Mesenteric Resistance Vessels Research Articles

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Reactivity of renal and mesenteric resistance vessels to angiotensin II is mediated by NOXA1/NOX1 and superoxide signaling.

Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2•- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2•- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2•- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Mas-related G protein-coupled receptor type D antagonism improves portal hypertension in cirrhotic rats.

Splanchnic vasodilatation contributes to the development and aggravation of portal hypertension (PHT). We previously demonstrated that in cirrhosis, angiotensin‐ mediates splanchnic vasodilatation through the Mas receptor (MasR). In this study, we investigated whether the recently characterized second receptor for angiotensin‐(1–7), Mas‐related G protein‐coupled receptor type D (MrgD), contributes to splanchnic vasodilatation in cirrhotic and noncirrhotic PHT. Splanchnic vascular hemodynamic and portal pressure were determined in two rat models of cirrhotic PHT and a rat model with noncirrhotic PHT, treated with either MrgD blocker D‐Pro7‐Ang‐(1‐7) (D‐Pro) or MasR blocker A779. Gene and protein expression of MrgD and MasR were measured in splanchnic vessels and livers of cirrhotic and healthy rats and in patients with cirrhosis and healthy subjects. Mesenteric resistance vessels isolated from cirrhotic rats were used in myographs to study their vasodilatory properties. MrgD was up‐regulated in cirrhotic splanchnic vessels but not in the liver. In cirrhotic rats, treatment with D‐Pro but not A779 completely restored splanchnic vascular resistance to a healthy level, resulting in a 33% reduction in portal pressure. Mesenteric vessels pretreated with D‐Pro but not with A779 failed to relax in response to acetylcholine. There was no splanchnic vascular MrgD or MasR up‐regulation in noncirrhotic PHT; thus, receptor blockers had no effect on splanchnic hemodynamics. Conclusion: MrgD plays a major role in the development of cirrhotic PHT and is a promising target for the development of novel therapies to treat PHT in cirrhosis. Moreover, neither MrgD nor MasR contributes to noncirrhotic PHT.

Abstract MP26: Angiotensin-(1-7) Increases Vascular Beta-2 Adrenergic Receptor Expression In Aging Mice

Aging is associated with increased sympathetic tone, which desensitizes vascular beta-2 adrenergic receptors (B2AR) to impair vasodilation and promote hypertension. We recently demonstrated that chronic treatment with angiotensin (Ang)-(1-7), a protective hormone of the renin-angiotensin system, decreases blood pressure and cardiac sympathetic tone in aging mice. In this study, we hypothesized that sympathoinhibitory effects of Ang-(1-7) would restore vascular B2AR expression to lower blood pressure in aging. To test this, aging (16-month-old) and young (2-month-old) male C57BL/6J mice received Ang-(1-7) [400 ng/kg/min, n=4] or saline (n=4) infusion for 6 weeks via subcutaneous osmotic mini-pump. At end of treatment, alpha- and beta-adrenergic receptor gene expression was measured in mesenteric arteries, thoracic aorta, and cardiac tissue by quantitative real-time PCR and quantified with 2-ΔΔCT methods. In a separate experiment, male C57BL/6J mice received a single subcutaneous injection of Ang-(1-7) (2 mg/mg, n=4), saline (n=3), or the B2AR antagonist ICI 118,551 (1 mg/kg) plus Ang-(1-7) (n=3). Blood pressure was measured via a carotid artery catheter at baseline and post-treatment. We found that aging mice have decreased mesenteric B2AR gene expression, which was restored by Ang-(1-7) (young saline: 1.04±0.35; aged saline: 0.46±0.17; aged Ang-(1-7): 1.04±0.31 A.U., p=0.026). As a control, Ang-(1-7) did not induce significant changes in B2AR mRNA in thoracic aorta or cardiac tissue, or changes in other adrenergic receptor subtypes (alpha1 or beta1) in any of the tissues studied in aging mice (p>0.05). We further found that depressor effects of acute Ang-(1-7) in mice are attenuated by B2AR blockade (saline: Δ -11±4 mmHg; Ang-(1-7): Δ -26±3 mmHg; ICI 118,551+Ang-(1-7): Δ -4±4 mmHg, p=0.009). Overall, these findings suggest that Ang-(1-7) restores B2AR expression in mesenteric resistance vessels in aging mice, and depressor effects of acute Ang-(1-7) are partially mediated by B2AR. These data support the concept that Ang-(1-7) decreases blood pressure in aging by restoring B2AR-mediated vasodilation. More broadly, Ang-(1-7) may provide a novel treatment target for age-related hypertension and cardiovascular disease.

The ability of baroreflex activation to improve blood pressure and resistance vessel function in spontaneously hypertensive rats is dependent on stimulation parameters.

Baroreflex activation by electric stimulation of the carotid sinus (CS) effectively lowers blood pressure. However, the degree to which differences between stimulation protocols impinge on cardiovascular outcomes has not been defined. To address this, we examined the effects of short- and long-duration (SD and LD) CS stimulation on hemodynamic and vascular function in spontaneously hypertensive rats (SHRs). We fit animals with miniature electrical stimulators coupled to electrodes positioned around the left CS nerve that delivered intermittent 5/25 s ON/OFF (SD) or 20/20 s ON/OFF (LD) square pulses (1 ms, 3 V, 30 Hz) continuously applied for 48 h in conscious animals. A sham-operated control group was also studied. We measured mean arterial pressure (MAP), systolic blood pressure variability (SBPV), heart rate (HR), and heart rate variability (HRV) for 60 min before stimulation, 24 h into the protocol, and 60 min after stimulation had stopped. SD stimulation reversibly lowered MAP and HR during stimulation. LD stimulation evoked a decrease in MAP that was sustained even after stimulation was stopped. Neither SD nor LD had any effect on SBPV or HRV when recorded after stimulation, indicating no adaptation in autonomic activity. Both the contractile response to phenylephrine and the relaxation response to acetylcholine were increased in mesenteric resistance vessels isolated from LD-stimulated rats only. In conclusion, the ability of baroreflex activation to modulate hemodynamics and induce lasting vascular adaptation is critically dependent on the electrical parameters and duration of CS stimulation.

Transglutaminases Are Active in Perivascular Adipose Tissue.

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model to test interactions of amines and TGs. We hypothesized that transglutaminases are active in PVAT. Real time RT-PCR determined that Sprague Dawley rat aortic, superior mesenteric artery (SMA), and mesenteric resistance vessel (MR) PVATs express TG2 and blood coagulation Factor-XIII (FXIII) mRNA. Consistent with this, immunohistochemical analyses support that these PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs when in a catalyzing calcium solution. Both TG2 and FXIII were active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT. Finally, experimentally added NE competitively inhibited incorporation of BAP into MRPVAT adipocytes. Further studies to determine the identity of amidated proteins will give insight into how these enzymes contribute to functions of PVAT and, ultimately, blood pressure.

Baroreceptor denervation reduces inflammatory status but worsens cardiovascular collapse during systemic inflammation

Beyond the regulation of cardiovascular function, baroreceptor afferents play polymodal roles in health and disease. Sepsis is a life-threatening condition characterized by systemic inflammation (SI) and hemodynamic dysfunction. We hypothesized that baroreceptor denervation worsens lipopolysaccharide (LPS) induced-hemodynamic collapse and SI in conscious rats. We combined: (a) hemodynamic and thermoregulatory recordings after LPS administration at a septic-like non-lethal dose (b) analysis of the cardiovascular complexity, (c) evaluation of vascular function in mesenteric resistance vessels, and (d) measurements of inflammatory cytokines (plasma and spleen). LPS-induced drop in blood pressure was higher in sino-aortic denervated (SAD) rats. LPS-induced hemodynamic collapse was associated with SAD-dependent autonomic disbalance. LPS-induced vascular dysfunction was not affected by SAD. Surprisingly, SAD blunted LPS-induced surges of plasma and spleen cytokines. These data indicate that baroreceptor afferents are key to alleviate LPS-induced hemodynamic collapse, affecting the autonomic control of cardiovascular function, without affecting resistance blood vessels. Moreover, baroreflex modulation of the LPS-induced SI and hemodynamic collapse are not dependent of each other given that baroreceptor denervation worsened hypotension and reduced SI.

IL‐10 Neither Causes Direct Vasorelaxation Nor Reduces Vasoconstriction in Rat Mesenteric Resistance Arteries

Perivascular adipose tissue (PVAT) is the fat that surrounds most of the peripheral blood vessels in the body. PVAT is protective and reduces contraction of blood vessels in health. We discovered that in health, PVAT around the rat small mesenteric resistance vessels (MRPVAT) contained a community of immune cells including T cells, B cells, macrophages, NK cells, neutrophils and mast cells. Interleukin (IL)‐10‐‐an anti‐inflammatory cytokine usually produced by T cells, B cells and macrophages‐‐was identified as one of the highly expressed (mRNA) cytokines in MRPVAT. One report suggests that IL‐10 causes relaxation of mouse mesenteric arteries. Hence, we hypothesized that IL‐10 directly causes vasorelaxation and/or reduces vasoconstriction in healthy rats. Male Sprague Dawley rats (12–14 weeks age) were used. Mesenteric resistance arteries expressed the receptor for IL‐10 (tunica intima and media) as determined by immunohistochemistry. Isolated tissue bath and third order mesenteric resistance arteries (without PVAT and with intact endothelium) were used for isometric contractility studies. Increasing concentrations [0.4–100 ng/mL] of recombinant rat (rr) IL‐10 or vehicle was directly added to half‐maximally constricted (phenylephrine PE or thromboxane A2 agonist U46619) vessels. IL‐10 did not reduce contraction, suggesting it was not a direct vasorelaxant. Further, the ability of rrIL‐10 to cause a rightward or downward shift of a vasoconstrictant‐response curve was tested. The vessels were incubated with rrIL‐10 [100 ng/mL or 10 ng/mL] or vehicle for 1.5 hours in the tissue bath followed by a cumulative PE [10−9–10−5 M] or U46619 [10−10–10−5 M] response curve. The maximal contractions or EC50 values (shown in table ) were not statistically different in rrIL‐10 incubated vessels vs vehicle. Thus, acute exposure of exogenous IL‐10 neither caused direct vasorelaxation nor reduced vasoconstriction. Further studies with neutralizing antibody for IL‐10 to vessels ± PVAT will help determine if IL‐10 produced in the PVAT causes vasorelaxation and/or reduction in local vasoconstriction indirectly, thus contributing to the anti‐contractile nature of PVAT.Support or Funding InformationNIH HL70687 −Log EC50 ± SEM (n=6–10 arteries/3–5 rats/curve) Vehicle rrIL‐10 (100 ng/mL) PE curve 5.7± 0.05 5.6±0.05 U46619 curve 7.6±0.28 7.2±0.59 Vehicle rrIL‐10 (10 ng/mL) PE curve 5.7±0.03 5.6±0.04 U46619 curve 7.1±0.12 7.2±0.13

Experimental preeclampsia results in post‐partum sensitivity to hypertensive stimuli

Preeclampsia (PE) is a syndrome of new onset high blood pressure (BP) with renal damage during late pregnancy that is associated with increased circulating anti‐angiogenic proteins, including soluble VEGF receptor (sFlt1). Women exposed to PE have increased risk of future cardiovascular disease, including hypertension, with evidence of a “hyper‐responsive” renin‐angiotensin‐aldosterone system (RAAS) persisting post‐partum. Aldosterone (Aldo) and angiotensin II (AngII) can activate vascular smooth muscle cell mineralocorticoid receptor (SMC‐MR), causing changes in gene transcription that contribute to increased BP. Thus, we hypothesized that exposure to increased sFlt1 during pregnancy may induce a state of persistent sensitivity to hypertensive stress by enhanced SMC‐MR activity post‐partum .We established a model that reproduces the hallmarks of PE by viral overexpression of sFlt1 compared to control virus in pregnant C57Bl6 mice. Using telemetry to measure blood pressure, we demonstrate that sFlt1 injection results in increased serum sFlt1, elevated BP and glomerular endotheliosis, the hallmark of PE‐induced renal damage, in late pregnancy that all resolve post‐partum. Two months post‐partum, wire myography studies performed on 3rd order mesenteric resistance vessels ex vivo reveal that sFlt1‐injected females (“prior‐PE”) have a vasoconstrictor sensitivity to AngII (1e‐9 dose: 0.16mN vs. 1.42mN, p=0.059) . To test whether prior PE results in increased sensitivity to hypertensive stimuli, post‐partum mice were exposed to various high salt diet conditions or to a moderate pressor dose of AngII (600mg/kg/day infusion) while BP was monitored via telemetry. Mice with prior PE had a trend toward an increased peak BP in response to 2% sodium (11.71mmHg vs. 3.62mmHg, p=0.09) and a significantly increased BP response to 4% sodium (8.88mmHg vs 5.34mmHg, p<0.05). Prior PE mice also had a significantly enhanced BP response to AngII versus those with normotensive pregnancies (22.83 mmHg vs 4.46 mmHg, p=0.0005). Plasma Aldo measured via radioimmunoassay tended to be higher in mice with prior PE (4.65nM vs 2.09nM, P=0.08). Since SMC‐MR can be activated by AngII, we investigated the extent of AngII‐induced SMC‐MR activation after PE using qPCR to measure known SMC‐MR target genes. In microvessels from mice with prior PE, mRNA expression showed increased AT1R (relative expression 3.69 vs 1.30, p<0.05), Cav1.2 (2.29 vs 1.11, p=0.06) and PlGF (1.92 vs 1.20, p=0.11). Studies are underway to determine whether SMC‐specific deletion of MR or post‐partum pharmacologic MR inhibition can protect against enhanced BP sensitivity to AngII and high salt diet after sFlt1‐induced PE.sFlt1‐induced PE produces a state of enhanced sensitivity to hypertensive stimuli that may be mediated by increased activation of SMC‐MR. If confirmed, these data support the use of spironolactone as a therapy to mitigate the increased risk of cardiovascular disease in women exposed to PE.Support or Funding InformationNIH T32HL007609 (LAB), NIH R25GM066567 (BVC)

Naïve, Regulatory, Activated, and Memory Immune Cells Co-exist in PVATs That Are Comparable in Density to Non-PVAT Fats in Health.

Perivascular adipose tissue (PVAT), the fat surrounding peripheral blood vessels, is protective and reduces the contraction of blood vessels in health. PVAT is composed of adipocytes, stromal cells, and immune cells. Recent work supports eosinophils as one of the cell types key to the anti-contractile nature of PVAT in health. Hence, we hypothesized that there exists a basally activated immune cell community in healthy PVAT that is distinctly different from non-PVAT fats. PVATs were from around mesenteric resistance vessels (MRPVAT – white fat) and thoracic aorta (APVAT – brown fat). Non-PVATs included retroperitoneal (RP fat – white fat) and subscapular (SS fat – brown fat) while the spleen was a positive control. Tissues were harvested from adult male and female Sprague Dawley rats. Six primary immune cell types were identified in PVATs. T cells (CD4 and CD8), B cells, natural killer (NK) cells, macrophages, mast cells, and neutrophils in the stromal vascular fraction of each fat were identified using nine-color flow cytometry. PVATs contained a higher number of total immune cells vs. their respective non-PVAT fats in females. Females had a higher number of T cells in MRPVAT vs. males. Females also had a greater number of T cells and total immune cells in APVAT vs. males. Further, activation, differentiation, and/or polarization of various immune cell types were similarly determined by flow cytometry. PVATs were similar to their respective non-PVAT fats in density of recently activated B cells (B220+ CD25+). However, MRPVAT in females had a higher number of naïve CD4 T cells vs. MRPVAT in males and APVAT in females. MRPVAT also had denser naïve CD8 T cells vs. APVAT in females. Overall, this research for the first time has identified a community of discrete populations of immune cells (naive/recently activated/regulatory/memory) in healthy PVATs. Contrary to our hypothesis, PVATs are more similar than different in density to their respective non-PVAT fats.

Abstract P138: Perivascular Adipose Tissues (pvats) Home Distinct 'Active' Immune Cell Niches, Denser Than Non-pvat Adipose Tissues of Healthy Rats

Perivascular adipose tissue (PVAT) is protective and reduces the contraction of blood vessels in health. Eosinophils are one of the key players to the anti-contractile nature of PVAT in health. This finding led to our hypothesis that an ‘active’ immune complement exists in healthy PVAT. Male and female Sprague Dawley rats (12-14 weeks age) were used. PVATs from the thoracic aorta (brown APVAT), mesenteric resistance vessels (white MRPVAT), non-PVATs: subscapular (brown SS fat), retroperitoneal (white RP fat) and spleen (positive control) were harvested from each animal. T cells (CD4 and CD8), B cells, NK cells, macrophages, mast cells and neutrophils in the stromal vascular fraction were quantified using 9-color flow cytometry. Activation status (effector/ memory/ regulatory) of macrophages, T and B cells were also determined by flow cytometry. The number of immune cells are normalized to tissue weight (table). MRPVAT has denser total immune population (both sexes) and active macrophage pool (females) vs RP fat. APVAT homes a denser immune niche (females) and active T cell, B cell pools (both sexes) vs SS fat. This is the first study to characterize ‘active’ immune populations in healthy PVATs vs non-PVATs in male and female rats. Further studies to understand the functions of basally active immune cells in healthy PVATs are warranted.

Abstract P172: Is the Elastin Receptor Complex a Bridge Between Perivascular Adipose Tissue and the Artery?

Elastin is a metabolically stable structural protein with a half-life known to last nearly a lifetime. This supporting peptide is essential for the artery to withstand the repetitive forces of pulsation. Vascular pathology and remodeling cause damage to this critical support system releasing elastin-derived peptides known as elastokines. Elastokines stimulate the Elastin Receptor Complex (ERC) via an Elastin Binding Peptide(EBP). We investigate the hypothesis that the EBP is present within perivascular adipose tissue (PVAT), providing a form of vessel-to-PVAT communication. Immunohistochemistry techniques were used to identify EBP and Neuraminidase-1, components of the ERC, in rat arteries. Additionally, mesenteric resistance vessel PVAT was isolated into adipocytes and stromal vascular fraction for determining the presence of GLB1 mRNA, encoding the EBP. GLB1 mRNA was observed in adipocytes and stromal vascular fraction respectively 2 -delta Ct 0.0220±0.00130 and 0.0237±0.00192 (N=5). EBP and Neuraminidase-1 immunofluorescently colocalized in vasculature and surrounding PVAT (figure 1, representative of N=5, scale bar 50μm). The EBP agonist VGVAPG [10 μM] (N=7) relaxed the isolated aorta to 65.8±1.67% of a half-maximal phenylephrine contraction. These results support the presence of the ERC in arteries, that PVAT may not be necessary for ERC activation, and that elastin may have multiple functions.

Perivascular Adipocytes Store Norepinephrine by Vesicular Transport.

Objective- Perivascular adipose tissue (PVAT) contains an independent adrenergic system that can take up, metabolize, release, and potentially synthesize the vasoactive catecholamine norepinephrine. Norepinephrine has been detected in PVAT, but the mechanism of its protection within this tissue is unknown. Here, we investigate whether PVAT adipocytes can store norepinephrine using VMAT (vesicular monoamine transporter). Approach and Results- High-performance liquid chromatography identified norepinephrine in normal male Sprague Dawley rat aortic, superior mesenteric artery, and mesenteric resistance vessel PVATs, and retroperitoneal fat. Real-time polymerase chain reaction revealed VMAT1 and VMAT2 mRNA expression in the adipocytes and stromal vascular fraction of mesenteric resistance vessel PVAT. Immunofluorescence demonstrated the presence of VMAT1 and VMAT2, and the colocalization of VMAT2 with norepinephrine, in the cytoplasm of adipocytes in mesenteric resistance vessel PVAT. A protocol was developed to capture real-time uptake of Mini 202-a functional and fluorescent VMAT probe-in live rat PVAT adipocytes. Mini 202 was taken up by freshly isolated and differentiated adipocytes from mesenteric resistance vessel PVAT and adipocytes from thoracic aortic and superior mesenteric artery PVATs. In adipocytes freshly isolated from mesenteric resistance vessel PVAT, addition of rose bengal (VMAT inhibitor), nisoxetine (norepinephrine transporter inhibitor), or corticosterone (organic cation 3 transporter inhibitor) significantly reduced Mini 202 signal. Immunofluorescence supports that neither VMAT1 nor VMAT2 is present in retroperitoneal adipocytes, suggesting that PVAT adipocytes may be unique in storing norepinephrine. Conclusions- This study supports a novel function of PVAT adipocytes in storing amines in a VMAT-dependent manner. It provides a foundation for future studies exploring the purpose and mechanisms of norepinephrine storage by PVAT in normal physiology and obesity-related hypertension.

Smoothelin-like 1 deletion enhances myogenic reactivity of mesenteric arteries with alterations in PKC and myosin phosphatase signaling

The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40 mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60–120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.

Abstracts from the NIH Office of Research on Women's Health 2018 Annual BIRCWH Meeting – Building Interdisciplinary Research Careers in Women's Health November 28, 2018

Abstracts from the NIH Office of Research on Women's Health 2018 Annual BIRCWH Meeting – Building Interdisciplinary Research Careers in Women's Health November 28, 2018

Abstract P162: Adiposity-Induced Hypertension is Associated With Sex-Specific Macrophage Infiltration of Mesenteric Perivascular Adipose Tissue in Dahl S Rats.

Perivascular adipose tissue (PVAT) may be a key player in mediating adiposity-associated hypertension because of its proximity to blood vessels. Although increased immune cell occupancy in adipose tissue is proposed to couple adiposity to hypertension, little is known about PVAT’s immune complement. We hypothesized that an immune complement exists in PVAT that would become pro-inflammatory with adiposity-induced hypertension. Male and female Dahl S rats were fed a regular (10% calories from fat) diet (control) or a high fat (60%) diet (HFD) from weaning through 24 weeks of age. HFD rats demonstrated significantly higher systolic blood pressure [mm Hg; control: 127±7 (M), 145±8 (F); HFD: 168±9 (M), 184±11 (F); tail cuff] and visceral adiposity (micro-CT scans). PVATs from the thoracic aorta (APVAT), mesenteric resistance vessels (MRPVAT), non-PVAT retroperitoneal fat (RP fat) and spleen (positive control) were harvested from each animal. T cells (CD4 and CD8), B cells, macrophages, mast cells and neutrophils in the stromal vascular fraction were quantified using 7-color flow cytometry. The type and number of immune cells are presented as a percentage (%) of total live singlet cells. Each immune cell type is reported as % of total immune cells, and CD4, CD8 cells as % of T cells. The table illustrates significant (P<0.05) differences with HFD vs respective controls. HFD-induced hypertension in Dahl S rats leads to greater macrophage infiltration of MRPVAT and RP fat in females vs males. Further studies are needed to gain mechanistic insights into how macrophage subtypes and their associated cytokines in PVAT may alter vascular tone and blood pressure.

Hepatology Basic Science

Hepatology Basic Science

Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries.

Background: Perivascular adipose tissue (PVAT) can decrease vascular contraction to NE. We tested the hypothesis that metabolism and/or uptake of vasoactive amines by mesenteric PVAT (MPVAT) could affect NE-induced contraction of the mesenteric resistance arteries.Methods: Mesenteric resistance vessels (MRV) and MPVAT from male Sprague-Dawley rats were used. RT-PCR and Western blots were performed to detect amine metabolizing enzymes. The Amplex® Red Assay was used to quantify oxidase activity by detecting the oxidase reaction product H2O2 and the contribution of PVAT on the mesenteric arteries' contraction to NE was measured by myography.Results: Semicarbazide sensitive amine oxidase (SSAO) and monoamine oxidase A (MAO-A) were detected in MRV and MPVAT by Western blot. Addition of the amine oxidase substrates tyramine or benzylamine (1 mM) resulted in higher amine oxidase activity in the MRV, MPVAT, MPVAT's adipocyte fraction (AF), and the stromal vascular fraction (SVF). Inhibiting SSAO with semicarbazide (1 mM) decreased amine oxidase activity in the MPVAT and AF. Benzylamine-driven, but not tyramine-driven, oxidase activity in the MRV was reduced by semicarbazide. By contrast, no reduction in oxidase activity in all sample types was observed with use of the monoamine oxidase inhibitors clorgyline (1 μM) or pargyline (1 μM). Inhibition of MAO-A/B or SSAO individually did not alter contraction to NE. However, inhibition of both MAO and SSAO increased the potency of NE at mesenteric arteries with PVAT. Addition of MAO and SSAO inhibitors along with the H2O2 scavenger catalase reduced PVAT's anti-contractile effect to NE. Inhibition of the norepinephrine transporter (NET) with nisoxetine also reduced PVAT's anti-contractile effect to NE.Conclusions: PVAT's uptake and metabolism of NE may contribute to the anti-contractile effect of PVAT. MPVAT and adipocytes within MPVAT are a source of SSAO.

Vascular mineralocorticoid receptor regulates microRNA-155 to promote vasoconstriction and rising blood pressure with aging.

Hypertension is nearly universal yet poorly controlled in the elderly despite proven benefits of intensive treatment. Mice lacking mineralocorticoid receptors in smooth muscle cells (SMC-MR-KO) are protected from rising blood pressure (BP) with aging, despite normal renal function. Vasoconstriction is attenuated in aged SMC-MR-KO mice, thus they were used to explore vascular mechanisms that may contribute to hypertension with aging. MicroRNA (miR) profiling identified miR-155 as the most down-regulated miR with vascular aging in MR-intact but not SMC-MR-KO mice. The aging-associated decrease in miR-155 in mesenteric resistance vessels was associated with increased mRNA abundance of MR and of predicted miR-155 targets Cav1.2 (L-type calcium channel (LTCC) subunit) and angiotensin type-1 receptor (AgtR1). SMC-MR-KO mice lacked these aging-associated vascular gene expression changes. In HEK293 cells, MR repressed miR-155 promoter activity. In cultured SMCs, miR-155 decreased Cav1.2 and AgtR1 mRNA. Compared to MR-intact littermates, aged SMC-MR-KO mice had decreased systolic BP, myogenic tone, SMC LTCC current, mesenteric vessel calcium influx, LTCC-induced vasoconstriction and angiotensin II-induced vasoconstriction and oxidative stress. Restoration of miR-155 specifically in SMCs of aged MR-intact mice decreased Cav1.2 and AgtR1 mRNA and attenuated LTCC-mediated and angiotensin II-induced vasoconstriction and oxidative stress. Finally, in a trial of MR blockade in elderly humans, changes in serum miR-155 predicted the BP treatment response. Thus, SMC-MR regulation of miR-155, Cav1.2 and AgtR1 impacts vasoconstriction with aging. This novel mechanism identifies potential new treatment strategies and biomarkers to improve and individualize antihypertensive therapy in the elderly.

Mechanism of Aging‐associated Hypertension: Role of Smooth Muscle Cell Mineralocorticoid Receptor Regulation of Vascular L‐type Calcium Channels

Hypertension (HTN), the most prevalent cardiovascular disease risk factor, increases with age. We found that mice with SMC-specific deletion of the mineralocorticoid receptor (MR KO) lack the aging-associated rise in blood pressure (BP). We hypothesize that SMC MR contributes to BP regulation with aging by regulating vascular L-type calcium channels (LTCC). Patch clamp studies on mesenteric resistance vessel (MRV) SMC from young (3-4 mo.) and aged (9-12 mo.) MR intact and MR KO reveal reduced LTCC current density in aged MR KO (p<0.05 vs. MR intact). Fura-2 photometry studies reveal decreased MRV Ca flux in aged MR KO (p<0.05 vs. MR intact) in response to BayK (LTCC agonist). Contraction to BayK is blunted in aged MR KO MRV (p<0.05 vs. MR intact). RNA expression of Cav1.2, the pore-forming subunit of LTCC, is reduced by 60% in aged MR KO vs. MR intact MRV (p<0.05). Micro-RNA expression profiling identified miR-155 as being modulated by SMC MR with aging. MRV miR-155 expression is increased in aged MR KO (p<0.05 vs. MR intact). Ingenuity pathway analysis identified Cav1.2 as a potential target of miR-155. Overexpression of miR-155 in mouse MRV SMC reduced Cav1.2 expression by 45% (p<0.05 vs. ctrl). These data suggest that SMC MR contributes to HTN through regulating Cav1.2 expression/function, potentially via miR-155. These results enhance our basic understanding of BP control with aging, and provide support for innovative therapeutic targets to treat aging-associated HTN. Supported by HL095590-05.