Intracavernosal OnabotulinumtoxinA Exerts a Synergistic Pro-Erectile Effect When Combined With Sildenafil in Spontaneously Hypertensive Rats.

BACKGROUND: Botulinum toxin A (BTX-A) has a variety of uses in medicine. Some evidence suggests that intracavernosal (ic) BTX-A injection administered in addition to phosphodiesterase type 5 inhibitors (PDE5-Is) could effectively treat erectile dysfunction (ED) in insufficient responders to PDE5-Is. AIM: To provide experimental pharmacological evidence for the use of onabotulinumtoxinA ic alone or in combination with PDE5-Is for difficult-to-treat ED. We thus compared the effects of BTX-A ic alone and BTX-A ic combined with PDE5-I iv, and a placebo treatment ic or iv. METHODS: Erectile function was evaluated following cavernous nerve electrical stimulation (6 V, 1-millisecond pulse, 45-second duration) at different frequencies (0, 2, 3, 4, 5, 7.5, and 10 Hz) in 4 groups (n = 8 / group) of anesthetized, spontaneously hypertensive rats, a robust animal model of ED of vascular origin. Rats were treated by onabotulinumtoxinA 10U or saline ic 1 week prior to erectile function testing and sildenafil (0.3 mg/kg) or saline iv 4 minutes prior to testing. Frequency-response curves were compared with a 2 way ANOVA. OUTCOMES: Both onabotulinumtoxinA ic, and sildenafil iv significantly improved erectile responses in spontaneously hypertensive rats, however the effect was greatly amplified when the treatments were combined. RESULTS: Intracavernosal pressure and/or mean arterial pressure ratios were significantly increased by sildenafil and onabotulinumtoxinA ic versus the control condition. OnabotulinumtoxinA 10U ic combined with sildenafil iv significantly potentiated erectile responses. Area under the curve and/or mean arterial pressure ratio increased by 19% with sildenafil iv, by 15% with onabotulinumtoxinA ic and by 58% with the combined treatment following cavernous nerve electrical stimulation at 6V, 1 ms, 10 Hz: these stimulation parameters elicited the maximal erectile response. CLINICAL TRANSLATION: These data provide a pharmacological rationale for the combined administration of onabotulinumtoxinA ic and sildenafil iv since the effects of both treatments were potentiated when their administration was combined. STRENGTHS & LIMITATIONS: First evidence of a synergistic pro-erectile effect of BTX-A combined with PDE5-I, however the mechanism behind the pro-erectile effect of BTX-A ic remains hypothetical. CONCLUSIONS: These results support further studies into the mechanisms behind the pro-erectile effect of BTX-A ic, as well as multicenter randomized control trials to evaluate the safety and efficacy of BTX-A ic combined with sildenafil for difficult-to-treat ED. Giuliano F., Joussain C., Denys P., et al. Intracavernosal OnabotulinumtoxinA Exerts a Synergistic Pro-Erectile Effect When Combined With Sildenafil in Spontaneously Hypertensive Rats. J Sex Med 2022;19:899-906.

BoNT/A1 Secondary Failure for the Treatment of Neurogenic Detrusor Overactivity: An Ex Vivo Functional Study.

Management of neurogenic detrusor overactivity (NDO) remains a clinical priority to improve patients' quality of life and prevent dramatic urological complications. Intradetrusor injection of onabotulinumtoxinA (BoNT/A1, botulinum neurotoxin A1) is approved as second therapeutic line in these patients, demonstrating a good efficacy. However, a loss of its efficacy over time has been described, with no clear understanding of the underlying mechanisms. This paper aims at shedding new light on BoNT/A1 secondary failure in NDO through functional and structural analysis. Three groups of patients (either non-NDO, NDO with no toxin history or toxin secondary failure) were investigated using an ex vivo bladder strip assay. Detrusor strips were tensed in organ baths and submitted to electrical field stimulation to generate contractions. Recombinant BoNT/A1 was then added at various concentrations and contractions recorded for 4 h. Histology exploring BoNT/A1 targets, fibrosis and neuronal markers was also used. Detrusor strips from patients with BoNT/A1 secondary failure displayed a smaller sensitivity to toxin ex vivo at 3 nM compared to the other groups. Histological evaluation demonstrated the presence of cleaved Synaptosomal-Associated Protein, 25 kDa (c-SNAP25) in the detrusor from the toxin-secondary failure population, indicating some remaining in vivo sensitivity to BoNT/A1 despite the therapeutic escape. Moreover, residual c-SNAP25 did not affect parasympathetic-driven contractions observed ex vivo. This study confirms the slightly lower efficacy of BoNT/A1 in the BoNT/A1 secondary failure NDO group, suggesting that the escape from BoNT/A1 efficacy in NDO occurs at least at the parasympathetic level and could imply compensatory mechanisms for detrusor contraction.

Silodosin improves functional consequences of lower urinary tract obstruction secondary to benign prostate hypertrophy, a proof of concept study in the spontaneously hypertensive rat supplemented with testosterone.

BACKGROUND: The main purpose of this study is to investigate the effect of silodosin on the urodynamic consequences in a previously established model of lower urinary tract symptoms associated with benign prostate hyperplasia, the spontaneously hypertensive rats (SHR) supplemented with testosterone. METHODS: Three groups of animals (8-week-old; n = 10/group) were considered: Wistar Kyoto (control) rats (WKY), SHR supplemented with testosterone at 3 mg/kg/day and treated with either vehicle (SHR-T, n = 10) or silodosin at 0.1 mg/kg/day (SHR-T + silodosin, n = 10) by oral gavage for 6 weeks. Cystometry experiments were performed. The bladder was harvested, weighed and paraffin-embedded for morphometric analysis. The prostate was also harvested and weighed. RESULTS: The number of animals included in the analysis were n = 10/10 for WKY and n = 7-8/10 for each SHR rats supplemented with testosterone group. SHR-T displayed a significant decrease in the intercontraction interval, infused volume and mean flow rate whereas the frequency of non-voiding contractions was increased. Silodosin improved the voiding behavior of SHR-T by significantly increasing the intercontraction interval, the infused volume and the mean flow rate and decreasing the number of non-voiding contractions. SHR-T displayed a significant increase in prostate and bladder weights and a 15% increase in the detrusor wall area compared to WKY. CONCLUSIONS: Chronic silodosin treatment relieved storage symptoms in SHR supplemented with testosterone and decreased the frequency of non-voiding detrusor contractions during the filling phase.

Extracorporeal Shock Waves Therapy Delivered by Aries Improves Erectile Dysfunction in Spontaneously Hypertensive Rats Through Penile Tissue Remodeling and Neovascularization.

BACKGROUND: Low-intensity extracorporeal shock wave therapy (Li-ESWT) has been reported to improve erectile function in patients with moderate-to-severe erectile dysfunction (ED) or even convert phosphodiesterase type 5 inhibitors nonresponders to responders. ED is highly prevalent in hypertensive patients. The effect of Li-ESWT on an animal model of hypertension-associated ED has not been reported. AIM: To investigate the effect of Li-ESWT on hypertension-associated ED and provide plausible mechanisms of action of Li-ESWT on local mechanisms of penile erection. METHODS: Spontaneously hypertensive rats (SHRs) in the active group (n = 13) received Li-ESWT at energy flux density 0.06 mJ/mm2 (Aries; Dornier MedTech, Wessling, Germany) twice weekly for 6 weeks. The emitter was set to zero for SHRs in the sham group (n = 12). Erectile function was assessed 4 weeks post-treatment by monitoring intracavernosal pressure (ICP) in response to electrical stimulation of cavernous nerve before and after single dose of 0.3 mg/kg intravenous sildenafil. Cavernosal tissue was then evaluated for collagen/smooth muscle content, neuronal nitric oxide synthase (nNOS), and vascular endothelial factor (CD31) expression. OUTCOMES: Erectile function was assessed with ICP, erectile tissue remodeling was studied by smooth muscle/collagen ratio, nNOS and CD31 were semiquantitatively evaluated on cavernosal sections. RESULTS: The improvement of ICP parameters was greater in Li-ESWT-treated rats compared with controls with and without sildenafil. Sildenafil led to 20% increase in area under the intracavernosal pressure curve measured during the entire response/mean arterial pressure at 10 Hz in ESWT_SHR + sildenafil compared with ESWT_SHR. The smooth muscle/collagen ratio increased 2.5-fold in Li-ESWT compared with sham. Expression of CD31 tended to be increased whereas nNOS was unchanged. CONCLUSIONS: Li-ESWT by Aries may represent an effective noninvasive therapeutic alternative and a relevant add-on therapy to phosphodiesterase type 5 inhibitors for ED in hypertensive patients, and it is suggested that it acts via remodeling of the penile tissue and promoting cavernosal vascularization. Assaly R, Giuliano F, Clement P, et al. Extracorporeal Shock Waves Therapy Delivered by Aries Improves Erectile Dysfunction in Spontaneously Hypertensive Rats Through Penile Tissue Remodeling and Neovascularization. Sex Med 2019;7:441-450.

The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes.

INTRODUCTION: Following the results of the EMPA-REG Outcome trial, we hypothesized that empagliflozin, a highly potent and specific sodium/glucose cotransporteur 2 inhibitor, could improve type 2 diabetes mellitus (T2DM)-associated erectile dysfunction (ED), a highly prevalent complication of T2DM, very often coexisting with cardiovascular complications and considered as a prognostic factor of cardiovascular disease in men with diabetes. AIM: To investigate the effects of chronic treatment with empagliflozin on ED in a T2DM rat model in the presence or absence of sildenafil. METHODS: Male Goto-Kakizaki (GK), a model of T2DM, and age-matched Wistar rats received placebo or empagliflozin treatment at 25.3 ± 0.9 mg/kg/d for 4 weeks. Then, the in vivo effect of empagliflozin on erectile function was assessed by electrical stimulation of the cavernous nerve at different frequencies under anesthesia in the presence or absence of acute intravenous injection of sildenafil. Endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from the rats were studied. MAIN OUTCOME MEASURES: Body weight, food consumption, metabolic parameters, plasma inflammation biomarkers, and in vivo erectile responses elicited by electrical stimulation of the cavernous nerve in empagliflozin-treated and untreated GK rats and control Wistar rats were assessed and followed by concentration or frequency response curves to endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from these rats. RESULTS: Chronic empagliflozin followed by acute sildenafil significantly improved erectile responses in adult GK rats (n = 12-15/group). Ratios of intracavernous pressure and area under the curve/mean arterial pressure during the electrical stimulation were significantly increased in empagliflozin-treated vs untreated GK rats. Nitrergic relaxations of cavernosal strips from GK rats were significantly increased with empagliflozin compared with placebo. Moreover, the effect of sildenafil on erectile function was not altered by empagliflozin treatment. CLINICAL IMPLICATIONS: Empagliflozin may benefit T2DM patient with ED. STRENGTHS & LIMITATIONS: The mechanism(s) by which empagliflozin shows favorable effect on erectile function in GK rats needs to be further elucidated. CONCLUSION: Empagliflozin shows favorable effect on erectile function in diabetic GK rats mediated by an improvement of nitrergic relaxation of erectile tissue. Whether this favorable effect on ED in the experimental context of T2DM is due to better glycemic control or to another effect of empagliflozin deserves further investigation. Assaly R, Gorny D, Compagnie S, et al. The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes. J Sex Med 2018;15:1224-1234.

Mitochondrial translocator protein (TSPO) ligands prevent doxorubicin-induced mechanical dysfunction and cell death in isolated cardiomyocytes.

Contractile dysfunction and subsequent development of cardiomyopathies are well known limiting factors in the treatment of cancer with doxorubicin and have been linked to mitochondrial dysfunction. Here, using adult isolated paced cardiomyocytes, we have demonstrated that ligands of translocator protein (TSPO) 4'-chlorodiazepam and TRO40303 prevented the doxorubicin-induced alterations in contractility and improved cardiomyocyte viability. This cardioprotective effect was closely associated with both a potent reduction in reactive oxygen species production and inhibition of mitochondrial permeability transition pore opening. Thus, preventive administration of TSPO ligands may represent a novel pharmacological strategy to protect the heart during doxorubicin treatment.

A model of hypoxia-reoxygenation on isolated adult mouse cardiomyocytes: characterization, comparison with ischemia-reperfusion, and application to the cardioprotective effect of regular treadmill exercise.

The use of in vitro experimental models of hypoxia-reoxygenation (H/R) that mimic in vivo ischemia-reperfusion represents a powerful tool to investigate cardioprotective strategies against myocardial infarction. Most in vitro studies are performed using neonatal cardiac cells or immortalized embryonic cardiac cell lines which may limit the extrapolation of the results. We developed an H/R model using adult cardiomyocytes freshly isolated from mice and compared its characteristics to the in vivo ischemia-reperfusion conditions. First, cell death was assessed at different values of pH medium during hypoxia (6.2 vs 7.4) to simulate extracellular pH during in vivo ischemia. Cardiomyocyte mortality was aggravated with hypoxia under acidic pH. We next evaluated the relationship between the duration of hypoxia and cell death. Hypoxia time-dependently reduced myocyte viability (-24%, -36%, -53%, and -74% with 1, 1.5, 2, and 3 hours of hypoxia followed by 17 hours of reoxygenation, respectively). We then focused on the duration of reoxygenation as cardioprotective strategies have been reported to have different effects with short and long durations of reperfusion. We observed that cardiomyocyte mortality was increased when the duration of reoxygenation was increased from 2 h to 17 hours. Finally, we used our characterized model to investigate the cardioprotective effect of regular treadmill exercise. Myocyte viability was significantly greater in exercised when compared to sedentary mice (44% and 26%, respectively). Similarly, mice submitted to in vivo ischemia-reperfusion elicited infarct sizes reaching 27%, 43%, and 55% with 20, 30, and 45 minutes of coronary artery occlusion. In addition, infarct size was significantly reduced by exercise. In conclusion, this H/R model of cardiomyocytes freshly isolated from adult mice shows similar characteristics to the in vivo ischemia-reperfusion conditions. The comparison of in vivo and in vitro settings represents a powerful approach to investigate cardioprotective strategies and to distinguish between direct and indirect cardiomyocyte-dependent mechanisms.

Oxidative stress, mitochondrial permeability transition pore opening and cell death during hypoxia-reoxygenation in adult cardiomyocytes.

Reactive oxygen species production is necessary to induce cell death following hypoxia/reoxygenation but the effect of reactive oxygen species produced during hypoxia on mitochondrial permeability transition pore (mPTP) opening and cell death is not established. Here we designed a model of hypoxia/reoxygenation in isolated cardiomyocytes measuring simultaneously reactive oxygen species production, mPTP opening and cell death in order (i) to establish a causal relationship between them, and (ii) to investigate the roles of various reactive oxygen species in mPTP opening. The percentage of cardiomyocytes exhibiting mPTP opening during reoxygenation increased with the duration of hypoxia. Antioxidants increased the time to mPTP opening when present during hypoxia but not at reoxygenation. This was associated with a drop in hydroxyl radical and hydrogen peroxide during hypoxia and the first minutes of reoxygenation. The increase in time to mPTP opening was accompanied by an improvement in cell viability reflected by maintenance of superoxide production at reoxygenation. Cyclosporin A delayed both the time to mPTP opening and cell death despite maintenance of reactive oxygen species production during hypoxia. These findings demonstrate that reactive oxygen species production precedes mPTP opening and that reactive oxygen species produced during hypoxia, particularly hydroxyl radicals and hydrogen peroxide, are necessary to induce mPTP opening which depends on hypoxia duration.

Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4.

BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition.

3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.

Cardioprotective effect of morphine and a blocker of glycogen synthase kinase 3 beta, SB216763 [3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via inhibition of the mitochondrial permeability transition pore.

Morphine has been shown to protect the myocardium against ischemia-reperfusion injury through inhibition of glycogen synthase kinase-3beta (GSK-3beta). Given that GSK-3beta is known to modulate the mitochondrial permeability transition pore (mPTP), we investigated the role of mPTP in the cardioprotective effect of morphine and the GSK-3beta inhibitor SB216763 [SB; 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] during ischemia-reperfusion. Both morphine (0.3 mg/kg) and SB (0.6 mg/kg) reduced infarct size in a model of regional myocardial ischemia-reperfusion in rats (13 +/- 1 and 14 +/- 3% of the area at risk versus 33 +/- 4% in controls; p < 0.05). Morphine and SB protected the ischemic myocardium against Ca(2+)-induced mPTP opening as demonstrated by the increased capacity of mitochondria to retain Ca(2+) when they were isolated from the ischemic zone 10 min after the onset of reperfusion (59 +/- 8 and 66 +/- 3 versus 29.5 +/- 6 nmol Ca(2+)/mg x protein, respectively; p < 0.05). This was associated with a restoration of mitochondrial oxidative phosphorylation parameters. In isolated adult rat cardiomyocytes subjected to anoxia-reoxygenation, morphine (2 microM), SB (3 microM), and the direct mPTP inhibitor cyclosporine A (3 microM) delayed mPTP opening as assessed by the calcein loading Co(2+)-quenching technique. This was accompanied by an increase in cell survival as measured by nuclear staining with propidium iodide. These in vitro effects of morphine on inhibition of mPTP opening during anoxia-reoxygenation were suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin (0.1 microM). These data indicate that the infarct-limiting effect of morphine and SB is linked by a cause-effect relationship, which leads to an increased mitochondrial resistance and inhibition of mPTP opening through the PI3-kinase pathway and subsequent inactivation of GSK-3beta.