ESSM 2022 Best of the best: basic research

Mario Lourenco

-Urology Department, Portuguese Oncology Institute, Coimbra, Portugal
– Medical Sciences Department, Beira-Interior University, Covilhã, Portugal

Erectile Dysfunction

Erectile dysfunction and altered contribution of kCa1.1 and kCa2.3 channels in penile tissue of type-2 diabetic db/db mice (#18)

  1. G. Comerma Steffensen, J. Prat Duran, S. Mogensen, R. S. Fais, E. Pinilla, U. Simonsen

Activation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. The authors hypothesized that in type-2 diabetic mice the function of KCa2.3/KCa1.1 channels is impaired in erectile tissue. Erectile function was measured, and corpus cavernosum strips were mounted for functional studies, and processed for qPCR and immunoblotting. diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1 channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. Those findings suggest that non-diabetic db/+ and diabetic db/db mice for translational purposes can be used for drug testing on, respectively, moderate and severe erectile dysfunction. Moreover, the results point towards the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation in the corpus cavernosum is markedly reduced, while the larger contribution of apamin-sensitive KCa2 channels to endothelium-dependent relaxation in the corpus cavernosum appears to be compensatory.

Intracavernosal onabotulinumtoxin a exerts a synergistic pro-erectile effect when combined with sildenafil in spontaneously hypertensive rats (#155)

  1. Giuliano, C. Joussain, P. Denys, M. Laurin, D. Behr-Roussel, R. Assaly

The authors aimed to provide experimental pharmacological evidence for the use of intracavernosal (ic) onabotulinumtoxinA alone or in combination with phosphodiesterase type 5 inhibitors for difficult-to-treat erectile dysfunction. They compared the effects of botulinum toxin A alone and botulinum toxin A combined with phosphodiesterase type 5 inhibitor, and a placebo treatment. Erectile function was evaluated following cavernous nerve electrical stimulation at different frequencies in 4 groups (n = 8 / group) of anesthetized, spontaneously hypertensive rats. 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. Intracavernosal pressure/mean arterial pressure ratios were significantly increased by sildenafil and onabotulinumtoxinA ic versus the control condition. OnabotulinumtoxinA 10U ic combined with sildenafil significantly potentiated erectile responses. Area under the curve/mean arterial pressure ratio increased by 19% with sildenafil, by 15% with onabotulinumtoxinA ic and by 58% with the combined treatment.

Transient receptor potential channel 5 (trpc5) inhibitor, ac1903, produces relaxation and improves endothelial function in human and rat penile vascular tissues. (#259)

  1. M. La Fuente, A. Fernández, E. García-Rojo, B. García-Gómez, E. Fernández-Pascual, R. Curvo, J. Romero-Otero, J. I. Martínez-Salamanca, J. Angulo

Transient receptor potential channels (TRPC) have been proposed to play a role in vascular function and vascular pathophysiology by regulating calcium signaling. The aim of this study was to evaluate the impact of TRPC inhibition on cavernosal tone and endothelial function in aged rats and ED patients. The effects of the inhibitors of TRPC3, Pyr3, TRPC4, ML204, and TRPC5, AC1903, were evaluated in precontracted strips of corpus cavernosum (RCC) from young (3-months-old, 3m) and aged (20-months-old, 20m) rats and in human corpus cavernosum (HCC) and penile resistance arteries (HPRA) from ED patients undergoing penile prosthesis insertion. Effects of AC1903 on endothelial relaxation were also determined. TRPC protein expression was determined in HCC from ED patients and organ donors (NoED). Pyr3, ML204, and AC1903 produced consistent relaxations of phenylephrine-contracted corpus cavernosum from old and young rats, being the TRPC5 inhibitor the most effective. Interestingly, AC1903-induced relaxations were significantly reduced by NO synthase inhibition and remained after contracting RCC with high K+. AC1903 was also effective in relaxing HCC and from ED patients. Pretreatment with AC1903 (3 µM) was able to enhance endothelium-dependent acetylcholine-induced relaxations in RCC from 20m rats and HCC from ED patients. TRPC1 and TRPC3-6 proteins were detected in HCC but TRPC4 and TRPC5 expressions were relatively weak. TRPC1 and TRPC3 were increased in ED patients with respect to NoED. In conclusion, putative TRPC5 inhibitor AC1903 reverses contractile tone and improves endothelial relaxation in cavernosal tissue from aged rats and ED patients. TRPC might be potential therapeutic targets in ED but mechanisms other than TRPC5 inhibition could contribute to AC1903-induced effects.

Isoforms of the cyclic gmp-dependent protein kinase i (cgki) are expressed in human penile erectile tissue (#8)

  1. Ückert, E. S. Waldkirch, V. Märker, P. Hedlund, M. A. Kuczyk

In animal models, a down-regulation of the cyclic GMP-dependent/binding protein kinase I (cGKI) has been linked to the on-set of diabetes mellitus and erectile dysfunction (ED). This study aimed to investigate in human penile erectile tissue (corpus cavernosum and cavernous arteries) the expression of cGKI (isoforms alpha and beta) in relation to smooth muscle alpha-actin, the second messenger molecule cyclic GMP and the endothelial nitric oxide synthase (eNOS). Cryostat sections of human penile erectile tissue were exposed to primary antibodies (ABs) directed against alpha-actin, cGKI, cGKI alpha and cGKI beta, cyclic GMP and eNOS, followed by the incubation with fluorochrome-labeled secondary ABs.Visualization was commenced by means of laser fluorescence microscopy. Western blot analysis was also performed. Abundant immunoreactivities indicating the presence of cGKI alpha and beta and the second messenger cyclic cGMP were observed in the trabecular smooth musculature as well as in the smooth muscle layer of tiny cavernous arteries. Double staining revealed the co-localization of cGKI and eNOS within the endothelium of small arterial vessels. The expression of cGKI alpha and beta was confirmed by Western blot analysis.

Prolonged pro-erectile facilitator effect of lib-01 in anesthetized wistar rats. (#189)

  1. Razanakolona, C. Bradesi, M. Laurin, K. Fugl-Meyer, D. Behr-Roussel, F. Giuliano, R. Assaly

This was a proof of concept study to investigate the effect of lib-01 on erection in anesthetized rats. The active substance lib-01 is a semi-synthetic molecule, originating from neobeguea mahafalensis roots with a long tradition in madagascar of ethnopharmacological use for the treatment of sexual disability. Adult Wistar rats (n=12 /group) were administered LIB-01 at 4 or 15 mg/kg by subcutaneous injection once daily for 3 consecutive days. Erection was assessed 1, 2 or 7 days after the last treatment by electrical stimulation of the cavernous nerve (ES CN) at different frequencies under isoflurane anaesthesia. LIB-01 treatment was compared to a single dose of sildenafil 0.3 mg/kg iv dispensed 4 minutes prior penile erection testing. There was no safety signal post LIB-01 treatment. Ratios of intracavernous pressure (ICP) and area under the curve (AUC)/mean arterial pressure (MAP) were increased in rats treated with LIB-01 sc 15 mg/kg/day for 3 days when erection was tested 1 day post last administration vs vehicle-treated rats (at 10 Hz, ICP/MAP: + 11 %) compared to a 19 % increase in sildenafil-treated group. The proerectile facilitator effects of LIB-01 at 15 mg/kg were improved when erection was tested 2 days compared to 1 day post-treatment, and further increased when evaluated 7 days post   treatment (at 10 Hz, ICP/MAP increased by 36 % and 48 % 2 and 7 days post-LIB-01 treatment respectively).

Is there a role of histamine in the pathophysiology of erectile dysfunction? (#4)

  1. Ückert, A. J. Becker, H. E. Rahardjo, A. Bannowsky, M. A. Kuczyk

Histamine [4-(2’-aminoethyl)-imidazol] is a bioactive amine related to the control of vascular tissue and the vasorelaxation mediated by nitric oxide (NO) and cyclic GMP. The role and function of histamine in the control of human penile erectile tissue (corpus cavernosum) is only poorly understood. The aim of this study was to evaluate the course of histamine plasma levels through different stages of sexual arousal in the systemic and cavernous blood of healthy males and in patients with erectile dysfunction (ED). 34 healthy adult males (mean age 26 y) and 46 patients with ED (mean age 52 y) were exposed to erotic stimuli in order to elicit sexual arousal. Blood was aspirated from the corpus cavernosum and a cubital vein during the penile conditions flaccidity, tumescence, rigidity (attained only by the healthy males) and detumescence. Plasma levels of histamine (ng/mL) were determined by means of a radioimmunoassay (RIA). In the healthy individuals, histamine slightly decreased in the cavernous blood with the beginning of sexual arousal, when the flaccid penis became tumescent (0.95 ± 0.5 to 0.87 ± 0.36), decreased further during rigidity (0.81 ± 0.3) and remained unaltered in the phase of detumescence. In the systemic circulation, no alterations in plasma histamine were observed with the initiation or termination of penile erection, In contrast, in the ED patients, a rise in cavernous histamine was registered from flaccidity/tumescence to detumescence (1.16 ± 0.33 to 1.33 ± 0.52). Moreover, at all penile conditions, plasma histamine was significantly higher in the cavernous blood than in the systemic circulation (p < 0.03). Based on the results, it seems unlikely that histamine has excitatory function in the control of penile erection.

Ejaculation Disorders

A new approach for on-demand premature ejaculation (#104)

  1. Olivier, J. Janssen, S. de Pretre, J. Olivier

The authors developed a model for male sexual behavior that is able to replicate the effects of SSRIs in men with lifelong premature ejaculation. After extensive weekly sexual training of large cohorts of male Wistar rats, a portion of them displays stable and high levels of sexual behavior in 30-min. tests with an estrus female, presented by very stable levels of high ejaculation frequencies and short ejaculation latencies. These fast-ejaculating rats were used for drug testing. The authors found that adding a very selective 5-HT1A receptor antagonist Atlas987 (10-mg/kg IP) to the SSRI paroxetine (5-mg/kg IP) led to acute (on-demand) inhibition of sexual behavior in male rats, evidenced by a strong reduction in the ejaculation frequency and doubling of ejaculation latency. A similar profile was found with the reference 5-HT1A-receptor antagonist WAY100,635 (0.3 mg/kg IP) plus 5 mg/kg paroxetine.

Preclinical evidence for a new on-demand treatment for lifelong premature ejaculation: enduro (#133)

  1. D. Olivier, J. A. Janssen, S. De Prêtre, B. Olivier

The aim of this study was to elucidate the effect of a new 5-HT1A-receptor antagonist Atlas987 in combination with the SSRI paroxetine in male rats. Male rats were trained in a 30-minute weekly sexual performance test, for ten weeks. A selection was made for rats that displayed short ejaculation latencies and high number of ejaculations. Atlas987 and Paroxetine (Enduro) were dose-dependently administered (oral) and acute responses on sexual behavior assessed. Next the effects of Enduro were assessed on sexual behavior one, two, three or four hours after administration. Blood was taken up to 8 hours after administration and plasma analyzed for paroxetine and Atlas987 concentrations. Enduro dose-dependently inhibited sexual behavior. It decreased the number of ejaculations and increased latency time to ejaculate. At each time point tested, Enduro showed the specific inhibitory effects on male rat sexual behavior. Plasma levels of Atlas987 and paroxetine nicely paralleled the ejaculation-reducing effects.

Peyronie’s Disease

Statins synergize with other drugs to prevent myofibroblast transformation in in vitro model of peyronie’s disease (#159)

  1. Ilg, D. Ralph, S. Cellek

Tunica albuginea (TA) of PD patients was used to isolate primary fibroblasts. TA-derived fibroblasts were exposed to Transforming Growth Factor-ß1 (TGF-β1) and a range of concentrations of statins, PDE5i, SERM, and their combinations for 72h before quantifying α-smooth muscle actin (α-SMA) using In-Cell ELISA (ICE). Statins (simvastatin, lovastatin) showed a concentration dependent anti-fibrotic effect to prevent TGF-ß1-induced myofibroblast transformation, with IC50 values of 0.77 µM and 0.8 µM for simvastatin and lovastatin, respectively. Addition of 0.3 µM of simvastatin to a full concentration response curve of vardenafil revealed drug synergy, as 0.3 µM of simvastatin showed 12% inhibition, 0.3 µM of vardenafil 1.5% inhibition, but their combination exerted 33% inhibition. Similarly, 0.18 µM of tamoxifen showed 2% inhibition, whilst a combination with 0.3 µM simvastatin resulted in 24% inhibition. Interestingly, combining simvastatin, vardenafil, and tamoxifen was not more effective than vardenafil and tamoxifen, or vardenafil and simvastatin only.

Defining a temporal gene expression profile of myofibroblast transformation in peyronie’s disease (#165)

  1. Ilg, S. Harding, A. Lapthorn, S. Kirvell, N. Schurman, S. Church, D. Ralph, S. Bustin, S. Cellek

This study aimed to examine differences in temporal gene expression patterns at various other stages of myofibroblast transformation. Human primary fibroblasts were isolated from tunica albuginea (TA) of PD patients. Myofibroblast transformation was induced in these cells by exposure to 10 ng/mL Transforming Growth Factor-ß1 (TGF-ß1). RNA was isolated from cells before stimulation and 24, 36, 48, and 72 h after and analysed using the Nanostring nCounter Fibrosis Panel. 770 genes from 51 pathways were examined with a minimum raw expression threshold. Genes with a >3-fold change were considered significant differential expression and further analysed. The top ten up- and down-regulated genes were confirmed by RT-qPCR. Out of 770 genes, 45 genes were differentially expressed with > 3-fold change in expression. 29 genes were up-regulated, and 16 genes were down-regulated. Altered gene expression patterns fell into three categories: those showing the strongest differential up-or down-regulation at 24h, at 72h or between 36h and 48h. Most changes in gene expression occurred within 24 hours and remained unchanged throughout the 72-hour period. Significant changes were observed in genes regulating inflammation, production and degradation of extracellular matrix proteins, chemoattraction, apoptosis, cell adhesion and proliferation. This study is the first to investigate the temporal expression of genes involved in myofibroblast transformation in PD. The three categories of genes identified might be responsible for different stages of the myofibroblast transformation process with those differentially expressed within 24h responsible for induction, those at 72h associated with maintenance of the myofibroblast phenotype, and those between 36h and 48h involved in a ‘point-of-no-return’ in myofibroblast transformation.

Investigating the role of extracellular vesicles in peyronie’s disease (#161)

  1. Ilg, P. Dyer, S. Bustin, D. Ralph, S. Cellek

Whilst the role of extracellular vesicles (EVs) and the secretome has been elucidated for other diseases, little is known about this in Peyronies’s Disease (PD). This study aimed to elucidate the role of EVs in myofibroblast transformation. Human primary fibroblasts were isolated from tunica albuginea (TA) of PD patients. Conditioned media was generated from cells treated with and without Transforming Growth Factor-ß1 (TGF-ß1). 10% Knockout Serum Replacement (KOSR) was used to avoid EV contamination from foetal calf serum (FCS). EVs were analysed via Tunable Resistive Pulse Sensing (TRPS) using the qNano Gold (Izon) to determine size, concentration, shape, and charge of the particles. Cells were treated with EVs in presence/absence of TGF-ß1 and myofibroblast transformation was quantified using In-Cell ELISA (ICE). TRPS analysis revealed that the TGF-ß1-treated cells produced more EVs (39.4×108 particles/mL) than untreated cells (7.24×108 particles/mL). Untreated cells showed a larger percentage population of particles >80nm compared to TGF-ß1-treated cells, which showed the highest concentration of particles at 70nm. Mean diameter was 92nm and 75nm for untreated and TGF-ß1-treated cells, respectively. There was no difference in charge of particles (zeta potential) between the groups. Slight differences in shape of particles could be detected, with the TGF-ß1-treated group showing more small but potentially elongated particles. No particles above 240nm were detected in either group. Preliminary ICE experiments revealed that isolated fractions impacted myofibroblast transformation. This is the first study to characterize the EV populations of fibroblasts and myofibroblasts in PD.

Orgasm/ Sexual desire

The role of multisensory integration and attention in sexual incentive salience (#273)

  1. B. Ruesink, T. M. Zorn, A. Pawlowska, S. M. Borsutzky, M.-C. Gerwert, S. Martens, J. R. Georgiadis

To become sexually aroused and eventually engage in sexual behavior, a sexual incentive must first capture our attention. A mechanism that can enhance the salience of such stimulus is multisensory integration (MSI), where input from different sensory modalities (e.g., visual, auditory, and tactile) that occurs close in time is integrated to form a single multimodal percept. This study main objective was to test whether the salience of sexual images would be enhanced by the simultaneous presentation of (task-irrelevant) sexual audio. Sixty-one heterosexual males took part in a modified attentional blink (AB) paradigm. They were instructed to detect the rotation of two target images (T1 and T2) amongst a rapid series of 17 images, presented for 100 ms each. T1 could show a nude (rated as highly erotic) or a dressed female (rated as neutral), whereas T2 displayed a control image (random animal). T1 and T2 were separated by a single (Lag-2) or seven (Lag-8) filler images. To investigate MSI, the authors added a task-irrelevant auditory stimulus (1,000 ms) that was always concurrently presented with T1 and either erotic (climatic female voice) or neutral (pseudo-words). Data showed that sexual images induced a larger AB than non-sexual images. However, the authors found no main effect of audio (sexual vs. non-sexual) on target detection, but a complex interaction between lag, T1 type, and audio type. For Lag-8, the sexual image-sexual audio combination resulted in the lowest target detection. For Lag-2, whenever the image was sexual, there was a substantially larger AB, regardless of the audio. This study provides the first evidence that MSI modulates the salience of sexual stimuli.