ACh-induced Relaxation Research Articles

Abstract P351: Obesity in Early Life Leads to Endothelial and Erectile Dysfunction in Young Adult Rats.

Background: Erectile dysfunction (ED) is largely associated with obesity, however, the consequences of early life obesity for ED in young adulthood are unknown. We hypothesize that obesity induced by preweaning overfeeding impairs vascular and erectile function and affects blood pressure in young adult male rats (5-month-old). Methods: At postnatal day (PND) 1, Wistar rats were divided into a normal litter (NL, 5 female + 5 male pups/dam) or small litter (SL, 2 male + 1 female pup/dam) during lactation. After weaning, SL pups had increased body weight. Next, rats from both groups returned to normal size colonies (4-5 rats per cage) and were fed standard chow. At PND160, we evaluated body weight and fat pad deposition, mean arterial pressure (MAP) by tail-cuff, and pudendal artery (PA) and corpus cavernosum (CC) reactivity. Concentration-response curves to phenylephrine (PE) or acetylcholine (ACh) were performed in both preparations. In the CC, contraction and relaxation to electrical field stimulation (EFS) were obtained. Concentration-response curves were analyzed using non-linear regression analysis. RNA sequencing (RNAseq) analyses were performed in both PA and CC. Data were analyzed by Student’s t-test and presented as mean ± S.E.M. Statistical significance was set at p<0.05. Results: At PND160 no differences in body weight were observed in SL (594±14g) compared to NL (559±13g), however, there was an increase in fat deposition in SL (retroperitoneal fat: NL 7.47±0.69g vs SL 11.25±1.23g; perigonadal fat: NL 10.05±0.55g vs SL 13.24±1.16g). MAP was higher in SL (120.5±1.20 mmHg vs 114.9±0.58 NL). In the PA, ACh-induced relaxation was reduced in SL (Emax: 28±6%) vs NL (Emax: 68±5.5%), with no changes in the contraction to PE. In the CC, EFS-induced relaxation was significantly decreased in SL rats compared to NL rats (16 Hz: NL 3.06 ± 0.28% vs SL 2.39 ± 0.53%). RNAseq revealed that, in PA from SL rats, 289 and 224 genes were down and upregulated, respectively, while in the CC from SL rats, 115 genes were downregulated, and 110 genes were upregulated. Among them, hcn2 gene (which encodes HCN2, the hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2) was strongly downregulated in both PA and CC, and this may affect mechanotransduction. Conclusion: Our data shows that early-life obesity causes long-lasting vascular and cavernous damage that might be associated with endothelial and erectile dysfunction in young adulthood.

Low Calcium-High Magnesium Krebs-Henseleit Solution Combined with Adenosine and Lidocaine Improved Rat Aortic Function and Structure Following Cold Preservation.

Background and objectives: The main problem of vascular preservation is the maintenance of vessel graft quality and function following extended storage. Conventional preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution, Phosphate-Buffer Solution (PBS), or sodium chloride 0.9% has been shown to be inadequate in preserving vascular physiological function after 3 days of cold storage. This study aimed to evaluate whether adenosine and lidocaine (AL) in a modified Krebs-Henseleit (KH) solution can preserve the function and histological structure of rat aortic rings after 6 days. Materials and Methods: Thirty-five aortic rings from male Wistar rats (200-300 g) were harvested and immediately immersed in one of the assigned cold preservation solutions: standard KH, modified KH (mod KH) with lower calcium (Ca2+) and higher magnesium content (Mg2+) with or without adenosine and lidocaine (mod KH-AL), and modified KH with AL, insulin, and melatonin (Mod KH-ALMI). The contraction and relaxation function of the aortic rings were examined using an isometric force transducer after 6 days of cold preservation. Hematoxylin and eosin staining were used to analyze the rings' histological structure. Results: Vascular contraction and relaxation functions were severely affected after a 6-day cold storage period in standard KH. Modifying the KH solution by reducing the Ca2+ and increasing the Mg2+ levels greatly recovered the vessel functions. The addition of AL or ALMI to the modified KH did not further recover vascular contractility. However, only the addition of AL to the modified KH increased the ACh-induced relaxation at 6 days when compared to the conventional KH, suggesting that endothelium preservation is improved. From histological analysis, it was found that the addition of AL but not ALMI further improved the endothelial lining and the structure of the elastic membrane layers of the preserved vessels after 6 days of cold preservation. Conclusions: The addition of AL to low calcium-high magnesium KH solution significantly enhanced endothelial preservation and improved endothelial-induced relaxation of preserved vessels after 6 days of cold storage.

Levamisole Impairs Vascular Function by Blocking α-Adrenergic Receptors and Reducing NO Bioavailability in Rabbit Renal Artery

Levamisole is an anthelmintic drug restricted to veterinary use but is currently detected as the most widely used cocaine cutting agent in European countries. Levamisole-adulterated cocaine has been linked to acute kidney injury, marked by a decrease in glomerular filtration rate, which involves reduced renal blood flow, but data on the alteration of renovascular response produced by levamisole are scarce. Renal arteries were isolated from healthy rabbits and used for isometric tension recording in organ baths and protein analysis. We provide evidence that depending on its concentration, levamisole modulates renovascular tone by acting as a non-selective α-adrenergic receptor blocker and down-regulates α1-adrenoceptor expression. Furthermore, levamisole impairs the endothelium-dependent relaxation induced by acetylcholine without modifying endothelial nitric oxide synthase (eNOS) expression. However, exposure to superoxide dismutase (SOD) partially prevents the impairment of ACh-induced relaxation by levamisole. This response is consistent with a down-regulation of SOD1 and an up-regulation of NADPH oxidase 4 (Nox4), suggesting that endothelial NO loss is due to increased local oxidative stress. Our findings demonstrate that levamisole can interfere with renal blood flow and the coordinated response to a vasodilator stimulus, which could worsen the deleterious consequences of cocaine use.Graphical EFS electric field stimulation, NA noradrenaline, AR adrenergic receptor, IP3 inositol 1, 4, 5-trisphosphate, cAMP cyclic adenosine monophosphate, mAChR muscarinic acetylcholine receptor, eNOS endothelial nitric oxide synthase, sGC soluble guanylyl cyclase, SOD superoxide dismutase, NOX4 NAPH oxidase 4

Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, andEDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 μM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 μM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

Oral curcumin phytosome supplementation improves anthropometric measures of adiposity and enhances endothelial function in rats on a high-fat-diet regimen

Objectives: Curcumin has a protective role in endothelial function and nitric oxide (NO) production in animal models of different diseases; however, the role of curcumin on aortic reactivity in rats placed on a high-fat diet (HFD) remains unclear. This study aims to determine whether oral curcumin phytosome supplementation can reduce adiposity and enhance endothelial function. Materials and Methods: Rats were assigned to one of three groups: normal diet (ND), HFD for 20 weeks, and HFD supplemented with curcumin phytosome (HFD + Curcumin). Anthropometric measures were recorded weekly for the three groups, until the end of the feeding regimen. After 20 weeks of feeding on HFD, myographic investigations were conducted on thoracic aortic rings dissected from HFD and HFD + Curcumin rats. The response to high potassium chloride (KCl), incremental doses of phenylephrine (Phe) before and after L-NAME treatment, acetylcholine (ACh), or sodium nitroprusside (SNP), was evaluated. ACh-induced relaxation was also assessed in HFD + Curcumin rats, after preincubation with chromium III-mesoporhyrin. Results: HFD rats exhibited increased adiposity measures, some of which were negatively correlated with vasorelaxation response to ACh. HFD + Curcumin rats had reduced anthropometric measures, compared to HFD rats. Aortic rings from HFD and HFD + Curcumin rats exhibited comparable contractile responses to KCl and Phe. The difference in contractile response to Phe before and after L-NAME incubation was greater for HFD + Curcumin rats. ACh induced greater vasorelaxant responses in HFD + Curcumin rats. There was no group difference in the relaxant response to SNP. In HD + Curcumin rats, chromium III mesoporphyrin significantly reduced ACh-induced relaxations. Conclusion: Oral curcumin phytosome supplementation could reduce adiposity in rats placed on an HFD and may have enhanced basal and stimulated NO release from the endothelium, and heme oxygenase-1 may partly mediate this curcumin protective role. This study provides evidence that this curcumin formulation, taken as a daily supplement, may be effective in providing some protection against adiposity-associated adverse cardiovascular disorders.

Influence of rutin and its combination with metformin on vascular functions in type 1 diabetes

The present work examined the effect of oral administration of rutin and its combination with metformin, an antidiabetic drug on blood glucose, total cholesterol and triglycerides level and vascular function in streptozotocin (STZ) -induced diabetic rats. Male Sprague Dawley rats were rendered diabetic by a single intraperitoneal injection of STZ (50 mg/kg). Rutin and metformin were orally administered to diabetic rats at a dose of 100 mg/kg and 300 mg/kg body weight/day, respectively, for 4 weeks. Plasma analysis was conducted to determine changes in the plasma glucose and lipid levels. Rat aortic ring reactivity in response to endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) relaxants, and to the α1-adrenergic agonist phenylephrine (PE) were recorded. Histology of pancreas, liver and kidney were evaluated. In results, rutin and metformin alone and in combination has led to significant improvements in blood glucose, cholesterol and triglyceride levels compared to diabetic group. Diabetic aortic rings showed significantly greater contraction in response to PE, and less relaxation in response to ACh and SNP. Treatment with rutin and metformin in combination significantly reduced PE-induced contraction and increased ACh-induced and SNP-induced relaxation in diabetes when compared to rutin or metformin alone. Significant histological improvements were seen with combination therapy. In conclusion, rutin and metformin combination therapy has the most potentiality for restoring blood glucose and lipid level as well as vascular function.

Endothelial Dysfunction in Superior Mesenteric Arteries Isolated from Adenine-Induced Renal Failure in Model Rats.

Endothelial dysfunction-a hallmark of chronic kidney disease (CKD)-is one of the major risk factors for cardiovascular diseases (CVD). Imbalances in endothelium-derived relaxing factors (EDRFs) and contracting factors (EDCFs) specific to endothelial dysfunction in CKD are yet to be studied. Therefore, using adenine-treated rats-a CKD rat model-we investigated the responsiveness of superior mesenteric artery (SMA) endothelium to acetylcholine (ACh) stimulation under different experimental conditions. Nine-week-old male Wistar rats were treated daily with adenine (200 and 600 mg/kg body weight) by oral gavage, for 10 d; the two groups were named adenine-200 (200 mg/kg body weight) and adenine-600 (600 mg/kg body weight). The systolic blood pressure (measured 1-, 8-, and 15 d post-treatment) was significantly increased in the adenine-600 group compared with that in the control group; whereas that in the adenine-200 group showed only a slight increase. Moreover, in the adenine-600 group the serum creatinine and blood urea nitrogen (BUN) levels (measured at 18 d post-treatment) were significantly elevated when compared with those in control or adenine-200 groups. The ACh-mediated relaxation was slightly reduced in the adenine-200 group. The ACh- and sodium nitroprusside (SNP)-mediated relaxations were impaired in the adenine-600 group. Although no ACh-mediated contraction was observed in the presence of a nitric oxide (NO) synthase inhibitor, ACh-induced endothelium-derived hyperpolarizing factor-mediated relaxation was largely impaired in the adenine-600 mg/kg group. This study revealed that in the SMA of adenine-induced CKD model rats, EDCF signaling remained unaltered while the NO and EDHF signaling were impaired.

(219) AUTOLOGOUS PLATELET-RICH PLASMA IMPROVES ENDOTHELIAL AND TADALAFIL-INDUCED RELAXATIONS IN CORPUS CAVERNOSUM FROM PATIENTS WITH ERECTILE DYSFUNCTION

Abstract Objectives Available clinical evidence suggests the potential therapeutic use of intracavernosal injections of platelet-rich plasma (PRP) to treat erectile dysfunction (ED). However, the effects of PRP on human cavernosal function have not been elucidated. Our aim was to evaluate the effects of acute exposure to autologous PRP on relaxant capacity of human corpus cavernosum (HCC) from patients with ED. Methods HCC strips were obtained from four ED patients undergoing penile prosthesis insertion and giving informed consent. Previous to intervention, PRP and platelet-poor plasma (PPP) were obtained from patients by apheresis and stored at -20°C until functional procedures. Endothelial relaxation to acetylcholine (ACh) was evaluated before and after 60 min exposure to PRP or PPP (1% v/v) in organ chamber. Relaxant response to the PDE5 inhibitor, tadalafil, was also evaluated in PRP or PPP (1%) treated HCC from the same patient. Results Average age of patients was 67-years-old (range 61-71) while hypertension and smoking history was present in 3/4, diabetes in 2/4, and obesity in 1/4. Mean platelet content in PRP was 1,096x10 9 /L (range 786 to 1,284 x109/L) while negligible platelet content was obtained in any PPP. Exposure to autologous PRP but not PPP significantly enhanced (p<0.0001 by two-factors ANOVA) ACh-induced endothelial relaxations (pEC50 5.75±0.65 vs. 7.16±0.50; Emax 57.3±14.8% vs. 88.5±2.0%; before and after 1%PRP, respectively). Additionally, HCC treated with autologous PRP displayed augmented relaxant responses to tadalafil (p=0.0018 by two-factors ANOVA) with respect to those treated with PPP (pEC50 6.08±0.5 vs. 6.82±0.51 for PPP and PRP, respectively). Conclusions Acute exposure to autologous PRP improves endothelial function and functional response to PDE5 inhibition. These results support the reported clinical improvement of erectile function in patients with ED and suggest that PRP injection might induce a functional effect on cavernosal tissue in addition to the claimed restorative/regenerative effect. Conflicts of Interest None

Chronic Unpredictable Stress Impairs Vascular Reactivity in Male and Female Mice

Background: Chronic stress is known to induce vascular inflammation causing endothelial and vascular smooth muscle dysfunction. Changes in the hypothalamic-pituitary-adrenal axis lead to increased sympathetic and decreased parasympathetic drive, causing the release of inflammatory cytokines in the vasculature, by mechanisms that are unclear. Hypothesis: We hypothesize that chronic unpredictable stress (CUS) increases reactive oxygen species and induces gasdermin pore formation and voltage-dependent anion channel (VDAC) oligomerization in the membrane of the mitochondria, which releases mitochondrial DNA (mtDNA) to the cytosol and induces TNF-α, IL-1β and IL-18 production through activation of the NLRP-3 inflammasome pathway. Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of chronic unpredictable stress (CUS) or handling (CTL). Systolic blood pressure (SBP) was measured at the end of the stress period by tail cuff plethysmography. Vascular function using myographs was assessed on isolated mesenteric resistance arteries (MRAs), pudendal artery (PA) and aorta from male and female CUS and CTL mice. The arterial rings were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration-response curves to phenylephrine (PE; 1nM-30μM) and acetylcholine (ACh; 1nM-30μM) were performed. Expression of gasderminD was measured in the MRA by western blot. Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data were analyzed with GraphPad Prism 9.2.0. Results: SBP was increased by 12.1±4.9% in CUS mice. In CUS mice, compared to the CTL, the potency of ACh-induced relaxation was decreased in the MRA (pEC50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC50 CUS 6.65±0.07 vs CTL 6.31±0.09; p=0.008) but not in the aorta of male mice. In females, the potency to ACh was decreased only in the MRA (pEC50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). There were no differences in the contractile responses to PE between controls and mice exposed to CUS in the MRA and PA of both males and females, while in the aorta of CUS males, there was a significant reduction in maximal response (EMAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23mN; p=0.007). The expression of gasderminD was increased in MRA of female mice only. Conclusion: Our results show that, in both sexes, CUS increased SBP and induced vascular dysfunction observed by impaired reactivity to ACh and PE, with increased expression of gasderminD in MRA of female mice only. These results suggest that the vascular adaptations to CUS are sex-dependent. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. 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.

Serum from Men with the Severe Form of COVID-19 Impairs the Nitric Oxide Signaling Pathway in Isolated Corpus Cavernosum from Mice: An In Vitro Study

Introduction. Erectile dysfunction (ED) is characterized by the inability to achieve and/or maintain an erection during sexual activity. Vascular-related diseases such as obesity, diabetes, aging, and hypertension are known risk factors that increase the prevalence of ED. The viral infection caused by SARS-CoV-2, the etiological agent of COVID-19, can evolve into mild to severe forms. Patients with comorbidities such as diabetes and obesity present a more severe state of the disease. The cytokine storm and reactive oxygen species associated with COVID-19 can lead to progressive systemic inflammation and vascular dysfunction. This study is aimed at assessing the in vitro effects of serum obtained from unvaccinated men who had the severe form of COVID-19 in isolated corpus cavernosum (CC) from healthy mice. Methods. Concentration-response curves to endothelium-dependent and endothelium-independent substances were carried out in isolated CC from mice after being incubated (3 hours) with serum obtained from patients with and without (control group) the severe form of COVID-19. qRT-PCR and western blotting were also carried out. Results. The relaxing responses induced by acetylcholine (ACh), a nitric oxide donor (sodium nitroprusside), soluble guanylate cyclase (sGC) stimulator (BAY 63-2521), and phosphodiesterase type 5 (PDE5) inhibitor (tadalafil) were significantly reduced in CC incubated with COVID-19 serum when compared with CC incubated with the control serum. Nonetheless, the relaxation induced by the sGC activator BAY 58-2667 was unaffected in CC stimulated with the COVID-19 serum. The coincubation of control or COVID-19 serum with a free radical scavenger (PEG-SOD; 150 UI/mL, 3 hours) significantly improved the relaxation induced by ACh. On the other hand, catalase did not improve ACh-induced relaxation in CC incubated with the sera. The gene expression for PDE5 and NADPH oxidase type 4 was increased in CC stimulated with COVID-19 serum in comparison to tissues stimulated with the control serum. The protein expression for sGC subunits was similar in both groups. Conclusion. Serum obtained from unvaccinated men who presented the severe form of COVID-19 impaired the relaxation induced by cyclic guanosine monophosphate-accumulating substances in CC from mice.

The Beneficial Effect of Swimming Training Associated with Quercetin Administration on the Endothelial Nitric Oxide-Dependent Relaxation in the Aorta of Rats with Experimentally Induced Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is related to the vascular oxidative and nitrosative stress, the trigger for atherosclerosis and cardiovascular complications. The effects of moderate swimming training associated with quercetin oral administration were evaluated in aorta of rats with experimentally induced type 1 diabetes mellitus (T1DM), by analysing the nitric oxide-endothelial dependent relaxation (NO-EDR). T1DM rats received daily quercetin 30 mg/kg and followed the protocol of 5-weeks swimming exercise (30 min/day; 5 days/week). Aorta relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were measured at the end of the experiment. Ach-induced endothelial dependent relaxation was significantly decreased in phenylephrine (PE) pre-contracted aorta of diabetic rats. Swimming exercise with quercetin administration preserved Ach-induced EDR but did not have any impact on SNP-induced endothelium-independent relaxation in the diabetic aorta. These findings suggest that quercetin administration associated with moderate swimming exercise could improve the endothelial NO-dependent relaxation in the aorta of rats with experimentally induced type 1 diabetes mellitus, showing that this therapeutical combination may improve and even prevent the vascular complications that occur in diabetic patients.

PS-BPB04-8: ANGIOTENSIN II FORMULATIONS AND THEIR IMPACT ON CARDAIC, VASCULAR AND INFLAMMATORY MECHANISMS OF HYPERTENSION

Angiotensin II (AngII) is a major effector of the renin-angiotensin system, with a critical role in BP regulation of vascular function, and end-organ damage. AngII infusion, using osmotic minipumps, is the most common animal model of hypertension, recapitulating key vascular and cardiac features of human hypertension. While different formulations of AngII are available, their impact on the key outcomes of the model remains unknown. Accordingly, we compared two most commonly used AngII formulations, and their impact on the key cardiovascular endpoints in a mouse model of hypertension. Male 12-week old C57BL/6J mice (n = 11–14) underwent Sham, AngII[Val5] or AngII[Ile5] (490 ng/min/kg) treatment for two weeks, using osmotic minipumps. Blood pressure was measured by tail-cuff, vascular function by wire myography, remodelling by picrosirius red staining, mRNA expression using real-time PCR and perivascular (PVAT) inflammation by flow cytometry. Data were analyzed using one-way or RM-two-way ANOVA. Both AngII formulations caused a significant BP elevation (p < 0.001) in comparison to sham animals, although AngII[Val5] had a more pronounced pressure effect than AngII[Ile5] (171 ± 4 vs 156 ± 6 mmHg, p = 0.03). Similarly, endothelium-dependent relaxation of resistance arteries and aorta was significantly impaired by AngII infusion (p < 0.001), however, a maximum Ach-induced relaxation was significantly lower in AngII[Val5] than in AngII[Ile5]. Both, AngII formulations increased intima-media thickness of the aorta (p < 0.0001), although the effect of AngII[Val5] was ∼2-fold higher than AngII[Ile5] (p = 0.001). While animals infused with AngII[Val5] had enhanced vascular collagen deposition (p < 0.001), this effect was lesser in the AngII[Ile5] group (p = 0.02). The total number of leukocytes infiltrating PVAT was induced by both AngII formulations, however, this effect was less pronounced in AngII[Ile5] (1002 ± 376 vs. 753 ± 242 cell/mg, p = 0.03). The same effect was found among CD11b+ and CD11c+ cells. While a significant increase of CD3+ cells was observed in both AngII formulations. While cardiac hypertrophy was evident in both AngII formulations this effect was significantly lower in AngII[Ile5] (p = 0.03). This was confirmed by cross-sectional area of cardiomyocytes. Histological analysis of heart sections showed a significant effect of both AngII formulation on cardiac fibrosis (p < 0.05). This effect was more pronounced upon AngII[Val5] infusion (p < 0.001) and was associated with higher expression of pro-fibrotic markers. AngII[Val5] formulation is a more efficient and consistent way to study key cardiovascular endpoints such as BP, inflammation, vascular and cardiac damage compared with AngII[Ile5]. Furthermore, the use of AngII[Val5] leads to a more pronounced phenotype and may be associated with reducing the number of animals needed in experiments.

Salusin-β, a TOR2A gene product, promotes proliferation, migration, fibrosis, and calcification of smooth muscle cells and accelerates the imbalance of vasomotor function and vascular remodeling in monocrotaline-induced pulmonary hypertensive rats.

Purpose: The hyper-proliferation, promoted migration, fibrosis, and calcification of pulmonary arterial smooth muscle cells (PASMCs) play critical roles in pulmonary artery (PA) continuous contraction and vascular remodeling, leading to elevated pulmonary arterial resistance and pulmonary hypertension (PH). In this study, we sought to ascertain the effects of a TOR2A gene product, salusin-β, on PASMCs’ proliferation, migration, fibrosis, calcification, and the imbalance of vasomotor function as well as pulmonary vascular remodeling in monocrotaline (MCT)-induced PH rats and their underlying mechanisms. Methods: Knockdown or overexpression of salusin-β in rats or PASMCs was performed through tail vein injection or cell transfection of virus. The right ventricular systolic pressure (RVSP) of the rat was measured by right ventricle catheterization. Sodium nitroprusside (SNP) or acetylcholine (ACh)-induced dose-dependent relaxation was used to evaluate the vasodilatation function. Primary PASMCs were isolated from the PAs of control and PH rats. Results: The salusin-β protein expressions were significantly increased in PAs and PASMCs isolated from PH rats compared with control rats. Knockdown of salusin-β in rats decreased high K+ solution-induced contraction, RVSP and RV hypertrophy index, improved SNP or ACh-induced vascular relaxation of PAs, and relieved vascular remodeling and calcification of PAs from PH rats. Silencing salusin-β in PASMCs isolated from PH rats alleviated the proliferation, migration, fibrosis, and calcification, as well as the NAD(P)H oxidase activity and reactive oxygen species (ROS) level. Overexpression of salusin-β exerted the opposite effects on vasomotor function and vascular remodeling, and PASMCs proliferation, migration, fibrosis and calcification. Conclusion: Increased salusin-β activity in PAs from PH rats contributes to PASMCs proliferation, migration, fibrosis, and calcification, leading to the imbalance of vascular contraction and relaxation and vascular remodeling through stimulating the production of NAD(P)H oxidase derived ROS.

Endothelial dysfunction of the mesenteric arteries of rats in the early obesity induced by high-fat diet

Background: Disturbance of the morphological and functional properties of the vascular bed in obesity are a serious clinical problem. Basis to their development is endothelial dysfunction. The developed models of obesity in animals using various diets indicate a change in vascular reactivity, however, questions about the stage at which this occurs and what mechanisms are involved in this process remain open, while they are decisive for choosing the correct tactics for correcting dysfunctions.Aim: The aim of the present study is to determine the changes in acetylcholine (ACh)-induced vasodilation of isolated arteries from rats after six weeks of administration of a high-fat diet (HFD).Materials and methods: The experiments were performed on Sprague-Dawley males, which at the age of 8 weeks were divided into 2 experimental groups that were treated for the next 6 weeks in the following manner: 1 - control) with standard dry food; 2 - a group fed with a HFD, the total amount of fat in which was 50%. At finish of the diet, the degree of obesity, biochemical parameters in the blood, and blood pressure were measured. Intravital microscopy of the rat mesentery with video recording was used to study the reactivity of the vessels. The contractile and relaxant responses of the vessels were determined by changes in their diameter.Results: The rats after treatment with the HFD (n=15) had higher body weight and amount of visceral fat, significantly increased blood triglycerides, moderate increases in glucose level in blood and systolic pressure compared with the control (n=15). Relaxation responses of mesenteric arteries, having a diameter of 140 to 300 μm in PSS, were recorded after precontraction by phenylephrine. A decrease in ACh-induced vasorelaxation was obtained, which manifests itself before the development of significant changes in carbohydrate metabolism. Incubation of drugs with the inhibitor of endothelial NO synthase L-NAME led to a pronounced weakening of relaxation in animals on a standard diet, and had little effect on vasodilation in the arteries of rats with the HFD. Vasodilation induced by the administration of sodium nitroprusside (NO donor) did not differ significantly in control and experimental animals, which indicates that the sensitivity of vascular smooth muscle to NO remained practically unchanged. ACh-induced relaxation of arteries in dietary rats did not change when the cyclooxygenase pathway was blocked by diclofenac.Conclusion: Functional changes in the contractile activity of the mesenteric arteries, manifested in the form of a decrease in ACh-induced vasorelaxation, occur after treatment with the HFD when animals had an early stage of obesity development before the onset of pronounced disorders of carbohydrate metabolism. This decrease is mainly due to the disruption of the NO-dependent mechanism underlying ACh-induced relaxation in the norm.

Does Dietary Methionine Alter Perivascular Adipose Tissue Function?

Perivascular adipose tissue (PVAT) is an adipose depot surrounding blood vessels and is a paracrine regulator of vascular tone. Obesity is associated with the loss of PVAT‐mediated anti‐contractile properties, thus contributing to obesity‐associated hypertension. Hydrogen sulfide (H2S) is a gaseous signaling molecule that is produced in the vasculature and is important for regulating vascular tone. In PVAT, cystathionine gamma lyase (Cse) is the primary source of H2S which acts as an important adipose‐derived relaxing factor. Dietary methionine restriction recapitulates many of the metabolic effects of caloric restriction and leads to preferential expression of activating transcription factor 4 (ATF4) which facilitates Cse transcription. We hypothesized that dietary methionine restriction will prevent high fat diet‐induced loss of PVAT anti‐contractile properties, through activation of the integrated stress response (ISR) and increased ATF4‐mediated transcription of Cse. Male Wistar rats were fed either a control (C, 10% calories from fat) or a high fat diet (HFD) (H, 60% calories from fat) that was either methionine replete (R, 0.86% methionine) or methionine depleted (D, 0.12% methionine) for 12 weeks such that there were four groups: CR, CD, HR, and HD. Dietary methionine restriction prevented body weight gain, while rats fed a methionine replete diet gained weight over the 12 week feeding period. There was no difference in weight gain between control or HFD fed rats. Arterial blood pressure, measured in conscious rats using tail cuff plethysmography, was not different between the four groups over the 12 week feeding period. After 12 weeks on diets, arterial contractility was assessed using wire myography in 2nd order mesenteric arteries (MAs) with and without attached PVAT. In our hands, the presence of PVAT was associated with reduced contraction to norepinephrine (NE, 10 µM) and greater relaxation to acetylcholine (ACh,10 µM) in MAs from all groups, and HFD was not associated with a loss of PVAT anti‐contractile properties. In the presence of PVAT, dietary methionine restriction increased relaxation to ACh in NE‐precontracted MAs, while HFD had no effect on ACh‐induced relaxation. In conclusion, in male Wistar rats, while HFD had no effect on weight gain, blood pressure, or PVAT modulation of MA contractility, dietary methionine restriction for 12 weeks had profound effects on body weight gain, no effect on arterial blood pressure, and a small effect on MA ACh‐induced relaxation in the presence of PVAT. Future studies include examining gene and protein expression of Cse and other ATF4 responsive genes using real time qPCR, Western blot analysis, and immunohistochemical staining of arterial cross‐sections. These studies will provide further insights into dietary interventions to minimize the cardiovascular co‐morbidities associated with obesity.

Endothelial connexin hemichannels as a novel endothelial regulatory mechanism of vasodilation

The vascular endothelium regulates vasomotor tone in arterioles through intercellular conduction of electrical signals by gap junctions. Endothelial vasodilation is mediated by nitric oxide (NO) and by a hyperpolarizing current initiated in the endothelium. Both signals are transmitted to smooth muscle cells evoking relaxation. Endothelial cells express connexin (Cx) 37,40,43. Cx hemichannels allow the exchange of ions and molecules between intracellular and extracellular compartments that may be associated with endothelial electrical activity. Whether or not Cx hemichannels regulate vasomotor tone is unknown. We evaluated the role of Cx‐hemichannels in the vasodilation process by measuring dye uptake in primary culture of endothelial cells and in isolated mouse mesenteric arterioles. We detected that both acetylcholine (ACh) and bradykinin (BK) increase dye permeability, which was inhibited by connexin mimetic peptides that block Cx43 hemichannels (Gap19). We determined that NO mediates Cx43 hemichannels activity inasmuch as blocking NOS inhibits hemichannel activity. Using biotin switch assay, we observed that ACh induced S‐nitrosylation only in Cx43 at cysteine 271. ACh and BK open endothelial Cx43 hemichannels activity and promote endothelial Ca2+entry from the extracellular space and lead to endothelium hyperpolarization signaling in isolated mesenteric arteries. Blocking Cx43‐hemichannels reduces calcium entry and hyperpolarization. To further assess the physiological role of Cx43 endothelial hemichannels activity, we cannulated isolated mesenteric arterioles and studied ACh‐induced arteriolar relaxation in the presence and absence of connexin mimetic peptides that block Cx43 hemichannels (Gap19) and Cx40 (40Gap 27, a connexin mimetic peptide). Gap19 attenuates ACh‐induced relaxation, but 40Gap27 fails to reduce relaxation. These results indicate that endothelial Cx43 hemichannels participate in arteriolar dilatation. In addition, we replaced cysteine 271 by serine to create a Cx43C271S mouse that failed to cause S‐nitrosylation of Cx43. We detected that this knockin mouse displayed hypertension compared to wild type mice, indicating that Cx43 hemichannels and S‐nitrosylated Cx43 play a role in vascular resistance. Our results suggest that Cx43 hemichannels are a potential therapeutic target in hypertension and vascular disease.

Comparison of del Nido and Histidine‐tryptophan‐ketoglutarate cardioplegic solutions on cardiac and endothelial function

ObjectivesThis study was designed to compare cardiac and endothelial protection of three clinically used cardioplegias del Nido (DNC), Histidine‐tryptophan‐ketoglutarate (HTK), and cold blood (BC) cardioplegia followed by HTK (BC+HTK) in ischemia/reperfusion(I/R)rat model.MethodsSixty male Wistar rats were subjected to either 120‐min global ischemia at 4°C followed by 90‐min reperfusion at 37°C or no I/R (Control) in Langendorff apparatus and were randomly allocated into five groups: Control, I/R, DNC, HTK, and BC+HTK. Hemodynamic parameters were continuously recorded. The endothelium‐dependent relaxation to acetylcholine (ACh) was measured in the left anterior descending artery thereafter in myograph. The protein expression of cardiac Troponin T (cTnT) and creatine Kinase MB (CKMB) were measured by western blotting.ResultsDuring reperfusion, HTK had higher LVSP whereas DNC had lower LVEDP, better LVDP, and best +dp/dtmax and ‐dp/dtmax than other two groups but the differences disappeared at the end of the reperfusion. HTK or BC+HTK preserves the ACh‐induced endothelium‐dependent relaxation better than DNC (Emax=48.2±8.0% in DNC vs. 75.0±8.0% in HTK, p<0.05; vs. 96.9±3.5% in BC+HTK, p<0.001). The protein level of cTnT and CKMB was downregulated in the three groups.ConclusionsDNC shows a better recovery in diastolic function, whereas HTK or BC+HTK exerts better protection in coronary endothelial function. All these three cardioplegias could ameliorate the myocardium injury at the protein level. These findings may suggest that searching for “perfect” cardioplegia is warranted.

Aortic Relaxation in Castrated Male Rats and Responses to Estrogen Treatment

Previous studies suggest that risk factors for cardiovascular diseases (CVD) have increased in the transgender population after they undergo cross‐sex hormone therapy (CSHT). Several studies, including ours in animal models as well as in humans, have shown that estrogen exerts direct beneficial effects on the vessel wall in healthy females. A growing body of evidence also suggests that androgen exhibits protective actions on the cardiovascular system. However, the effects of CSHT in cardiovascular physiology in males or females are less understood.The current study was, therefore, undertaken to evaluate the effects of estrogen treatment on aortic reactivity in castrated (CAS) male rats. Specifically, we investigated the effects of in vivo treatment with 17β‐estradiol (E2) on the aortic responses of CAS male as well as ovariectomized (OVX) female rats.Age‐matched CAS male and OVX female rats were implanted subcutaneously with either 17β‐estradiol (E2,1.5 mg) or a placebo‐containing pellet for 30 days. Endothelium‐dependent vasorelaxation (EDV) in response to acetylcholine (ACh, 10‐8 to 10‐5 M) in aorta precontracted with phenylephrine (PE, 2 µM) was measured before and after pretreatment with indomethacin (10 µM, cyclooxygenase inhibitor), followed by N‐nitro‐ L‐arginine methyl ester [L‐NAME, 200 µM, a nonselective nitric oxide synthase (NOS) inhibitor]. Endothelium‐independent vasorelaxation was assessed by measuring sodium nitroprusside (SNP)‐induced relaxation response in endothelium‐denuded rings in addition to the contractile response curves to PE before and after pretreatment with L‐NAME in aortic rings.EDV to ACh was significantly impaired in aortic rings from E2‐treated CAS rats compared to their placebo‐treated counterparts and E2‐treated OVX rats. The impairment of EDV to ACh in E2‐treated CAS rats was accompanied by a significant decrease in nitric oxide (NO) contribution to ACh‐induced relaxation in this group. The smooth muscle sensitivity to NO as measured by SNP relaxation response curve was not altered, whereas the responsiveness to PE was slightly enhanced in the aortas of E2‐treated CAS group compared to those in other experimental groups. Endothelium‐derived NO release during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was not different in the aortas of experimental groups.Overall, our data demonstrate that aortic function was altered in E2‐treated CAS male rats. Here, we provide the first evidence of impairment in aortic relaxation in E2‐treated CAS male rats, possibly via a significant decrease in the relative contribution of NO to the regulation of vascular relaxation response. Furthermore, the comparison of current data in the CAS male rats with our recently published data on the aortic reactivity in the intact males appears to rule out any effect of testosterone deprivation on the stimulated release of NO in male rat aortas. Additional studies will be needed to document the direction and magnitude of CSHT interaction with vasculatures.

Sarco/Endoplasmic Reticulum Ca2+ ATPase 2 Activator Ameliorates Endothelial Dysfunction; Insulin Resistance in Diabetic Mice.

Background: Sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic (db/db) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db/db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db/db mice. In isometric tension measurements of aortic rings from db/db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db/db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db/db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db/db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db/db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

Abdominal Aortic Endothelial Dysfunction Occurs in Female Mice With Dextran Sodium Sulfate-Induced Chronic Colitis Independently of Reactive Oxygen Species Formation.

Background and ObjectiveInflammatory bowel disease (IBD) produces significant local and systemic inflammation with increased reactive oxygen species (ROS) formation. IBD Patients are at an increased risk for developing endothelial dysfunction and cardiovascular diseases. The present study tested the hypothesis that IBD impairs aortic endothelial function via ROS formation and investigate potential sex-related differences.Methods and ResultsAcute and chronic colitis models were induced in male and female C57BL/6 mice with dextran sodium sulfate (DSS) treatment. Aortic wall stiffness, endothelial function, and ROS levels, as well as serum levels of pro-inflammatory cytokines were evaluated. Acetylcholine (Ach)-induced endothelium-dependent relaxation of abdominal aorta without perivascular adipose tissue (PVAT) was significantly reduced in female mice, not males, with chronic colitis without a change in nitroglycerin-induced endothelium-independent relaxation. PVAT effectively preserved Ach-induced relaxation in abdominal aorta of female mice with chronic colitis. Aortic peak velocity, maximal intraluminal diameters, pulse wave velocity, distensibility and radial strain were preserved in mice with both acute and chronic colitis. Although pro-inflammatory cytokines levels were increased in mice with acute and chronic colitis, aortic ROS levels were not increased.ConclusionThe data demonstrate that abdominal aortic endothelial function was attenuated selectively in female mice with chronic colitis independent of ROS formation. Further, PVAT played an important role in preserving endothelial function in female mice with chronic colitis.