Maximal Relaxation Response Research Articles

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

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

Functional and Histological Changes in Umbilical Artery and Myometrium Isolated from IUGR Complicated Pregnancies

Objective: To investigate the relaxation responses mediated by L-type Ca2+ channels and big-conductance Ca2+-activated K+ (BKCa) channels and histological changes in the human umbilical artery (HUA) and myometrium smooth muscle isolated from pregnancies complicated with intrauterine growth restriction (IUGR).Methods: The muscle reactivity and the histology of the smooth muscle of the HUA and myometrium retrieved from 14 women with IUGR and 14 controls were investigated by the isolated tissue bath and immunohistochemical method.Results: In HUA, the maximum relaxation responses and pD2 values of nifedipine and NS11021 (BKCa channel opener) were significantly increased and significant histopathological changes are observed in the IUGR group.Conclusions: The pathogenesis of IUGR might be associated with the impairment in the functional responses of L-type Ca2+ channels and BKCa channels in HUA smooth muscle. The increased staining of myometrium and UC with HIF-1α in IUGR may indicate apoptosis, histological damage, and impaired fetal growth.

In vitro effect of relaxin in the rat corpus cavernosum under hyperglycemic and normoglycemic conditions.

Relaxin, an endogenous peptide hormone, elicits vascular relaxation by its direct effect or by modulating the endothelium-dependent relaxation response and is clinically evaluated for the treatment of coronary artery disease. However, its effect on penile tissue has not been explored yet. This study aimed to investigate the effect of serelaxin, recombinant human relaxin-2, on rat corpus cavernosum (CC) under healthy and hyperglycemic conditions. Strips of CC obtained from thirty-nine male Wistar rats weighing 300-350 g were used in organ baths for isometric tension studies to investigate the serelaxin-mediated relaxation (10-12-10-7 M) under normoglycemic conditions and the effect of serelaxin on endothelium-dependent [nitric oxide (NO)- and prostacyclin-mediated] relaxation responses under hyperglycemic conditions. The in vitro hyperglycemia model was created by 3 h of incubation with 44 mM glucose monohydrate +120 μM methylglyoxal. NO-dependent relaxation responses were evaluated by cumulative acetylcholine (10-9-10-4 M) administration in the presence of indomethacin (10-6 M). Prostacyclin-mediated relaxation was evaluated by cumulative administration of iloprost (10-9-10-6 M), a prostacyclin analog. Maximum relaxation responses to serelaxin were not significantly different compared to the time-control (p = 0.480). Three hours of incubation of rat CC in hyperglycemic conditions impaired NO- and prostacyclin-mediated relaxation responses (p = 0.032 and p = 0.047, respectively). Serelaxin coincubation worsened NO-mediated relaxation responses (p = 0.016) but did not affect prostacyclin-mediated responses (p = 0.425). Together, our results demonstrate that in vitro administration of serelaxin does not cause relaxation in penile tissue and short-term in vitro serelaxin treatment in hyperglycemic conditions mimicked diabetes modulates endothelium-dependent responses by worsening NO-mediated responses. Serelaxin exerts different effects via different mechanism on endothelium-dependent responses depending on the dose and duration of exposure. Therefore, proper timing and dosing of serelaxin administration in the penile tissue need to be investigated in further studies in diabetic animal models.

Parabiosis Improves Endothelial Dysfunction in Aged Female Mice

IntroductionThe study aims to investigate the effect of parabiosis method on endothelial dysfunction in naturally aging mice and determine the time projections for predicted improvement in the mentioned target group. MethodsThe balb/c mice were separated into six groups, these being; isochronic old, heterochronic old (HP-O), isochronic young, heterochronic young, young control, and old control. After parabiosis protocol, animals were sacrificed at the third, fifth, seventh, and ninth weeks, and their thoracic aortas were isolated. The vasodilatation and vasoconstriction responses of the vessels were detected using potassium chloride and phenylephrine, acetylcholine (ACh), and sodium nitroprusside. ResultsAging had a significant decreasing effect on maximum ACh relaxation responses (P < 0.01). However, in the HP-O group, the maximum ACh relaxation response in the third week was significantly lower (P < 0.05), but this effect disappeared in the ninth week. Maximum phenylephrine contraction responses were lower in the heterochronic parabiosis group (P < 0.05). ConclusionsACh responses increased at the end of the ninth week in the HP-O group, therefore, the parabiosis model may have an improving effect on endothelial dysfunction seen in aging.

In Utero Morphine Exposure Induces Hypertension And Opioid Receptor‐Mediated Angiotensin II Sensitization

Opioid use disorder (OUD) is associated with nearly two‐fold increased risk of cardiovascular disease, metabolic syndrome and diabetes. OUD by pregnant women is an understudied area related to the opioid epidemic leading to increased incidence of neonatal abstinence syndrome (NAS) and neonatal opioid withdrawal syndrome (NOWS). There is an interaction between the Renin‐Angiotensin System (RAS) and the endogenous opioid system (EOS) commonly associated with the development of hypertension. This study tests the hypothesis that in utero opioid exposure induces hypertension and increases vascular reactivity to Angiotensin‐II (AngII) in the adult offspring. Sprague Dawley dams were treated with escalating doses of a mu‐opioid receptor agonist morphine (MOR, 5‐20mg/kg/day, s.c) or saline (VEH). Weight gain was not different between MOR and VEH‐exposed pregnant rats (F‐MOR 100.2±9.5 vs F‐VEH 95±10 g, n=4, p=NS). Additionally, litter size was not different (F‐MOR13.75±0.2 vs F‐VEH 12.25±1.8 pups, n=4, p=NS). However, MOR‐exposed females exhibited low birth weight (F‐MOR 5.6±0.2 vs F‐VEH 7.2±0.6 g, n=20‐22, p&lt;0.05). Both MOR‐exposed male and female offspring weighed less at weaning compared to VEH‐exposed offspring (p&lt;0.05). At 16 weeks on CHOW diet, only MOR‐exposed female offspring continue to weigh significantly less than VEH‐exposed offspring (F‐MOR 264.5±9.7 vs F‐VEH 306.5±14 g, n=14‐16, p&lt;0.05). Furthermore, both MOR‐exposed male and female rats are hypertensive (M‐MOR 127±0.84 vs M‐VEH 110±1.45 mmHg, p&lt;0.05; F‐MOR 115.2±0.49 vs F‐VEH 93.5±0.50 mmHg, p&lt;0.05). MOR‐exposed offspring displayed exacerbated acute pressor response to Angiotensin II (AngII, 0.01‐1 ng/Kg, intraarterial), the effect was greater in MOR‐exposed males than in females. Additionally, MOR‐exposed females show impaired maximal relaxation in response to acetylcholine (Ach, 10‐5 to 10‐9 M, F‐MOR 22.2±6 vs F‐VEH 44.7±4 %, p&lt;0.05) but not MOR‐exposed males. However, only MOR‐exposed males show exacerbated maximal AngII‐induced vasoconstriction (M‐MOR 49.4±4 vs M‐VEH 35.8±3.9 %, p&lt;0.05), which was blunted by an opioid receptor antagonist preincubation (naloxone, 1mM, 10 min, M‐MOR=49.4±4 vs M‐MOR+NAL 34.1±2.2 % max constriction, p&lt;0.05; M‐VEH=35.8±3.9 vs M‐VEH+NAL=45.7±4 % max constriction, p=NS). At 16 weeks on high fat diet (HFD, 60% kcal from fat), male MOR‐exposed offspring displayed weight gain in response to HFD (M‐MOR 556.4±27 vs M‐VEH 598.3±12.4 g, p=NS), while female MOR‐exposed offspring were resistant to show diet‐induced obesity (F‐MOR 295.1±13 vs F‐VEH 350.6±22 g, p&lt;0.05). HFD impaired maximal relaxation in response to acetylcholine in VEH‐exposed females (F‐VEH 44.7±4 vs. F‐VEH+HF 19±5 %, p&lt;0.05) and MOR‐exposed males relaxation (M‐MOR 49.4±4 vs M‐MOR+HF 31.2±3 %, p&lt;0.05) but not VEH‐exposed males or MOR‐exposed females. Furthermore, circulating AngII was increased in MOR‐exposed males (p&lt;0.05 vs VEH). Maternal MOR exposure increases the risk of outcomes linked to cardiovascular and metabolic disease in adult offspring. The effects appeared to be mediated through a sex‐specific mechanism in hypertension development involving potential crosstalk between AngII and the EOS.

Contribution of Genetic Background to Vascular Adaptation to Exercise Training Based on Critical Speed of Different Intensities

An impaired endothelial function is a fundamental component of the pathogenesis of cardiovascular disease. In mice, exercise training is well‐known to improve cardiorespiratory fitness and endothelial function. However, the post exercise training effect on endothelial function varies among different strains of mice, suggesting that genetic background contributes to the heterogeneous effect of exercise on vascular health. NZW/LacJ (NZW) mice are known as relatively low responders to exercise training compared to high responder strains such as FVB/NJ (FVB). NZW mice also have relatively poor endothelial function in thoracic aorta when compared with other mouse strains. Therefore, we aimed to determine the contribution of genetic background to endothelial adaptation to exercise training in FVB and NZW mice. Vasoreactivity was assessed in femoral arteries from female mice from both strains (NZW and FVB) after 6 weeks of training using two different critical speed (CS) treadmill training intensities. Mice from each strain were randomly assigned to one of three groups: sedentary (SED), 80% of CS training (CS80), and 90% of CS (CS90) training. After training, endothelium‐dependent vasorelaxation to acetylcholine (ACh) and endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) were measured as well as the vasocontractile responses to phenylephrine (PE) and potassium chloride (KCl). Maximal relaxation responses and IC50 for ACh and SNP were similar between NZW groups (SED, CS80, and CS90). In femoral arteries from FVB mice, there were no differences for maximal relaxation to ACh, but there was significant difference in IC50 for ACh‐induced vasorelaxation between SED vs. CS90 groups (p=0.05). Maximal responses to ACh were similar between NZW and FVB; however, IC50 was significantly lower in FVB compared with NZW, indicating that sensitivity to ACh is greater in arteries from FVB. There was a significant strain‐dependent difference for IC50 for SNP despite no strain differences for maximal responses. There was no effect of exercise or strain on contractile responses to PE and KCl, although the EC50 for KCl induced vasocontraction was higher (p=0.0329) in NZW. Treadmill exercise training at 80% or 90% CS did not contribute to improved vasomotor function in NZW while higher intensity exercise improved sensitivity to ACh in arteries from FVB. Genetic background did not significantly impact vasomotor function or endothelial responses to exercise training in FVB and NZW mice.

Genetic Background Influences Endothelium‐dependent Vasomotor Function in Large Arteries

Introduction Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. Previous research by our laboratory has shown that vasomotor function, and more specifically endothelium-dependent vasorelaxation responses, differ across large arteries from mice with distinct genetic backgrounds. However, it is unclear how much of the observed variation in endothelium-dependent vasorelaxation is due to nitric oxide (NO) mediated pathways. Objective The purpose of this study was to test the hypothesis that strain-dependent vasorelaxation differences were primarily the result of differences in reliance on NO-mediated pathways. Methods Vasomotor function was assessed in thoracic aorta, abdominal aorta, carotid arteries, and femoral arteries from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Strains were chosen based on previously documented vasomotor variation and susceptibility to clinically significant pathologies. Arteries were dissected, cut into 2mm segments, and mounted in a wire myograph system. Arterial segments were stretched to a resting tension equivalent to a pressure of 90mmHg for all strains based on a passive stretch and relaxation tension-force assessment. Increasing concentrations of the endothelium-dependent vasodilator acetylcholine (ACh, 1x10-9 – 1x10-5M) and endothelium-independent vasodilator sodium nitroprusside (SNP, 1x10-9 – 1x10-5M) were used to assess vasorelaxation responses. To assess reliance on NO, vasorelaxation responses to Ach and SNP were assessed in the presence of a nitric oxide synthase inhibitor (NOS). One arterial segment from each group was treated with the NOS inhibitor N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME: 10x4 M) for thirty minutes. Results Two way ANOVA revealed a significant main effect of strain in vasorelaxation responses to ACh for all arteries tested (P <.0001) and no significant differences in response to SNP. Maximal relaxation responses to ACh were significantly inhibited in all arterial segments treated with L-NAME, while no differences were detected for L-NAME treated vessels in response to SNP. These findings indicate that large arteries have a strong dependence on NO-mediated, endothelium-dependent pathways to maintain vascular regulation. Furthermore, strain-dependent vasorelaxation differences were abolished in the presence of L-NAME indicating that strain differences were NO-dependent. Conclusion These findings indicate a strong influence of genetic background on endothelial function and suggest that these differences are primarily due to strain related reliance on NO for endothelium-dependent vasodilation.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 μM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 μM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Diabetes Attenuates the Contribution of Endogenous Nitric Oxide but Not Nitroxyl to Endothelium Dependent Relaxation of Rat Carotid Arteries.

Introduction:Endothelial dysfunction is a major risk factor for several of the vascular complications of diabetes, including ischemic stroke. Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO•), is resistant to scavenging by superoxide, but the role of HNO in diabetes mellitus associated endothelial dysfunction in the carotid artery remains unknown. Aim: To assess how diabetes affects the role of endogenous NO• and HNO in endothelium-dependent relaxation in rat isolated carotid arteries. Methods: Male Sprague Dawley rats were fed a high-fat-diet (HFD) for 2 weeks prior to administration of low dose streptozotocin (STZ; 35 mg/kg i. p./day) for 2 days. The HFD was continued for a further 12 weeks. Sham rats were fed standard chow and administered with citrate vehicle. After 14 weeks total, rats were anesthetized and carotid arteries collected to assess responses to the endothelium-dependent vasodilator, acetylcholine (ACh) by myography. The combination of calcium-activated potassium channel blockers, TRAM-34 (1 μmol/L) and apamin (1 μmol/L) was used to assess the contribution of endothelium-dependent hyperpolarization to relaxation. The corresponding contribution of NOS-derived nitrogen oxide species to relaxation was assessed using the combination of the NO• synthase inhibitor, L-NAME (200 μmol/L) and the soluble guanylate cyclase inhibitor ODQ (10 μmol/L). Lastly, L-cysteine (3 mmol/L), a selective HNO scavenger, and hydroxocobalamin (HXC; 100 μmol/L), a NO• scavenger, were used to distinguish between NO• and HNO-mediated relaxation. Results: At study end, diabetic rats exhibited significantly retarded body weight gain and elevated blood glucose levels compared to sham rats. The sensitivity and the maximal relaxation response to ACh was significantly impaired in carotid arteries from diabetic rats, indicating endothelial dysfunction. The vasorelaxation evoked by ACh was abolished by L-NAME plus ODQ, but not affected by the apamin plus TRAM-34 combination, indicating that NOS-derived nitrogen oxide species are the predominant endothelium-derived vasodilators in sham and diabetic rat carotid arteries. The maximum relaxation to ACh was significantly decreased by L-cysteine in both sham and diabetic rats, whereas HXC attenuated ACh-induced relaxation only in sham rats, suggesting that diabetes impaired the contribution of NO•, whereas HNO-mediated vasorelaxation remained intact. Conclusion: Both NO• and HNO contribute to endothelium-dependent relaxation in carotid arteries. In diabetes, NO•-mediated relaxation is impaired, whereas HNO-mediated relaxation was preserved. The potential for preserved HNO activity under pathological conditions that are associated with oxidative stress indicates that HNO donors may represent a viable therapeutic approach to the treatment of vascular dysfunction.

Constitutive LH receptor activity impairs NO-mediated penile smooth muscle relaxation.

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1–3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%–5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound–microbubble–tissue interactions in the optimization of targeted endothelial drug delivery.

Is Blue Light the Next Little Blue Pill?: Effects of Light‐Stimulation on Erectile Dynamics

INTRODUCTIONMany major risk factors for erectile dysfunction (ED) are associated with depressed nitric oxide (NO) availability. Phosphodiesterase type 5 inhibitors are the current first line therapy for ED and are ineffective in 30–50% of patients, largely due to impaired NO production. New therapeutic approaches for ED are needed under such conditions. The phenomenon of light‐stimulated vasodilation has recently been described in aortic tissue, which is enhanced by G‐protein coupled receptor kinase 2 (GRK2) inhibition and particular for blue light. The objective of this project was to determine if this GRK2 sensitive photorelaxation response is active in erectile tissue and the contribution of the NO signaling pathway to the response.METHODSYoung male mice (n = 4–8 / experiment) were sacrificed and erectile tissue was harvested from the shaft of the penis. Ex vivo constriction and relaxation responses were assessed in a muscle strip myograph. The GRK2‐sensitive photorelaxation response was assessed ± 1 μM GRK2 inhibitor using nine different wavelengths (365–700 nm) of light at constant intensity (100 Lux), and then at nine different light intensities (1–1000 Lux) at 455 nm. The contribution of the NO signaling pathway to the GRK2‐sensitive response was tested with stimulations of 20 and 100 Lux at 455 nm with 1 μM GRK2 inhibitor ± the NO synthase inhibitor L‐NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, the protein kinase G (PKG) inhibitor Rp‐8‐Br‐cGMPS, or the NO scavenger cPTIO, as well as in tissue from mice with a deletion of endothelial + neuronal NO synthase. Differences in responses were determined by two‐way repeated measures ANOVA followed by Sidak’s post hoc test where appropriate.RESULTSMaximal relaxation responses at 455 nm increased with increasing light intensity in the absence (2–10%) and presence (57–103%) of the GRK2 inhibitor, demonstrating a stark increase in photorelaxation under GRK2 inhibition. However, this response waned more rapidly at intensities &gt; 100 Lux. Significant GRK2‐sensitive responses were observed at 365, 395, 430, and 455 nm at 100 Lux intensity, with no significant responses observed in the 490–700 nm range. Both L‐NAME and deletion of endothelial/neuronal NO synthase increased the magnitude of relaxation at all time‐points at 20 Lux, and prolonged maximal relaxation at 100 Lux. CPTIO prolonged the relaxation response at both intensities. ODQ drastically blunted the photorelaxation response at all time points and intensities. Rp‐8‐Br‐cGMPS had no effect on photorelaxation.CONCLUSIONSGRK2‐sensitive photorelaxation was determined to be optimal in the 430–455 nm (violet‐blue) range at ~100 Lux. Depletion of NO by scavenging, enzyme inhibition or genetic deletion enhanced the response, which is sGC‐dependent but PKG‐independent. These findings have implications for treatment of ED due to low NO availability if the GRK2‐sensitive photorelaxation pathway can be harnessed therapeutically.

Effects of Oleamide on the Vasomotor Responses in the Rat

Introduction: Cardiovascular effects of endocannabinoids (eCBs) have generated considerable interest since it has been suggested that the eCB system could become the new pharmacological target, either by blocking its activity or by promoting its effects on several cardiovascular diseases such as hypovolemic and septic shock or hypertension. The purpose of this study was to examine the effects of oleamide on several vasomotor responses in adult rats. Materials and Methods: Blood pressure (BP) was measured both directly and indirectly. Coronary flow was quantified with Langendorf preparation, and the vasomotor responses induced by oleamide were analyzed in the aortic rings. Results: Oleamide induced a decrease in BP, by both direct and indirect methods, which were dose dependent. An increase in coronary flow was observed with Langendorf preparation depending on the dose. Oleamide produced a vasodilator response in aortic rings pre-contracted with phenylephrine (10-5 M), which was concentration and endothelium dependent. This relaxing effect was of minor magnitude than that induced with the same dose on BP. L-NAME did not modify these effects. However, indomethacin induced a shift to the left of the concentration-response curve to oleamide and an increase in the magnitude of maximum vasodilation in rings with endothelium. Oleamide produced the maximal relaxant response at 10-5 M concentration. Conclusions: Oleamide has both in vivo and in vitro vasodilator effects. Vasodilator effects could be mediated by compounds synthesized/released by the endothelium (hyperpolarizing factor) or acting directly on vascular smooth muscle in aortic rings. The TRPV1 and CB1R receptors could mediate these effects. Finally, the results suggest that oleamide probably induces the synthesis/release of a vasoconstrictor prostanoid.

Red raspberry (Rubus idaeus) consumption restores the impaired vasoconstriction and vasorelaxation response in the aorta of the obese Zucker rat, a model of the Metabolic Syndrome

Metabolic Syndrome (MetS) increases the risk of cardiovascular disease. Whole red raspberry (WRR) consumption on vascular function was investigated in the obese Zucker rat (OZR), model of MetS. Male OZR and their lean littermates (LZR) were placed on a control (C) or an 8% w/w WRR-enriched diet for 8 weeks. Phenylephrine (Phe)-induced vasoconstriction and acetylcholine (Ach)-induced vasorelaxation were measured in aortic rings in the presence or absence of L-N-monomethyl-arginine (L-NMMA) and mefenamic acid (MFA). Phe-induced vasoconstriction was lower in the OZR-C compared to LZR-C (p &lt; 0.05). The WRR diet partially restored aortic response in the OZR-WRR aorta (p &lt; 0.05) compared to OZR- C. The OZR-WRR group pre-treated with L-NMMA increased compared to OZR-C (p &lt; 0.05). Pre-treatment with L-NMMA, maximal relaxation response was higher in the OZR compared to the LZR (p &lt; 0.05). With L-NMMA, maximal relaxation response in OZR-WRR (p &lt; 0.05) was lower compared to the OZR-C. Prostacyclin I2 concentration was higher in the OZR compared to the LZR (p &lt; 0.05) and was attenuated in the OZR-WRR (p &lt; 0.05). Aortic expression of eNOS and COX-2 were downregulated in the OZR-WRR (p &lt; 0.05). In conclusion, WRR restores the impaired vascular tone of the OZR by enhancing Phe-induced vasoconstriction and attenuating Ach-induced vasorelaxation.

Abstract 122: Cardiac Hypertrophy and Vascular Function are Directly Proportional to Mitochondrial Variations in Intrinsic Exercise Capacity

Exercise is a primary lifestyle intervention to control cardiovascular diseases such as hypertension. Exercise capacity and its health benefits are inheritable. However, the mechanisms by which exercise capacity differences influence overall health state remain unclear. We first hypothesized that low and high intrinsic exercise capacity lead to distinct heart weight and vascular function. Animal model (male, 20-24-weeks old, n = 6): heterogeneous stock selectively bred for over 20 years to produce low-capacity running rats (LCR) and high-capacity running rats (HCR), and an intrinsic control, high response trainer rats (HRT, control). Statistics: t-test: *p&lt;0.05. Left ventricular (LV) weight was normalized by tibia length. We observed that HCR presented with LV hypertrophy compared to LCR and control (Control: 0.0155 ± 0.0006 vs. HCR: 0.0192 ± 0.0007* vs. LCR: 0.0164 ± 0.0004). Performing wire myography with 3 rd -order mesenteric resistance arteries (MRA), we observed that LCR MRA had an impaired maximum relaxation response (E max ) to acetylcholine (ACh) compared to HCR and control (Control: 94.99 ± 1.79 vs. HCR: 93.49 ± 12.17 vs. LCR: 72.95 ± 12.40*). Next, we questioned if these differences were the result of mitochondria. We used two reciprocal conplastic strains created by utilizing maternal mitochondrial inheritance (male, 30-36-weeks old, n = 4): LCR nuclear genome with HCR mitochondria (LCR.HCR mt ) and the reciprocal HCR.LCR mt . We observed that LCR.HCR mt presented with LV hypertrophy compared to LCR (0.0195 ± 0.0002* vs 0.0164 ± 0.0004) while HCR.LCR mt abolished HCR LV hypertrophy (0.0151 ± 0.0002* vs. 0.0192 ± 0.0007). Interestingly, in a separate group of animals (n = 4 - 5), LCR had higher mean arterial pressure (MAP) compared to HCR, and LCR.HCR mt had reduced MAP compared to LCR (LCR: 119 ± 2 vs. LCR.HCR mt : 111 ± 1*). No changes were observed in HCR.LCR mt compared to HCR. Further, LCR.HCR mt MRA presented with improved E max to ACh compared to LCR MRA (LCR: 72 ± 12 vs. LCR.HCR mt : 93 ± 2*) while no changes were observed in HCR.LCR mt MRA compared to HCR MRA. Overall, we conclude that intrinsic exercise capacity is directly proportional to cardiovascular effects. Similar to the nuclear genome, mitochondria are the key to this hereditary predisposition.

Effect of Extracts of the Aerial Parts and Roots from Four Ferulago Species on Erectile Dysfunction in Rats with Streptozotocin-Induced Diabetes.

The extracts of Ferulago species are used as aphrodisiacs in Turkey and so we aimed to demonstrate in vivo and in vitro the relaxant effect of four Ferulago species' extracts on the corpus cavernosum (CC). A total of 30 adult male Sprague Dawley rats were divided into control and diabetic groups. Diabetes was induced by a single intraperitoneal injection of 40 mg/kg streptozotocin. In vivo erectile responses were obtained by stimulation of the cavernosal nerves and repeated after intracavernosal injection of extracts in rats, and the data were expressed as intracavernosal pressure (ICP)/mean arterial pressure and total ICP. The relaxant and contractile responses of CC strips were analyzed in the presence or absence of extracts. The extracts were active in both control and diabetic rats. The extract-induced maximum relaxation responses (especially of methanol extract of the root of Ferulago bracteata) (98.30±2.6%) were decreased after incubation with L-NAME (44.8±1.8). ODQ, a soluble guanylate cyclase inhibitor, inhibited 77% of extract-induced maximum relaxation in the CC from the control rats. These species can be utilized in erectile dysfunction and may be an herbal alternative to synthetic drugs.

PO-01-064 Testosterone relaxes human corpus cavernosum tissue of patients with erectile dysfunction

To investigate the effect of testosterone (T) on relaxation of human corpus cavernosum tissue. Human corpus cavernosum tissue samples were obtained after consent from men undergoing penile prosthesis implantation (n=46) for organ bath experiments. Tissue strips were precontracted with phenylephrine after which increasing doses of T, DHT, and androstenedione were added. Blockade of androgen receptor as well as hydrogen sulfide and nitric oxide biosynthesis was studied. Fifteen minutes after addition of the different compounds, tissue relaxation was measured. Results are expressed as mean+/-SD. Statistical analysis was performed using unpaired t-test. T relaxed precontracted tissues with 70.7+/-16.9 % (p = 0.003) at 300 microM compared to DMSO vehicle only. Addition of 300 microM of androstenedione (p = 0.17) and DHT (p=0.09) resulted in no significant difference in relaxation compared to T. Maximum relaxation response to T were not inhibited by N (G)-nitro-L-arginine methyl ester (p = 0.16), beta-cyanoalanine (p = 0.16), propargylglycine (p=0.34) and glibenclamide (p=0.73). The addition of 350 nanoM flutamide (androgen receptor inhibitor) did not alter the effect of T on tissue relaxation with 80.2 +/- 26.5% of relaxation compared to T alone (p = 0.25).

Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Cullin3 (Cul3) is a critical subunit of the Cul3‐Ring‐Ligase (CRL3) ubiquitin ligase complex that targets proteins for proteasomal degradation. Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9, termed Cul3Δ9) cause hypertension in humans. Evidence suggests that the hypertension in these subjects is likely to be caused by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with Tek‐CREERT2 mice specifically expressing tamoxifen‐inducible Cre‐recombinase in the endothelium. The resultant mice (E‐Cul3Δ9) exhibited impaired endothelial‐dependent relaxation in carotid artery (maximal relaxation in response to 30 μM acetylcholine, 69% vs 84% in littermate control mice, n=5, p&lt;0.05, two‐way ANOVA repeated measurements) and in the cerebral basilar artery (maximal relaxation in response to 100 μM acetylcholine, 41% vs 77% in littermate control mice, n=5, p&lt;0.01). No difference in smooth muscle function was observed. Moreover, E‐Cul3Δ9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak systolic BP, 138±3 mmHg n=7 vs 122±4 mmHg n=5, p&lt;0.01). However no difference was seen in daytime pressure. Preliminary data indicated that treatment with nitric oxide synthase (NOS) inhibitor Nω‐Nitro‐L‐arginine methyl ester markedly elevated nocturnal peak SBP in control mice (133±5 mmHg, n=3) but not in E‐Cul3Δ9 mice (143±3 mmHg, n=3), suggesting that NOS activity is low in E‐Cul3Δ9 mice. E‐Cul3Δ9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s n=5 vs control mice 2.7±0.1 m/s n=7, p&lt;0.01). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3Δ9 construct and Cul3Δ9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which negatively regulates eNOS phosphorylation, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin‐3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.Support or Funding InformationDr. Jing Wu is supported by an American Heart Association Postdoctoral Fellowship (17POST33660685). This project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P189: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause hypertension in humans. The hypertension phenotype is unlikely to be driven by renal tubular mechanisms, as kidney-specific deletion of Cullin3 (Cul3) in mice results in hypotension, not hypertension. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3α9 mice with Tek-CRE ERT2 mice specifically expressing tamoxifen-inducible Cre-recombinase in the endothelium. The resultant mice (E-Cul3α9) exhibited impaired endothelial-dependent relaxation in the basilar artery (maximal relaxation in response to 30 μM acetylcholine, 45% vs 85% in control mice) and carotid artery. No difference in smooth muscle function was observed. Moreover, E-Cul3α9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak BP, 141±3 mmHg vs 122±3 mmHg). However no difference was seen in daytime pressure. E-Cul3α9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s vs 2.7±0.1 m/s). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3α9 construct and Cul3α9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro . Cul3α9 acted in a dominant negative manner by interfering with expression and function of wild type Cul3, leading to impaired turnover of a Cul3 substrate protein phosphatase 2A, marked reduction in phosphorylated eNOS, and decreased nitric oxide production. Treatment with a selective PP2A inhibitor Okadaic Acid (1 nM) rescued Cul3α9-induced impairment of eNOS activity. These data define a novel pathway involving Cullin-3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3α9 partially phenocopies the hypertension observed in Cul3α9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

‐LCZ696, the First‐in‐Class Angiotensin Receptor Neprilysin Inhibitor, Improves Vascular Reactivity in Hypertensive Rats in the Setting of Heart Failure ‐

BackgroundLCZ696 combines an angiotensin receptor blocker (ARB) with a neprilysin inhibitor (NEPi). This inhibits RAAS over activity while simultaneously augmenting the natriuretic peptide system. LCZ696 has been shown to improve cardiovascular function in the setting of heart failure, however, its effects on vascular function have not been defined.MethodsMale spontaneously hypertensive rats (SHRs) at 18–20 weeks of age were subjected to 45 minutes of transient LAD coronary artery ligation followed by reperfusion for 8 weeks to induce heart failure with reduced ejection fraction. At 4 weeks post‐reperfusion animals received daily oral therapy of either vehicle (VEH, n=14) or 68 mg/kg LCZ696 (LCZ, n=14). Myocardial infarct size as measured by plasma troponin levels at 4 hours post‐reperfusion were similar between the study groups. After 4 weeks of therapy, thoracic aorta was collected and evaluated for vascular reactivity in a tissue chamber apparatus connected to force transducers. Vasoreactivity was assessed for the endothelium‐dependent vasodilator acetylcholine (ACh, 0.1nM–10mM) and the direct smooth muscle vasodilator sodium nitroprusside (SNP, 0.1nM–10 mM). In addition, we also studied the vasoconstriction response to the endothelial nitric oxide synthase (eNOS) inhibitor N(omega)‐nitro‐L‐arginine methyl ester (L‐NAME, 30mM). Effects on heart function were also determined via 2‐D echocardiography and measurements of left ventricle (LV) pressures.ResultsLCZ improved LV function with a significant reduction in LV end‐diastolic pressure (3.31 ± 0.39 mmHg vs 7.86 ± 1.10mmHg; p&lt;0.01). LCZ also attenuated the decrease in ejection fraction at 8 weeks post reperfusion (−6.1 ± 2.5% vs −14.3 ± 3.6%; p=0.06). LCZ treatment significantly improved vascular reactivity in the dose response relaxation curve for both ACh and SNP. There was a significant improvement in maximal relaxation in response to ACh (76.1 ± 3.6% vs 52.9 ± 3.6%; p&lt;0.0001) as well as ACh EC50 (11.0 ± 4.0 nM vs 97.3 ± 9.8 nM, p&lt;0.0001) suggesting augmented endothelium mediated vasorelaxation. Similar changes were seen in SNP maximal relaxation (90.9 ± 3.5% vs 69.1 ± 5.9%; p&lt;0.01) and SNP EC50 (26.8 ± 4.4nM vs 125.7 ± 13.4nM, p&lt;0.0001) suggesting improvements in vascular smooth muscle function. Lastly, there was an increased contraction in response to administration of L‐NAME following maximal relaxation by ACh suggesting the potential role of nitric oxide in the improvements seen in vascular reactivity (60 ± 6mg vs 40 ± 6mg, p=0.06).ConclusionLCZ696 significantly improved LV function during heart failure in the setting of hypertension with concomitant improvements in vascular reactivity. These improvements in vascular reactivity are likely a result of increased activity of eNOS and increased bioavailability of nitric oxide (NO). Studies are currently underway to evaluate eNOS function and NO levels following LCZ administration in heart failure.