Endothelium-dependent Relaxation In Response Research Articles

Proximal convoluted tubule cells-derived exosomes attenuate aging-related vascular dysfunction

Aging is an identified risk factor for cardiovascular disease. Aging is associated with increased prevalence of endothelial dysfunction and arterial stiffening. Exosomes are bi-lipid nanoscale vesicles that play important roles in intercellular communications. The purpose of this study was to determine whether administration of renal proximal convoluted tubule cells (PCT)-derived exosomes affects vascular and renal function in aging mice. Aging (24 months old) and young mice (3 months old) were treated with PCT-derived exosomes via non-invasive injection into the kidney 3 times a week for 2 weeks. Administration of PCT-derived exosomes did not affect blood pressure or urinary Na+, K+, and Ca2+ excretions, indicating that PCT-derived exosomes did not affect kidney function. Pulse wave velocity (PWV) was increased in aging mice, indicating arterial stiffening. Interestingly, treatment with PCT-exosomes effectively reduced aging-associated arterial stiffening. In addition, aortic endothelium-dependent relaxing (EDR) responses to acetylcholine (Ach) were reduced in aging mice, indicating endothelial dysfunction. Treatment with PCT-derived exosomes rescued aging-associated aortic endothelial dysfunction. Mechanistically, aortic expression of VCAM1, ICAM1, TNFα, and MCP-1 was upregulated in aging mice, indicating vascular inflammation. Notably, aging-associated vascular inflammation was largely attenuated by delivery of PCT-derived exosomes. Our results suggest that PCT-derived exosomes improves aging-associated arterial stiffening and vascular dysfunction partly via inhibiting vascular inflammation. The findings may help develop novel therapeutic strategies for aging-associated vascular dysfunction using PCT-derived exosomes. This work is supported by NIH research grants R01 HL154147 and AG062375. This is the full abstract presented at the American Physiology Summit 2024 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.

Regular transient limb ischemia improves endothelial function and inhibits endothelial cell apoptosis to prevent atherosclerosis in rabbit

AimsRegular transient limb ischemia (RTLI) can prevent atherosclerosis (AS) progression in hypercholesterolemic rabbits. This study aimed to investigate the minimum effective intensity and possible mechanisms of RTLI for preventing atherosclerosis.MethodsEighty rabbits were divided into eight groups: normal (N), high cholesterol (H), three RTLI [three RTLI cycles every other day (R3qod), three RTLI cycles daily (R3qd), and six RTLI cycles daily (R6qd), each cycle of RTLI included 5 min of limb ischemia followed by 5 min limb reperfusion], and three correlated sham RTLI [sham ischemia for 30 min once every other day (S3qod), sham ischemia for 30 min once daily (S3qd), and sham ischemia for 60 min once daily (S6qd)]. Rabbits in group N were kept normally, while the others were fed 1% cholesterol diet for 12 weeks. The RTLI and sham RTLI groups were received RTLI or sham RTLI procedure, respectively. The plaque area in the thoracic aorta was determined by oil red O staining, and quantifying the ratio of plaque area to intimal area (PA/IA). Endothelium-dependent and -independent relaxation were also determined. Endothelial cell were isolated from abdominal aorta of rabbits, and the apoptosis ratio was detected using flow cytometry.ResultsThe PA/IA and early apoptotic cell ratio was significantly lower as well as the endothelium-dependent relaxation response was higher in group R6qd than those in groups H and S6qd, while those in the R3qod group was not significantly different from those in groups H and S3qod, as well as those in the R3qd group showed no significant difference compared to those in groups H and S3qd.ConclusionsSix cycles of RTLI daily was the optimal effective intensity to prevent AS progression in rabbits. Endothelial function improvement and apoptosis inhibition might contribute to the anti-AS effects.

Short-term Effects of Cadmium Exposure on Blood Pressure and Vascular Function in Wistar Rats.

Chronic cadmium exposure is known to be associated with vascular changes and increased blood pressure, but its short-term effects on the cardiovascular system remain poorly understood. This study aimed to investigate the pressoric and vascular effects of a 7-day exposure to CdCl2 in Wistar rats. The rats were divided in control group (Ct), which received tap water, and the Cd group, which received a 100mg/L CdCl2 solution via drinking water for 7days. We analyzed body weight, plasma Cadmium concentration, systolic blood pressure (SBP), and vascular responses. Despite relatively low plasma Cadmium concentration, the Cd group exhibited elevated SBP and increased contractile response to phenylephrine. Endothelium removal and NOS inhibition increased contractions in both groups. In the Cd group's aorta, we observed enhanced levels of phospho-eNOS (Ser1177) and basal NO release. Cd group showed reduced Catalase expression and increased basal release of H2O2, with catalase reducing the contractile response. In arteries pre-contracted with phenylephrine, Cd group showed impaired endothelium-dependent (Acetylcholine) and independent (sodium nitroprussiate-SNP) relaxation responses. However, responses to SNP were similar after pre-contraction with KCl in both groups. These data suggest early effects of Cadmium on blood pressure and aortic function, indicating impaired H2O2-scavenging by catalase. Increased H2O2 due to Cadmium exposure might explain heightened responses to phenylephrine and weakened relaxation responses mediated by the NO-K+-channels pathway. Our findings shed light on Cadmium's short-term impact on the cardiovascular system, providing insights into potential mechanisms underlying its effects on blood pressure regulation and vascular function.

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis

AimsVascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. Methods and resultsThe case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17–13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated β-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE−/− mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. ConclusionsSAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

Combined levothyroxine and testosterone treatment for restoring erectile dysfunction in propylthiouracil-induced hypothyroid rats.

Sexual dysfunction may indicate severe endocrine diseases. Recent research has suggested a link between hypothyroidism, low testosterone (T) levels, and erectile dysfunction (ED); however, the exact cause is unknown. We sought to investigate possible beneficial effects of levothyroxine and T alone or in combination on ED in propylthiouracil (PTU)-induced hypothyroid rats. Adult Wistar rats (n = 35) were divided into 5 groups: control, PTU-induced hypothyroidism, PTU + levothyroxine, PTU + Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, and decanoate) and PTU + levothyroxine + Sustanon. PTU was given in drinking water for 6 weeks. Four weeks after PTU administration, levothyroxine (20μg microgram kg/day, oral) and Sustanon (10mg/kg/week, intramuscular) were given for 2weeks. Serum levels of total T, triiodothyronine (T3), and thyroxine (T4) were determined. In vivo erectile response and in vitro relaxant responses were measured. Localization of neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and phosphodiesterase type 5 (PDE5) were determined using immunohistochemical analysis. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Outcome variables included in vivo erectile function, in vitro relaxant and contractile responses of corpus cavernosum (CC) strips; protein localization of eNOS, nNOS, and PDE5; and smooth muscle content in penile tissue. The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3, and T4. Levothyroxine increased T3 and T4 levels, whereas Sustanon normalized only total T levels. Combined treatment enhanced all hormone levels. Rats with hypothyroidism displayed the lowest erectile response (P < 0.001 vs controls). Combined treatment returned reduced responses, while partial amelioration was observed after levothyroxine and Sustanon treatment alone. Acetylcholine (P < 0.01 vs controls), electrical field stimulation (P < 0.001 vs controls), and sildenafil-induced relaxant responses (P < 0.05 vs controls) were decreased in the CC strips from hypothyroid rats. The combined treatment increased the reduction in relaxation responses. Levothyroxine and Sustanon restored decreases in eNOS and nNOS expression in the hypothyroid group. There was no significant difference in PDE5 expression among groups. Monotreatment partially enhanced reduced smooth muscle mass, while combined therapy completely recovered. The combination of thyroid hormones and T is likely to be a therapeutic approach for treatment of hypothyroidism-induced ED in men. Beneficial effects of levothyroxine and Sustanon treatment were shown in vitro and in vivo in PTU-induced hypothyroid rats. The main limitation of the study was the lack of measurement of androgen-sensitive organ weights and luteinizing hormone, follicle-stimulating hormone, and prolactin levels. These findings demonstrate that neurogenic and endothelium-dependent relaxation responses are reduced by hypothyroidism, which is detrimental to T levels and erectile responses. Levothyroxine and Sustanon combination medication was able to counteract this effect.

Effect of Chronically Suppressed Plasma Angiotensin II on Regulation of the CYP4A/20-HETE Pathway in the Dahl Salt-Sensitive Rat.

In Dahl salt-sensitive (SS) rats, impaired vascular relaxation can be restored by: (1) minipump infusion of a low (sub-pressor) dose of angiotensin II (ANG II) to restore physiological levels of plasma ANG II, (2) inhibition of 20-HETE production, and (3) introgression of a normally functioning renin allele from the Brown Norway rat (SS-13BN consomic rat). Unlike SS rats, SS-13BN rats have normal levels of ANG II on a normal-salt diet and suppressed ANG II on a high-salt (HS) diet. This study tested whether chronically low ANG II levels in SS rats upregulate cytochrome P450-4A (CYP4A) increasing the production of the vasoconstrictor 20-HETE. Although salt-induced suppression of ANG II levels increased reactive oxygen species (ROS) in basilar arteries from SS-13BN rats in previous studies, this study showed no change in vascular 20-HETE levels in response to ANGII suppression. CYP4A inhibition significantly reduced vascular ROS levels and restored endothelium-dependent relaxation in response to acetylcholine in the middle cerebral artery (MCA) of SS rats and HS-fed SS-13BN rats. These data demonstrate that both the renin-angiotensin system and the CYP4A/20-HETE pathway play a direct role in the vascular dysfunction of the Dahl SS rat but are independent of each other, even though they may both contribute to vascular dysfunction through ROS production.

Evaluation of coronary function in female rats with severe type 1 diabetes: Effects of combined treatment with insulin and pyridoxamine.

This study aimed to evaluate the coronary function, myocardium, and epicardial adipose tissue (EAT) in female rats with severe type 1 diabetes and the effects of combined treatment with insulin and pyridoxamine (AGEs inhibitor). Female Wistar rats were divided into groups: control (CTR, n=13), type 1 diabetes (DM1, n=12), type 1 diabetes treated with insulin (DM1+INS, n=11), and type 1 diabetes treated with insulin and pyridoxamine (DM1+INS+PDX, n=14). The vascular responsiveness was performed in the septal coronary artery and the protein expressions of AGE, RAGE, GPER, NF-kB was evaluated in the left ventricle (LV), as well as the reactive oxygen species (ROS) was measured in LV and in EAT. We analyzed plasma levels of glucose, estradiol, Nε-carboxymethylisine (CML), thiobarbituric acid reactive substances (TBARS), catalase (CAT), and superoxide dismutase (SOD). The maximal responses to ACh were reduced in the DM1 compared with the CTR group, accompanied by an increase in circulating glucose, CML, and TBARS. Additionally, the expression of NF-kB in LV and generation of ROS in the presence of MnTMPyP (SOD mimetic) were increased in the DM1 group compared with CTR. Only the combined treatment was effective for fully re-establish ACh relaxation response, NF-kB protein expression, ROS generation, and increased SOD activity in the DM1+INS+PDX group. The reduction of the endothelium-dependent relaxation response in the septal coronary artery of female rats with severe type 1 diabetes was normalized with the combined treatment with insulin and pyridoxamine, associated with reduced inflammation and oxidative stress in the myocardium and increased circulating antioxidant activity.

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.

Effects of Cold Storage in Krebs-Henseleit Solution at +4 °C on Vasoreactivity of the Rat Thoracic Aorta

Aim: This study aimed to investigate whether endothelium-dependent and -independent relaxation responses can be preserved intact in the rat thoracic aorta after storage for 3, 6, and 18 hours in Krebs-Henseleit solution at +4 °C.&#x0D; Methods: Isolated organ bath model and 10-12 weeks old male Wistar rats were used to perform the experiments. To investigate the effect of endothelium-dependent relaxation factors, the cyclooxygenase inhibitor INDO was added to the Krebs-Henseleit solution to inhibit endogenous prostanoid synthesis. Submaximal contraction response was obtained with a single dose of PE and then ACh was administered cumulatively (10-9-10-4 M) to induce endothelium-dependent relaxation responses. Besides, smooth muscle-dependent relaxation responses were obtained by applying SNP cumulatively (10-9-10-5 M) following precontraction induced by PE. The statistical significance level was considered as p0.05). Besides, cumulatively administered ACh did not cause a significant change in endothelium-dependent relaxation responses (p&gt;0.05). Similarly, SNP did not modulate the endothelium-independent relaxation responses in aortic segments after storage for 3, 6, or 18 hours (p&gt;0.05).&#x0D; Conclusion: In the present study, the first physiological findings have been obtained that the endothelium-dependent and -independent contraction-relaxation responses of rat thoracic aortas can be preserved intact after storage periods of 3, 6, or 18 hours in Krebs-Henseleit solution at +4°C.

Sodium butyrate ameliorates erectile dysfunction through fibrosis in a rat model of partial bladder outlet obstruction.

In different animal models, a histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu) reduced inflammation, oxidative stress and fibrosis which were involved in the pathogenesis of erectile dysfunction (ED), but whether NaBu could improve ED in an experimental animal model of benign prostate hyperplasia (BPH) was not known. To investigate the preventive effect of NaBu on ED in a partial bladder outlet obstruction (PBOO) rat model. PBOO was induced by partial urethral obstruction. NaBu (20mg/kg/day) was administered orally to rats for 6 weeks after creation of PBOO. In vivo erectile responses, in vitro relaxation and contraction responses in cavernosal tissue were measured. Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to determine the gene and protein expression. Inflammation, fibrosis, and localization of proteins were evaluated using histological techniques. HDAC activity and tumor necrosis factor (TNF)-α levels were measured in penile tissues. NaBu improved decreased intracavernosal pressure/mean arterial pressure, nitrergic and endothelium-dependent relaxation responses, and contractile responses to phenylephrine and electrical field stimulation in the PBOO group without affecting increased bladder weight. Increased endothelial nitric oxide synthase (eNOS), transforming growth factor (TGF)-β1, and nuclear factor kappa B (NF-κB) gene levels in PBOO group were ameliorated by NaBu treatment. The administration of NaBu to PBOO rats significantly increased neuronal NOS (nNOS) and decreased TGF-β1 protein expression. The nuclear/cytosolic ratio of NF-κB demonstrated a decrease in PBOO and all treatment groups compared to control. A significant increase in the nuclear-to-cytoplasmic ratio of nuclear factor erythroid 2-related factor 2 (Nrf2) after PBOO was reduced by the treatment. Both eNOS and inducible NOS (iNOS) protein expression, together with TNF-α levels did not differ in the penile tissue of all groups. In histological analysis, increased TGF-β1 protein expression and fibrosis, as well as decreased nNOS protein in PBOO, were reversed by the treatment. NaBu did not normalize moderate inflammation in obstructed rats. An increase in the HDAC activity in PBOO was significantly suppressed by NaBu. Inhibition of the HDAC activity by NaBu in penile tissue could ameliorate fibrosis-associated changes induced by PBOO. NaBu promotes recovery of erectile function, and also significantly prevents penile fibrosis and normalizes TGF-β1 and nNOS protein expression in a rat model of PBOO. The HDAC pathway may present a promising target to prevent ED in patients with BPH.

Maternal perfluorooctane sulfonic acid exposure during rat pregnancy causes hypersensitivity to angiotensin II and attenuation of endothelium-dependent vasodilation in the uterine arteries †.

Epidemiological studies show a strong association between environmental exposure to perfluorooctane sulfonic acid (PFOS) and preeclampsia and fetal growth restriction; however, the underlying mechanisms are unclear. We tested the hypothesis that gestational PFOS exposure leads to pregnancy complications via alterations in uterine vascular endothelium-independent angiotensin II-related mechanisms and endothelium-derived factors such as nitric oxide. Pregnant Sprague-Dawley rats were exposed to PFOS 0.005, 0.05, 0.5, 5, 10, and 50μg/mL through drinking water from gestational day 4 to 20, and dams with PFOS 50μg/mL were used to assess mechanisms. PFOS exposure dose dependently increased maternal blood pressure but decreased fetal weights. Uterine artery blood flow was lower and resistance index was higher in the PFOS dams. In PFOS dams, uterine artery contractile responses to angiotensin II were significantly greater, whereas contractile responses to K+ depolarization and phenylephrine were unaffected. Plasma angiotensin II levels were not significantly different between control and PFOS dams; however, PFOS exposure significantly increased Angiotensin II type 1 receptor (AGTR1) and decreased AGTR2 protein levels in uterine arteries. Endothelium-dependent relaxation response to acetylcholine was significantly reduced with decreased endothelial nitric oxide synthase expression in the uterine arteries of PFOS dams. Left ventricular hypertrophy and fibrosis were observed, along with increased ejection fraction and fractional shortening in PFOS dams. These results suggest that elevated maternal PFOS levels decrease uterine blood flow and increase vascular resistance via heightened angiotensin II-mediated vasoconstriction and impaired endothelium-dependent vasodilation, which provides a molecular mechanism linking elevated maternal PFOS levels with gestational hypertension and fetal growth restriction.

Treatment with levothyroxine and testosterone ameliorated erectile dysfunction in a rat model of propylthiouracil-induced hypothyroidism

ABSTRACT Introduction The endocrine disease frequently interrupts sexual function, and sexual dysfunction may signal serious endocrine disease. Recent studies have claimed an association with hypothyroidism, low testosterone (T) levels and erectile dysfunction (ED), but causality is unclear. There is no study on the experimental model of hypothyroidism that was induced in rats by administration of propylthiouracil (PTU). Objective The purpose of this study was to investigate the deleterious effects of PTU-induced hypothyroidism on ED and T levels. In addition, Levothyroxine is known worldwide as the most common and least effective drug for controlling hypothyroidism. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups; control, PTU-induced hypothyroidism, PTU+ Levothyroxine, PTU+ Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, decanoate) and PTU+ Levothyroxine + Sustanon. PTU was given in drinking water (0.05%) for 6 weeks. 4 weeks after the beginning of PTU treatment, Levothyroxine (20 mg/kg/day, oral) and Sustanon (10 mg/kg, I.M., once/week) were given for 2 weeks. Serum levels of total T, triiodothyronine (T3) and thyroxine (T4) were determined. The ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) and total ICP were measured by cavernous nerve stimulation. The relaxant and contractile responses were evaluated on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) and phosphodiesterase type 5 (PDE5​) were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3 and T4. Levothyroxine increased T3 and T4 levels while Sustanon normalized only total T levels. In addition, combined treatment of Levothyroxine and Sustanon enhanced all hormone levels. Rats with hypothyroidism displayed the lowest ICP/MAP ratio and total ICP for 7.5 V (P &amp;lt; .001 vs controls). Combined treatment returned reduced ICP/MAP and total ICP responses, while partial amelioration was observed after Levothyroxine and Sustanon treatment alone. In vitro endothelium-dependent acetylcholine-caused relaxation at 1 nM (P &amp;lt; .001 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P &amp;lt; .001 vs controls) and sildenafil-induced relaxation at 10 µM (P &amp;lt; .05 vs controls) were decreased in the CC strips from hypothyroid rats. The treatment with Levothyroxine and Sustanon increased the reduction in relaxation responses. Decreased in the expressions of eNOS and nNOS and increased in the expressions of PDE5 in the hypothyroid group were restored by Levothyroxine and Sustanon. Mono-treatment partially enhanced reduced smooth muscle mass while combined therapy completely recovered. Conclusions These results show that hypothyroidism adversely affects T levels and erectile responses via decreasing neurogenic and endothelium-dependent relaxation responses which were reversed by the combination therapy with Levothyroxine and Sustanon. Even though the associated alterations in T levels, ED and its reversibility indicate an exact role of thyroid hormones in the erection physiology. Disclosure Work supported by industry: no.

Treatment with levothyroxine and testosterone ameliorated erectile dysfunction in rat model of propylthiouracil-induced hypothyroidism

ABSTRACT Introduction The endocrine disease frequently interrupts sexual function, and sexual dysfunction may signal serious endocrine disease. Recent studies have claimed an association with hypothyroidism, low testosterone (T) levels and erectile dysfunction (ED), but causality is unclear. There is no study on the experimental model of hypothyroidism that was induced in rats by administration of propylthiouracil (PTU). Objective The purpose of this study was to investigate the deleterious effects of PTU-induced hypothyroidism on ED and T levels. In addition, Levothyroxine is known worldwide as the most common and least effective drug for controlling hypothyroidism. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups; control, PTU-induced hypothyroidism, PTU+ Levothyroxine, PTU+ Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, decanoate) and PTU+ Levothyroxine + Sustanon. PTU was given in drinking water (0.05%) for 6 weeks. 4 weeks after the beginning of PTU treatment, Levothyroxine (20 mg/kg/day, oral) and Sustanon (10 mg/kg, I.M., once/week) were given for 2 weeks. Serum levels of total T, triiodothyronine (T3) and thyroxine (T4) were determined. The ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) and total ICP were measured by cavernous nerve stimulation. The relaxant and contractile responses were evaluated on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) and phosphodiesterase type 5 (PDE5) were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3 and T4. Levothyroxine increased T3 and T4 levels while Sustanon normalized only total T levels. In addition, combined treatment of Levothyroxine and Sustanon enhanced all hormone levels. Rats with hypothyroidism displayed the lowest ICP/MAP ratio and total ICP for 7.5 V (P &amp;lt; .001 vs controls). Combined treatment returned reduced ICP/MAP and total ICP responses, while partial amelioration was observed after Levothyroxine and Sustanon treatment alone. In vitro endothelium-dependent acetylcholine-caused relaxation at 1 nM (P &amp;lt; .001 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P &amp;lt; .001 vs controls) and sildenafil-induced relaxation at 10 µM (P &amp;lt; .05 vs controls) were decreased in the CC strips from hypothyroid rats. The treatment with Levothyroxine and Sustanon increased the reduction in relaxation responses. Decreased in the expressions of eNOS and nNOS and increased in the expressions of PDE5 in the hypothyroid group were restored by Levothyroxine and Sustanon. Mono-treatment partially enhanced reduced smooth muscle mass while combined therapy completely recovered. Conclusions These results show that hypothyroidism adversely affects T levels and erectile responses via decreasing neurogenic and endothelium-dependent relaxation responses which were reversed by the combination therapy with Levothyroxine and Sustanon. Even though the associated alterations in T levels, ED and its reversibility indicate an exact role of thyroid hormones in the erection physiology. Disclosure No

Gestational Intermittent Hypoxia Programs Hypertensive Response in Female Rat Offspring: Impact of Ovaries.

Obstructive sleep apnea (OSA) is a chronic condition frequently observed in pregnant women. We have shown that gestational intermittent hypoxia (GIH), a hallmark of OSA, leads to sex-specific impairment in the endothelium-dependent relaxation response and an increase in blood pressure in adult male but not female rat offspring. The present study tested the hypothesis that functional ovaries normalize GIH-induced hypertensive response in female offspring. Experiments were done in female offspring of pregnant rats exposed to normoxia or GIH (FIO2 21-10.5% from gestational days 10 to 21). Ovariectomy and sham surgery were performed at 5 weeks of age. Pups born to GIH dams were significantly smaller than the controls, but they exhibited catch-up growth and were similar to controls by 5 weeks of age. Ovariectomy significantly exacerbated bodyweight gain to a similar extent in both control and GIH offspring. Marked increases in blood pressure were observed in pre-pubertal GIH offspring compared to controls; however, after puberty, blood pressure in GIH offspring progressively decreased and became normotensive at adulthood. Ovariectomy led to the maintenance of higher blood pressure in post-pubertal GIH offspring with no significant effect in controls. Vascular contractile and relaxation responses were not affected in the GIH and control offspring; however, ovariectomy selectively decreased endothelium-dependent relaxation response along with a decrease in endothelial nitric oxide synthase expression in the GIH offspring. These findings suggest that functional ovaries are crucial in protecting females against GIH-mediated endothelial dysfunction and hypertension in adulthood.

Perivascular adipose tissue-derived nitric oxide compensates endothelial dysfunction in aged pre-atherosclerotic apolipoprotein E-deficient rats

Background and aimsAtherosclerosis is a major contributor to global mortality and is accompanied by vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is an established regulator of vascular function with emerging implications in atherosclerosis. We investigated the modulation of aortic relaxation by PVAT in aged rats with apolipoprotein E deficiency (ApoE−/−) fed a high-fat diet as a model of early atherosclerosis. Methods and resultsApoE−/− rats (N = 7) and wild-type Sprague-Dawley controls (ApoE+/+, N = 8) received high-fat diet for 51 weeks. Hyperlipidemia was confirmed in ApoE−/− rats by elevated plasma cholesterol (p < 0.001) and triglyceride (p = 0.025) levels. Early atherosclerosis was supported by increased intima/media thickness ratio (p < 0.01) and ED1-positive macrophage influx in ApoE−/− aortic intima (p < 0.001). Inflammation in ApoE−/− PVAT was characteristic by an increased [18F]FDG uptake (p < 0.01), ED1-positive macrophage influx (p = 0.0003), mRNA expression levels of CD68 (p < 0.001) and IL-1β (p < 0.01), and upregulated iNOS protein (p = 0.011). The mRNAs of MCP-1, IL-6 and adiponectin remained unchanged in PVAT. Aortic PVAT volume measured with micro-PET/CT was increased in ApoE−/− rats (p < 0.01). Maximal endothelium-dependent relaxation (EDR) to acetylcholine in ApoE−/− aortic rings without PVAT was severely impaired (p = 0.012) compared with controls, while ApoE−/− aortic rings with PVAT showed higher EDR than controls. All EDR responses were blocked by L-NMMA and the expression of eNOS mRNA was increased in ApoE−/− PVAT (p = 0.035). ConclusionUsing a rat ApoE−/− model of early atherosclerosis, we capture a novel mechanism by which inflammatory PVAT compensates severe endothelial dysfunction by contributing NO upon cholinergic stimulation.

Which proteinase-activated receptor-1 antagonist is better?: Evaluation of vorapaxar and parmodulin-2 effects on human left internal mammary artery endothelial function

ObjectiveEndothelial dysfunction occurs as an early event in cardiovascular disease. Previously, vorapaxar, a proteinase-activated receptor-1 antagonist, was shown to cause endothelial damage in a cell culture study. Therefore, our study aimed to compare the effects of vorapaxar and parmodulin-2, proteinase-activated receptor-1 biased agonist, on human left internal mammary artery endothelial function in vitro. MethodIsolated arteries were hung in the organ baths. Acetylcholine responses (10−11–10−6 M) were obtained in endothelium-intact tissues the following incubation with vorapaxar/parmodulin-2 (10−6 M) to determine the effects of these molecules on the endothelium-dependent relaxation. Subsequently, endothelium-dependent relaxation responses of tissues were investigated in the presence of L-NAME (10−4 M), L-arginine (10−5 M), indomethacin (10−5 M), and charybdotoxin-apamin (10−7 M) in addition to vorapaxar/parmodulin-2 incubation. Besides, the effect of these molecules on endothelium-independent relaxation response was evaluated with sodium nitroprusside (10−11–10−6 M). Finally, the sections of human arteries were imaged using a transmission electron microscope, and the integrity of the endothelial layer was evaluated. ResultsWe found that vorapaxar caused significant endothelial dysfunction by disrupting nitric oxide and endothelium-derived hyperpolarizing factor-dependent relaxation mechanisms. Parmodulin-2 did not cause endothelial damage. Neither vorapaxar nor parmodulin-2 disrupted endothelium-independent relaxation responses. The effect of vorapaxar on the endothelial layer was supported by the transmission electron microscope images. ConclusionParmodulin-2 may be a better option than vorapaxar in treating cardiovascular diseases since it can inhibit PAR-1 without caused endothelial dysfunction.

The effects of folic acid on vascular reactivity in a hyperhomocysteinemic rat model

Abstract&#x0D; Objective: This study aimed to investigate the antioxidant effects of folic acid and its effects on contraction and relaxation responses in rat aorta in hyperhomocysteinemic rats.&#x0D; Methods: Thirty-four male Wistar Albino rats were allocated into four groups. Rats in the hyperhomocysteinemia group (Group 1, n=9) received 1g/kg/day methionine via orogastric gavage for 30 days and then injected with intraperitoneal saline for the next 7 days. In the hyperhomocysteinemia+folic acid group (Group 2, n=9), following the administration of methionine for 30 days, folic acid (4mg/kg/day) was delivered intraperitoneally for 7 days. Sham group rats (Group 3, n=8) received orogastric saline for 30 days, which was followed by an IP injection of saline for another 7 days. Animals allocated into the folic acid group (Group 4, n= 7) had orogastric saline for 30 days and intraperitoneal folic acid for 7 days. After 5 weeks of treatment, blood samples were obtained, all animals were sacrificed, and hearts were harvested. Thoracic aortic segments were suspended on individual organ baths, and acetylcholine-induced (endothelium-dependent) relaxation responses of isolated aortic rings were evaluated. &#x0D; Results: Relaxation responses in Group 1 thru 4 were 73.889.96, 76.159.28, 76.618.83, and 69.2615.68, respectively. There was no significant difference in the organ bath in terms of relaxation response to acetylcholine at a dose of 10-9 mM between the groups (F=0.716, p=0.550). &#x0D; Conclusion: Folic acid therapy failed to produce a significant improvement in vascular reactivity. &#x0D; Keywords: Hyperhomocysteinemia, folic acid, vascular reactivity

Restorative effects of red onion (Allium cepa L.) juice on erectile function after-treatment with 5α-reductase inhibitor in rats.

Benign prostatic hyperplasia (BPH) is one of the most prevalent conditions among aged men. The use of 5α-reductase inhibitors (5-ARIs) to treat BPH was linked to erectile dysfunction (ED). Many medicinal plants and secondary metabolites are used in the management of ED. Onion (Allium cepa L.) is an economically affordable vegetable with vital phytochemicals and biological functions. The study aimed to identify the beneficial effects of onion juice on dutasteride (a 5-ARI)-induced ED. Rats were divided into two groups (n = 5 per group): control and dutasteride-treated rats (0.5 mg/kg/day). Dutasteride was administered in drinking water for 12 weeks. Experiments were performed at the end of the 12th week. In vivo erectile responses were measured before and after intracavernosal injection of onion. Relaxant responses to onion juice were examined in the corpus cavernosum (CC). Acetylcholine (ACh)-, electrical field stimulation (EFS)-, sodium nitroprusside (SNP)-induced relaxation responses in CC tissues were evaluated in the absence and presence of onion juice. Total intracavernosal pressure (ICP) and ICP/ mean arterial pressure were significantly reduced in dutasteride-treated rats (1881.14 ± 249.72 mmHg, P < 0.001;0.26 ± 0.03, P < 0.01) as compared to control rats (4542.60 ± 429.19 mmHg, 0.51 ± 0.05), which was normalized after the intracavernous administration of onion (3288.60 ± 185.45 mmHg, 0.58 ± 0.04). Onion markedly induced relaxant responses in control (72.5 ± 4.7) and dutasteride-treated (66.5 ± 2.7) groups after precontraction with phenylephrine. Relaxation responses to onion were partially inhibited after precontraction with KCl (32.5 ± 3.1, P < 0.001). The relaxant responses to ACh (14.9 ± 4.2, P < 0.01) were diminished in dutasteride-treated CC) compared to control CC(59.8 ± 3.4), which was enhanced after the incubation with onion (36.6 ± 4.8). There were no differences in relaxation response to SNP among all groups. However, relaxation response to SNP was reduced in dutasteride-treated CC at 1 μM(P < 0.05) and 10 μM dosages (P < 0.001), which was partially increased after the incubation with onion at 10 μM dosage (P < 0.01). The presence of onion did not change the reduction in EFS-caused relaxation in the dutasteride-treated group. The current data suggest that red onion juice has a restorative effect on erectile function and endothelium-dependent relaxation response following the treatment of dutasteride.

Chronic Inhibition of mROS Protects Against Coronary Endothelial Dysfunction in Mice With Diabetes.

Diabetes is associated with coronary endothelial dysfunction. Persistent oxidative stress during diabetes contributes to coronary endothelial dysfunction. The mitochondria are main sources of reactive oxygen species (ROS) in diabetes, and mitochondria-targeted antioxidant mito-Tempo can prevent mitochondrial reactive oxygen species (mROS) generation in a variety of disorders. Inhibition/inactivation of small-conductance Ca2+-activated K+ (SK) channels contribute to diabetic downregulation of coronary endothelial function/relaxation. However, few investigated the role of mROS on endothelial dysfunction/vasodilation and endothelial SK channel downregulation in diabetes. The aim of present study was to investigate the chronic administration of mito-Tempo, on coronary vasodilation, and endothelial SK channel activity of mice with or without diabetes. Mito-Tempo (1 mg/kg/day) was applied to the mice with or without diabetes (n = 10/group) for 4 weeks. In vitro relaxation response of pre-contracted arteries was examined in the presence or absence of the vasodilatory agents. SK channel currents of the isolated mouse heart endothelial cells were measured using whole-cell patch clamp methods. At baseline, coronary endothelium-dependent relaxation responses to ADP and the selective SK channel activator NS309 and endothelial SK channel currents were decreased in diabetic mice compared with that in non-diabetic (ND) mice (p < 0.05). After a 4-week treatment with mito-Tempo, coronary endothelium-dependent relaxation response to ADP or NS309 and endothelial SK channel currents in the diabetic mice was significantly improved when compared with that in untreated diabetic mice (p < 0.05). Interestingly, coronary relaxation responses to ADP and NS309 and endothelial SK channel currents were not significantly changed in ND mice after mito-Tempo treatment, as compared to that of untreated control group. Chronic inhibition of endothelial mROS appears to improve coronary endothelial function/dilation and SK channel activity in diabetes, and mROS inhibitors may be a novel strategy to treat vascular complications in diabetes.

Hydrogen sulfide as a mediator of endothelium-dependent relaxation evoked by Moringa oleifera leaf extract in mesenteric arterial beds isolated from L-NAME hypertensive rats.

Aqueous extract of Moringa oleifera leaves (MOE) is a potent inducer of endothelium-dependent relaxation of mesenteric resistance arteries of rats induced to be hypertensive using Nω-nitro-L-arginine methyl ester (L-NAME). Hydrogen sulfide (H2S) has been shown to participate in endothelium-dependent relaxation of small resistance arteries. Therefore, this study aimed to investigate whether endothelial H2S-dependent signaling plays a role in the vasorelaxation in response to MOE. Mesenteric arterial beds isolated from L-NAME hypertensive rats were set up in an exvivo perfusion system for measurement of vasoreactivity. All experiments were performed in the presence of the nitric oxide synthase inhibitor, L-NAME (100µM) and the cyclooxygenase inhibitor, indomethacin (10µM) to prevent the formation of nitric oxide and prostanoids, respectively. In the presence of the nitric oxide synthase inhibitor, L-NAME and the cyclooxygenase inhibitor, indomethacin, the endothelium-dependent vasorelaxation induced by MOE (0.001-3mg) was completely inhibited by DL-propargylglycine (100µM), which inhibits the H2Sgenerating enzyme, cystathionine γ-lyase. This H2Sdependent response was reduced by the KATP channel blocker; glibenclamide (10µM), the KCa channel blocker; tetraethylammonium (1µM), and the myo-endothelial gap-junctional uncoupler; 18α-glycyrrhetinic acid (10µM). In contrast, the muscarinic receptor antagonist, atropine (100µM), did not affect the response to MOE. The results may suggest that H2S is the likely mediator of endothelium-dependent relaxation in response to MOE in mesenteric arterial beds of L-NAME-induced hypertensive rats. MOE-induced H2S-dependent vasorelaxation involves activation of KATP and KCa channels and requires myo-endothelial gap-junctional communication.