Function Of Mesenteric Arteries Research Articles

Aged Gut Microbiome Induces Metabolic Impairment and Hallmarks of Vascular and Intestinal Aging in Young Mice.

Aging, an independent risk factor for cardiometabolic diseases, refers to a progressive deterioration in physiological function, characterized by 12 established hallmarks. Vascular aging is driven by endothelial dysfunction, telomere dysfunction, oxidative stress, and vascular inflammation. This study investigated whether aged gut microbiome promotes vascular aging and metabolic impairment. Fecal microbiome transfer (FMT) was conducted from aged (>75 weeks old) to young C57BL/6 mice (8 weeks old) for 6 weeks. Wire myography was used to evaluate endothelial function in aortas and mesenteric arteries. ROS levels were measured by dihydroethidium (DHE) staining and lucigenin-enhanced chemiluminescence. Vascular and intestinal telomere function, in terms of relative telomere length, telomerase reverse transcriptase expression and telomerase activity, were measured. Systemic inflammation, endotoxemia and intestinal integrity of mice were assessed. Gut microbiome profiles were studied by 16S rRNA sequencing. Some middle-aged mice (40-42 weeks old) were subjected to chronic metformin treatment and exercise training for 4 weeks to evaluate their anti-aging benefits. Six-week FMT impaired glucose homeostasis and caused vascular dysfunction in aortas and mesenteric arteries in young mice. FMT triggered vascular inflammation and oxidative stress, along with declined telomerase activity and shorter telomere length in aortas. Additionally, FMT impaired intestinal integrity, and triggered AMPK inactivation and telomere dysfunction in intestines, potentially attributed to the altered gut microbial profiles. Metformin treatment and moderate exercise improved integrity, AMPK activation and telomere function in mouse intestines. Our data highlight aged microbiome as a mechanism that accelerates intestinal and vascular aging, suggesting the gut-vascular connection as a potential intervention target against cardiovascular aging and complications.

Vγ6/Vδ1+ γδ T cells protect from angiotensin II effects on blood pressure and endothelial function in mice.

γδ T cells mediate angiotensin II (AngII)-induced hypertension and vascular injury. γδ T cells expressing specific T-cell receptor (TCR) variable (V) γ chains develop in several waves in the thymus and migrate to specific or diverse tissues. We hypothesized that γδ T cells expressing specific Vγ subtypes in perivascular tissue mediate AngII hypertensive effects. C57BL/6J male mice were infused or not with AngII (490 ng/kg/min, subcutaneously) for 14 days. γδ T-cell Vγ subtypes were profiled by flow cytometry in the spleen, descending thoracic aorta with adherent perivascular adipose tissue (DTAo/PVAT) and mesenteric vessels (MV)/PVAT. Other sets of AngII-infused mice were injected with control or specific anti-Vγ6 or Vγ4 antibodies. Blood pressure (BP) was determined by telemetry, and mesenteric artery function and remodeling by pressurized myography. Vγ6/Vδ1+ γδ T cells represented more than 50% of the γδ T-cell Vγ subtypes in DTAo/PVAT and MV/PVAT, whereas Vγ1/2+, Vγ4+ and Vγ6/Vδ1+ γδ T cells were the most abundant Vγ subtypes in the spleen. The frequency of Vγ6/Vδ1+ γδ T cells was increased at least 1.5-fold in the spleen and DTAo/PVAT, and tended to increase in MV/PVAT by AngII. A majority of Vγ6/Vδ1+ γδ T cells were activated in perivascular tissues. Vγ6/Vδ1+ γδ T-cell neutralization caused a steeper BP elevation and greater mesenteric artery endothelial dysfunction in mice infused with AngII. This was associated with more than three-fold increase in activated Vγ6/Vδ1- γδ T cells in perivascular tissues. Depletion of Vγ4+ γδ T cells did not alter AngII detrimental effects. Vγ6/Vδ1+ γδ T cells reduce the BP elevation and endothelial dysfunction induced by AngII infusion.

Abstract 32: Vγ6 + γδ T cells protect against angiotensin II-induced hypertension and vascular injury in male mice

Background: γδ T cells mediate angiotensin II (AngII)-induced blood pressure (BP) elevation, vascular injury and activated T cell infiltration in mesenteric vessels with adherent perivascular adipose tissue (MV/PVAT). γδ T cells expressing specific T cell receptor (TCR) variable (V) chain γ develop in several waves in the thymus and migrate to diverse tissues. We hypothesized that γδ T cells expressing specific Vγ subtypes in perivascular tissue are implicated in AngII detrimental hypertensive effects. Methods: C57BL/6J male mice were infused or not with AngII (490 ng/kg/min, SC) for 14 days. Vγ1 and Vγ2 (Vγ1/2) + , Vγ4 + , Vγ5 + , Vγ6 + , and Vγ7 + γδ T cell frequencies and T cell phenotypes were determined by flow cytometry in the spleen, descending thoracic aorta (DTAo)/PVAT, and MV/PVAT (n=7-13). Other sets of AngII-infused mice were injected with control or anti-Vγ6 (n=7-12) or Vγ4 antibodies (n=5-8). BP was determined by telemetry, mesenteric artery (MA) function and remodeling by pressurized myography, and T cell phenotypes by flow cytometry. Results: In DTAo/PVAT and MV/PVAT, respectively, 43±5% and 51%±6% of γδ T cells were Vγ6 + , and 17±2% and 23±4% Vγ4 + , whereas in the spleen, 36±2% of the γδ T cells were Vγ1/2 + , 32±1% Vγ4 + and 24±1% Vγ6 + . AngII increased the frequency of Vγ6 + γδ T cells in the spleen (0.8±0.1 vs 0.5±0.0% of CD3, P <0.001) and DTAo/PVAT (5.9±0.8 vs 3.6±0.5% of CD3, P <0.05), and tended to increase their frequency in MV/PVAT. The majority of Vγ6 + γδ T cells were activated (CD69 + ) in DTAo/PVAT (75±5%) and MV/PVAT (84±2%). Vγ6 + γδ T cell depletion caused a steeper BP elevation ( P <0.05) and worsened MA endothelial dysfunction (maximal acetylcholine relaxation response: 28±3 vs 55±8%, P <0.05) in mice infused with AngII. This was associated with increased CD69 + non-Vγ6 + γδ T cells in DTAo/PVAT (3.0±0.7 vs 0.8±0.4% of CD3, P <0.01) and MV/PVAT (15.2±2.6 vs 4.9±1.0% of CD3, P <0.01). Depletion of Vγ4 + γδ T cells in perivascular tissues, did not alter the AngII detrimental effects. Conclusions: Vγ6 + γδ T cells are the most abundant γδ T cell Vγ subtype in perivascular tissues. AngII increased Vγ6 + γδ T cells in the spleen and perivascular tissue. Vγ6 + γδ T cell depletion in AngII-infused mice induced a steeper BP elevation, worsened MA endothelial dysfunction, and activated non-Vγ6 + γδ T cells. Vg6 + γδ T cells may play a protective role in AngII-induced hypertension and vascular injury.

Eplerenone Reduces Blood Pressure and Improves Vascular Function in a Mouse Model of Leptin-induced Preeclampsia

Preeclampsia is a hypertensive pregnancy disease that increases the risks for adverse outcomes of pregnancy for both mother and baby, notably by reducing fetal growth in utero. We have shown that mid-late gestation leptin infusion induces preeclampsia characteristics in pregnant mice in inducing hypertension, endothelial dysfunction, and fetal growth restriction, which is ablated in mice with endothelial mineralocorticoid receptor deletion (ECMR). However, there is a lack of preclinical data and rationale for MR antagonism for use in preeclampsia in the existing literature. Therefore, we hypothesized that leptin increases ECMR expression and that MR antagonism decreases hypertension, endothelial dysfunction, and fetal growth restriction in leptin-induced preeclamptic mice. We infused timed-pregnant Balb/C mice with saline (sham) or leptin (LEP, 0.9 mg/kg/day) via s.c. osmotic minipump and administered vehicle or eplerenone (EPL, 200mg/kg/day) in drinking water from gestation day (GD)11-18 (n=7-12). We measured mean arterial blood pressure (BP) via radiotelemetry, vascular function in 2nd order mesenteric arteries by wire myography, and pup/placental weights on GD18. We isolated endothelial cells from placentas of our mice via magnetic cell sorting. Pairwise comparisons were by student’s t test and myography curves analyzed by 2-W ANOVA w/RM. Leptin increased ECMR mRNA expression (3.1±0.32 WT LEP vs 1.1±0.48 WT sham, fold change, *p<0.05) compared to WT sham mice in placenta indicating a direct role for leptin to upregulate ECMR in pregnancy. EPL ablated leptin-induced increases in BP (98±4 LEP/EPL vs 113±3 lep/veh mmHg, *p<0.05), and when treated with EPL leptin did not induce endothelial dysfunction, as assessed as a lack of decrease in vascular relaxation to acetylcholine (p>0.05 LEP/EPL vs sham/EPL). Interestingly, EPL treatment did not prevent leptin from decreasing placental effciency in pregnant mice (7.42±0.17 LEP/veh vs 6.71±0.15 LEP/EPL, pup/placenta weight ratio, p>0.05). Collectively, these data show that leptin infusion from GD11-18 in pregnant mice increases ECMR activation by increasing expression. Furthermore, MR antagonism by eplerenone ablates the maternal hypertension and endothelial dysfunction in leptin-induced preeclampsia mice, however, fetal growth is not restored by eplerenone. Therefore, eplerenone may be a promising therapeutic for the maternal cardiovascular syndrome of preeclampsia patients, however, MR antagonism therapies may need additional agents to improve fetal outcomes. 1R01HL169576, 4R00HL146948. 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.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II–hypertensive mice

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II–induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg−1 min−1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg−1 min−1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10−9 to 10−6 mol L−1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10−9 mol L−1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II–mediated hypertension. Aldosterone contributes little to angiotensin II–induced hypertension in mice.

Abstract P104: EXAMINING THE EFFECTS OF FOLLISTATIN ON CONTRACTION OF VESSELS FROM NORMOTENSIVE AND HYPERTENSIVE RATS

The lab has previously shown that follistatin (FST), a glycoprotein inhibitor of activins, reduced blood pressure in the Spontaneously Hypertensive Rat (SHR) model of Essential Hypertension. Short-term exposure (30 minutes) to FST also reduced vessel contraction induced by potassium (KCl). This study aims to illustrate the mechanisms through which FST inhibits potassium-induced contraction in SHR and normotensive (WKY) rats. Function of SHR or WKY resistance mesenteric arteries was tested in response to KCl and ion channel modulators using wire myography. Cultures of primary smooth muscle cells (SMC) were also established from these vessels using cell explant technique. The calcium sensor dye Fluo-4 AM was used to examine calcium influx on a cellular level. To test whether FST effects were due to activin neutralization or activin-receptor inhibition, neutralizing antibodies against activin A and B or an activin receptor inhibitor (SB 431542) were used, respectively. Only activin A neutralization in the SHR reduced KCl-induced contraction similarly to FST. Activin receptor inhibition also reduced KCl constriction similarly to FST. When directly stimulated with Activin A, both WKY and SHR vessels displayed augmented KCl contraction compared to controls but the effect was more pronounced in the SHR. Preliminary Fluo-4 results showed that FST decreased KCl-induced calcium influx in SMC. Since activin A was shown to increase L-type voltage-gated Ca2+channel activity, an agonist (FPL 64176) of this channel was used to induce contraction. Although FST reduced KCl-induced contraction, it did not affect FPL-induced contraction in either SHR or WKY. Thus, Activin A neutralization reproduces FST reduction of KCl-induced vessel contraction. FST did not reduce FPL-induced contraction, suggesting that its inhibition of KCl-induced contraction is not through effects on this channel but rather an alternate Ca2+ or K-ATP channel.

P2RX7 gene knockout or antagonism reduces angiotensin II-induced hypertension, vascular injury and immune cell activation.

Extracellular ATP is elevated in hypertensive mice and humans and may trigger immune activation through the purinergic receptor P2X7 (P2RX7) causing interleukin-1β production and T-cell activation and memory T-cell development. Furthermore, P2RX7 single nucleotide polymorphisms (SNP) are associated with hypertension. We hypothesized that P2RX7 activation contributes to hypertension and cardiovascular injury by promoting immune activation. Male wild-type and P2rx7-/- mice were infused or not with angiotensin II (AngII) for 14 days. A second group of AngII-infused wild-type mice were co-infused with the P2RX7 antagonist AZ10606120 or vehicle. BP was monitored by telemetry. Cardiac and mesenteric artery function and remodeling were assessed using ultrasound and pressure myography, respectively. T cells were profiled in thoracic aorta/perivascular adipose tissue by flow cytometry. Associations between SNPs within 50 kb of P2RX7 transcription, and BP or hypertension were modeled in 384 653 UK Biobank participants. P2rx7 inactivation attenuated AngII-induced SBP elevation, and mesenteric artery dysfunction and remodeling. This was associated with decreased perivascular infiltration of activated and effector memory T-cell subsets. Surprisingly, P2rx7 knockout exaggerated AngII-induced cardiac dysfunction and remodeling. Treatment with a P2RX7 antagonist reduced BP elevation, preserved mesenteric artery function and reduced activated and effector memory T cell perivascular infiltration without adversely affecting cardiac function and remodeling in AngII-infused mice. Three P2RX7 SNPs were associated with increased odds of DBP elevation. P2RX7 may represent a target for attenuating BP elevation and associated vascular damage by decreasing immune activation.

Maternal exercise represses Nox4 via SIRT1 to prevent vascular oxidative stress and endothelial dysfunction in SHR offspring.

Maternal exercise during pregnancy has emerged as a potentially promising approach to protect offspring from cardiovascular disease, including hypertension. Although endothelial dysfunction is involved in the pathophysiology of hypertension, limited studies have characterized how maternal exercise influences endothelial function of hypertensive offspring. In this study, pregnant spontaneously hypertensive rats and Wistar-Kyoto rats were assigned either to a sedentary lifestyle or to swimming training daily, and fetal histone deacetylase-mediated epigenetic modification and offspring vascular function of mesenteric arteries were analyzed. Maternal exercise ameliorated the impairment of acetylcholine-induced vasodilation without affecting sodium nitroprusside-induced vasodilation in mesenteric arteries from the hypertensive offspring. In accordance, maternal exercise reduced NADPH oxidase-4 (Nox4) protein to prevent the loss of nitric oxide generation and increased reactive oxygen species production in mesenteric arteries of hypertensive offspring. We further found that maternal exercise during pregnancy upregulated vascular SIRT1 (sirtuin 1) expression, leading to a low level of H3K9ac (histone H3 lysine 9 acetylation), resulting in the transcriptional downregulation of Nox4 in mesenteric arteries of hypertensive fetuses. These findings show that maternal exercise alleviates oxidative stress and the impairment of endothelium-dependent vasodilatation via SIRT1-regulated deacetylation of Nox4, which might contribute to improved vascular function in hypertensive offspring.

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease.

Introduction: Inflammatory bowel disease involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how inflammatory bowel disease affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with Inflammatory bowel disease. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. Methods: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H. hepaticus to induce disease (inflammatory bowel disease) or left non-gavaged (Control). For all other studies, Control and Inflammatory bowel disease mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Leukocyte populations, and perivascular nerves, and adventitial neurotransmitter receptors were labeled using fluorescent immunolabeling. Results: Inflammatory bowel disease was associated with increases in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in inflammatory bowel disease. Acetylcholine-mediated dilation was impaired in inflammatory bowel disease and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. Conclusion: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in Inflammatory bowel disease patients.

RSK2 deletion alters vascular function and hypertension in resistance mesenteric arteries of aged mice

Hypertension (HT), a condition that becomes increasingly prevalent with age, affects millions of Americans, and can lead to heart disease, stroke, and kidney failure. Pathophysiological mechanisms contributing to the increased peripheral vascular resistance associated with HT, include arterial remodeling, increased vascular contraction and reduced vasodilation. Vascular structural and functional changes of aging closely resemble the classical HT phenotype. Current antihypertensive medications are less than 50% effective at controlling blood pressure, highlighting the need for better therapeutic options. Recently, our group has demonstrated that p90 ribosomal s6 kinase (Rsk2) plays a significant physiological role in SM contractility. We have shown that young adult global Rsk2 KO mice had more dilated mesenteric arteries (MAs), with reduced vessel tone, stiffness and RLC20 phosphorylation, and significantly lower basal blood pressure compared to WT. However, it is unknown whether Rsk2 plays a functional role in age-related HT. We found that >24-month-old WT mice had a significantly higher mortality rate compared to aged-match Rsk2 KO. Baseline Systolic blood pressure (SBP) was significantly lower in KOs compared to WT. L-NAME elevated SBP in WT but not in KO mice. There were no differences in echo parameters in WT and Rsk2 KO aged mice. Histology of 3rd and 4th order WT and Rsk2 KO MA cross-sections revealed no differences in lumen diameter, wall, thickness, or ratio of wall area to total area in aged mice. Thus, Rsk2 deletion did not alter vascular remodeling in resistance arteries of aged mice. Pressure myography studies assessed vascular reactivity and arterial stiffness in MAs isolated from Rsk2 KO and WT mice. Myogenic tone was significantly lower in Rsk2 KO 3rd and 4th order resistance arteries. Vascular stiffness, in aged WT and aged Rsk2 KO mice did not differ but was significantly increased when compared to young WT mice. Resistance MAs (<200 μm) from young and aged WT mice were examined for Ca2+ signals, and relaxation to spermine NONOate after 88 wk of age. Arteries from aged mice experienced an attenuated vasodilatory response to the NO donor at 1 and 3 μM when compared to arteries from young mice. Spontaneous Ca2+ frequency was significantly increased in aged mice compared to young mice. Ca2+ frequency in young mice was attenuated by the RSK inhibitor LJH685 (5 μM), but unchanged in aged mice. The mean Ca2+ amplitude in MAs from aged mice was significantly upregulated when compared to young mice, and significantly attenuated by LJH685 in both young and aged arteries. Our data suggest that in the absence of Rsk2, vessel remodeling and changes in BP are dissociated in aged mice. Furthermore, we have shown that Rsk2 serves as a mediator of downstream signaling elements that alter vascular function in aged mice by decreasing NO sensitivity and enhancing [Ca2+]i resulting in an increase in vascular tone that contributes to HT. NIH grant for AVS: HL147555; Research Supplements for RJA. 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.

S-40-4: ANGIOTENSIN II-INDUCED BLOOD PRESSURE ELEVATION WAS POTENTIATED TOGETHER WITH INCREASES IN INTERFERON-GAMMA PRODUCING T CELLS IN INTERLEUKIN-23 RECEPTOR-DEFICIENT MICE

Objectives: We previously demonstrated a role for γδ T cells in hypertension and vascular injury. In inflammatory conditions γδT17 are a prominent producer of IL-17A, which has been shown to contribute to hypertension. The development and expansion of γδT17 cells is regulated in part through IL-23 receptors (IL-23R). We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in mice with dysfunctional IL-23R. Design and method: Wild-type (WT) and Il23r knock-in (Il23r gfp/gfp) mice were infused or not with Ang II (490ng/kg/min, SC) for 7 or 14 days. BP was monitored via telemetry, mesenteric artery function and remodeling using pressurized myography, and T cell profiling in mesenteric artery perivascular adipose tissue (PVAT) by flow cytometry. Results: Il23r gfp/gfp mice exhibited a greater BP elevation in response to Ang II than WT by day 3 (152 ± 5 vs 144 ± 7 mm Hg, P < 0.05), which was sustained through day 9 (169 ± 2 vs 155 ± 5 mm Hg, P < 0.05), but eventually similar to that of WT mice by the end of the Ang II infusion (167 ± 2 vs 167 ± 4 mm Hg). Il23r gfp/gfp mice were not protected from vascular dysfunction and remodeling after 14 days of Ang II. Il23r gfp/gfp mice had less γδT17 cells in PVAT than WT mice (40 ± 8 vs 108 ± 15 cells/PVAT, P < 0.05). Ang II increased interferon-γ producing γδ T cells in WT (13 ± 3 vs 5 ± 1 cells/PVAT, P < 0.05) and Il23r gfp/gfp mice (14 ± 3 vs 3 ± 1 cells/PVAT, P < 0.05), and interferon-γ producing CD4+ (125 ± 27 vs 38 ± 13 cells/PVA, P < 0.05) and CD8+ T cells (76 ± 13 vs 33 ± 8 cells/PVA, P < 0.05) only in Il23r gfp/gfp mice. Conclusion: Functional IL-23R deficiency exaggerated BP elevation during the initiation of Ang II-induced hypertension, potentially due to the increased number of interferon-γ producing T cells.

PS-BPB04-2: P2RX7 DEFICIENCY OR P2RX7 ANTAGONISM BLUNTS ANGIOTENSIN II-INDUCED HYPERTENSION, VASCULAR INJURY AND CD8+ T CELL ACTIVATION

Objectives: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β; release, which drives further immune activation. Elevated plasma ATP levels was observed in hypertensive patients. We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Design and method: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7-/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: Plasma ATP was 2.2 fold higher in AngII-infused compared to sham-treated mice (P < 0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (12 mm Hg, P < 0.05) or P2RX7 antagonism (27 mm Hg, P < 0.01). AngII induced a 26% decrease in LV fractional shortening (FS, P < 0.05) and a 1.5 fold increase in LV mass/body weight (BW) in WT mice (P < 0.001), which were exaggerated in P2rx7-/- mice (FS: 38% lower and LVmass/BW: 1.14 fold higher, P < 0.05), but not in mice receiving AZ10606120. AngII treatment caused a 26% reduction in the dilatation response of mesenteric arteries to acetylcholine in WT mice (P < 0.05), but not in P2rx7-/- or AZ10606120-treated mice. AngII promoted a 3.8 fold increase in CD69+CD8+ T cell infiltration in aortic PVAT of WT mice (P < 0.001), but not in P2rx7-/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8+ T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

Maternal exercise upregulates the DNA methylation of Agtr1a to enhance vascular function in offspring of hypertensive rats.

The angiotensin II signaling system regulates vascular dysfunction and is involved in the programming of hypertension. Maternal exercise has been linked to both short-term and long-term benefits for the mother and fetus. However, the impacts of maternal exercise on the intravascular renin-angiotensin system (RAS) in hypertensive offspring remain unexamined. This study examined whether maternal exercise has an epigenetic effect in repressing angiotensin II type 1 receptor (AT1R) expression, which leads to favorable alterations in the mesenteric artery (MA) function of spontaneously hypertensive offspring. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) pregnant rats were randomly divided into an exercise group and a control group. Blood pressure, vascular tone, AT1R protein and mRNA expression, and AT1R gene (Agtr1a) promoter methylation status were examined in the MAs of 3-month-old male offspring. Maternal exercise significantly reduced the resting blood pressure and cardiovascular reactivity of offspring from SHRs. Furthermore, Ang II-AT1R activity in regulating vascular tone and AT1R expression was decreased in the MAs of the SHR offspring from the exercise groups. Importantly, exercise during gestation suppressed AT1R expression via hypermethylation of the Agtr1a promoter region and upregulated DNA methyltransferase (DNMT) expression in MAs of SHR offspring. These results suggest that maternal exercise upregulates DNMT expression, resulting in hypermethylation and repression of the Agtr1a gene, which may prevent MA dysfunction in the offspring of SHRs. A mechanistic model on the epigenetics of exercise during pregnancy. Maternal exercise during pregnancy triggers hypermethylation and transcriptional suppression of the Agtr1a gene via increased DNMT1 and DNMT3B expression in MAs of SHR offspring. Downregulation of AT1R expression reduces the contribution of Ang II to vascular tone, ultimately improving vascular structure and function. VSMC vascular smooth muscle cell; Ang II angiotensin II; AT1aR angiotensin type 1 receptor (AT1R) alpha subtypes; Agtr1a AT1R alpha isoform gene; MAs mesenteric arteries; BP blood pressure.

The Synthetic Flavonoid Hidrosmin Improves Endothelial Dysfunction and Atherosclerotic Lesions in Diabetic Mice.

In diabetes, chronic hyperglycemia, dyslipidemia, inflammation and oxidative stress contribute to the progression of macro/microvascular complications. Recently, benefits of the use of flavonoids in these conditions have been established. This study investigates, in two different mouse models of diabetes, the vasculoprotective effects of the synthetic flavonoid hidrosmin on endothelial dysfunction and atherogenesis. In a type 2 diabetes model of leptin-receptor-deficient (db/db) mice, orally administered hidrosmin (600 mg/kg/day) for 16 weeks markedly improved vascular function in aorta and mesenteric arteries without affecting vascular structural properties, as assessed by wire and pressure myography. In streptozotocin-induced type 1 diabetic apolipoprotein E-deficient mice, hidrosmin treatment for 7 weeks reduced atherosclerotic plaque size and lipid content; increased markers of plaque stability; and decreased markers of inflammation, senescence and oxidative stress in aorta. Hidrosmin showed cardiovascular safety, as neither functional nor structural abnormalities were noted in diabetic hearts. Ex vivo, hidrosmin induced vascular relaxation that was blocked by nitric oxide synthase (NOS) inhibition. In vitro, hidrosmin stimulated endothelial NOS activity and NO production and downregulated hyperglycemia-induced inflammatory and oxidant genes in vascular smooth muscle cells. Our results highlight hidrosmin as a potential add-on therapy in the treatment of macrovascular complications of diabetes.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p<0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

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 131: Interleukin-23 Receptor Deficiency Increased Interferon-gamma Producing T Cells And Potentiated Blood Pressure Elevation In Angiotensin II-infused Mice

Introduction: We previously demonstrated a role for γδ T cells in hypertension and vascular injury. In inflammatory conditions γδT17 are a prominent producer of IL-17A, which has been shown to contribute to hypertension. The development and expansion of γδT17 cells is regulated in part through IL-23 receptors (IL-23R). Hypothesis: We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in mice with dysfunctional IL-23R. Methods: Wild-type (WT) and Il23r knock-in ( Il23r gfp/gfp ) mice were infused or not with Ang II (490ng/kg/min, SC) for 7 or 14 days. BP was monitored via telemetry, mesenteric artery function and remodeling using pressurized myography, and T cell profiling in mesenteric artery perivascular adipose tissue (PVAT) by flow cytometry. Results: Il-23r gfp/gfp mice exhibited a greater BP elevation in response to Ang II than WT by day 3 (152±5 vs 144±7 mm Hg, P <0.05), which was sustained through day 9 (169±2 vs 155±5 mm Hg, P <0.05), but eventually similar to that of WT mice by the end of the Ang II infusion (167±2 vs 167±4 mm Hg). Il-23r gfp/gfp mice were not protected from vascular dysfunction and remodeling after 14 days of Ang II. Il23r gfp/gfp mice had less γδT17 cells in PVAT than WT mice (40±8 vs 108±15 cells/PVAT, P <0.05). Ang II increased interferon-γ producing γδ T cells in WT (13±3 vs 5±1 cells/PVAT, P <0.05) and Il23r gfp/gfp mice (14±3 vs 3±1 cells/PVAT, P <0.05), and interferon-γ producing CD4 + (125±27 vs 38±13 cells/PVA, P <0.05) and CD8 + T cells (76±13 vs 33±8 cells/PVA, P <0.05) only in Il23r gfp/gfp mice. Conclusion: Functional IL-23R deficiency exaggerated BP elevation during the initiation of Ang II-induced hypertension, potentially due to the increased number of interferon-γ producing T cells.

Abstract 118: P2X7 Receptor Contributes To Angiotensin II-induced Hypertension, Vascular Injury And Cd8 + T Cell Activation

Introduction: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β release, which drives further immune activation. Hypothesis: We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: AngII increased plasma ATP in WT mice (4.4±1.2 vs 2.0±0.9 μM, P <0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (164±3 vs 176±2 mm Hg, P <0.05) or P2RX7 antagonism (143±5 vs 170±5 mm Hg, P <0.01). AngII decreased LV fractional shortening (FS, 32.5±3.1% vs 43.8±2.4%, P <0.05) and increased LV mass/body weight (BW) in WT mice (LVmass/BW, 6.3±0.2 vs 4.2±0.2 mg/g, P <0.001), which were exaggerated in P2rx7 -/- (FS: 20.2±3.1% and LVmass/BW: 7.2±0.5 mg/g, P <0.05), but not in mice receiving AZ10606120. AngII reduced the dilatation response of mesenteric arteries to acetylcholine in WT (61±7 vs 83±4%, P <0.05), but not in P2rx7 -/- or AZ10606120-treated mice. AngII increased CD69 + CD8 + T cell infiltration in aortic PVAT of WT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), but not in P2rx7 -/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

17β-Estradiol Treatment Improves Acetylcholine-Induced Relaxation of Mesenteric Arteries in Ovariectomized UC Davis Type 2 Diabetes Mellitus Rats in Prediabetic State.

We recently reported sex differences in mesenteric arterial function of the UC Davis type-2 diabetes mellitus (UCD-T2DM) rats as early as the prediabetic state. We reported that mesenteric arteries (MA) from prediabetic male rats exhibited a greater impairment compared to that in prediabetic females. However, when females became diabetic, they exhibited a greater vascular dysfunction than males. Thus, the aim of this study was to investigate whether the female sex hormone, estrogen preserves mesenteric arterial vasorelaxation in UCD-T2DM female rats at an early prediabetic state. Age-matched female Sprague Dawley and prediabetic (PD) UCD-T2DM rats were ovariectomized (OVX) and subcutaneously implanted with either placebo or 17β-estradiol (E2, 1.5 mg) pellets for 45 days. We assessed the contribution of endothelium-derived relaxing factors (EDRF) to acetylcholine (ACh)-induced vasorelaxation, using pharmacological inhibitors. Responses to sodium nitroprusside (SNP) and phenylephrine (PE) were also measured. Additionally, metabolic parameters and expression of some targets associated with vascular and insulin signaling were determined. We demonstrated that the responses to ACh and SNP were severely impaired in the prediabetic state (PD OVX) rats, while E2 treatment restored vasorelaxation in the PD OVX + E2. Moreover, the responses to PE was significantly enhanced in MA of PD OVX groups, regardless of placebo or E2 treatment. Overall, our data suggest that 1) the impairment of ACh responses in PD OVX rats may, in part, result from the elevated contractile responses to PE, loss of contribution of endothelium-dependent hyperpolarization (EDH) to vasorelaxation, and a decreased sensitivity of MA to nitric oxide (NO), and 2) the basis for the protective effects of E2 may be partly attributed to the elevation of the NO contribution to vasorelaxation and its interaction with MA as well as potential improvement of insulin signaling. Here, we provide the first evidence of the role of E2 in protecting MA from early vascular dysfunction in prediabetic female rats.

Dietary Blueberry Ameliorates Vascular Complications in Diabetic Mice Possibly through NOX4 and Modulates Composition and Functional Diversity of Gut Microbes.

In diabetes, endothelial inflammation and dysfunction play a pivotal role in the development of vascular disease. This study investigates the effect of dietary blueberries on vascular complications and gut microbiome in diabetic mice. Seven-week-old diabetic db/db mice consume a standard diet (db/db) or a diet supplemented with 3.8% freeze-dried blueberry (db/db+BB) for 10 weeks. Control db/+ mice are fed a standard diet (db/+). Vascular inflammation is assessed by measuring monocyte binding to vasculature and inflammatory markers. Isometric tension procedures are used to assess mesenteric artery function. db/db mice exhibit enhanced vascular inflammation and reduced endothelial-dependent vasorelaxation as compared to db/+ mice, but these are improved in db/db+BB mice. Blueberry supplementation reduces the expression of NOX4 and IκKβ in the aortic vessel and vascular endothelial cells (ECs) isolated from db/db+BB compared to db/db mice. The blueberry metabolites serum reduces glucose and palmitate induced endothelial inflammation in mouse aortic ECs. Further, blueberry supplementation increases commensal microbes and modulates the functional potential of gut microbes in diabetic mice. Dietary blueberry suppresses vascular inflammation, attenuates arterial endothelial dysfunction, and supports the growth of commensal microbes in diabetic mice. The endothelial-specific vascular benefits of blueberries are mediated through NOX4 signaling.