Relaxation In Mesenteric Arteries Research Articles

Alteration of Piezo1 signaling in type 2 diabetic mice: focus on endothelium and BKCa channel.

Piezo1 mechanosensitive ion channel plays a important role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Ten- to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db-/db-) were used. The second-order mesenteric arteries (~ 150 μm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db-/db- mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db-/db- mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db-/db- mice, while there was no effect in control mice. Co-immunofluorescence staining showed co-localization of Piezo1 and BKCa channel was more pronounced in db-/db- mice than in control mice. These results indicate that the vascular responses induced by Piezo1 activation are different in the mesenteric resistance arteries in type 2 diabetic mice.

Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

Smooth Muscle-Specific Knockout of LRRC8A Anion Channels Improves Vasodilation in Renal Arteries from Diabetic Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise volume-regulated anion channels (VRACs). We previously have shown that knockout (KO) or block of LRRC8A anion channel attenuates reactive oxygen species (ROS) production by Nox1, impairs TNFα-induced inflammation, and increases vascular relaxation in mesenteric arteries. These changes are associated with reduced RhoA activation. Diabetic (db/db−/−) mice have previously been shown to develop vascular dysfunction that is characterized by increased ROS production and enhanced RhoA activation. Diabetic nephropathy causes chronic renal disease and can lead to end-stage renal failure. Oxidative stress plays a role in both the initiation and progression of this process. We hypothesized that LRRC8A KO would protect vascular function in db/db−/− mice and promote vascular relaxation in renal arteries. Smooth-muscle specific LRRC8A KO mice were created in a db/db−/− background. Renal arteries were isolated from db/db−/− WT and db/db−/− LRRC8A KO mice vascular contraction and relaxation were measured. Contractile responses to phenylephrine (PE) were unaltered in LRRC8A KO compared to WT vessels. However, both endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings that were contracted with PE (Emax ACh: WT 76.8 ± 5.5 vs. KO 92.2 ± 3.3 %, LogEC50 SNP: WT -7.6 ± 0.14 vs. KO -8.4 ± 0.14, n = 4-5, * p<0.05). These data demonstrate preserved vasodilation in smooth muscle-specific LRRC8A KO blood vessels. This may lead to increased renal blood flow in the setting of db/db−/−-related diabetes. Ongoing work will determine if improved vasodilation is associated with preserved renal function in these animals. If so, VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of diabetes-induced renal dysfunction. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

XX Sex Chromosome Complement Promotes Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases precipitously during menopausal transition due to estradiol decline. Longitudinal studies show a steep increase in PWV of postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized and middle-aged female mice. Additionally, we showed increased oxidative stress in female than male vascular cells treated with angiotensin II in hormone-free charcoal-stripped media. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. In this study, we used the four core genotype mice (n=6-10/group) comprising females with either XX or XY sex chromosome complement that were left intact or ovariectomized for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2nd order mesenteric artery were assessed by pressure myography. To induce hypertension, ovariectomized XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline as drinking water. PWV was significantly increased in ovariectomized than in gonadal intact XX and XY mice (main effect: p<0.001). However, an interaction effect was indicated with higher PWV in XX than XY ovariectomized mice (4.9±0.1 vs. 4.2±0.2 m/s; p<0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY ovariectomized mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY ovariectomized mice (30±0.01 vs. 26±0.02 %; P=0.005). Higher systolic blood pressure (SBP) was indicated in ovariectomized XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). DOCA-induced hypertension blunted the PWV difference between OVX XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.3), (Week 2: 148±4 vs.140±1 mmHg; P=0.08), and (Week 3: 137±9 vs.127±4 mmHg; P=0.4). We observed impaired acetylcholine-mediated relaxation in mesenteric arteries of hypertensive ovariectomized XX than XY mice (47±6 % vs. 24±6 %; P=0.03). Our data suggested that ovariectomized female mice with XX sex chromosome complement promote arterial stiffening and blood pressure. However, DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice. Future studies will identify elements in the XX sex chromosome complement contributing to arterial stiffening and high blood pressure. R00HL155841 and R25HL105400. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Knockout of LRRC8A Anion Channels in Vascular Smooth Muscle Cells Impairs NF-κB and Hif1α-mediated Signaling

Vascular smooth muscle cell (VSMC) inflammation associated with the development of vascular disease, including hypertension. We have demonstrated that knockout (KO) or inhibition of LRRC8A anion channels in VSMCs attenuates reactive oxygen species (ROS) production by NADPH oxidase 1, impairs TNFα-induced inflammation, and promotes vascular relaxation in murine mesenteric arteries. To explore the mechanism of these effects we performed RNA sequencing (RNAseq) on LRRC8A wild-type (WT) and knockout (KO) VSMCs (N = 3 pairs) under control conditions and 6hrs following TNFα treatment (10ng/ml). The data were normalized by Median ratio (DESeq2) and differential expression was determined by DESeq2 method using Partek software and pathway enrichment analysis was done by KEGG pathway. Differential expression of genes of interest was confirmed by quantitative PCR (qPCR) in both cultured cells and in whole aortic tissues (endothelium-denuded, N = 3) from LRRC8A WT and KO mice. HIF1α transcriptional activity was quantified by luciferase reporter assays in cells exposed to hypoxia (1% oxygen) or cobalt chloride (300μM) for 6 hrs. RNAseq demonstrated broad impairment of pro-inflammatory signaling in KO cells. This was consistent with previous observations of impaired nuclear factor kappa B (NF-κB) reporter activation by TNFα in the absence of LRRC8A. Compared to WT VSMCs, resting LRRC8A KO VSMCs exhibited statistically significant downregulation of 33 NF-κB target genes including the IL-6 (Il6) and IL-1β (Il1r1) receptors. TNFα treatment increased the expression of 37 NF-κB target genes in WT cells. Most of these genes (34 genes) were not significantly activated by TNFα in KO cells, while 3 genes (chemokine (C-C motif) ligand 20; CCL20, prostaglandin E synthase; PTGES and vascular endothelial growth factor C; VEGFC) were significantly downregulated. Pathway analysis data revealed that Hif1α signaling appear in the top downregulated pathway and it was impaired as 14 Hif1α target genes were downregulated in unstimulated KO VSMCs. In addition, expression of Rps4i, a long non-coding mRNA associated with inhibition of HIF1α signaling was significantly upregulated. Luciferase reporter assays following exposure to hypoxia or cobalt chloride confirmed impaired Hif1α activation in LRRC8A KO cells. Important target genes for HIF1α, including HK2, LDHB, ACE, BMP4 and PFKP, were less activated, while RPS4i remained upregulated compared to WT cells. These findings demonstrate disruption of both NF-κB and HIF1α signaling in VSMCs lacking LRRC8A. This anion channel represents a novel target for anti-inflammatory therapy in vascular disease. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alteration of Piezo1 signaling in type 2 diabetic mice: Focus on endothelium and BKca channel

Aim: Piezo1, a mechanosensitive ion channel, plays a significant role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Methods: Ten-to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db−/db−) were used. The second-order mesenteric arteries (~150 mm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Results: Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db−/db− mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db−/db− mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db−/db− mice, while there was no effect in control mice. Co-immunofluorescence staining showed more structural coupling between Piezo1 and BKCa channel was observed in db−/db− mice than in control mice. Conclusions: The present study is the first report on the distinct aspects of vascular response induced by Piezo1 activation in the mesenteric resistance arteries in type 2 diabetic mice. National Research Foundation of Korea (RS-2023-00272628). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stimulation of the calcium-sensing receptor induces relaxations of rat mesenteric arteries by endothelium-dependent and -independent pathways via BKCa and KATP channels.

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.

Aging related decreases in NM myosin expression and contractility in a resistance vessel.

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

Follistatin-like 1 protects endothelial function in the spontaneously hypertensive rat by inhibition of endoplasmic reticulum stress through AMPK-dependent mechanism

ABSTRACT Objective Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs). Methods Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography. Results We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor). Conclusions These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.

Impaired vascular β-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries

It was suggested that impaired β-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased β-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of β-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (β2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (β1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated β-adrenergic vasodilatation of conduit arteries of SHR but similar β-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to β-adrenergic agonists do not support major role of altered β-adrenergic vasodilatation for high BP in genetic hypertension.

Abstract P181: Tcell Derived Hiv Proteins Promote Oxidative Stress, Endothelial Dysfunction And Hypertension Via Interleukin 1a-mediated Mechanisms In Mice.

Antiretroviral therapy (ART) has markedly extended the life of people living with HIV (PLWH). However, PLWH now experience accelerated development of hypertension and cardiovascular disease (CVD), the etiology of which, including the individual contributions of ART and repressed viral infection, remain ill-defined. While ART prevents viral replication, viral reservoirs persist, and viral proteins remain in the circulation leading us to hypothesize that Tcell-derived HIV proteins contribute to endothelial dysfunction and hypertension. We utilized the Tg26 transgenic mouse model which ubiquitously express 7/9 viral proteins and reported increased blood pressure (BP) and impaired endothelium-dependent relaxation (EDR) in aorta and mesenteric arteries in both sexes. Bone marrow transplant (BMT) from Tg26 mice into WT (Tg26->WT) increased BP (WT->WT: 141.6 Tg26->WT 160.3 mmHg; P<0.05) and impaired EDR in conduit and resistance vessels (P<0.05). To determine whether Tcells, the main target of HIV, are responsible for CVD in Tg26 mice, thoracic aortas from WT male and female mice were co-cultured with either WT or Tg26 primary CD3+ Tcells, and yielded endothelial dysfunction. Moreover, inhibition of Tcell activation via Abatacept treatment in intact Tg26 mice lowered BP, restored vasorelaxation and reduced sympatho-activation to WT levels in Tg26 mice (P<0.05). A cytokine panel revealed increased production of interleukin 1α (IL-1α) in Tg26 CD3+ Tcells and plasma of Tg26->WT BMT (P<0.05). Abatacept decreased plasma IL-1α levels in intact Tg26 mice (P<0.05). Treatment with anti-IL-1α antibody abolished Tcell-mediated endothelial dysfunction and primary human microvascular endothelial cells (hMVEC) treated with IL-1α for 24 hours increased ROS mediated DNA damage (8-OHdG). IL-1α exposed hMVEC displayed increases in NOX1, NOXO1, and NOX4 while BMT aortas showed increased NOX1, NOXO1, and NOXA1. Inhibition of NOX1 via GKT771 ex vivo restored vasorelaxation. Additionally, anti-IL-1α restored vasorelaxation and BP in intact Tg26 mice. Ultimately, we found that Tcells expressing viral proteins produce high IL-1α levels which increase endothelial NOX1 expression and ROS production, leading to endothelial dysfunction and hypertension

Cl− induces endothelium-dependent mesenteric arteriolar vasorelaxation through the NKCC1/TRPV4/NCX axis

AimsAlthough absorbed NaCl increases intestinal blood flow to facilitate absorption and transportation, it is unclear if it can directly mediate mesenteric arterial relaxation. We aimed to investigate and test our hypothesis that Cl− induces mesenteric arterial vasorelaxation via endothelium-dependent hyperpolarization (EDH). Main methodsWe used wire myograph to study NaCl-induced vasorelaxation of mesenteric arteries isolated from mice. Cl−, Ca2+ and K+ imaging was performed in human vascular endothelial cells pre-treated with pharmacological agents. Key findingsThe Cl− concentration-dependently induced vasorelaxation of mesenteric arteries likely through EDH. The Cl−-induced vasorelaxation was attenuated in TRPV4 KO mice and inhibited by selective blockers of Na+-K+-2Cl− cotransporter 1 (NKCC1) (bumetanide, 10 μM), transient receptor potential vanilloid 4 (TRPV4) (RN-1734, 40 μM), and small conductance Ca2+-activated K+ channels (SKCa) (apamin, 3 μM)/ intermediate conductance Ca2+-activated K+ channels (IKCa) (TRAM-34, 10 μM) and myoendothelial gap junction (18α-glycyrrhetinic acid, 10 μM), but enhanced by a selective activator of IKCa/SKCa (SKA-31, 0.3 μM). Cl− decreased intracellular K+ concentrations in endothelial cells, which was reversed by apamin (200 nM) plus TRAM-34 (500 nM). Extracellular Cl− raised intracellular Cl− concentrations in endothelial cells, which was attenuated by bumetanide (10 μM). Finally, Cl− induced a transient Ca2+ signaling via TRPV4 in endothelial cells, which became sustained when the Ca2+ exit mode of Na+-Ca2+ exchanger (NCX) was blocked. SignificanceCl− induces a pure EDH-mediated vasorelaxation of mesenteric arteries through activation of endothelial NKCC1/TRPV4/NCX axis. We have provided a novel insight into the role of Cl−-induced vasorelaxation via EDH mechanism.

Piezo1 Mediates Vasodilation Induced by Acute Hyperglycemia in Mouse Renal Arteries and Microvessels.

Acute hyperglycemia is a risk factor for developing acute kidney injury and poor renal outcome in critically ill patients, whereby the role of renal vasculature remains unclear. We hypothesize that hyperglycemia-associated hyperosmolarity facilitates vasodilation through Piezo1-mediated eNOS (endothelial NO synthase) activation. Vasoreactivity was analyzed using wire myography in isolated mouse mesenteric arteries and renal interlobar, and using microvascular perfusion in renal afferent arterioles and efferent arterioles, and vasa recta. Immunofluorescence and Western blot were used for molecular analyses of isolated mouse blood vessels and human umbilical vein endothelial cells. Pretreatment with hyperglycemia (44 mmol/L glucose; 4 hours) increased acetylcholine-induced relaxation in interlobar arteries and mesenteric arteries, which was prevented by eNOS inhibition using Nω-nitro-L-arginine methylester hydrochloride. Hyperosmotic mannitol solution had a similar effect. Hyperglycemia induced an immediate, Nω-nitro-L-arginine methylester hydrochloride-inhibitable dilation in afferent arterioles, efferent arterioles, and vasa recta, whereby stronger dilation in afferent arterioles compared to efferent arterioles. Hyperglycemia also increased glomerular filtration rate in mice. In human umbilical vein endothelial cells, hyperglycemia, and the Piezo1 activator Yoda-1 increased levels of Piezo1 protein, p-CaMKII (phosphorylated Ca2+/Calmodulin-dependent protein kinase type II), Akt, and p-eNOS (phosphorylated eNOS). The hyperglycemia effect could be prevented by inhibiting Piezo1 using GsMTx4 (Grammostola spatulata mechanotoxin 4) and CaMKII using KN93. Furthermore, in arteries and microvessels, inhibition of Piezo1 using GsMTx4 prevented the hyperglycemia -effect, while Yoda-1 caused relaxation and dilation, respectively. Results reveal that Piezo1 mediates renal vasodilation induced by hyperosmolarity in acute hyperglycemia. This mechanism may contribute to the pathogenesis of renal damage by acute hyperglycemia.

Intravenous administration of apeling-13 induces a depressor response by releasing an unidentified substance

Apelin and APJ receptor play an important role in the regulating cardiovascular function; however, conflicting results have been reported regarding the effect of apelin on cardiovascular regulation. In this study, blood pressure and heart rate were measured by femoral arterial catheterization; and cardiac contractility was recorded by left ventricular catheterization through the right carotid artery in rats before and after intravenous administration of [pyr1]-apelin-13. The results show that intravenous administration of apelin-13 caused a dramatic reduction in BP but did not significantly alter heart rate and contractility. To study the mechanism of the apelin-induced depressor response, isometric tension was measured in isolated mesenteric arteries using a myograph approach. Surprisingly, treatment of the arteries with [pyr1]-apelin-13 did not cause relaxation of mesenteric arteries preconstricted with norepinephrine; however, treatment with plasma collected from rats that received intravenous administration of [pyr1]-apelin-13 caused pronounced relaxation of isolated arteries. Incubation with the guanylyl cyclase inhibitor, ODQ, blocked NO-induced relaxation, but did not significantly alter the relaxation response to the plasma from apelin-treated rats. Taken together, these findings demonstrate that intravenous injection of apelin causes a significant depressor response that is mediated by a NO-independent mechanism involving an unidentified substance released into the bloodstream leading to vasodilation.

TRPM8 channels do not regulate endothelium-dependent and -independent vascular reactivity in mice

Foremost among the ion channels that control vascular tone is the transient receptor potential (TRP) cation channels. The TRPM subfamily of the TRP channels includes eight members, expressed in various excitable and non-excitable cells and involved in diverse functions, such as sensing membrane potential, cold, taste, redox, osmolarity, and cell survival. Previous analysis of TRPM gene expression showed the highest expression of TRPM8 in intact blood vessels compared with other isoforms (TRPM1-7). Studies have also revealed that activating TRPM8 by menthol and icilin modulates endothelium-dependent and independent vascular reactivity. However, the non-selectivity of pharmacological TRPM8 modulators constitutes a significant limitation in the prior studies aimed at delineating TRPM8-dependent vasoregulation. In this study, we demonstrate that acetylcholine (ACH)- and sodium nitroprusside (SNP)-induced reduction in arterial pressure was unaltered in mice lacking TRPM8. We generated endothelial-specific knockout (KO) of TRPM8 (ecTRPM8-/-) by breeding TRPM8 floxed (TRPM8fl/fl) with Cdh5-Cre (VE-Cadherin-Cre) mice. Unlike global TRPM8 KO, ecTRPM8-/- mice were not resistant to cold temperatures. Phenylephrine-induced contraction of renal and mesenteric arteries isolated from TRPM8fl/fl and ecTRPM8-/- mice were similar. Also, ACH- and SNP-induced relaxation of phenylephrine-precontracted renal and mesenteric arteries from TRPM8fl/fl and ecTRPM8-/- mice were comparable. These findings suggest that TRPM8 channels do not regulate endothelium-dependent and -independent vasorelaxation in mice. Disclosure of Funding: NHLBI: R01 HL151735-01 and R01HL151735-S This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Moderate-Intensity Exercise Improves Mesenteric Arterial Function in Male UC Davis Type-2 Diabetes Mellitus (UCD-T2DM) Rats: A Shift in the Relative Importance of Endothelium-Derived Relaxing Factors (EDRF)

The beneficial cardiovascular effects of exercise are well documented, however the mechanisms by which exercise improves vascular function in diabetes are not fully understood. This study investigates whether there are (1) improvements in blood pressure and endothelium-dependent vasorelaxation (EDV) and (2) alterations in the relative contribution of endothelium-derived relaxing factors (EDRF) in modulating mesenteric arterial reactivity in male UC Davis type-2 diabetes mellitus (UCD-T2DM) rats, following an 8-week moderate-intensity exercise (MIE) intervention. EDV to acetylcholine (ACh) was measured before and after exposure to pharmacological inhibitors. Contractile responses to phenylephrine and myogenic tone were determined. The arterial expressions of endothelial nitric oxide (NO) synthase (eNOS), cyclooxygenase (COX), and calcium-activated potassium channel (KCa) channels were also measured. T2DM significantly impaired EDV, increased contractile responses and myogenic tone. The impairment of EDV was accompanied by elevated NO and COX importance, whereas the contribution of prostanoid- and NO-independent (endothelium-derived hyperpolarization, EDH) relaxation was not apparent compared to controls. MIE 1) enhanced EDV, while it reduced contractile responses, myogenic tone and systolic blood pressure (SBP), and 2) caused a shift away from a reliance on COX toward a greater reliance on EDH in diabetic arteries. We provide the first evidence of the beneficial effects of MIE via the altered importance of EDRF in mesenteric arterial relaxation in male UCD-T2DM rats.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signalingnor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

Vanillin Induces Relaxation in Rat Mesenteric Resistance Arteries by Inhibiting Extracellular Ca2+ Influx.

Vanillin is a phenolic aldehyde, which is found in plant species of the Vanilla genus. Although recent studies have suggested that vanillin has various beneficial properties, the effect of vanillin on blood vessels has not been studied well. In the present study, we investigated whether vanillin has vascular effects in rat mesenteric resistance arteries. To examine the vascular effect of vanillin, we measured the isometric tension of arteries using a multi-wire myograph system. After the arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM), vanillin was administered. Vanillin induced concentration-dependent vasodilation. Endothelial denudation or treatment of eNOS inhibitor (L-NNA, 300 μM) did not affect the vasodilation induced by vanillin. Treatment of K+ channel inhibitor (TEA, 10 mM) or sGC inhibitor (ODQ, 10 μM) or COX-2 inhibitor (indomethacin, 10 μM) did not affect the vanillin-induced vasodilation either. The treatment of vanillin decreased the contractile responses induced by Ca2+ addition. Furthermore, vanillin significantly reduced vascular contraction induced by BAY K 8644 (30 nM). Vanillin induced concentration-dependent vascular relaxation in rat mesenteric resistance arteries, which was endothelium-independent. Inhibition of extracellular Ca2+ influx was involved in vanillin-induced vasodilation. Treatment of vanillin reduced phopsho-MLC20 in vascular smooth muscle cells. These results suggest the possibility of vanillin as a potent vasodilatory molecule.

Dynein Coordinates β2-Adrenoceptor-Mediated Relaxation in Normotensive and Hypertensive Rat Mesenteric Arteries.

The voltage-gated potassium channel (Kv)7.4 and Kv7.5 channels contribute to the β-adrenoceptor-mediated vasodilatation. In arteries from hypertensive rodents, the Kv7.4 channel is downregulated and function attenuated, which contributes to the reduced β-adrenoceptor-mediated vasodilatation observed in these arteries. Recently, we showed that disruption of the microtubule network, with colchicine, or inhibition of the microtubule motor protein, dynein, with ciliobrevin D, enhanced the membrane abundance and function of Kv7.4 channels in rat mesenteric arteries. This study aimed to determine whether these pharmacological compounds can improve Kv7.4 function in third-order mesenteric arteries from the spontaneously hypertensive rat, thereby restoring the β-adrenoceptor-mediated vasodilatation. Wire and intravital myography was performed on normotensive and hypertensive male rat mesenteric arteries and immunostaining was performed on isolated smooth muscle cells from the same arteries. Using wire and intravital microscopy, we show that ciliobrevin D enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline. This effect was inhibited partially by the Kv7 channel blocker linopirdine and was dependent on an increased functional contribution of the β2-adrenoceptor to the isoprenaline-mediated relaxation. In mesenteric arteries from the spontaneously hypertensive rat, ciliobrevin D and colchicine both improved the isoprenaline-mediated vasorelaxation and relaxation to the Kv7.2 -7.5 activator, ML213. Immunostaining confirmed ciliobrevin D enhanced the membrane abundance of Kv7.4. As well as an increase in the function of Kv7.4, the functional changes were associated with an increase in the contribution of β2-adrenoceptor following isoprenaline treatment. Immunostaining experiments showed ciliobrevin D prevented isoprenaline-mediated internalizationof the β2-adrenoceptor. Overall, these data show that colchicine and ciliobrevin D can induce a β2-adrenoceptor-mediated vasodilatation in arteries from the spontaneously hypertensive rat as well as reinstating Kv7.4 channel function.

Abstract 003: Treatment With Ligilactobacillus Improves Gut Permeability And Hypertension In Spontaneously Hypertensive Rats

Emerging evidence provide that a microbial imbalance (dysbiosis) is linked to several diseases including cardiovascular diseases. One of the features in hypertension is increased gut permeability. Supporting this, plasma endotoxin level is increased in hypertension and our previous study indicated that the plasma level of exotoxin from Streptococcus was increased in spontaneously hypertensive rats (SHR). Thus, we hypothesized that improvement of gut permeability in hypertension might be expected to prevent development of hypertension. In this study, we performed a 16S metagenome analysis using a synthetic long read sequencing approach by LoopSeq in feces from 10- to 15-week old SHR and Wistar-Kyoto rat (WKY). The analysis revealed that two difference species of anti-inflammatory microbiome were strongly decreased in SHR compared with WKY (n=4). We focus on the decreased Ligilactobacillus sp. (LAB) in SHR and oral administration of LAB to SHR for 2 weeks. SHR (10-15-week-old) exhibited increased gut permeability in proximal colon (P<0.05, n=4), which was improved by treatment with LAB for 2 weeks (P<0.05, n=4). The expression level of tight junction protein Claudin4 was decreased in SHR proximal colon (SHR vs WKY, 1.0±0.1 vs 1.7±0.2-fold increase to SHR, P<0.05, n=8), which was significantly increased by LAB treatment (SHR vs SHR+LAB, 1.0±0.1 vs 2.8±0.5-fold increase to SHR, P<0.05, n=10). Proximal colon in SHR increased the expression level of TNF-α mRNA (1.0±0.2 vs 0.2±0.1, P<0.05, n=8), which was reversed by LAB (1.0±0.2 vs 0.3±0.1, P<0.05, n=10). The level of mucin 2 was lower in SHR proximal colon (1.0±0.2 vs 4.3±0.8, P<0.05, n=8), which was not reversed by LAB (1.0±0.1 vs 0.8±0.3, n=10). We observed that high blood pressure in SHR was attenuated by treatment with LAB for 2 weeks (194.0±5.9 vs 149.7±4.8 mmHg, P<0.05, n=10). LAB also improved decrease in acetylcholine-induced relaxation in SHR mesenteric artery (P<0.05, n=5-8). We conclude that treatment of SHR with the decreased anti-inflammatory microbiota, LAB improves gut permeability in proximal colon possibly through inhibition of inflammation, resulting in lowering blood pressure. These results suggest that anti-inflammatory microbiome might be a new therapeutic target for hypertension.