Dysfunction In Resistance Arteries Research Articles

The Vascular Function of Resistance Arteries Depends on NADPH Oxidase 4 and Is Exacerbated by Perivascular Adipose Tissue.

The NADPH oxidase NOX4 that releases H2O2 can mediate vasoprotective mechanisms under pathophysiological conditions in conductive arteries. However, the role of NOX4 in resistance arteries and in perivascular adipose tissue is not well understood. We hypothesized that NOX4 is of functional importance in resistance arteries and perivascular adipose tissue under dyslipidemia conditions. We detected elevated NOX4 expression in murine and human vessels under dyslipidemia. Diminishing Nox4 under these conditions led to endothelial dysfunction in resistance arteries. The mesenteric arteries of Nox4-/-/Ldlr-/- mice revealed decreased eNos mRNA expression. Inhibition of eNOS in those vessels did not affect vascular function, while in Ldlr-/- mice endothelial function was significantly altered. Anticontractile properties of perivascular adipose tissue at resistance arteries were diminished in Nox4-/-/Ldlr-/- compared with Ldlr-/- mice. In addition, the presence of perivascular adipose tissue further worsened endothelial dysfunction in mesenteric arteries under dyslipidemia conditions. Perivascular adipose tissue from mesenteric arteries revealed a higher expression of markers of white adipocytes compared to markers of beige/brown adipocytes. Among those white adipocyte markers, leptin was significantly less expressed in perivascular adipose tissue from Nox4-/-/Ldlr-/- mice compared with Ldlr-/- mice. Furthermore, in human perivascular adipose tissue with a profound pattern of white adipocyte marker genes, we detected a correlation of NOX4 and LEP expression. In addition, incubating arterial vessels with leptin induced nitrite release, indicating increased eNOS activity. In humans, a higher expression of leptin in perivascular adipose tissue correlated with eNOS expression in the corresponding left internal mammary artery. In conclusion, vascular function of resistance arteries was dependent on Nox4-derived H2O2, especially under dyslipidemia conditions. Perivascular adipose tissue of the mesenteric arteries with white adipose tissue characteristics further aggravated endothelial function through reduced leptin-eNOS signaling.

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.

Cardiac Hypertrophy and Vascular Dysfunction in Resistance Arteries Occurs Prior to the Onset of Hypertension in Schlager (BPH/2J) Mice

The Schlager inbred hypertensive (BPH/2J) mouse strain was developed by Gunther Schlager more than 45 years ago and have been recognized as a model of neurogenic hypertension. Activation of the sympathetic nervous system contributes to the maintainance of high blood pressure levels in BPH/2J male mice with the systolic blood pressure (SBP) starting to increase at seven weeks of age until reaching the highest value at 20 weeks of age. Several studies have shown cardiovascular dysfunction in these hypertensive mice, but no one has demonstrated whether heart and vascular dysfunction are present prior to the elevation in SBP. Our objective was to determine if cardiovascular dysfunction in male BPH/2J mice happens prior to the elevation of blood pressure. We hypothesized that cardiovascular remodeling along wih vascular dysfunction in mesenteric resistance arteries (MRA) occurs prior to the hypertension in BPH/2J mice, supporting the notion that the mechanism of hypertension in BPH/2J mice is not predominantly neurogenic. We evaluated intact-endothelium MRA function from 6-weeks-old male BPH/2J and normotensive control mice (BPN/3J). Intact-endothelium MRA were mounted on wire myographs. SBP was measured directly in the left carotid artery, and cardiac function and morphology was evaluated by echocardiography. Second harmonic generation was used to evaluate collagen deposition in MRA, and hematoxin and eosin staining done for cardiomyocyte hypertrophy. These data were analyzed using the Student t test ( p<0.05). SBP was not different between 6-weeks-old male BPH/2J and BPN/3J mice (78±2.33mmHg vs. 89±1.20 mmHg; n=3-4, respectively, p>0.05). BPH/2J presented increased left ventricle anterior wall thickness during systole (1.79±0.1mm) and diastole (1.13±0.06mm, n=4) compared to BPN/3J (systole: 1.46±0.08mm; diastole: 0.93±0.05mm, n=5, p<0.05). In addition, cardiomyocyte area values was higher in BPH/2J (1,188±51μm2, n=8) than BPN/3J (820±53μm2, n=4, p<0.05), suggesting that cardiac hypertrophy occurs prior to the onset of hypertension. BPH/2J mice presented increased phenylphrine-induced contraction (Rmax: BPH/2J: 6.25±0.54 vs. BPN/3J: 4.23±0.28 mN, n=4, p<0.05) and decreased endothelium-dependent relaxation (Rmax: BPH/2J: 76±5, n=4 vs. BPN/3J: 93±2%, n=3, p<0.05), and BPH/2J MRA presented a tendency to increase arterial collagen content compared to BPN/3J mice. These data reveal that cardiac remodeling and vascular dysfunction and fibrosis occurs prior to the elevation of SBP in BPH/2J mice, suggesting novel contributive causes of hypertension in Schlager mice. R00GM118885, R01HL149762, R00HL151889, and NHLBI (R00HL151889) 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.

Abstract P058: Endothelial Dysfunction And Increased Contractility In Resistance Arteries Occurs Prior To Onset Of Hypertension In Schlager (BPH/2J) Mice

Genetically hypertensive mice (BPH/2J) were developed by Gunther Schlager more than 45 years ago, and these mice have neurogenic hypertension. Seven-weeks-old male BPH/2J mice present elevations in systolic blood pressure (SBP), and its highest value is observed in 20-weeks-old mice. Several studies have showed vascular dysfunction in these mice, but no one has demonstrated whether resistance artery dysfunction is present prior to the elevation in SBP. We hypothesized that hypercontractility and endothelial dysfunction is associated with the cause of hypertension in BPH/2J mice. We evaluated mesenteric resistance arteries (MRA) function from 5-weeks-old male BPH/2J and 9-weeks-old normotensive mice (BPN/3J), prior to the elevation of SBP. Intact-endothelium MRA were mounted in wire myographs. KCl-induced contraction (120 mM), or phenylephrine (PE) and acetylcholine (ACh) cummulative concentration curves were performed. Non-linear curve regressions were performed and maximum response (Rmax) was analyzed by Student t test ( p <0.05). BPH/2J mice presented increased KCl contraction (BPH/2J: 6.47±0.51 vs. BPN/3J: 2.95±0.13 mN/mm, n=4, p <0.05) and PE-induced contraction (Rmax: BPH/2J: 6.25±0.54 vs. BPN/3J: 4.23±0.28 mN/mm, n=4, p <0.05). ACh-dependent relaxation was decreased in BPH/2J (Rmax: BPH/2J: 76±5, n=4 vs. BPN/3J: 93±2%, n=3, p <0.05). ACh promoted contraction at high concentrations (>0.3 μM) in BPH/2J mice but not BPN/3J mice (Fig.1), suggesting increased cyclooxygenase-induced inflammation. These data reveal that vascular dysfunction contributes to the cause of hypertension in Schlager mice.

Microsomal prostaglandin E synthase-1 is involved in the metabolic and cardiovascular alterations associated with obesity.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible isomerase responsible for prostaglandin E2 production in inflammatory conditions. We evaluated the role of mPGES-1 in the development and the metabolic and cardiovascular alterations of obesity. mPGES-1+/+ and mPGES-1-/- mice were fed with normal or high fat diet (HFD, 60% fat). The glycaemic and lipid profile was evaluated by glucose and insulin tolerance tests and colorimetric assays. Vascular function, structure and mechanics were assessed by myography. Histological studies, q-RT-PCR, and western blot analyses were performed in adipose tissue depots and cardiovascular tissues. Gene expression in abdominal fat and perivascular adipose tissue (PVAT) from patients was correlated with vascular damage. Male mPGES-1-/- mice fed with HFD were protected against body weight gain and showed reduced adiposity, better glucose tolerance and insulin sensitivity, lipid levels and less white adipose tissue and PVAT inflammation and fibrosis, compared with mPGES-1+/+ mice. mPGES-1 knockdown prevented cardiomyocyte hypertrophy, cardiac fibrosis, endothelial dysfunction, aortic insulin resistance, and vascular inflammation and remodelling, induced by HFD. Obesity-induced weight gain and endothelial dysfunction of resistance arteries were ameliorated in female mPGES-1-/- mice. In humans, we found a positive correlation between mPGES-1 expression in abdominal fat and vascular remodelling, vessel stiffness, and systolic blood pressure. In human PVAT, there was a positive correlation between mPGES-1 expression and inflammatory markers. mPGES-1 inhibition might be a novel therapeutic approach to the management of obesity and the associated cardiovascular and metabolic alterations.

Pathophysiological adaptations of resistance arteries in rat offspring exposed in utero to maternal obesity is associated with sex-specific epigenetic alterations

Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little is known about resistance arteries. They are of particular interest because of their specific role in the regulation of local blood flow. Then our study aims to determine if maternal obesity can directly program fetal vascular dysfunction of resistance arteries, independently of metabolic disorders. With a model of rats exposed in utero to mild maternal diet-induced obesity (OMO), we investigated third-order mesenteric arteries of 4-month old rats in absence of metabolic disorders. The methylation profile of these vessels was determined by reduced representation bisulfite sequencing (RRBS). Vascular structure and reactivity were investigated using histomorphometry analysis and wire-myography. The metabolic function was evaluated by insulin and glucose tolerance tests, plasma lipid profile, and adipose tissue analysis. At 4 months of age, small mesenteric arteries of OMO presented specific epigenetic modulations of matrix metalloproteinases (MMPs), collagens, and potassium channels genes in association with an outward remodeling and perturbations in the endothelium-dependent vasodilation pathways (greater contribution of EDHFs pathway in OMO males compared to control rats, and greater implication of PGI2 in OMO females compared to control rats). These vascular modifications were detected in absence of metabolic disorders. Our study reports a specific methylation profile of resistance arteries associated with vascular remodeling and vasodilation balance perturbations in offspring exposed in utero to maternal obesity, in absence of metabolic dysfunctions.

Abstract 126: Role of V γ6 + γδ T Cells in Angiotensin Il-Induced Hypertension and Vascular Injury

Introduction: Both innate and adaptive immune cells (such as T lymphocytes) have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subpopulation of T cells considered "innate-like", expressing the γδ T-cell receptor (TCR) rather than the much more frequent αβ TCR, plays a key role in hypertension and vascular injury. The number and activation of γδ T cells were increased after 7 days of angiotensin (Ang) II infusion, and absence of γδ T cells prevented the development of hypertension, endothelial dysfunction of resistance arteries, and activation of CD4 + and CD8 + T cells in mice infused with Ang II for 14 days. γδ T cells can be subdivided according to the TCR variant (V) subtype that is generally specific for a tissue. A subpopulation of γδ T cells in the lungs and skin that are Vγ6 + and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown. We hypothesized that γδ T cells involved in hypertension are Vγ6 + . Methods: Eleven- to 13-week old C57BL/6J male mice were infused or not with Ang II (490 ng /kg /min, sc) for 14 days (n = 5-14), and γδ T cell Vγ subtypes were profiled by flow cytometry in the spleen, mesenteric lymph nodes (MLNs), thoracic aortic (TA), perivascular adipose tissue (PVAT) and mesenteric artery (MA) PVAT. Results: In spleen and MLNs the most abundant γδ T cell Vγ subtypes were Vγ1,2 + and Vγ4 + followed by Vγ6 + , Vγ5 + and Vγ7 + . In TA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ1,2 + , Vγ5 + and Vγ7 + . In MA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ7 + , Vγ5 + and Vγ1,2 + . Ang II infusion increased the frequency of Vγ6 + γδ T cells in the spleen (% of CD3 + T cells: 0.77±0.09 vs. 0.5±0.02, P <0.01) and TA PVAT 5.9±0.8 vs. 3.6±0.4, P <0.01), whereas it only tended to increase in MA PVAT 14.5±1.7 vs. 10.2± 1.9, P =0.07). The Vγ6 + γδ T cell frequency in MLNs was unaffected by Ang II infusion. Conclusion: A differential distribution of γδ T cell Vγ subtypes was observed in lymphoid organs compared to PVAT. Vγ6 + γδ T cells may play a role in hypertension. Targeting Vγ6 + γδ T cells could be a therapeutic approach to reduce inflammation in hypertension.

Exercise ameliorates endoplasmic reticulum stress-mediated vascular dysfunction in mesenteric arteries in atherosclerosis

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

Erratum: Decreased EDHF-mediated relaxation is a major mechanism in endothelial dysfunction in resistance arteries in aged mice on prolonged high-fat sucrose diet.

[This corrects the article DOI: 10.14814/phy2.13502.].

Regulation of voltage-gated potassium channels in vascular smooth muscle during hypertension and metabolic disorders.

Voltage-gated potassium (KV ) channels are key regulators of vascular smooth muscle contractility and vascular tone, and thus have major influence on the microcirculation. KV channels are important determinants of vascular smooth muscle membrane potential (Em ). A number of KV subunits are expressed in the plasma membrane of smooth muscle cells. Each subunit confers distinct kinetics and regulatory properties that allow for fine control of Em to orchestrate vascular tone. Modifications in KV subunit expression and/or channel activity can contribute to changes in vascular smooth muscle contractility in response to different stimuli and in diverse pathological conditions. Consistent with this, a number of studies suggest alterations in KV subunit expression and/or function as underlying contributing mechanisms for small resistance artery dysfunction in pathologies such as hypertension and metabolic disorders, including diabetes. Here, we review our current knowledge on the effects of these pathologies on KV channel expression and function in vascular smooth muscle cells, and the repercussions on (micro)vascular function.

Decreased EDHF-mediated relaxation is a major mechanism in endothelial dysfunction in resistance arteries in aged mice on prolonged high-fat sucrose diet.

High‐fat sucrose (HFS) diet in aged individuals causes severe weight gain (obesity) with much higher risk of cardiovascular diseases such as hypertension or atherosclerosis. Endothelial dysfunction is a major contributor for these vascular disorders. We hypothesize that prolonged ingestion of HFS diet by aged mice would accentuate endothelial dysfunction in the small resistance arteries. Male C57BL/6J mice at 12 weeks of age were divided into four groups and fed either normal chow (NC) or high‐fat sucrose diet (HFS). Young group received NC for 4 months, and high‐fat diet (HFD) for 3 months and 1 month HFS + 10% Sucrose (HFS diet). Aged mice received NC for 12 months. Aged HFS group received HFD for 4 months + 1 month HFD + 10% sucrose + 8 months HFD. Total body weight, plasma blood glucose levels, and glucose tolerance were determined in all groups. Isolated mesenteric arteries were assessed for arterial remodeling, myogenic tone, and vasomotor responses using pressure and wire myography. Both young and aged HFS mice showed impaired glucose tolerance (Y‐NC, 137 ± 8.5 vs. Y‐NC HFS, 228 ± 11.71; A‐NC, 148 ± 6.42 vs. A‐HFS, 225 ± 10.99), as well as hypercholesterolemia (Y‐NC 99.50 ± 6.35 vs. Y‐HFS 220.40 ± 16.34 mg/dL; A‐NC 108.6 ± vs. A‐HFS 279 ± 21.64) and significant weight gain (Y‐NC 32.13 ± 0.8 g vs. Y‐HFS 47.87 ± 2.18 g; A‐NC 33.72 vs. A‐HFS 56.28 ± 3.47 g) compared to both groups of mice on NC. The mesenteric artery from mice with prolonged HFS diet resulted in outward hypertrophic remodeling, increased stiffness, reduced myogenic tone, impaired vasodilation, increased contractility and blunted nitric oxide (NO) and EDH‐mediated relaxations. Ebselen, a peroxinitrite scavenger rescued the endothelium derived relaxing factor (EDHF)‐mediated relaxations. Our findings suggest that prolonged diet‐induced obesity of aged mice can worsen small resistance artery endothelial dysfunction due to decrease in NO and EDHF‐mediated relaxation, but, EDHF‐mediated relaxation is a major contributor to overall endothelial dysfunction.

Antiperoxynitrite Treatment Ameliorates Vasorelaxation of Resistance Arteries in Aging Rats: Involvement With Protection of Circulating Endothelial Progenitor Cells.

Numerous studies have found that the age-associated structural and functional alterations in arteries were characterized by increased endothelial dysfunction. In this study, young (3 months), adult (9 months), and aging (20 months) male Sprague-Dawley rats were randomly divided into 6 groups, including control groups and FeTMPyP (peroxynitrite scavenger) groups receiving saline and FeTMPyP, respectively, for 5 administrations once every 3 days through intraperitoneal injection. The aged-related proteins beta-galactosidase, p53, and p16 as well as the nitrotyrosine and endothelial marker endothelial nitric oxide synthase and von Willebrand factor (vWF) in vascular tissues were measured by immunohistochemistry. Endothelium-dependent vasorelaxation and endothelium-independent vasorelaxation of rat thoracic aortas and mesenteric arteries were measured by acetylcholine and sodium nitroprusside, respectively. The amount of circulating endothelial progenitor cells (EPCs) was determined by flow cytometry. The endothelium-dependent/independent relaxation in mesenteric arteries and the amount of circulating EPCs (CD31/CD34) in peripheral blood of aging rats were reduced significantly compared with young and adult rats. Immunohistochemistry results showed that the nitrotyrosine levels and morphological damage in mesenteric arteries were increased significantly in aging rats. Adoption of peroxynitrite scavenger FeTMPyP intervention may not only improve the endothelium-dependent relaxation and the amount of circulating EPCs in aging rats but also reverse endothelial injury. In conclusion, this study demonstrates that enhanced nitrative stress may aggravate the endothelial injury and vascular dysfunction of resistance arteries in aging rats. Antiperoxynitrite treatment can ameliorate the vasorelaxation and may be involved with the protection of circulating EPCs.

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries.

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20% v/v) for 6weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O2-) generation and decreased nitrate/nitrite (NO x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H2O2) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1β. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1β, and IL-10, whereas it decreased NO x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.

Endothelial Dysfunction in Resistance Arteries of Hypertensive Humans: Old and New Conspirators.

A large body of homogeneous reports documented that endothelial dysfunction, which characterizes human essential hypertension is largely dependent on an impaired endothelial nitric oxide availability and an increased production of endothelium-derived contracting factors. These factors include endothelin, prostanoids, and reactive oxygen species (ROS). In particular, it was evidenced that acute intraarterial administration of indomethacin, a nonselective cyclooxygenase (COX)-inhibitor, and ascorbic acid, an antioxidant, normalized the blunted endothelial dysfunction by restoring nitric oxide availability at the level of peripheral microcirculation, thus demonstrating that COX-derived endothelium-derived contracting factors are one of the major sources of ROS. Recent studies put in evidence new lights on the mechanisms involved in endothelial dysfunction in human hypertension, identified as "new conspirators." In particular, functional and immunohistochemical experiments with selective COX inhibitors identified the isoform COX-2 as the main source of intravascular ROS generation in isolated small vessels from essential hypertensive patients. In addition, important vascular protective properties by human ghrelin have been demonstrated in human hypertension, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, while a normalization of its levels may restore vascular homeostasis.

Toll‐like receptor 9 contributes to increased vascular tone via small GTPase activation

Toll‐like receptor (TLR)9 is a pattern recognition receptor of the innate immune system. We have previously reported that TLR9 stimulation causes resistance artery dysfunction via increased sensitivity to norepinephrine, and this contributed to the pathogenesis of hypertension. However, the mechanisms underlying this increased vascular tone after TLR9 activation need to be further clarified. We hypothesized that TLR9 activation would increase expression of small GTPases, indicative of actin cytoskeleton remodeling. Isolated mesenteric resistance arteries from Sprague‐Dawley rats (n=4–6) were divided into three sections for Western blot analysis of Rho family small GTPases. Each section was incubated for 20 minutes in Krebs solution with either vehicle (endotoxin free water), TLR9 agonist ODN2395 (2 μM), or ODN2395 after a 5‐minute pre‐incubation with TLR9 antagonist ODN2088 (20 μM). ODN2395 alone increased protein expression of cdc42 (1.7 fold vs. Veh, p<0.05) and RhoA (1.4 fold vs. Veh, p<0.05), and this increase in expression was inhibited by pre‐incubation with ODN2088 (p>0.05). On the other hand, Rac1 was unchanged after incubation with ODN2395 (p>0.05). These results show that TLR9 stimulation leads to activation of some Rho family small GTPases and suggests another mechanism by which TLR9 causes increased vascular tone is actin cytoskeleton remodeling.Support or Funding InformationAmerican Heart Association (AHA) and National Institutes of Health (NIH)

Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/.

Greater impairments in cerebral artery compared with skeletal muscle feed artery endothelial function in a mouse model of increased large artery stiffness.

Increased large artery stiffness is a hallmark of arterial dysfunction with advancing age and is also present in other disease conditions such as diabetes. Increased large artery stiffness is correlated with resistance artery dysfunction in humans. Using a mouse model of altered arterial elastin content, this is the first study to examine the cause-and-effect relationship between large artery stiffness and peripheral resistance artery function. Our results indicate that mice with genetically greater large artery stiffness have impaired cerebral artery endothelial function, but generally preserved skeletal muscle feed artery endothelial function. The mechanisms for impaired cerebral artery endothelial function are reduced nitric oxide bioavailability and increased oxidative stress. These findings suggest that interventions that target large artery stiffness may be important to reduce disease risk associated with cerebral artery dysfunction in conditions such as advancing age. Advancing age as well as diseases such as diabetes are characterized by both increased large artery stiffness and impaired peripheral artery function. It has been hypothesized that greater large artery stiffness causes peripheral artery dysfunction; however, a cause-and-effect relationship has not previously been established. We used elastin heterozygote mice (Eln(+/-) ) as a model of increased large artery stiffness without co-morbidities unrelated to the large artery properties. Aortic stiffness, measured by pulse wave velocity, was ∼35% greater in Eln(+/-) mice than in wild-type (Eln(+/+) ) mice (P=0.04). Endothelium-dependent dilatation (EDD), assessed by the maximal dilatation to acetylcholine, was ∼40% lower in Eln(+/-) than Eln(+/+) mice in the middle cerebral artery (MCA, P<0.001), but was similar between groups in the gastrocnemius feed arteries (GFA, P=0.79). In the MCA, EDD did not differ between groups after incubation with the nitric oxide (NO) synthase inhibitor N(ω) -nitro-l-arginine methyl ester (P>0.05), indicating that lower NO bioavailability contributed to the impaired EDD in Eln(+/-) mice. Superoxide production and content of the oxidative stress marker nitrotyrosine was higher in MCAs from Eln(+/-) compared with Eln(+/+) mice (P<0.05). In the MCA, after incubation with the superoxide scavenger TEMPOL, maximal EDD improved by ∼65% in Eln(+/-) (P=0.002), but was unchanged in Eln(+/+) mice (P=0.17). These results indicate that greater large artery stiffness has a more profound effect on endothelial function in cerebral arteries compared with skeletal muscle feed arteries. Greater large artery stiffness can cause cerebral artery endothelial dysfunction by reducing NO bioavailability and increasing oxidative stress.

Effects of the use of assisted reproductive technologies and an obesogenic environment on resistance artery function and diabetes biomarkers in mice offspring.

Maternal obesity affects the incidence of cardiovascular disease and diabetes in offspring. Also the use of assisted reproductive technologies (ART) has been associated with cardiovascular deficiencies in offspring. Obese women often suffer from infertility and use ART to achieve a pregnancy, but the combined effects of maternal obesity and ART on cardiovascular health and incidence of diabetes in the offspring is not known. Here, we report the effects of the use of ART within an obesogenic environment, consisting of feeding a western diet (WD) to dams and offspring, on resistance artery function and presence of diabetes biomarkers in juvenile mice offspring. Our results indicate that WD and ART interacted to induce endothelial dysfunction in mesenteric resistance arteries isolated from 7-week-old mice offspring. This was determined by presence of a reduced acetylcholine-induced dilation compared to controls. The arteries from these WD-ART mice also had greater wall cross-sectional areas and wall to lumen ratios indicative of vascular hypertrophic remodeling. Of the diabetes biomarkers measured, only resistin was affected by a WD×ART interaction. Serum resistin was significantly greater in WD-ART offspring compared to controls. Diet and sex effects were observed in other diabetes biomarkers. Our conclusion is that in mice the use of ART within an obesogenic environment interacts to favor the development of endothelial dysfunction in the resistance arteries of juvenile offspring, while having marginal effects on diabetes biomarkers.

Vasoactive effects of prostaglandins from the perivascular fat of mesenteric resistance arteries in WKY and SHROB rats

AimsWe have studied the vasoactive role of prostaglandins derived from perivascular adipose tissue (PVAT) and their effects on endothelial function in healthy rats and rats with metabolic syndrome (SHROB). Main methodsMesenteric resistance arteries (MRA) from SHROB and control rats (WKY) were mounted on wire myographs: a) together with a sphere of naturally occurring perivascular adipose tissue (with-PVAT group), or b) dissecting all the adventitial tissue (without-PVAT group). Key findingsEndothelial function, tested by acetylcholine reactivity of SHROB arteries with PVAT, was significantly lower than that of WKY. With-PVAT arteries, especially the SHROB, showed lower responses than those without PVAT. NO synthase inhibition diminished the acetylcholine responses in every group except the with-PVAT SHROB group. Blockade of cyclooxygenase-2, PGI2-IP, TXA2-TP, or TXA2 synthase increased to different extents the arterial responses in the SHROB with-PVAT group. PVAT from both rat strains revealed cyclooxygenase-2 activity and immunoassay confirmed the release of PGE2, PGI2 and TXA2. SignificanceOur major finding is that PVAT is a source of vasoactive prostaglandins in WKY and SHROB. We also report that the presence of visceral PVAT causes endothelial dysfunction of resistance arteries in the SHROB. The vascular responses to prostaglandins partly underlie the endothelial dysfunction of SHROB arteries.

Abstract 194: Low Levels of Angiotensin Converting Enzyme (ace) Do Not Prevent From Metabolic Alterations And Endothelial Dysfunction of Resistance Arteries Induced by High Fructose Intake

Purpose: ACE gene polymorphism may be associated with different responses to cardiovascular and metabolic disturbances. Therefore, we tested whether mice with low vs. normal levels of circulating and tissue ACE differently responds to chronic and excessive fructose (F) consumption. Methods: Male mice (22±1g) harboring 1 or 2 copies of the ACE gene received F (100mg/l) or tap water for 8 weeks. Blood pressure (BP) was measured by plethysmography. Glycemia, lipids and glucose tolerance (0 to 120 min after glucose load=1.5 g/Kg ip) were also evaluated. After euthanasia, the white adipose tissue was weighed and the mesenteric resistance artery (MRA) was dissected out. Relaxation of MRA rings was evaluated in an isometric myograph in response to acetylcholine (Ach;0.01nM-30μM) or sodium nitroprusside (SNP; 0.01nM-300μM) after pre-constriction with noradrenaline. Results: Caloric intake (~ 12.7 kcal/day) and body weight gain (1 copy: ~40%; 2 copies: ~30%) were similar among all groups. However, adipose tissue deposition was 43% higher in 1-copy F-fed mice and 36% higher in 2-copy F-fed mice in relation to the controls (p&lt;0.05), with no influence of ACE gene copy number. F consumption increased systolic BP in 2-copy mice (1 copy: 114±2 vs. F=116±2; 2 copies: 113±2 vs. F=122±1 mmHg) while diastolic BP was increased in both F-fed groups (1 copy: 79±1 vs. F= 88±2; 2 copies: 81±2 vs. F=93±2 mmHg). This augment in BP was significantly higher in 2-copy than in 1-copy mice (p&lt;0.05). F-fed mice also presented hyperglycemia (1 copy: 84±3 vs. F=128±5; 2 copies: 85±4 vs. F=133±8 mg/dl) as well as glucose intolerance (increase of ~67% in the area under time course curve); with no alterations in total cholesterol nor triglycerides. MRA % of relaxation was unaltered in response to SNP, while F-fed groups showed reduced relaxation response to Ach (1 copy: 87% vs. F=51%; 2 copies: 92% vs. F=57%). Conclusions: F intake provokes glucose intolerance, increases visceral adipose tissue deposition and decreases endothelium-dependent relaxation of resistance arteries at a similar magnitude for both, 1- and 2-copy ACE gene mice, while F-induced BP increase seems to be more dependent on ACE gene dosage.