Combination of LIM-kinase 2 and Jun Amino-terminal Kinase Inhibitors Improves Erectile Function in a Rat Model of Cavernous Nerve Injury.

OBJECTIVE: To determine if combined administration of LIMK2 and JNK inhibitors in a rat model of erectile dysfunction induced by cavernosal nerve (CN) injury could restore erectile function by suppressing both cavernosal apoptosis and fibrosis via rectification of molecular pathways related to the structural alterations. METHODS: Sixty 12-week-old male Sprague-Dawley rats were categorized into 4 groups: (1) Sham-surgery (Sham) group, (2) CN-crush-injury (CNCI), (3) CNCI group (CNCI+L+1.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and low-dose (1.0 mg/kg) JNK-inhibitors, and (4) CNCI group (CNCI+L+10.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and a high dose (10.0 mg/kg) of JNK-inhibitors. Ten days after surgery, erectile response, histological-studies, and Western-blot was investigated. RESULTS: The CNCI group showed a reduced maximal ICP/MAP or AUC/MAP, decreased immunohistochemical-staining of α-SMA, decreased SM/collagen ratio, increased phospho-cJun-positive apoptotic cells, increased phospho-LIMK2-positive fibroblasts, increased cJun-phosphorylation, increased LIMK2/Cofilin-phosphorylation, decreased Bcl-2/Bax ratio, and increased protein-expression of fibronectin, compared to the Sham group. Both the CNCI+L+1.0J and CNCI+L+10.0J groups showed improvements in erectile-responses, content of cavernosal α-SMA, number of phospho-cJun-positive apoptotic cells, Bcl-2/Bax ratio and cJun phosphorylation. Their improvements in the CNCI+L+10.0J group showed a tendency to be greater than those in the CNCI+L+1.0J group. Also, in the 2 treatment groups, rectification of SM/collagen ratio, number of phospho-LIMK2-positive fibroblasts, LIMK2/Cofilin-phosphorylation, and protein-expression of fibronectin was observed. CONCLUSION: This study suggests that combined inhibition of JNK and LIMK2 may improve erectile function by suppressing cavernosal apoptosis and fibrosis via restoration of cJun/Bcl-2/Bax and LIMK2/Cofilin pathways at 10 days after CN injury.

The effects of single versus combined therapy using LIM-kinase 2 inhibitor and type 5 phosphodiesterase inhibitor on erectile function in a rat model of cavernous nerve injury-induced erectile dysfunction.

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg-1 LIMK2i and 20.0 mg kg-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.

Chronic administration of LIMK2 inhibitors alleviates cavernosal veno-occlusive dysfunction through suppression of cavernosal fibrosis in a rat model of erectile dysfunction after cavernosal nerve injury.

We evaluated whether chronic administration of LIMK2-inhibitors could improve erectile function by alleviating CVOD through suppressing cavernosal fibrosis in a rat model of cavernosal nerve crush-injury (CNCI). Forty-two 12-week-old rats were equally categorized into the three groups: sham-surgery (S), CNCI (I), and CNCI treated with LIMK2-inhibitors (L). The L-group was treated with daily intraperitoneal injection of LIMK2-inhibitors (10.0 mg/kg) for 30-days after surgery. Erectile function was assessed using dynamic-infusion-cavernosometry (DIC). Penile tissue was processed for Masson's-trichrome staining, Western-blotting, and double immunofluorescence. The I-group showed significantly higher maintenance and drop rates as well as lower papaverine response, compared to the S-group. Chronic inhibition of LIMK2 in the L-group significantly improved the DIC parameters compared to those in the I-group, although the parameters were not completely restored to normal control values. Also, the I-group showed a reduced smooth muscle (SM)-to-collagen ratio, decreased immunohistochemical staining for α-SM-actin, increased number of fibroblasts positive for phosphorylated Cofilin, increased LIMK2/Cofilin phosphorylation and increased protein expression of Collagen-1 or Fibronectin, compared to the S-group. The L-group showed significant improvements in SM/collagen ratio and the deposition of Collagen-1 or Fibronectin compared to the I-group, although not completely normalized. According to the densitometry and confocal microscopy results, the L-group showed restoration of LIMK2/Cofilin phosphorylation and amount of fibroblasts positive for phosphorylated Cofilin to the normal control value. In conclusion, chronic inhibition of LIMK2 can improve CVOD and ED by alleviating cavernosal fibrosis via normalizing the LIMK2/Cofilin pathway.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury.

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

Inhibition of Jun N-terminal Kinase Improves Erectile Function by Alleviation of Cavernosal Apoptosis in a Rat Model of Cavernous Nerve Injury.

OBJECTIVE: To determine whether Jun N-terminal kinase (JNK) inhibition could alleviate erectile dysfunction (ED) through suppressing cavernosal apoptosis in a rat model of carvernosal nerve crush injury (CNCI), thereby providing potential therapeutic strategy for alleviating postradical prostatectomy ED. MATERIALS AND METHODS: Fifty-six 11-week-old male Sprague-Dawley rats were categorized equally into the following 4 groups: (1) sham surgery (S), (2) CNCI (I), (3) CNCI treated with low-dose JNK inhibitor (L), and (4) CNCI treated with high-dose JNK inhibitor (H). The L and H groups received daily intraperitoneal injection of JNK inhibitors (1.0 mg/kg for the L group and 10.0 mg/kg for the H group) for 2 weeks starting from the following day after surgery. Erectile response, Western blot, and immunohistochemistry were assessed. RESULTS: At 2 weeks after surgery, intracavernous pressure-mean arterial pressure and area under the curve-mean arterial pressure in group I were significantly decreased compared with those in group S. Erectile responses in group H were significantly improved compared with those in group I. Group I showed decreased smooth muscle (SM) content, increased apoptosis, increased apoptotic or SM cells positive for phosphorylated c-Jun, increased c-Jun phosphorylation, and decreased Bcl2-to-Bax ratio compared with group S. Group H showed significant improvements in histologic alterations and dysregulation of the JNK-driven pathway. CONCLUSION: Our data suggest that JNK inhibition can improve erectile function by alleviating cavernosal apoptosis through restoring the JNK-related pathway toward normal. Thus, an early therapeutic strategy targeting the JNK pathway might be able to alleviate cavernosal SM apoptosis and postradical prostatectomy ED caused by cavernous nerve injury.

Investigation of the effects of the level of glycemic control on erectile function and pathophysiological mechanisms in diabetic rats.

INTRODUCTION: Poor glycemic control is associated with erectile dysfunction (ED); however, differences in ED according to the level of glycemic control have been poorly investigated. AIM: The aim of this paper is to investigate the change in erectile function according to the level of glycemic control and to clarify the pathophysiological mechanism of diabetes-associated ED. METHODS: Streptozotocin was injected into 55 male Sprague-Dawley rats classified into four groups: control (group 1), diabetes with multiple insulin injections (group 2), diabetes with a single injection (group 3), and untreated diabetes (group 4). Daily insulin injections in groups 2 and 3 were administered for 4 weeks after 10 weeks of diabetic induction. MAIN OUTCOME MEASURES: The main outcome measures are the anova or Kruskal-Wallis tests to evaluate glycosylated hemoglobin (HbA1c), testosterone levels, the ratios of intracavernosal pressure to mean arterial pressure (ICP/MAP), area under the ICP curve to MAP (AUC/MAP), and changes in cavernous tissue and protein expression related to Rho kinase and nitric oxide pathways. RESULTS: HbA1c levels were different between pairs of groups. Group 4 showed the lowest erectile parameters and group 2 showed near normal level. No differences in erectile parameters were found between groups 1 and 2 or between groups 3 and 4, except the ratio of AUC to MAP for group 1 was significantly higher than that of group 2 (20 Hz stimulation). Decrease in erectile function of group 2 was related to decreased expression of nitrergic nitric oxide synthase or decreased testosterone level compared with group 1. Groups 2 and 3 showed significant differences in erectile parameters, which were associated with difference in apoptotic index. Groups 3 and 4 showed no differences in erectile parameters, although these groups had significant differences in apoptotic index, smooth muscle component, and protein expression ratios of phosphorylated to total myosin phosphatase target subunit 1, endothelial nitric oxide synthase, and Akt. CONCLUSIONS: Improvement in glycemic control assists recovery from diabetes-associated ED; however, only tight glycemic control can provide recovery from ED to a near normal status.

Involvement of sphingosine-1-phosphate/RhoA/Rho-kinase signaling pathway in corporal fibrosis following cavernous nerve injury in male rats.

INTRODUCTION: Postprostatectomy erectile dysfunction (ED) is thought to be due primarily to injury to cavernous nerve (CN) during surgery. The molecular mechanisms leading to ED after CN injury are poorly understood. AIM: We determined whether transforming growth factor-ß(1) (TGF-ß(1)), sphingosine-1-phosphate (S1P) and RhoA/Rho-kinase (ROCK) signaling pathways were involved in corporal fibrosis after bilateral CN injury in rats. METHODS: Forty-eight 10-week-old male Sprague-Dawley rats were equally divided into the following four groups: normal control group (C); sham surgery group (S); bilateral CN crush injury group (I); and bilateral CN transection group (T). Within each of the four groups, two subgroups were analyzed as a function of time (1 and 8 weeks postoperatively). MAIN OUTCOME MEASURES: Penile tissue was processed for immunoblot (RhoA, ROCK1, phospho-myosin phosphatase target subunit [MYPT1]), reverse transcription-polymerase chain reaction (RT-PCR) (TGF-ß(1), sphingosine kinase type 1 [SphK1], and S1P(2)), immunohistochemistry (alpha smooth muscle actin [α-SMA]), and Masson's trichrome staining. RESULTS: At 1 and 8 weeks postoperatively, the I and T groups had a significantly decreased smooth muscle cell/collagen ratio, the expression of α-SMA and phospho-MYPT1 compared to the C group. Densitometry revealed a significantly higher expression of RhoA and ROCK1 in the T group compared to the C group at 1 and 8 weeks postoperatively. For the I group, the expression of RhoA significantly increased starting from 1 week postoperatively, but the expression of ROCK1 significantly increased as late as 8 weeks following injury. The expression of TGF-ß(1) and S1P(2) mRNA in the I or T group remained significantly increased up to 8 weeks compared to the C group, despite significant reduction at 8 weeks compared to 1 week postoperatively. The expression of SphK1 mRNA in the I and T groups was significantly increased at 1 week but not 8 weeks postoperatively. CONCLUSIONS: Our data suggest that S1P and RhoA/ROCK1 signaling may be involved in corporal fibrosis associated with loss of smooth muscle through coordination with TGF-ß(1) after CN injury.