Isometric Tension Studies Research Articles

O47 THE ROLE OF IL-33 AND ST2 IN THE MODULATION OF TARGET ORGAN DAMAGE IN ANGIOTENSIN II-DEPENDENT HYPERTENSION

Introduction: Hypertension is associated with a chronic inflammatory state and aberrant immune system activity. Interleukin-33/suppression of tumorigenicity 2 (IL-33/ST2) signalling axis is important in the modulation of atherosclerosis and cardiovascular fibrosis. We aimed to evaluate the modulation of IL-33 and ST2 in hypertensive mouse models and patients. Methods and Results: Hypertension was induced by a 14-day infusion of Ang-II (490ng/min/kg) in 12-week-old WT C57BL/6, ST2KO, IL33KO mice. IL-33 and ST2 mRNA and protein were evaluated using qPCR and Western blot in WT mice. Ang II-infusion significantly increased IL-33 and ST2 protein expression in target organs related to hypertension, particularly the aorta (1.0±0.1 vs 2.8±0.2, p<0.0001, and 0.963±0.068 vs 2.094±0.231, p=0.002, respectively). Further, mRNA expression of IL-33 and ST2 was elevated in hypertensive aortas when compared with control (1.0±0.053 vs 1.76±0.271, p=0.0185 and 1.0±0.039 vs 1.978±0.221, p=0.0039, respectively). IHC from WT mice illustrated the localisation of IL-33 and ST2 in Ang-II aortas in ECs and SMCs. Further in human hypertensive mammary arteries, the localisation was. In-vivo, IL33KO and ST2KO mice showed no altered BP at baseline or after 14 days of Ang-II compared to WT littermates. ST2KO Ang-II mice showed protection against endothelial dysfunction compared to WT Ang-II mice through isometric tension studies (p=0.0317). Additionally, Ang-II-induced IL33KO and ST2KO mice worsen cardiac fibrosis compared to WT littermates (p=0.0005,p<0.0001, respectively). Conclusion: Both IL-33 and its receptor, ST2 are increased in the vascular and perivascular tissues in hypertension. Additionally, the IL-33/ST2 axis has a role in regulating cardiac fibrosis.

Involvement of ObRb receptor, nitric oxide, and BKCa channel signaling pathways in leptin-induced relaxation of pregnant mouse uterus

The purpose of this study was to determine the receptor subtype and the underlying mechanisms involved in the relaxant effect to leptin in mid- and late-pregnant mouse uterus. We determined the relative mRNA expression of receptor subtypes, eNOS, and BKCa channel by quantitative PCR and also the overall receptor expression by immunohistochemistry. Isometric tension studies were conducted to evaluate the effects of leptin and to delineate its mechanisms. A selective siRNA for the ObRb receptor was used to determine the participation of the receptor subtype in biochemical and molecular effects of leptin.The relaxant response to leptin was greater in mid-pregnancy compared to late pregnancy and was mediated by the activation of BKCa channels by eNOS-derived nitric oxide in an ObRb receptor-dependent manner. In comparison to mid-pregnancy, expression of short forms (mainly ObRa receptor) of the receptor was significantly increased in late pregnancy, whereas ObRb receptor expression was similar in both phases.The results of the study suggest that ObRb receptor mediates leptin-induced increase in eNOS expression and NO synthesis. Leptin-induced eNOS expression and activation cause cGMP-independent stimulation of BKCa channels causing uterine relaxation. Increased short forms of the receptors and reduced BKCa channels exert a negative effect on uterine relaxation in late pregnancy. Leptin may have a physiological role in maintaining uterine quiescence in mid-pregnancy and its reduced relaxant response in late gestation may facilitate labor. Further, ObRb receptor agonists may be useful in the management of preterm labor.

Short-term effects of suprapubic catheterization on detrusor muscle contraction in a rat model of postoperative urinary dysfunction

Catheterization is commonly performed in pelvic surgery to manage postoperative urinary dysfunction. However, little is known about the effects of catheterization on bladder function, although catheterization can drain urine from the bladder. In the present study, we evaluated the short-term effects of catheterization on detrusor muscle contraction in a rat model of postoperative urinary dysfunction due to bilateral accessory nerve injury (BACNI). Ten-week-old male Wistar/ST rats were divided into sham, sham＋catheterization (sham＋C), BACNI, and BACNI＋catherterization (BACNI＋C) groups. The sham＋C and BACNI＋C groups underwent catheter placement in the bladder dome prior to sham or BACNI surgery and intermittent bladder drainage during the observation period. Four hours after surgery, the bladders were excised and subjected to isometric tension study and immunofluorescence staining. Bladder overdistension and overflow urinary incontinence were observed in the BACNI group, not the BACNI＋C group. The contractile responses of the detrusor muscle to 80 mM KCl, carbachol, and electrical field stimulation (EFS) were enhanced in the BACNI group compared to the sham group; the enhanced contractile responses to EFS were improved in the BACNI＋C group. The ratio of the detrusor muscle layer area to the total bladder area increased with increasing interstitial area ratio in the BACNI and BACNI＋C groups, while the total bladder area increased in the sham＋C and BACNI＋C groups. These results suggest that catheterization does not improve the augmentation of detrusor muscle contraction and increase in the detrusor muscle layer area ratio immediately after BACNI and that catheterization causes bladder hypertrophy.

Empagliflozin, an SGLT2i, relaxes coronary smooth muscle at pharmacological concentrations

Morbidity and mortality due to heart failure with preserved ejection fraction (HFpEF) is increasing and there are no universally recognized strategies for treatment. Sodium-glucose co-transporter type 2 inhibitors (SGLT2i), such as Empagliflozin, have been shown to reduce myocardial stiffness and improve coronary microvascular performance, but mechanisms are unclear. Previous studies in rabbit aorta and rat mesentery suggest Empagliflozin relaxes smooth muscle through activation of K+ channels. This study investigated the effects of therapeutic and pharmacological concentrations of Empagliflozin (10-300 μM) on the isometric tension of left circumflex (CFX) coronary arteries from Yorkshire swine. We tested the hypothesis that Empagliflozin would relax coronary smooth muscle via a concentration-dependent manner, involving K+ channels. CFX arteries were dissected, cleaned of adventitia, cut into 3 mm rings (n = 16), and mounted in organ baths containing Krebs-Henseleit buffer (37 °C) for isometric tension studies. Each CFX segment was adjusted to optimal preload (~3 g passive tension) as determined by <10 % change in active tension in response to 60 mM KCl. In order to determine whether Empagliflozin caused relaxation and, if so, by what mechanism, rings were contracted with either 30 mM K+ or the thromboxane mimetic U46619 (1 μM). Once tension plateaued, Empagliflozin (10-300 μM) was added in increasing concentrations. Therapeutic concentrations of Empagliflozin (10-30 μM), elicited no significant relaxation. Pharmacologic concentrations of Empagliflozin (≥100 μM) caused significant relaxation from ~15 ± 11% to ~45 ± 10% in response to 100 μM vs. 300 μM Empagliflozin, respectively. Relaxation was not different between rings contracted with K+ or U46619, suggesting membrane hyperpolarization via K+ channels is not involved. Our results represent the first study of the effects of Empagliflozin on coronary smooth muscle reactivity. Data support the idea that relaxation of coronary smooth muscle by Empagliflozin occurs at supra-pharmacologic concentrations in vitro, but K+ channel activation appears to be an unlikely mechanism. This project was supported by National Institutes of Health grant R01 HL158723. 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.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

Co-activation of Mas and pGCA receptors suppresses Endothelin-1-induced endothelial dysfunction via nitric oxide/cGMP system

BackgroundThe aortic endothelium is crucial in preserving vascular tone through endothelium-derived vasodilators and vasoconstrictors. Dysfunction in the endothelium is an early indicator of cardiovascular diseases. Our study explores the therapeutic potential of a dual-acting peptide (DAP) to co-activate Mas and pGCA receptors and restore the balance between vasodilators and vasoconstrictors on endothelial dysfunction in DOCA-salt-induced hypertensive rats. MethodsDOCA-salt was administered to male wistar rats to induce hypertension, and various parameters, including blood pressure (BP), water intake and body weight were monitored. DAP, Ang1–7, BNP, and losartan were administered to hypertensive rats for three weeks. Histological analysis and isometric tension studies were carried out to assess endothelial function. In addition to this, we used primary aortic endothelial cells for detailed mechanistic investigations. ResultsDOCA-salt administration significantly elevated systolic, diastolic, mean arterial BP, and water intake whereas, downregulated the gene expression of Mas and pGCA receptors. However, DAP co-administration attenuated BP increase, upregulated the gene expression of Mas and pGCA receptors, normalized serum and urinary parameters, and effectively reduced fibrosis, inflammation, and vascular calcification. Notably, DAP outperformed the standard drug, Losartan. Our findings indicate that DAP restores aortic function by balancing the NO and ET1-induced pathways. ConclusionCo-activating Mas and pGCA receptors with DAP mitigates vascular damage and enhances endothelial function, emphasizing its potential to maintain a delicate balance between vasodilatory NO and vasoconstrictor ET1 in endothelial dysfunction.

(059) Effects of a Red-Light–Controllable Nitric Oxide Donor, NORD-1, on Diabetes Mellitus-Induced Erectile Dysfunction in Rats

Abstract Introduction Diabetes mellitus (DM) often causes erectile dysfunction (ED). Phosphodiesterase-5 inhibitor has limited effects on diabetes mellitus-induced erectile dysfunction (DMED). One contributing factor to this reduced effect is the decreased production of nitric oxide owing to neuropathy and endothelial impairment. To address this issue, we developed a red-light–controllable nitric oxide donor, NORD-1, that successfully enhanced erectile function in intact and cavernous nerve injury model rats. Objective In this study, we investigated whether NORD-1 and red-light irradiation could improve DMED in rats. Methods Seven-week-old Sprague–Dawley male rats were used. Rats in the DM and sham groups were intravenously administered streptozotocin 50 mg/kg and saline, respectively. One week after treatment, the blood glucose levels of rats in the DM group were found to be over 250 mg/dL. Five weeks after the treatment, we performed a functional study using the measurements of intracavernous pressure (ICP) under the stimulation of the cavernous nerve. The study was performed before and after NORD-1 treatment, both with and without light irradiation. Additionally, we performed an isometric tension study using corpus cavernosum from rats treated with either NORD-1 or a control compound, SiR650. Results The ICP/MAP ratio of the DM group was significantly lower than that of the sham group before and after NORD-1 treatment without light irradiation (both P<0.05). However, after NORD-1 treatment with light irradiation, the ICP/MAP ratios of the Sham and DM groups were substantially enhanced compared with that before and after NORD-1 treatment without light irradiation (all P<0.05). The ICP/MAP ratio of the DM group was similar to that of the Sham group under the normal condition before NORD-1 treatment by light irradiation. Moreover, systemic blood pressure was not affected by NORD-1 and light irradiation. In the isometric tension study, rat corpus cavernosum from rats treated with SiR650 showed no changes under red light in both the Sham and DM groups. However, the corpus cavernosum from rats treated with NORD-1 was strongly relaxed by red light in both groups. Conclusions NORD-1 and red-light irradiation could improve DMED in STZ-induced DM rats without lowering blood pressure. Disclosure No.

(057) Influence of Circadian Disruption on Erectile Function in Chronic Jet-Lagged Young Rats

Abstract Introduction Sleep is crucial during the developing stage of one’s life and has been associated with physical activity. However, as the number of 24-hour stores such as convenience stores and supermarkets have increased, along with increased internet usage, people have developed the habit of staying up late, resulting in sleep deprivation due to disruption in the light-dark cycle. Jet lag is widely used in animal models for inducing irregular circadian rhythms. Chronic jet lagged model animals are known to develop a variety of disorders. Objective To investigate the influences of chronic jet lag on erectile function in rats in order to elucidate the effects of irregular circadian rhythms on future sexual function. Methods 4-week-old rats underwent randomly assigned to remain either under the control light/dark cycle (Cont group) or under chronic light/dark shift schedule to simulate the conditions of chronic jet lag (CJL group). The shift-lag protocol consisted of 8 h light/dark phase advances repeated every 3 days for 4 weeks. Erectile function was examined at 1, 2, and 4 weeks post CJL treatment. After 4 weeks of CJL treatment, the rats were returned to control light/dark cycle for 4 and 8 weeks, as CJL(4w)+Rest(4w) group and CJL(4w)+Rest(8w) group, and erectile function was examined correspondingly. Erectile function was tested using intracavernous pharmacotherapy (ICP) measurements, and endothelial function was tested through an isometric tension study. Western blot analysis was used to assess the levels of endothelial nitric oxide (eNOS) and phosphorylated eNOS (p-eNOS). Additionally, mRNA expressions of clock genes (Bmal1 and Cry 1) and growth factors (TGF-β1 and ICAM-1) were tested using real-time PCR. Results The ICP/MAP ratio was significantly lower in the CJL group (0.35 ± 0.05) at 4 weeks than in the control group (0.66 ± 0.05) (P < 0.05). CJL group exhibited significantly lower testosterone levels, Bmal1, Cry 1, TGF-β1, and ICAM-1 expression, and p-eNOS and cGMP levels. The ICP/MAP ratio in the CJL(4w)+Rest(4w) group (0.47 ± 0.06) was lower than that in the control group (P < 0.01) whereas the ratio in the CJL(4w)+Rest(8w) group (0.81 ± 0.03) was similar to that in the control group (P > 0.05). Conclusions In this study, chronic jet lagged (4 weeks) rats exhibited a decreased ICP/MAP ratio, reduced testosterone level, and decreased p-eNOS expression, indicating the development of erectile dysfunction (ED). Upon 8 weeks of normal light/dark shift schedule, erectile function was recovered. This study suggests that irregular circadian rhythms induced by repeated jet lag, especially in childhood, may affect the production of sex hormones and erectile function in the future. Therefore, it is necessary for children to follow a regular circadian rhythm and healthy lifestyle. Disclosure No.

(370) Overview of Nonclinical Studies of LIB-01, a Novel Promising Oral Drug Under Development for Treatment of Erectile Dysfunction

Abstract Introduction LIB-01 is a novel molecule being developed as an oral drug for the treatment of erectile dysfunction (ED). It is a semi-synthetic analog, developed based on research on active compounds identified in root bark that had been used for treatment of male sexual disability since time immemorial. Objective In preparation for clinical development, a number of nonclinical studies on erectile function were conducted in normal and diabetic rats with ED. Studies to investigate the mechanism supporting the pro-erectile effect of LIB-01 were also conducted. A summary of the results from the nonclinical research program is presented. Methods Erectile response elicited by electrical stimulation of the cavernous nerve (ES CN) in anaesthetized rats was assessed. Intracavernous and arterial blood pressures were monitored according to a standardized experimental procedure. Mechanism of action studies were conducted with rat cavernosal tissue samples using immunohistochemistry and in ex vivo/organ chamber experiments. The isometric tension studies were conducted on cavernosal strips from LIB-01 treated and untreated Wistar rats. Cavernosal strips from untreated rats were incubated with either LIB-01 or DMSO (control) and then pre-contracted by phenylephrine (PHE). Concentration- or frequency response curves were performed on PHE pre-contracted strips in 3 consecutive steps: (i) cumulative addition of increasing acetylcholine (ACh) concentrations, (ii) electrical field stimulation (EFS) with increasing frequencies, and (iii), cumulative addition of increasing sodium nitroprusside (SNP) concentrations. Results A statistically significant pro-erectile facilitatory effect of LIB-01 has been repeatedly evidenced in all the studies conducted in anaesthetized control Wistar rats, by oral or subcutaneous administration of LIB-01 at 15 mg/kg/d, with an effect in the similar range as iv sildenafil 0.3mg/kg, when compared. The pro-erectile effect of LIB-01 was not acute, instead, the effect gradually increased and remained for at least 7 days post treatment. In addition, LIB-01 significantly improved erectile responses in anaesthetized diabetic Goto-Kakizaki ED rats compared to vehicle-treated rats, demonstrating the pro-erectile effect of LIB-01 in a robust validated pathophysiological rat model of type 2 diabetes. Immunohistochemistry experiments on cavernosal tissue from LIB-01 treated normal rats showed that neither CD31 nor nNOS expression was altered, thus likely ruling out any effect of LIB-01 on cavernosal micro-vascularization or cavernosal nNOS. Furthermore, only at a high concentration (10-5 M) was there a slight increase in the endothelium dependent relaxations induced by ACh compared to vehicle while LIB-01 did not exert any effect neither on endothelium-independent relaxations induced by SNP nor on nitrergic relaxation induced by EFS of untreated cavernosal strips exposed ex-vivo to different concentrations of LIB-01. Conclusions LIB-01 is a novel molecule which displays a significant pro-erectile effect in anesthetized normal rats as well as in diabetic rats with ED. The pro-erectile effect of LIB-01 is not acute, instead, the effect gradually increases and remains for at least 7 days post-treatment. Results from several mechanistic studies lead to postulate that the mechanism of LIB-01 does not recruit the NO-cGMP pathway thereby differing from phosphodiesterase type 5 inhibitors. A first-in-human clinical trial with LIB-01 is planned to start in 2023. Disclosure Yes, this is sponsored by industry/sponsor: Dicot AB. Clarification: Industry initiated, executed and funded study. Any of the authors act as a consultant, employee or shareholder of an industry for: Dicot AB.

The Effects of a Red-Light Controllable Nitric Oxide Donor, NORD-1, on Erectile Dysfunction in Rats with Streptozotocin Induced Diabetes Mellitus.

Patients with diabetes mellitus (DM) often exhibit refractory erectile dysfunction (ED). Red-light-controllable nitric oxide donor (NORD-1) and red-light irradiation have successfully enhanced erectile function in intact rats. In this study, we investigated whether the combination of NORD-1 and red-light irradiation effectively treated ED in streptozotocin (STZ)-treated rats with DM. Seven-week-old male Sprague-Dawley rats were used in this study. Rats in the DM and sham groups received intravenous STZ (50 mg/kg) and saline, respectively. One week after treatment, the blood glucose level of rats in the DM group was >250 mg/dL. Five weeks after the treatment, we performed a functional study by measuring intracavernous pressure (ICP) under cavernous nerve stimulation before and after NORD-1 treatment with and without light irradiation. Additionally, we performed an isometric tension study using the corpus cavernosum of rats treated with NORD-1 or the control compound, SiR650. The ICP/mean arterial pressure (MAP) ratio was significantly lower in the DM group than in the sham group before and after NORD-1 treatment without light irradiation (both p<0.05). After NORD-1 treatment with light irradiation, the ICP/MAP ratio in the sham and DM groups was significantly enhanced than before and after NORD-1 treatment without light irradiation (all p<0.05). The ICP/MAP ratio in the DM group after NORD-1 with light irradiation was similar to that in the sham group under normal conditions before NORD-1 treatment. Moreover, the systemic blood pressure was not affected by NORD-1 or light irradiation. In the tension study, the corpus cavernosum of rats treated with SiR650 was not changed by red light in the sham or DM groups. However, the rats treated with NORD-1 were strongly relaxed by red light in both groups. NORD-1 and red-light irradiation could improve ED in the presence of DM without lowering blood pressure.

A review of experimental techniques for erectile function researches and development of medical technology using animal erectile dysfunction models in sexual and reproductive medicine.

Erectile dysfunction (ED) is one of the causes of male infertility and is a disease that requires treatment. The first-line drugs for ED are phosphodiesterase 5 (PDE-5) inhibitors, and further treatment options are currently limited. Medical technologies, such as genetic control and regenerative medicine, are developing rapidly. Research on erectile function is progressing rapidly, coupled with technological innovations in other areas. A PubMed search using the keywords "animal (rat, mouse, rabbit, dog, and monkey)" and "erectile" was conducted, and all relevant peer-reviewed English results were evaluated. The methods for evaluating erectile function include intracavernous pressure (ICP) measurements, isometric tension studies, and dynamic infusion cavernosometry. Papers also reported various disease model animals for the study of diabetes mellitus, cavernous nerve injury, and drug-induced ED. Basic research on ED treatment has progressed rapidly over the past 20 years. In particular, research on the mechanism of ED has been accelerated by the publication of a study on the evaluation of erectile function using ICP measurements in rats. In addition, molecular biological experimental methods such as polymerase chain reaction (PCR) and western blotting have become relatively easy to perform due to technological progress, thus advancing research development.

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.

Effect of DPP-4 inhibitors on erectile dysfunction caused by vincristine in rats

ABSTRACT Introduction Various chemotherapeutic agents are administered to cancer patients worldwide. Previously, an analysis of the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database showed that vincristine (VCR) increased the risk of erectile dysfunction (ED), as reported in the world meeting on Sexual Medicine 2018. As such, countermeasures for late complications after anticancer drug treatment are required. Neuropathy is a well-known side effect of VCR; however, in recent years, it has been reported that DPP-4 inhibitors improve neuropathy. Objective To investigate the effect of DPP-4 inhibitors to ED caused by VCR in rats. Methods Twelve-week-old male Wistar ST rats were separated into Control, VCR, and VCR+DPP-4i groups. VCR (0.1 mg/kg) was intravenously administered on days 1, 8, 15, and 22 in the VCR and VCR+DPP-4i groups. Two types of long-term preparations, toleragliptin (TGP; 3 mg/kg/week, po.) and omaligliptin (OGP; 3 mg/kg/week, po.), were examined for DPP-4i. Erectile function was tested using intracavernous pharmacotherapy (ICP) measurements, and endothelial function was tested through an isometric tension study. Additionally, mRNA expressions were tested using real-time PCR. Results After the 4-week observation period, the ICP/mean arterial pressure (MAP) ratio in the VCR group (0.42 ± 0.04) was significantly decreased compared to the Control group (0.72 ± 0.05) (P &amp;lt;0.01). The ICP/MAP ratio in the VCR+TGP group (0.64 ± 0.02) increased significantly compared to the VCR group (P &amp;lt;0.05), however the ICP/MAP ratio in the VCR+OGP (0.50 ± 0.08) did not increase significantly compared to the VCR group (P &amp;gt; 0.05). Real-time PCR analysis showed that the catalase, VEGF, MCP-1, and PAI-1 mRNA expressions were increased, while the IL-6 mRNA expression decreased significantly in the VCR+TGP and VCR+OGP groups. In contrast, the nNOS mRNA expression level was higher in the VCR+TGP group than in the VCR+OGP group. Conclusions In this study, we determined that DPP-4 inhibitors may improve ED by VCR administration. For ED associated with VCR administration, long-term DPP-4 inhibitor TGP administration suggested a significant improvement in erectile function, but OGP did not show a significant improvement effect. An increase in antioxidant activity and a decrease in inflammatory cytokines were observed for both drugs, including a surge in vascular increase factors. In contrast, the nNOS expression level was higher in TGP, suggesting that there may be a difference in the amount of NO production via nerves. Therefore, TGP may be useful for male sexual dysfunction associated with vincristine administration. Disclosure Work supported by industry: no.

Oxaliplatin, an Anticancer Agent, Causes Erectile Dysfunction in Rats due to Endothelial Dysfunction

Chemotherapeutics, one of the standard treatment options for cancer worldwide, have various adverse effects, including erectile dysfunction (ED). To investigate erectile function in an animal model after administration of the anticancer agent oxaliplatin (L-OHP). Male Wistar/ST rats were divided into 2 groups: L-OHP rats (n = 21), which were intravenously administered L-OHP (4 mg/kg; twice a week for 4 weeks), and Control rats (n = 21), which were injected with the same volume of 5% glucose solution, using the same dosing schedule. At the end of the study period, erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) after cavernous nerve stimulation (n = 9-10). Endothelial function was evaluated with an isometric tension study using corpus cavernosum strips (n = 11). Western blot analysis was used to assess neuronal nitric oxide (nNOS) and endothelial NO synthase (eNOS) protein levels (n = 7). Real-time quantitative polymerase chain reaction (qRT-PCR) was used to assess the expression of inflammation- and oxidative stress-related markers (nicotinamide adenine dinucleotide phosphate oxidase-1, p22phox, interleukin [IL]-6, and nuclear factor-kappa B) (n = 6). Statistical significance was determined using the Student's t-test. The L-OHP group had a significantly lower ICP:MAP ratio than the control group (P < .05). Compared to the Control group, the L-OHP group exhibited significantly lower responses to ACh and eNOS protein levels and significantly higher inflammatory biomarker levels. The results based on this animal model indicate that use of the anticancer agent L-OHP should be considered as a risk factor for ED occurring via reduction of NO bioavailability in humans; our results provide possible treatment strategies for maintaining the erectile function of cancer survivors. Our study showed that the anticancer agent L-OHP has the propensity to cause ED in rats. A major limitation of this study is the lack of an established cure for ED associated with L-OHP and the lack of clinical evidence. L-OHP causes ED in rats via reduction of NO bioavailability caused by endothelial dysfunction.

Effects of High Salt Intake on Detrusor Muscle Contraction in Dahl Salt-Sensitive Rats.

High salt intake has been reported as a risk factor for urinary storage symptoms. However, the association between high salt intake and detrusor muscle contraction is not clear. Therefore, we investigated the effects of high salt intake on the components of detrusor muscle contraction in rats. Six-week-old male Dahl salt-resistant (DR; n = 5) and Dahl salt-sensitive (DS; n = 5) rats were fed a high salt (8% NaCl) diet for one week. The contractile responses of the detrusor muscle to the cumulative administration of carbachol and electrical field stimulation (EFS) with and without suramin and atropine were evaluated via isometric tension study. The concentration–response curves of carbachol were shifted more to the left in the DS group than those in the DR group. Contractile responses to EFS were more enhanced in the DS group than those in the DR group (p < 0.05). Cholinergic component-induced responses were more enhanced in the DS group than those in the DR group (p < 0.05). High salt intake might cause urinary storage symptoms via abnormalities in detrusor muscle contraction and the enhancement of cholinergic signals. Excessive salt intake should be avoided to preserve bladder function.

Hyperuricemia impaired nitric oxide bioavailablity and deteriorated pulmonary arterial hypertension via a uric acid transporter, URATv1 in xanthine oxidoreductase (XOR)-independent manner

Abstract Background Hyperuricemia occurs in approximately 80% in patients with pulmonary arterial hypertension (PAH) and is positively correlated with pulmonary arterial pressure (PAP). It has been reported that uric acid (UA) reduced endothelium derived nitric oxide (NO) production in porcine pulmonary arterial endothelial cells (PAEC). However, the effects of UA and xanthine oxidoreductase (XOR), catalytic enzyme of UA, on the development of PAH have not been fully elucidated. Purpose We examined the followings; (1) the effects of hyperuricemia on the endothelial function and the development of PAH in rats (2) the therapeutic effects of UA transporter inhibitor on PAH in rats, and (3) the role of XOR in PAH in mice. Methods We used normal and 5-wk Sugen5416/Hypoxia/Normoxia-exposed (SU/Hx/Nx) rats. Gene expression levels of URATv1, a UA transporter, were measured by RT-PCR. We determined the isometric tension of PA rings isolated from normal rats. The study with the isolated perfused lung preparation was performed in SU/HX/Nx rats. To investigate the chronic effect of UA on the development of PAH, hyperuricemia was induced by the administration of 2% oxonic acid (OA) in diet for 6-wk. Benzbromarone (BBR, 10mg/kg/day, diet, from weeks 0 to 5), a URATv1 transporter inhibitor, was administered in the SU/Hx/Nx-rats with or without 2%OA. To examine the role of XOR in PAH, XOR+/− and wild type (WT) mice were exposed to 3-wk Nx or Hx (10% O2). Results The mRNA of URATv1 was detected in the normal lungs. Isometric tension study showed that UA (8 mg/dl) inhibited acetylcholine-induced vasorelaxation. In perfused lung preparations, UA acutely increased estimated PVR in a dose-dependent manner (1.6–16.0mg/dl) with reducing cGMP levels in the lungs. BBR significantly attenuated the pressor response to UA. UA levels in the plasma and the lung tissues were significantly elevated in SU/Hx/Nx-rats with 2%OA (normal vs. vehicle vs. 2%OA, plasma: 0.24±0.01 vs. 0.80±0.14 and 1.44±0.17 mg/dl; lung tissues: 68±3 vs. 142±3 and 377±46 pmol/g tissue). They exhibited further elevation of right ventricle systolic pressure (RVSP) (31±2 vs. 72±6 vs. 101±3 mmHg) and Ea (a marker of RV afterload) (0.24±0.04 vs. 0.97±0.15 vs. 2.36±0.49 mmHg/μL) with the exacerbation of occlusive lesions of PAs. BBR had no changes in the UA levels in the plasma (1.93±0.30 mg/dL), but significantly reduced the UA levels in the lung tissues (101±10 pmol/g tissue) and attenuated the increase in RVSP (53±8mmHg) and Ea (0.21±0.05 mmHg/mL) in the SU/Hx/Nx-rats with 2%OA. On the other hand, BBR had no effects on RVSP (76±7 mmHg) and Ea (0.91±0.15 mmHg/mL) in the SU/Hx/Nx-rats without 2%OA. There were no significant differences in RVSP between XOR+/− mice with Hx and WT with Hx (26±2 vs. 26±2 mmHg). Conclusions Hyperuricemia itself impairs endothelial function and deteriorates PAH via URATv1 in a XOR-independent manner. UA can be a novel therapeutic target for PAH. Funding Acknowledgement Type of funding source: None

PKR inhibitor imoxin prevents hypertension, endothelial dysfunction and cardiac and vascular remodelling in L-NAME-treated rats

AimsHypertension is one of the leading causes of cardiovascular mortality and morbidity. It is associated with severe cardiac and vascular dysfunction. Double-stranded RNA-dependent protein kinase (PKR), is a known inducer of inflammation and apoptosis. However, no research has been done to elucidate the role of the PKR in an experimental model of hypertension, and related cardiovascular complications. Main methodsL-NAME (NG-Nitro-L-arginine-methyl ester) was used to induce the hypertension. Imoxin treatment was given to Wistar rats for the four weeks along with the L-NAME, to investigate the influence on the hypertension. Changes in physiological parameter were assessed by recording non-invasive blood pressure. Expression of PKR and downstream markers for inflammation, fibrosis, and vascular damage in rat heart and aorta was determined by western blot and immunohistochemistry. Histological examination and fibrosis assessment were done by using assay kits. Vascular reactivity was determined by ex-vivo isometric tension studies on rat aortic rings. Key findingsL-NAME-treated rats showed a significant increase in PKR expression followed by cardiac damage and vascular alterations compared to that of control animals. Results of western blot and immunohistochemistry indicate a significant increase in the inflammatory markers downstream to PKR. Endothelium-dependent vascular relaxation was significantly impaired in L-NAME administered rats. All effects of the L-NAME were attenuated by selective inhibition of PKR by imoxin. SignificanceAlterations in the heart and vasculature could be mediated in part by activation of the PKR pathway. Hence selective inhibition of PKR has therapeutic potential for combating hypertension and associated cardiovascular complications.

Hyperbaric oxygen therapy prevents subarachnoid hemorrhage-induced apoptosis and impaired contractility of the rabbit bladder.

To explore the effects of experimental subarachnoid hemorrhage (SAH) on rabbit urinary bladder and to assess the potential protective effects of hyperbaric oxygen therapy (HBOT). A total of 15 male New Zealand whiterabbits were divided randomly to one of three groups: group I was spared as the control group (n = 5), group II was exposed to SAH, received no treatment, and acted as the SAH group (n = 5) and group III was exposed to SAH and received fivesessions of HBOT (started 12 hours after SAH induction and was given twice daily for the first 2 days and once on the third day) and acted as the treatment group (n = 5). At 72 hours after the SAH induction, bladders from all animals were removed for in vitro organ bath experiments and biochemical analyses. İsometric tension studies revealed that compared to group I, the contractile responses of the strips to carbachol in group II were significantly decreased whereas HBOT restored the contractile responses (P < .05). Caspase-3 and nitric oxide synthase (NOS) activities of bladder tissues were significantly increased in group II when compared with group I, whereas caspase-3 and NOS activities were significantly decreased in the tissues of group III (P < .01). Subarachnoid hemorrhage stimulates apoptosis of the rabbit bladder and impairs the contractile response of the rabbit bladder to carbachol. HBOT creates a protective effect in rabbit bladder tissues and restores SAH-induced changes.

Effects of short‐term therapy with high dose nutritional inorganic nitrite and nitrate on vascular dysfunction, oxidative damage and epigenetic regulation in experimental arterial hypertension

BackgroundArterial hypertension is one of the major risk factors leading to highly relevant diseases like coronary artery disease, stroke or peripheral artery disease. Even short‐term application of angiotensin‐II (AT‐II) in animal models of arterial hypertension is leading to vascular dysfunction, enhanced production of reactive oxygen species (ROS) and modification of epigenetic processes. Nitric oxide (NO) represents an important opponent to AT‐II induced vasoconstriction and high blood pressure. Dietary uptake (e.g. vegetables) of inorganic nitrite (NO2−) and nitrate (NO3−) leads to enhanced NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2− and NO3− co‐therapy in hypertensive mice.Methods and resultsHigh‐dose AT‐II (1mg/kg/d for 1 week) was used to induce arterial hypertension in male C57BL/6 mice. In parallel high dose inorganic nitrite (7.5mg/kg/d) and inorganic nitrate (150mg/kg/d) was administered via drinking water. Hemodynamic effects were evaluated by blood pressure measurements and isometric tension studies. To elucidate underlying epigenetic regulation processes gene expression analyses and quantification of ROS formation were performed.Administration of AT‐II induced hypertension, cardiac hypertrophy and impaired endothelial function. Co‐therapy with high dose inorganic nitrite, but not with nitrate, normalised vascular function and arterial hypertension. In addition, oxidative stress markers and inflammatory pathways were enhanced in hypertensive mice and mostly normalized by nitrite therapy. We observed hypertension induced changes, at least by trend, of epigenetic proteins like histone deacetylase 2 or lysine demethylase 3A in cardiac tissue, which were mostly normalized by nitrite therapy.ConclusionNutritional nitric oxide precursors represent complementary therapy to classical antihypertensive drugs, representing a dietary treatment applicable to the general population (e.g. by eating certain vegetables). Short‐term therapy with inorganic nitrite showed beneficial effects on vascular function, oxidative stress and epigenetic regulation in experimental hypertension, which were less pronounced with inorganic nitrate. The observed differences may rely on superior bioconversion of nitrite to NO or intoxication through methaemoglobinaemia because of high dose inorganic nitrate therapy. Therefore, in particular inorganic nitrite seems to represent a cost‐efficient, drug‐free, alternative in antihypertensive therapy. Further clinical research is necessary to further support these preclinical findings and to demonstrate whether the incidence and severity of hypertension in particular and cardiovascular outcome in general can be influenced by inorganic nitrite therapy.Support or Funding InformationWe gratefully acknowledge the financial support by Stiftung Mainzer Herz (to M.O. and A.D.).

Relaxation Effect of Patchouli Alcohol in Rat Corpus Cavernous and Its Underlying Mechanisms.

In this study, we investigated the relaxation effect and mechanisms of patchouli alcohol (PA) on rat corpus cavernosum. Corpus cavernosum strips were used in organ baths for isometric tension studies. The results showed that PA demonstrated concentration-dependent relaxation effect on rat corpus cavernosum. The relaxant response to PA was not influenced by tetrodotoxin and atropine while it was significantly inhibited by removal of endothelium. L-NG-nitroarginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor) significantly inhibited relaxation response to PA, whereas indomethacin (COX inhibitor) had no effect on PA-induced relaxation. The treatment of endothelium-deprived corpus cavernosum with several potassium channel blockers including tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glibenclamide had no effect on PA-induced relaxation. Endothelium-deprived corpus cavernosal contractions induced by cumulative addition of Ca2+ to high KCl solution without CaCl2 were significantly inhibited by PA. Also, PA improved relaxant capacity of sildenafil in rat corpus cavernosum. In addition, the perfusion with PA significantly increased the levels of cGMP and expression of mRNA and protein of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). Furthermore, intracavernous injection of PA enhanced the rise in intracavernous pressure in rats during cavernosal nerve electric stimulation. In conclusion, PA relaxed the rat corpus cavernosum attributed to both endothelium-dependent and -independent properties. While the former component was mostly involved in nitric oxide signaling pathway, the endothelium-independent mechanism involved in PA-induced relaxation was probably linked to calcium antagonism.