance size arteries (<100 μm) is greater than the dilation sensitivity in large arteries (>300 μm). Although H 2 S can induce vasodilation in different size arteries, it is not clear how H 2 S signaling mechanisms differ in large and small arteries, garnering H 2 S-induced dilation prominent and important in small resistance arteries. We previously demonstrated that within endothelial cells, H 2 S-mediated vasodilation involves activation of transient receptor potential cation channel subfamily V member 4 (TRPV4)-dependent Ca 2+ influx and large conductance Ca 2+ -activated potassium (BK) channel. Based on previous reports that membrane cholesterol negatively regulates TRPV4 mobility and BK activity, we hypothesized that H 2 S-mediated vasodilation is diminished by membrane cholesterol and that lower sensitivity in large arteries might be due to higher levels of membrane cholesterol in endothelial cells. Isolated pressurized mesenteric arteries (300-380 μm) were treated luminally with vehicle (HEPES) or the cholesterol-depleting agent, methyl β cyclodextran (MBCD, 100 μM) and exposed to H 2 S donor, NaHS (10 μM). Analysis of H 2 S-induced dilation (expressed as % Ca 2+ free diameter) showed that MBCD treatment significantly enhanced H 2 S-mediated vasodilation (vehicle: 1.2% ± 1.2, MBCD: 8.9% ± 2.3, p = 0.012, n = 5 animals/group). Additionally, mesenteric arteries of different orders were luminally loaded with the fluorescent cholesterol-binding agent, filipin III (20 μg/ml, ex/em: 405/461 nm) the endothelial glycocalyx marker DyLight 594-tomato lectin (20 μg/ml, ex/em: 561/613 nm) and the nuclei stain SYTOX green (ex/em: 488/518 nm). Analysis of endothelial membrane cholesterol content (filipin III /tomato lectin fluorescence) was significantly greater in larger arteries (2 nd order: 1.019 ± 0.158, 5 th order: 0.344 ± 0.179, p = 0.0054, n = 3-5 animals/group). These studies suggest that higher membrane cholesterol decreases H 2 S-mediated vasodilation.

The availability of sympathetic neurotransmitter release for nerve stimulation is enhanced in mesenteric arteries from long‐term paraplegic and tetraplegic rats

Loss of supraspinal inputs to sympathetic preganglionic neurons causes a marked reduction in post‐ganglionic sympathetic vasoconstrictor nerve activity and sympathetic support of blood pressure in both humans and rats with spinal cord injury (SCI). The long‐term impact of this reduced sympathetic outflow on sympathetic neurotransmission is unknown. We investigated this question using established long‐term animal models of paraplegic (P, T2–3) and tetraplegic (T, C6–7) SCI in male Sprague Dawley rats (26 weeks in total age). Sixteen weeks after SCI, P and T rats showed significantly lower blood pressure than sham operated rats (P 103±2mmHg, T 97±4mmHg, sham 117±6mmHg) (P<0.05 P or T vs sham). P rats had faster heart rate (410±6bpm), while T rats had slower heart rate (299±10bpm) than sham rats (321±14bpm) (P<0.05, P or T vs sham). P and T rats also showed lower body weight than sham rats (P 380±5g, T 340±12g, sham 459±18g) (P<0.05, P or T vs sham). Small mesenteric arteries (MA) were collected from these rats for detailed examination of sympathetic neurotransmission. Norepinephrine (NE) release from sympathetic nerve terminals was measured using amperometry. ATP release was determined indirectly by measuring excitatory junction potentials (EJPs) in MA smooth muscle cells. Frequency dependent NE release curves in response to nerve stimulation were slightly leftward‐shifted in MA from P and T rats compared to sham rats. Interestingly, frequency dependent EJP response curves to nerve stimulation in MA of P and T rats were shifted markedly leftward and upward compared to sham rats; and responses in these rats also showed significant initially facilitation of EJPs during long‐train nerve stimulation. In pressurized MA, exogenous NE and ATP‐induced contractions were comparable in all rats. Immune staining of tyrosine hydroxylase in MA showed comparable sympathetic nerve density and distribution in all rats. NE content in tissues from left ventricle, kidney, spleen and MA were measured by HPLC. Compared to sham rats, NE content in left ventricle from P and T rats, and in MA from T rats, was significantly increased. NE content in MA from P rats was not different from sham rats, and NE content in kidney and spleen from P and T rats did not differ from sham animals. Our studies show that peripheral vascular NE, and especially ATP, release is enhanced during nerve stimulation 16 weeks after SCI, whereas post‐ganglionic nerve density and arterial reactivity to sympathetic neurotransmitters are unchanged. Even though overall sympathetic support of blood pressure is reduced after SCI, improvement in sympathetic neurotransmitter release may help maintain cardiovascular stability in long‐term SCI subjects. (Supported by NHLBI 2P01HL070687 and HL122223).Support or Funding InformationNHLBI 2P01HL070687 and HL122223This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholesterol Inhibits H2S‐Mediated Vasodilation

Hydrogen sulfide (H2S), the most recently described endothelium‐derived vasodilator, is predominantly produced in the endothelium by cystathionine γ‐lyase (CSE). We previously demonstrated that within endothelial cells (ECs), H2S‐mediated vasodilation involves activation of transient receptor potential cation channel subfamily V member 4 (TRPV4)‐dependent Ca2+ influx and large conductance Ca2+‐activated potassium (BK) channel. In addition, a single concentration of H2S donor NaHS (1 μM) dilated small mesenteric arteries (<100 μm) but had no effect in large mesenteric arteries (>300 μm). Furthermore, inhibiting CSE generation of H2S with β‐cyanoalanine (BCA, 100 μM) impaired acetylcholine (ACh)‐induced dilation in small mesenteric arteries, but had no effect in large mesenteric arteries. These data suggest that H2S‐induced dilation is prominent and important in small resistance arteries but not in larger arteries. It is possible that decreased availability of TRPV4 and/or BK in larger arteries is responsible for the diminished sensitivity to H2S‐induced vasodilation. Previous reports suggest that membrane cholesterol negatively regulates TRPV4 mobility and BK activity. Our current study tested the hypothesis that H2S‐mediated vasodilation is inhibited by membrane cholesterol. Mesenteric arteries induced vasodilation was assessed in pressurized large rat mesenteric arteries (>300 μm). Isolated pressurized mesenteric arteries (320 μm – 360 μm) were pretreated luminally with vehicle (HEPES) or the cholesterol‐depleting agent, methyl β cyclodextran (MBCD, 100 μM) and exposed to NaHS (100 μM). NaHS‐induced dilation in U46619 pre‐constricted arteries was significantly enhanced by MBCD pretreatment (+vehicle: 6.78% ± 1.6, +MBCD: 17.10% ± 6.8, p < 0.05, n=3–4 animals/group). These studies suggest that membrane cholesterol disrupts H2S‐mediated vasodilation. Ongoing studies in isolated mesenteric arteries of different sizes are investigating endothelial cell membrane cholesterol content and the effect of cholesterol depletion on TRPV4‐dependent Ca2+ influx in large and small mesenteric arteries.Support or Funding InformationFunding provided by the NIH HL12330 & HL7736‐23This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P279: Regulation Of H 2 S Production By Cystathionine Gamma-lyase In Endothelial Cells.

Hydrogen sulfide (H 2 S) is the most recently described endothelium-derived vasodilator. In the endothelium, H 2 S is predominantly produced by cystathionine γ-lyase (CSE). We and others have previously shown CSE inhibition diminishes acetylcholine (ACh)-induced dilation in aortic ring segments. However, the mechanism of CSE regulation is not well-defined. The goal of this study was to identify endogenous regulators of CSE in endothelial cells. Previous reports suggest that [Ca 2+ ] and phosphorylation regulate CSE activity. We previously reported that there was an increase in H 2 S production in rat aortic endothelial cells loaded with an H 2 S fluorescence indicator, sulfide fluor-7 acetoxymethylester (SF7-AM, 10 μM) after exposure to either ACh (10 μM) or an H 2 S donor (NaHS, 10 μM). Pretreatment with the CSE inhibitor, β-cyanoalanine (BCA, 100 μM) prevented the ACh-induced but not the NaHS-induced increases in SF 7 flourescence. Our current study tested the hypothesis that increasing intracellular calcium stimulates CSE-dependent H 2 S production. Cultured rat aortic endothelial cells were loaded with SF 7 and a nuclear dye (Nuclear-ID Red DNA stain, to correct for cell number) and fluorescence was measured at baseline and in response to a calcium ionophore (ionomycin, 100 nM). Ionomycin increased H 2 S production (Control: 0.8293, +ionomycin: 1.0365, p = 0.0001, n=3 of 7-15 wells per replicate). Pretreatment with BCA significantly decreased ionomycin-induced H 2 S production (+ionomycin: 1.0365, +BCA: 0.6600, p < 0.0001, n=3 of 7-15 wells per replicate). Preliminary ongoing studies in pressurized mesenteric arteries suggest a tendency of CSE inhibition to reduce ACh-induced dilation (Control: 77.06%, +BCA: 42.04%, p=0.08, n=3-4 arteries per group). However, a larger sample size is needed. Taken together, these studies suggest that ACh elicits vasodilation through a Ca 2+ -dependent activation of CSE.

Chronic administration of an endothelial KCa channel activator (SKA‐31) improves agonist evoked vasodilation in mesenteric arteries of aged rats

Endothelial dysfunction occurs in healthy ageing, and may contribute to the development of cardiovascular complications (e.g. hypertension, atherosclerosis) that are more prevalent in aged individuals. One cause of age‐related endothelial dysfunction is an imbalance in the generation and/or release of vasodilatory and constrictor agents by the vascular endothelium (e.g. reduced NO bioavailability). Ageing may also impact the expression of endothelial proteins involved with vasoactive signaling to the surrounding smooth muscle.The goal of our study was to assess the effects of long term, in vivo administration of SKA‐31, a small molecule activator of KCa 2.x and KCa 3.1 channels, on the functional reactivity of mesenteric arteries in healthy aged rats. Young (225–250 g, 12 weeks of age) and aged (600–700 g, 18 months of age) male Sprague Dawley (SD) rats were treated daily with either vehicle or 10 mg/kg SKA‐31 for 8 weeks, after which mesenteric arteries (i.e. 200–400 μm internal diameter) were isolated and cannulated in an arterial pressure myography chamber. Experimentally, arteries were pressurized to 70 mmHg, preconstricted with 1 μM phenylephrine (PE) and superfused at 6–7 ml/min with Krebs' solution at ~36°C using a peristaltic pump. Vasoactive agents were added directly to the superfusate.The PE‐induced decrease in the intraluminal diameter of pressurized mesenteric arteries (i.e. % change of maximal passive diameter) was significantly greater in aged (44.3 ± 2.7%, n=8) vs. young animals (38.1 ± 3.6%, n=12), whereas chronic SKA‐31 treatment of aged rats reduced the level of vasoconstriction to 36.5 ± 4.0% (n=8).Aged mesenteric arteries dilated less in response to 0.3 μM acetylcholine (ACh), 0.3 μM bradykinin (BK) and 10 μM sodium nitroprusside (SNP) (i.e. inhibition of active tone = 37.0 ± 1.9%, 44.6 ± 4.5%, 55.5 ± 4.8%, respectively; n=4) compared with young arteries (48.3 ± 6.8%, 62.0 ± 7.1%, 68.0 ± 7.9%, respectively, n=4). Chronic treatment of aged SD rats with SKA‐31 improved vasodilation evoked by ACh, BK and SNP (58.5 ± 5.5%, 64.3 ± 5.5%, 76.7 ± 4.4%, respectively; n=4), however, SKA‐31 treatment had no effect on the dilations evoked by either 10 μM adenosine or 5 μM pinacidil.Molecular analysis confirmed that expression of KCa 2.3 and KCa 3.1 channels was significantly decreased in aged mesenteric arteries compared with young vessels. Chronic SKA‐31 administration significantly increased the expression of these channels relative to vehicle‐treated animals (n=3).Collectively, these data demonstrate that chronic SKA‐31 treatment can improve evoked vasodilation in the aged mesenteric microcirculation, in part by enhancing the expression of endothelial KCa 2.3 and KCa 3.1 channels.Support or Funding InformationSupported by research funding to APB from the Canadian Institutes of Health Research. RKM is a recipient of a postdoctoral scholarship from the Libin Cardiovascular Institute and Cumming School of Medicine, Univ. of Calgary.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic Disease.

Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (HdbWnox1), lean Nox1 knockout (HdbKnox1), obese (KdbWnox1), and obese KK (KdbKnox1). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW (P<0.01), whereas deletion of Nox1 in KW mice normalized dilation. Vasodilator responses after inhibition of NO synthase blunted acetylcholine responses in KK and lean controls, but had no impact in KW, attributing recovered dilatory capacity in KK to normalization of NO. Acetylcholine responses were improved (P<0.05) with Tempol, and histochemistry revealed oxidative stress in KW animals, whereas Tempol had no impact and reactive oxygen species staining was negligible in KK. Blunted dilatory responses to an NO donor and loss of myogenic tone in KW animals were also rescued with Nox1 deletion. Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.

Activation of TRPM3 in Perivascular Sensory Nerves Induces Dilation of Mouse Resistance Arteries

The vascular tone is determined by a complex interplay of vasodilator and vasoconstrictor stimuli that modulate the contractile state of vascular smooth muscle cells (VSMCs). Activation of the cation channel Transient Receptor Potential Melastatin 3 (TRPM3) has been shown to induce contraction of VSMCs in aorta. However, the contribution of this channel to the vascular tone in resistance arteries remains unknown. Real-time qPCR and immunochemistry experiments showed Trpm3 expression in mesenteric arteries isolated from wild type (WT) C57BL/6J mice. Myography experiments carried out in intact pressurized mesenteric arteries from WT mice showed that the TRPM3 agonist PS induces vasodilation at concentrations above ∼5 µM, with a concentration-dependency featuring two distinct increasing phases. In contrast, PS only induced vasodilation above 10 µM following a single Hill-type behavior in preparations from Trpm3 knockout (KO) mice. Recordings in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096 recapitulated the results of Trpm3 KO preparations, indicating that the TRPM3-mediated effect of PS entails CGRP release from perivascular nerve endings. The effect of 10 µM PS was inhibited to about 50% by the combination of potassium channel blockers (500 nM paxilline, 10 µM correolide and 50 nM stromatoxin). Electrophysiological recordings in freshly isolated mesenteric VSMCs, revealed that basal currents are not sensitive to PS (10 µM and 30 µM) and that PS has no effect on potassium currents. Our data indicates that activation of TRPM3 channels in perivascular sensory nerves induces CGRP release, which leads to activation of potassium channels in smooth muscle cells, resulting in dilation of mesenteric arteries. These findings reveal a potential role of TRPM3 in vascular tone regulation, and support the recent notion that this channel may play roles in neurogenic inflammation.

Inwardly rectifying K+ channels are major contributors to flow-induced vasodilatation in resistance arteries.

Endothelial inwardly rectifying K+ (Kir2.1) channels regulate flow-induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries. Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance. Flow-induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K+ channels. This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow. Inwardly rectifying K+ (Kir) channels are known to be sensitive to flow, but their role in flow-induced endothelial responses is not known. The goal of this study is to establish the role of Kir channels in flow-induced vasodilatation and to provide first insights into the mechanisms responsible for Kir signalling in this process. First, we establish that primary endothelial cells isolated from murine mesenteric arteries express functional Kir2.1 channels sensitive to shear stress. Then, using the Kir2.1+/- heterozygous mouse model, we establish that downregulation of Kir2.1 results in significant decrease in shear-activated Kir currents and inhibition of endothelium-dependent flow-induced vasodilatation (FIV) assayed in pressurized mesenteric arteries pre-constricted with endothelin-1. Deficiency in Kir2.1 also results in the loss of flow-induced phosphorylation of eNOS and Akt, as well as inhibition of NO generation. All the effects are fully rescued by endothelial cell (EC)-specific overexpression of Kir2.1. A component of FIV that is Kir independent is abrogated by blocking Ca2+ -sensitive K+ channels. Kir2.1 has no effect on endothelium-independent and K+ -induced vasodilatation in denuded arteries. Kir2.1+/- mice also show increased mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a tail-cuff. Importantly, blocking Kir channels also inhibits flow-induced vasodilatation in human subcutaneous adipose microvessels. Endothelial Kir channels contribute to FIV of mouse mesenteric arteries via an NO-dependent mechanism, whereas Ca2+ -sensitive K+ channels mediate FIV via an NO-independent pathway. Kir2 channels also regulate vascular resistance and blood pressure. Finally, Kir channels also contribute to FIV in human subcutaneous microvessels.

High fat‐diet induced obesity does not necessarily promote vascular remodeling/fibrosis in rats

Inflammation and hyperglycemia are responsible for obesity associated vascular remodeling/fibrosis. Our lab has previously observed that high fat (HF) diet‐induced obesity and hyperglycemia promote vascular remodeling/fibrosis, but HF diet does not necessarily promote hypertension in male mice. However, it is unclear if vascular remodeling/fibrosis and inflammation are unique changes in all HF diet‐induced obese rodent models. In current studies, we compared the blood pressure, vascular and tissue histological structures of heart, mesentery, and kidney in male and female Sprague Dawley rats fed with HF (60 kcal% fat) or control (10 kcal% fat) diet up to 20 or 48 weeks. Vascular responsibilities to norepinephrine and wall thickness/distensibility were evaluated in pressurized mesenteric arteries (MA) and veins (MV) in vitro. The plasma levels of TNA‐α, monocyte chemoattractant protein‐1 (MCP‐1), insulin, glucose and leptin were also evaluated in these rats. HF fed female (20 weeks) and male (48 weeks) rats were obese and hyperleptinemia, but normotensive and with unchanged vascular contractility. These rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, and systemic inflammation, when compared with control diet fed rats. After 48 weeks of exposure to HF diet, female rats were obese, hyperleptinemia, hypertensive and with reduced vascular reactivity to norepinephrine, however, these rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, or systemic inflammation either. Our findings suggested that obesity is not the determinant in the development of vascular fibrosis/remodeling. HF diet induced hyperglycemia and tissue inflammation may be the predominant mediators in obesity associated vascular remodeling/fibrosis.Support or Funding InformationSupported by Undergraduate Research in Biomedical Sciences Program (NIH grant HL103156, for Dr. Ewart to support Carter‐Taylor) and NIH grant 2P01HL070687 (For Drs Galligan and Xu)

0343 : Importance of the membrane estrogen receptor alpha (ER) in the vascular response to shear stress in mice

Resistance arteries are sensitive to mechanical forces exerted on the vessel wall. While endothelial shear stress triggers flow-mediated dilation (FMD), chronic increase in shear forces drives expansive arterial remodeling. We showed previously that endothelial estrogen receptor alpha (ER‹) controlled this adaptive remodeling. We aimed to evaluate ER‹contribution in acute response to flow, by using various genetic models of ER‹deficiency in male mice, reducing estrogen hormonal influence. FMD was evaluated following step increase in flow applied to pressurized mesenteric arteries (arteriography). Arteries were isolated from wild-type (WT), (i) ER‹KO, or mice invalidated for (ii) the ligand-dependent transactivation function AF2 of ERa (AF2°) and (iii) the plasma membrane-located ER‹(mutated for the palmitoylation site C451A). In mice deficient in ER‹, FMD was attenuated (dilation 50μl/min WT: 59+/–4% vs. ER‹KO: 41+/–4% p<0.01) without significant modification in response to acetylcholine. Invalidation of AF2 (ER‹nuclear transcriptional action) only partially impacted FMD (WT: 61+/–5% vs. AF2°: 51+/–5%). Ex vivo ligand-dependent activation of ER‹with 17®estradiol or blockade by ICI 182,780 had no significant effect on FMD in WT arteries. Importantly, invalidation of membrane ER‹(C451A) markedly altered FMD (WT: 56+/–7% vs. C451A: 34+/–6% p<0.05). In contrast to ER‹KO and AF2°, C451A arteries showed a reduced dilating response to acetylcholine but only a decreased sensitivity to insulin, which shares common intracellular pathways triggered by shear stress, without altering maximal vasodilation (WT: 31+/–7% vs. C451A: 15+/–3% p<0.05). We show for the first time that membrane ER‹contributes to arterial shear-sensing irrespective of the presence of its agonist. Cell membrane ER‹participates to vascular homeostasis and could constitute a novel therapeutical target in peripheral vascular diseases. The author hereby declares no conflict of interest

Abstract 18791: Immune Cell Derived Nerve Growth Factor Initiates Neurogenic Hypertension in Obesity

Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological and immunological antecedents, however the relationship between these is unclear. Methods and Results: Here we used a mouse model of diet-induced obesity, pressure myography, immunohistochemistry, flow cytometry and radiotelemtry to demonstrate that immune cells, located within the vascular adventitia of arteries important for controlling blood pressure, produce nerve growth factor (NGF) and rapidly initiate growth of the perivascular sympathetic nerve plexus; which increases vascular tone and thereby elevates blood pressure. We also show that Rag1-/- mice lacking T and B cells do not develop hypertension when obesity is induced with a high fat diet (HFD) and show that their sympathetic innervation and vascular tone remains normal, suggesting that neurogenic hypertension in the obese likely occurs independently of weight gain. Furthermore, we show that the adoptive transfer of wildtype CD3+ T cells restores the sympathetic hyperinnervation and associated hypertension whereas the transfer of CD3+ T cells deficient in NGF had no effect on sympathetic innervation or blood pressure. Conclusion: These results not only demonstrate the role of T cells in the genesis of obesity-related hypertension but also demonstrate the pivotal role of NGF in linking dysfunction of the immune and sympathetic nervous systems that characterize hypertension in the obese. a, Change in body weight b, nerve-mediated contraction amplitude recorded from pressurized small mesenteric arteries (1Hz, 30s) and c, systolic BP in mice in RAG-1-/- mice, which lack B and T cells, fed NCD or HFD. RAG-1-/-Tcell mice are recipients of CD3+ T cells via adoptive transfer. d, Perivascular nerve density (synaptophysin) over wholemount small mesenteric arteries from RAG-1-/- mice. Scale, 50μm.

Hepatic oxidative stress, genotoxicity and vascular dysfunction in lean or obese Zucker rats.

Metabolic syndrome is associated with increased risk of cardiovascular disease, which could be related to oxidative stress. Here, we investigated the associations between hepatic oxidative stress and vascular function in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24 and 37 weeks of age. Obese Zucker rats had more hepatic fat accumulation than their lean counterparts. Nevertheless, the obese rats had unaltered age-related level of hepatic oxidatively damaged DNA in terms of formamidopyrimidine DNA glycosylase (FPG) or human oxoguanine DNA glycosylase (hOGG1) sensitive sites as measured by the comet assay. There were decreasing levels of oxidatively damaged DNA with age in the liver of lean rats, which occurred concurrently with increased expression of Ogg1. The 37 week old lean rats also had higher expression level of Hmox1 and elevated levels of DNA strand breaks in the liver. Still, both strain of rats had increased protein level of HMOX-1 in the liver at 37 weeks. The external and lumen diameters of mesenteric arteries increased with age in obese Zucker rats with no change in media cross-sectional area, indicating outward re-modelling without hypertrophy of the vascular wall. There was increased maximal response to acetylcholine-mediated endothelium-dependent vasodilatation in both strains of rats. Collectively, the results indicate that obese Zucker rats only displayed a modest mesenteric vascular dysfunction, with no increase in hepatic oxidative stress-generated DNA damage despite substantial hepatic steatosis.

Chronic deficit in nitric oxide elicits oxidative stress and augments T-type calcium-channel contribution to vascular tone of rodent arteries and arterioles

As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo. We used pressurized cerebral and mesenteric arteries in vitro and skeletal muscle arterioles in vivo to study the contribution of L-type (1 µmol/L nifedipine) and T-type (1 µmol/L mibefradil, 3 µmol/L NNC 55-0396) calcium channels to vascular tone, following acute or chronic inhibition of nitric oxide. Acute inhibition with l-NAME (10 µmol/L) significantly increased the T-type, but not the L-type, channel contribution to vascular tone in vitro and in vivo, and altered the smooth muscle expression of the Cav3.1 and Cav3.2 T-type channels. In pressurized mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with l-NAME, the contribution of T-type channels relative to L-type channels was significantly reduced, compared with arteries from wild-type mice.Chronic l-NAME treatment (40 mg/kg/day; 14-18 days) increased blood pressure, vascular superoxide, and the contribution of T-type channels to vascular tone in vivo. The latter was reversed by acute scavenging of superoxide with tempol (1 mmol/L), or inhibition of NADPH oxidase with apocynin (500 µmol/L) or DPI (5 µmol/L). We conclude that nitric oxide deficit produces a significant increase in the contribution of Cav3.1 and Cav3.2 T-type calcium channels to vascular tone, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function.

Hydrogen sulphide–mediated vasodilatation involves the release of neurotransmitters from sensory nerves in pressurized mesenteric small arteries isolated from rats

Hydrogen sulphide (H(2)S) is a gas that has recently been shown to have biological activity. In the majority of blood vessels studied so far, H(2)S has been shown to cause vasorelaxation, although contractile responses have been reported. In the present study, we have made a pharmacological assessment of the effects of H(2)S in mesenteric small arteries isolated from rats. Rat mesenteric small arteries were studied using pressure myography. In pressurised arteries, responses were obtained to the H(2)S donor, sodium hydrogen sulphide (NaHS), in the absence and presence of the NOS inhibitor L-NAME, raised extracellular potassium, the K(ATP) channel inhibitor glibenclamide, the Cl- channel blockers DIDS, NPPB and A9C, the TRPV1 receptor desensitizing agent, capsaicin, the CGRP antagonist, olcegepant, the TRPV1 channel blocker capsazepine and the TRPA1 channel blocker HC-030031. NaHS produced a vasodilator response in rat mesenteric small arteries held at 90 mmHg. Responses to NaHS were not reproducible. Neither, glibenclamide nor, L-NAME inhibited responses to NaHS. DIDS abolished vasodilator responses to NaHS, but these were unaffected by the chloride channel blockers, NPPB and A9C. Responses to NaHS were attenuated after capsaicin pre-treatment, by a CGRP receptor antagonist and an inhibitor of TRPA1 channels. In small arteries isolated from the rat mesentery, NaHS caused a vasodilatation. This response was not reproducible in vitro, since it was mediated by the release of sensory neurotransmitters in a capsaicin-like action. This release was mediated by a H(2)S-induced activation of TRPA1 channels.

The effect of streptozotocin-induced diabetes on the EDHF-type relaxation and cardiac function in rats

The endothelium-derived hyperpolarizing factor (EDHF) response is a critical for the functioning of small blood vessels. We investigated the effect of streptozotocin-induced diabetes on the EDHF response and its possible role in the regulation of cardiac function. The vasorelaxant response to ACh- or NS309- (direct opener endothelial small- (SKCa)- and intermediate-conductance (IKCa) calcium-activated potassium channels; main components of EDHF response) were measured in pressurized mesenteric arteries (diameter 300–350μm). The response to 1μM ACh was reduced in diabetes (84.8±2.8% control vs 22.5±5.8% diabetics; n⩾8; P<0.001). NS309 (1μM) relaxations were also decreased in diabetic arteries (78.5±8.7% control vs 32.1±5.8% diabetics; n⩾5; P<0.001). SKCa and IKCa-mediated EDHF relaxations in response ACh or NS309 were also significantly reduced by diabetes. Ruthenium red, RuR, a blocker of TRP channels, strongly depress the response to ACh and NS309 in control and diabetic arteries. RuR decreased SKCa and IKCa-mediated EDHF vasodilatation in response to NS309 but not to ACh. An elevation in systolic blood pressure was observed in diabetic animals. ECG recording of control hearts showed shortening of PR interval. RuR reduced PR interval and R wave amplitude in diabetic hearts. In conclusion, the reduced EDHF-type relaxations in STZ-induced diabetes is due impairment of KCa channels function. TRP channels possibly contribute to EDHF vasodilatation via direct opening of endothelial KCa. It is possible that EDHF and TRP channels contribute to the regulation of cardiac function and therefore can be considered as therapeutic targets to improve cardiovascular complications of diabetes.