Endothelium-dependent Dilation Research Articles

Role of the circulating milieu in age-related arterial dysfunction: a novel ex vivo approach.

The circulating milieu, bioactive molecules in the bloodstream, is altered with aging and interfaces constantly with the vasculature. This anatomic juxtaposition suggests that circulating factors may actively modulate arterial function. Here, we developed a novel, translational experimental model that allows for direct interrogation of the influence of the circulating milieu on age-related arterial dysfunction (aortic stiffening and endothelial dysfunction). To do so, we exposed young and old mouse arteries to serum from young and old mice and young and midlife/older (ML/O) adult humans. We found that old mouse and ML/O adult human, but not young, serum stiffened young mouse aortic rings, assessed via elastic modulus (mouse and human serum, P = 0.003 vs. young serum control), and impaired carotid artery endothelial function, assessed by endothelium-dependent dilation (EDD) (mouse serum, P < 0.001; human serum, P = 0.006 vs. young serum control). Furthermore, young mouse and human, but not old, serum reduced aortic elastic modulus (mouse serum, P = 0.009; human serum, P < 0.001 vs. old/MLO serum control) and improved EDD (mouse and human serum, P = 0.015 vs. old/MLO serum control) in old arteries. In human serum-exposed arteries, in vivo arterial function assessed in the human donors correlated with circulating milieu-modulated arterial function in young mouse arteries (aortic stiffness, r = 0.634, P = 0.005; endothelial function, r = 0.609, P = 0.004) and old mouse arteries (aortic stiffness, r = 0.664, P = 0.001; endothelial function, r = 0.637, P = 0.003). This study establishes novel experimental approaches for directly assessing the effects of the circulating milieu on arterial function and implicates changes in the circulating milieu as a mechanism of in vivo arterial aging.NEW & NOTEWORTHY Changes in the circulating milieu with advancing age may be a mechanism underlying age-related arterial dysfunction. Ex vivo exposure of young mouse arteries to the circulating milieu from old mice or midlife/older adults impairs arterial function whereas exposure of old mouse arteries to the circulating milieu from young mice or young adults improves arterial function. These findings establish that the circulating milieu directly influences arterial function with aging.

Retraction: Bacillus Subtilis DE111 improves endothelial dependent vascular dilation in mice fed a Western diet

Risk BD. Bacillus Subtilis DE111 improves endothelial dependent vascular dilation in mice fed a Western diet. The FASEB Journal, 2022;36. https://doi.org/10.1096/fasebj.2022.36.S1.L8035.The above abstract, published online on May 13, 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the author; the journal Editor‐in‐Chief, Loren E. Wold; the Federation of American Societies for Experimental Biology; and Wiley Periodicals LLC. The retraction has been agreed upon since the abstract was submitted and approved for publication by Briana D. Risk without authorization and consent from the other co‐authors: Elliot L. Graham, Mingue Zhang, Yuren Wei, Grace Stark, Gaby Brown, Christopher L. Gentile, and Tiffany L. Weir. In addition, the authors asked to retract this article as they found that the strain of bacteria listed in the abstract (Bacillus subtilis DE111) is incorrect. Strain NRRL B‐67989 was used. The author apologizes for any inconvenience this may have caused.

Acute inspiratory resistance training enhances endothelium-dependent dilation and retrograde shear rate in healthy young adults.

Inspiratory resistance training (IRT) yields significant reductions in resting blood pressure and improves vascular endothelial function. Our objective was to quantify the acute effects of IRT on brachial artery flow-mediated dilation (FMD) and shear rates (SRs) in healthy men and women. Twenty young adults (22.9 ± 3.4 years; 10 male, 10 female) completed a single bout of IRT or Rest condition in a randomized crossover design. Brachial artery FMD was performed before, 10 min after, and 40 min after the assigned condition. Brachial artery blood flow velocities were collected during IRT, separated by breathing cycle phase, and converted into SRs. FMD improved 10 min post-IRT (+1.86 ± 0.61%; p = 0.025) but returned to baseline by 40 min post-IRT (p = 0.002). Anterograde SR decreased by 10% and retrograde SR increased 102% during resisted inspiration, relative to baseline SR (p < 0.001). Anterograde SR increased by 7% in men and women (p < 0.001) and retrograde SR decreased by 12% in women but not men (p = 0.022) during unresisted expiration, relative to baseline SR. A single bout of IRT elicits a transient enhancement in FMD in both men and women. Acute IRT-related enhancements in SRs may contribute to sustained improvements in FMD that have been reported previously.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that exvivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Exvivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Endothelial cell-specific reduction in mTOR ameliorates age-related arterial and metabolic dysfunction.

Systemic inhibition of the mammalian target of rapamycin (mTOR) delays aging and many age-related conditions including arterial and metabolic dysfunction. However, the mechanisms and tissues involved in these beneficial effects remain largely unknown. Here, we demonstrate that activation of S6K, a downstream target of mTOR, is increased in arteries with advancing age, and that this occurs preferentially in the endothelium compared with the vascular smooth muscle. Induced endothelial cell-specific deletion of mTOR reduced protein expression by 60-70%. Although this did not significantly alter arterial and metabolic function in young mice, endothelial mTOR reduction reversed arterial stiffening and improved endothelium-dependent dilation (EDD) in old mice, indicating an improvement in age-related arterial dysfunction. Improvement in arterial function in old mice was concomitant with reductions in arterial cellular senescence, inflammation, and oxidative stress. The reduction in endothelial mTOR also improved glucose tolerance in old mice, and this was associated with attenuated hepatic gluconeogenesis and improved lipid tolerance, but was independent of alterations in peripheral insulin sensitivity, pancreatic beta cell function, or fasted plasma lipids in old mice. Lastly, we found that endothelial mTOR reduction suppressed gene expression of senescence and inflammatory markers in endothelial-rich (i.e., lung) and metabolically active organs (i.e., liver and adipose tissue), which may have contributed to the improvement in metabolic function in old mice. This is the first evidence demonstrating that reducing endothelial mTOR in old age improves arterial and metabolic function. These findings have implications for future drug development.

Advanced glycated end-products inhibit dilation through constitutive endothelial RAGE and Nox1/4 in rat isolated skeletal muscle arteries.

This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA). Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography. High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619. These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation.

Patients with Chronic Coronary Syndrome Can Benefit from Consumption of Enriched Chicken Eggs: The Effects on Microvascular Function, Inflammatory Biomarkers, and Oxidative Status—Randomized Clinical Study

The aim of this clinical study was to determine the impact of the consumption of chicken eggs enriched with n-3 polyunsaturated fatty acids, selenium, vitamin E, and lutein on micro- and macrovascular endothelium-dependent dilation, inflammation biomarkers, and oxidative stress levels in participants with chronic coronary syndrome (CCS). This was a double-blind, placebo-controlled clinical study that included 30 CCS participants (9 women, 21 men) randomized into the control group (N = 15), who ate ordinary chicken eggs (three per day), and the Nutri4 group (N = 15), who ate enriched eggs (three per day) for 21 days. Microvascular and macrovascular endothelium-dependent vasodilation was evaluated by measuring forearm skin post-occlusive reactive hyperemia (PORH) and acetylcholine-induced dilation (AChID) and the flow-mediated dilation (FMD) of the brachial artery, respectively. The serum lipid profile, anti- and proinflammatory cytokine levels, serum concentration of nitric oxide synthase (NOS) isoforms, and oxidative stress biomarkers were measured before and after the diet protocols. Enriched, but not regular, chicken eggs significantly improved microvascular PORH and AChID and macrovascular FMD, increased the serum concentration of inducible NOS, decreased serum triglyceride levels, and decreased proinflammatory cytokine IL-17A and TGF-1β levels compared to initial measurements. Patients with CCS can benefit from the consumption of enriched chicken eggs due to improved lipid biomarkers, a more favorable anti-inflammatory milieu, and improved vascular relaxation at micro- and macrovascular levels.

Abstract 17565: Sympathetic Neural Overactivity, Endothelial Dysfunction, Aortic Stiffening, and Diminished Exercise Capacity in Breast Cancer Survivors Treated With Doxorubicin and Trastuzumab-Based Chemotherapy

Introduction: Previous evidence demonstrates that breast cancer (BC) survivors who were treated with doxorubicin and trastuzumab-based chemotherapy have an increased risk of developing cardiovascular (CV) disease at least 7 years after completion of cancer treatment. The reasons for the increased CV disease risk are not completely understood, a constellation of physiological changes may contribute. We hypothesized that BC survivors compared with age- and body mass index (BMI), sex -matched healthy controls, exhibit sympathetic neural overactivity, vascular endothelial dysfunction, aortic stiffening, and diminished exercise capacity. Methods: Twelve women BC survivors, treated with doxorubicin and trastuzumab (age=48±2 years and BMI=28±2 Kg/m 2 ) and fourteen women control subjects (age=46±1 years and BMI=27±1 Kg/m 2 ) were studied. Muscle sympathetic nerve activity (MSNA, Microneurography), endothelium-dependent dilation (brachial artery flow-mediated dilation, BAFMD), aortic stiffness (carotid-femoral pulse wave velocity, CFPWV), blood pressure (BP, Finometer), heart rate (HR, Electrocardiogram), and peak oxygen uptake (V˙O 2 peak, Cardiopulmonary exercise testing) were measured. Results: BC survivors were evaluated 8±2 years after the completion of cancer treatment. Burst frequency and burst incidence of MSNA were higher and BAFMD and V˙O 2 peak were lower in BC survivors than in controls ( p &lt;0.001). There were not differences between the groups in CFPWV, BP, and HR ( p &gt;0.05). MSNA burst frequency and burst incidence were inversely associated with V˙O 2 peak or BAFMD ( p &lt;0.05). Conclusion: Our findings demonstrate that BC survivors have an augmented sympathetic neural activity, endothelial dysfunction, and attenuated exercise capacity, which may help to explain, at least in part, the increased CV risk in this population. Therefore, it is urgent to establish therapeutic strategies (e.g., physical exercise rehabilitation) to restore or alleviate these cardiovascular changes in BC survivors.

Abstract 17170: Association of Plasma Homocysteine Levels in Cancer Survivors - Data From Framingham Heart Study Offspring Cohort

Introduction: Cancer survivors experience a high prevalence of vascular remodeling from their anti-cancer therapies, which can lead to arterial stiffness. High plasma homocysteine (Hcy) is associated with increased cardiovascular disease risk via its role in decreasing nitric oxide bioavailability and impairing vascular function. However, there is limited work on its role in cancer survivorship. We, therefore, hypothesized that survivors of breast, gastrointestinal, and prostate cancer with elevated plasma Hcy values have higher arterial stiffness and lower endothelial function compared to those with low plasma Hcy. Methods: We evaluated cardiovascular disease-free cancer survivors in the Framingham Heart Study Offspring Cohort (n=196, 66±8 years old, 55.1% women, 26.4±1.2 years from cancer diagnosis, Range: [23.8 - 29.6 years]) with plasma Hcy measurements. The cohort median plasma Hcy was used to define low (&lt;8.16 μmol/L) and high Hcy (≥8.16 μmol/L) groups. Arterial stiffness was assessed via carotid-femoral pulse wave velocity (cfPWV). Peripheral vascular endothelial function was assessed via endothelium-dependent brachial artery flow-mediated dilation (FMD). Differences in cfPWV, FMD, and cardiovascular risk factors were assessed via independent sample t-test or chi-square test. Differences in cfPWV and FMD were further assessed via one-way ANCOVA with age, sex, and cardiovascular medications included as covariates. Results: cfPWV was significantly higher in the high Hcy (9.9±1.5m/s) vs. low Hcy group (8.5±1.9m/s, p=0.005); however, there were no differences in FMD between groups (2.4±1.7 vs. 2.7±2.0%, p=0.42). After controlling for our covariates, cfPWV was still significantly higher in the high Hcy group (Mean[95% CI]): (9.8[9.0-10.61]) compared to the low Hcy group (8.5[8-9.1]) (p=0.015). Conclusions: Our findings suggest an association between elevated Hcy and an increase in arterial stiffness in cancer survivors. This supports the potential adverse consequences of vascular stiffening following cancer diagnosis and treatment which could have long-term cardiovascular health implications.

Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

The effects of water-based circuit exercise training on vascular function in people with coronary heart disease.

Impaired endothelial function in people with coronary heart disease (CHD) is associated with increased mortality. Water immersion can increase peripheral artery shear stress which may provide an additional stimulus to the endothelium during exercise. This study compared the effects of water-based circuit exercise training (WEX) and gym-based circuit exercise training (GEX) on vascular function in people with stable CHD. Participants were randomised to 12 weeks of WEX (n=20), GEX (n=20) or a control group (usual activities; n=12). Endothelium-dependent flow-mediated dilation (FMD) and glyceryl trinitrate-mediated dilation (GTN) of the brachial artery were assessed pre- and post-intervention. FMD increased following WEX [4.0% (3.0-5.1%) to 5.3% (4.1-6.5%); p=0.016], but was unchanged following GEX [4.9% (3.8-5.9%) to 5.0% (3.8-6.1%); p=0.822]. There were no between-group differences in the change in FMD and no significant changes in GTN-mediated dilation percentage. Triglycerides decreased following GEX [1.2 mmol.L-1 (1.0-1.4 mmol.L-1) to 1.0 mmol.L-1 (0.8-1.3 mmol.L-1); p=0.022], but there were no further differences in lipid profiles. WEX improved endothelial function of the brachial artery in people with stable CHD, suggesting that WEX is an effective alternative to gym-based exercise in people living with CHD, which may specifically address vascular health.

Sex Differences in Oxidative Stress-Mediated Reductions in Microvascular Endothelial Function in Young Adult e-Cigarette Users.

Chronic electronic-cigarette (EC) use is reported to decrease vascular endothelial function. However, the mechanism(s) mediating this reduction remain unclear. In this study, we examined endothelium- and NO-dependent dilation, and the role of oxidative stress in attenuating these responses, in healthy young EC users (n=20, 10 males/10 females) compared with healthy controls (n=20, 10 males/10 females). We hypothesized that EC would have reduced endothelium- and NO-dependent dilation and administration of the superoxide scavenger tempol would increase these responses in EC. We further hypothesized that female EC would have the greatest reductions in endothelium- and NO-dependent dilation. We assessed microvascular endothelium-dependent vasodilator function in vivo by measurement of cutaneous vascular conductance (%CVCmax) responses to a standardized local heating protocol in control and 10 μM tempol-treated sites. After full expression of the local heating response, 15 mM NG-nitro-L-arginine methyl ester (NO synthase inhibition) was perfused. EC had significantly reduced endothelium- (73±15 versus 87±9%CVCmax; P<0.001) and NO-dependent (48±17% versus 62±15%; P=0.011) dilation. Tempol perfusion increased endothelium-dependent (84±12%CVCmax P=0.01) and NO-dependent (63±14% P=0.005) dilation in EC but had no effect in healthy control. Within female sex, EC had lower endothelium-dependent (71±13 versus 89±7%CVCmax; P=0.002) and NO-dependent (50±6 versus 69±11%; P=0.005) dilation compared with healthy control, and tempol augmented endothelium-dependent (83±13%CVCmax; P=0.002) and NO-dependent (62±13%; P=0.015) dilation. There were no group or treatment differences within male sex. Healthy young adult EC users have reduced microvascular endothelium-dependent and NO-dependent dilation, driven by greater reductions in female EC users, and mediated in part by superoxide.

Reduction of double-strand DNA break repair exacerbates vascular aging.

Advanced age is the greatest risk factor for cardiovascular disease (CVD), the leading cause of death. Arterial function is impaired in advanced age which contributes to the development of CVD. One underexplored hypothesis is that DNA damage within arteries leads to this dysfunction, yet evidence demonstrating the incidence and physiological consequences of DNA damage in arteries, and in particular, in the microvasculature, in advanced age is limited. In the present study, we began by assessing the abundance of DNA damage in human and mouse lung microvascular endothelial cells and found that aging increases the percentage of cells with DNA damage. To explore the physiological consequences of increases in arterial DNA damage, we evaluated measures of endothelial function, microvascular and glycocalyx properties, and arterial stiffness in mice that were lacking or heterozygous for the double-strand DNA break repair protein ATM kinase. Surprisingly, in young mice, vascular function remained unchanged which led us to rationalize that perhaps aging is required to accumulate DNA damage. Indeed, in comparison to wild type littermate controls, mice heterozygous for ATM that were aged to ~18 mo (Old ATM +/-) displayed an accelerated vascular aging phenotype characterized by increases in arterial DNA damage, senescence signaling, and impairments in endothelium-dependent dilation due to elevated oxidative stress. Furthermore, old ATM +/- mice had reduced microvascular density and glycocalyx thickness as well as increased arterial stiffness. Collectively, these data demonstrate that DNA damage that accumulates in arteries in advanced age contributes to arterial dysfunction that is known to drive CVD.

Abstract 122: Cerebral Artery Function And Stiffness Is Dependent On Apoe Genotype And Ovariectomy Status In Mice

The major risk factors for late-onset Alzheimer’s Disease (LOAD) include old age, female sex and the APOE4 allele. Post-menopausal females with an APOE4 allele have higher rates of LOAD compared with age-matched males with an APOE4 allele, suggesting an interaction between APOE genotype and estrogen deficiency. Importantly, cerebrovascular dysfunction is a contributor to LOAD risk. However, it is unknown if APOE genotype and estrogen deficiency interact to impact cerebrovascular function.Young female homozygous APOE3 and APOE4 mice (n=4-6 per group; ~6 months old) fed a high-fat, high-cholesterol diet were intact (sham) or ovariectomized (OVX). We assessed endothelium-dependent dilation to increasing doses of acetylcholine (ACh) in pressurized posterior cerebral arteries (PCAs). There was an interaction effect of APOE genotype and OVX status for endothelium-dependent dilation (p=0.03), such that APOE3 -Sham mice had a greater endothelium-dependent dilation compared to the APOE3 -OVX group (70±9.1% vs. 39±7.6%, p=0.003). These differences were mediated by nitric oxide as indicated by the absence of dilation in the presence of N omega-Nitro-L-arginine methyl ester hydrocholoride (L-NAME, Sham: 3.7±3.3% vs OVX: 4.1±4.0%, p= 0.99). In contrast, APOE4 -OVX had preserved endothelium-dependent dilation compared with APOE4 -Sham (49.9±14.8% vs 56.1±9.2%, p=0.8). Endothelium-independent dilation was assessed by the response to increasing doses of sodium nitroprusside and did not differ among groups (all p&gt;0.05). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. For the elastic modulus at low pressures, there was an interaction effect of APOE genotype and OVX status (p=0.03), with APOE4 -OVX having a higher elastic modulus compared to APOE4 -Sham (0.93±0.3 vs. 0.47±0.1, p=0.03). The elastic modulus was similar in APOE3 groups (sham: 0.64±0.01, OVX: 0.62±0.09, p=0.99). Overall, we found that estrogen deficiency impacts cerebral artery endothelial function in APOE3 , but not APO E4 mice, while estrogen deficiency impacts cerebral artery stiffness in APOE4 , but not APOE3 mice. These results shed light on the impact of APOE genotype on the cerebral vasculature, suggesting a potential mechanism for LOAD.

Abstract P209: Six Weeks Oral Losartan Treatment Improves Endothelium-dependent Dilation In Normotensive Women With A History Of Preeclampsia

Women with a history of preeclampsia have a 4-fold increased risk of cardiovascular disease compared to women who had a healthy pregnancy. These women demonstrate microvascular endothelial dysfunction, mediated by reduced nitric oxide (NO)-dependent dilation and increased sensitivity to angiotensin II. We hypothesized that systemic angiotensin II type 1 receptor (AT 1 R) inhibition would improve endothelium- and NO-dependent dilation in the microvasculature of women with a history of preeclampsia. Seven normotensive (seated SBP 118±6; DBP 78±6 mmHg) women (33±6 years of age, 11±7 months postpartum) with a history of preeclampsia participated in a double-blind placebo-controlled crossover study. Following 6 weeks of placebo and losartan treatment (50 mg/day), participants wore a 24-hour blood pressure monitor and completed 1 study visit in which 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusions of acetylcholine (ACh, 10 -7 -10 2 mM) or ACh + 15 mM N G nitro L-arginine methyl ester (L-NAME; NO synthase inhibitor) to assess endothelium- and NO-dependent dilation, respectively. Red blood cell flux was measured over each microdialysis site by laser-Doppler flowmetry. Cutaneous vascular conductance was calculated (CVC=Doppler flux/mean arterial pressure) and normalized to maximum (%max; 28 mM SNP + 43°C). Systemic losartan treatment augmented endothelium-dependent (losartan: 96±4 vs placebo: 70±14%max; P &lt;0.001) and NO-dependent (losartan: 35±14 vs placebo: 23±8%; P &lt;0.001) dilation. There was no effect of treatment on 24-hour systolic blood pressure (losartan: 117±4 vs placebo: 117±8 mmHg), diastolic blood pressure (losartan: 72±5 vs placebo: 71±5 mmHg) or mean arterial pressure (losartan: 92±4 vs placebo: 92±6 mmHg; all P&gt;0.05 ). Losartan treatment increased endothelium-dependent vasodilation via NO-mediated pathways in the absence of changes in blood pressure in women with a history of preeclampsia. These data suggest that systemic AT 1 R inhibition may be a viable therapeutic approach to treat persistent microvascular dysfunction in otherwise healthy women who have had preeclampsia.

Abstract P217: The Role Of Angiotensin-(1-7) In Regulating Vascular Endothelial Function In Postmenopausal Women

Postmenopausal women (PMW) have reduced endothelial function, however, the mechanisms contributing to this decline remain unclear. Angiotensin-(1-7) [Ang-(1-7)] is a vasoactive peptide of the vasodilatory arm of the Renin-Angiotensin system and increasing bioavailable Ang-(1-7) enhances endothelial function in young women (YW) with pre-clinical cardiovascular disease. Circulating Ang-(1-7) decreases with aging and may be impacted by menopause. However, whether increasing bioavailable Ang-(1-7) improves endothelial function in PMW is unknown. The purpose of this study was to test the hypothesis that increasing bioavailable Ang-(1-7) improves endothelial function in PMW. We assessed skin blood flow responses to local heating in 8 healthy YW (27±9 yrs) and 5 healthy PMW (56±4 yrs). Cutaneous vasodilation was measured using laser-Doppler flowmetry directly over microdialysis perfusions of lactated Ringer’s (control) in YW and PMW, and in PMW only, 100 μM Ang-(1-7). Following 45 minutes of perfusion of lactated Ringer’s and Ang-(1-7), skin was heated to 42°C (0.1°C·sec -1 ) until laser-Doppler flux reached a stable plateau, indicative of endothelium-dependent dilation. Following the plateau, 28mM sodium nitroprusside was perfused while skin was heated to 43°C to achieve maximal vasodilation. Cutaneous vascular conductance (CVC) was calculated as laser-Doppler flux/MAP, and normalized to maximal dilation (%CVC max ). Women were normotensive (MAP: YW 86±7 mmHg; PMW 85±9 mmHg) and not obese (BMI: YW 23±3 kg/m 2 ; PMW 23±4 kg/m 2 ). YW had greater dilation during local heating (control: YW 94.54±5.4 vs. PMW 82.59±10.72 %CVC max ; P=0.021). Contrary to our hypothesis, Ang-(1-7) did not enhance heating-induced vasodilation in PMW (80.10±9.43 %CVC max ; P=0.69). These preliminary results suggest that increasing bioavailable Ang-(1-7) may not improve endothelial function in PMW. Additional studies are needed to understand if these findings are due to estrogen deficiency in PMW.

Cellular Senescence Contributes to Large Elastic Artery Stiffening and Endothelial Dysfunction With Aging: Amelioration With Senolytic Treatment.

Here, we assessed the role of cellular senescence and the senescence associated secretory phenotype (SASP) in age-related aortic stiffening and endothelial dysfunction. We studied young (6-8 mo) and old (27-29 mo) p16-3MR mice, which allows for genetic-based clearance of senescent cells with ganciclovir (GCV). We also treated old C57BL/6N mice with the senolytic ABT-263. In old mice, GCV reduced aortic stiffness assessed by aortic pulse wave velocity (PWV; 477±10 vs. 382±7 cm/s, P<0.05) to young levels (old-GCV vs. young-vehicle, P=0.35); ABT-263 also reduced aortic PWV in old mice (446±9 to 356±11 cm/s, P<0.05). Aortic adventitial collagen was reduced by GCV (P<0.05) and ABT-263 (P=0.12) in old mice. To show an effect of the circulating SASP, we demonstrated that plasma exposure from Old-vehicle p16-3MR mice, but not from Old-GCV mice, induced aortic stiffening assessed ex vivo (elastic modulus; P<0.05). Plasma proteomics implicated glycolysis in circulating SASP-mediated aortic stiffening. In old p16-3MR mice, GCV increased endothelial function assessed via peak carotid artery endothelium-dependent dilation (EDD; Old-GCV, 94±1% vs. Old-vehicle, 84±2%, P<0.05) to young levels (Old-GCV vs. young-vehicle, P=0.98), and EDD was higher in old C57BL/6N mice treated with ABT-263 vs. vehicle (96±1% vs. 82±3%, P<0.05). Improvements in endothelial function were mediated by increased nitric oxide (NO) bioavailability (P<0.05) and reduced oxidative stress (P<0.05). Circulating SASP factors related to NO signaling were associated with greater NO-mediated EDD following senescent cell clearance. Cellular senescence and the SASP contribute to vascular aging and senolytics hold promise for improving age-related vascular function.

Postnatal development of extracellular matrix and vascular function in small arteries of the rat.

Introduction: Vascular extracellular matrix (ECM) is dominated by elastic fibers (elastin with fibrillin-rich microfibrils) and collagens. Current understanding of ECM protein development largely comes from studies of conduit vessels (e.g., aorta) while resistance vessel data are sparse. With an emphasis on elastin, we examined whether changes in postnatal expression of arteriolar wall ECM would correlate with development of local vasoregulatory mechanisms such as the myogenic response and endothelium-dependent dilation. Methods: Rat cerebral and mesenteric arteries were isolated at ages 3, 7, 11, 14, 19days, 2months, and 2years. Using qPCR mRNA expression patterns were examined for elastin, collagen types I, II, III, IV, fibrillin-1, and -2, lysyl oxidase (LOX), and transglutaminase 2. Results: Elastin, LOX and fibrillar collagens I and III mRNA peaked at day 11-14 in both vasculatures before declining at later time-points. 3D confocal imaging for elastin showed continuous remodeling in the adventitia and the internal elastic lamina for both cerebral and mesenteric vessels. Myogenic responsiveness in cannulated cerebral arteries was detectable at day 3 with constriction shifted to higher intraluminal pressures by day 19. Myogenic responsiveness of mesenteric vessels appeared fully developed by day 3. Functional studies were performed to investigate developmental changes in endothelial-dependent dilation. Endothelial-dependent dilation to acetylcholine was less at day 3 compared to day 19 and at day 3 lacked an endothelial-derived hyperpolarizing factor component that was evident at day 19. Conclusion: Collectively, in the rat small artery structural remodeling and aspects of functional control continue to develop in the immediate postnatal period.

Effects of aurantiamide on a rat model of renovascular arterial hypertension.

Asperglaucide (ASP) is an aurantiamide, an effective constituent of purslane (Portulaca oleracea L.), a safe to eat greenery. Effects of ASP on endothelial function, endothelial nitric oxide synthase (eNOS) expression, vascular fluidity, renal and vascular reactive oxygen, and nitrogen species (ROS/RNS) production was examined in the two-kidney one-clip (2K-1C) rat model of renovascular arterial hypertension. ASP toxicity, dose dependent eNOS gene expression and protein levels were also analyzed in human umbilical vein endothelial cells (HUVEC). The 2K-1C model of hypertension was created via surgery and mean blood pressure (MBP) was measured by tail-cuff method during four weeks of ASP treatment. Erythrocyte deformability was monitored by rotational ektacytometry, while vascular constrictor and dilator responses were determined in organ baths. eNOS gene expression and protein levels were assessed in thoracic aorta and HUVEC. MBP was significantly decreased in hypertensive rats treated with ASP. Endothelium dependent vascular dilator and constrictor responses were also considerably improved following ASP treatment. There was a notable increase in red blood cell deformability in hypertensive rats treated with ASP as compared to hypertensive rats alone. A significant increase was observed in eNOS gene expression and protein levels in both normotensive and hypertensive rats treated with ASP. Treatment of HUVEC with 3µM ASP notably increased eNOS mRNA and protein levels. In conclusion, ASP lowered blood pressure, improved endothelium-mediated relaxation, decreased renovascular ROS/RNS production in hypertensive rats. ASP also increased eNOS protein expression in aorta and HUVEC at nontoxic doses. ASP may have future potential as an anti-hypertensive agent.

Dapagliflozin-affected endothelial dysfunction and altered gut microbiota in mice with heart failure.

To investigate the potential microbiome profile of a mouse model with heart failure (HF) during dapagliflozin treatment. An HF model was constructed in 8-week-old male mice, and cardiac tissues were analyzed using histological staining. Hemodynamic indexes were measured, and fecal samples were collected for 16S rDNA sequencing. Chao1, Shannon, and Simpson were used for α-diversity analysis. b-Diversity analysis was conducted using principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) based on the Bray-Curtis distance. Linear discriminant analysis coupled with effect size measurements (LEfSe) was used to identify signature gut microbiota, and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict the function of altered gut microbiota. Dapagliflozin treatment reduced inflammation, infarction area, and cardiac fibrosis in HF mice. It also increased endothelial-dependent dilation and inflammation in mice with HF. Dapagliflozin decreased the ratio of Firmicutes/Bacteroidetes, which was increased in HF mice. There was no significant statistical difference in α-diversity among the control, HF, and HF+dapagliflozin groups. Desulfovibrio, AF12, and Paraprevotella were enriched in HF+dapagliflozin, while Rikenella and Mucispirillum were enriched in HF based on LEfSe. KEGG analysis revealed that altered gut microbiota was associated with fermentation, amino acid biosynthesis, nucleoside and nucleotide biosynthesis/degradation, fatty acid and lipid biosynthesis, carbohydrate biosynthesis/degradation, and cofactor/prosthetic group/electron carrier/vitamin biosynthesis. Understanding the microbiome profile helps elucidate the mechanism of dapagliflozin for HF. The signature genera identified in this study could be used as a convenient method to distinguish between HF patients and healthy individuals.