Yang Luan

Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1

Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson′s trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson′s trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

AR Pathway Is Involved in the Regulation of CX43 in Prostate Cancer

CX43 plays a critical role in tumor progression. Previous studies imply that AR pathway may be involved in regulation of CX43. This study was focused on determining the relationship between AR pathway and CX43. The result showed that the expression of CX43 in malignant cells was higher than that in normal cells, and in nonmalignant and malignant cells, not only is the expression level of CX43 different, but the localization of CX43 can also be changed. After androgen stimulation and inhibition of AR pathway, expression of CX43 can also be changed. Thus, AR pathway plays an important role in regulation of CX43 expression in prostate cancer cells. AR may be the upstream signal of CX43.

MP43-18 HUMAN TISSUE KALLIKREIN 1 RESCUES ERECTILE FUNCTION IN RATS WITH HYPERHOMOCYSTEINAEMIA BY PROTECTING ENDOTHELIAL FUNCTION AND INHIBITING FIBROSIS

INTRODUCTION AND OBJECTIVES: To investigate the detailed mechanism of erectile dysfunction (ED) induced by hyperhomocysteinaemia (HHcy) in rats and determine whether the Human Tissue Kallikrein 1 (hKLK1) might improve it, as we have proved the protective role of hKLK1 on erectile function in aged rats. METHODS: We established a rat model of HHCy through dietary-rich methionine (Met) in male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 10 weeks of age. Then 24 WTRs were divided into control (n1⁄48), the low-dose (4% Met, n1⁄48), and the highdose (7% Met, n1⁄48). Another 8 age-matched TGRs with the highdose formed the TGR+7%Met group. 30 days later, erectile function, level of total homocysteinaemia (tHcy), oxidative stress, endothelial function, cavernous nerve function and fibrosis of all groups were determined. RESULTS: hKLK1 in the TGR+7%Met group could greatly decrease the tHcy levels and improve ED induced by HHcy in rats. For the endothelial function, hKLK1 could preserve the endothelial cell-cell junction, enhance endothelial regeneration and activated the Akt/eNOS signaling pathway. Together with the promotion of hKLK1 on nNOS expression, the NO/cGMP signaling pathway activity was also increased. For the fibrosis, hKLK1 could preserve normal corpus cavernosum structure through inhibiting apoptosis and promoting autophagy on corpus cavernosum smooth muscle cells. In addition, hKLK1 also inhibited the fibrosis-related signaling pathway activity. CONCLUSIONS: hKLK1 might effectively improve ED induced by HHcy in rats by protecting endothelial function, promoting cavernous nerve function and inhibiting fibrosis, which suggested hKLK1 might be a potential treatment method for ED.

Human tissue kallikrein 1 ameliorates erectile function via modulation of macroautophagy in aged transgenic rats

Previously, we have demonstrated that human tissue kallikrein 1 (hKLK1) improves age‐related erectile dysfunction (ED). Autophagy has been implicated in age‐related diseases, including ED. However, the molecular mechanisms underlying hKLK1‐mediated amelioration of age‐related ED via regulation of autophagy remains unknown. To explore the potential mechanism, male wild‐type Sprague‐Dawley rats (WTR) and transgenic rats harboring human KLK1 (TGR) were bred till 4 or 18 months of age and divided into three groups: young WTR (yWTR) as the control group, aged WTR (aWTR) group, and aged TGR (aTGR) group. The erectile function of each rat was evaluated using cavernous nerve electrostimulation. The ratio of intracavernous pressure/mean arterial pressure (ICP/MAP) and total ICP were also measured. Western blotting, immunohistochemistry, and transmission electron microscopy were performed to detect the levels of autophagy. The expression levels of related signaling pathways were determined by western blotting and immunohistochemistry. We found that hKLK1 improved the impaired erectile function of aged rats. Compared to the yWTR and aTGR groups, the aWTR group showed reduced smooth muscle/collagen ratio, fewer autophagosomes, and lower expression of Beclin 1 and LC3‐II, which indicate impaired smooth muscle function and low level of autophagy in the smooth muscle cells. Moreover, the PI3K/Akt/mTOR signaling pathway, which is considered to be a negative regulator of autophagy, was upregulated in the aWTR group. hKLK1 may partially restore erectile function in aged transgenic rats by upregulating protective autophagy via the PI3K/Akt/mTOR pathway. These observations indicate that hKLK1 is a potential gene therapy candidate for age‐related ED.

AB087. Preserved erectile function in the hyperhomocysteinaemia transgenic rats harboring human tissue kallikrein

Background To investigate the role of human tissue kallikrein 1 (hKLK1) gene on the erectile dysfunction (ED) of induced by hyperhomocysteinaemia (HHcy) in rats. Methods The HHcy rat model was formed by a methionine (Met)-rich diet in SD rats. Here, 32 rats, 10-week-age, were divided in four groups: control (n=8), low-dose (4% Met; n=8), high-dose (7% Met; n=8) and transgenic rats (TGR +7% Met; n=8). Thirty days later, erectile function and related targets were tested. Results ED, impaired endothelial and smooth muscle function, and pathological changes (a higher apoptosis level and a lower autophagy level) were showed in the 4% Met and 7% Met groups compared with the control, while were all markedly diminished by the hKLK1 gene in the TGR +7% Met group. Conclusions These data suggested that hKLK1 might play an inhibition role on HHcy-induced ED in rats by protection of endothelial function and inhibition of oxidative stress and corporal fibrosis.

MP81-14 HUMAN TISSUE KALLIKREIN 1 AMELIORATES ERECTILE FUNCTION VIA MODULATING AUTOPHAGY AND ACTIVATING HIF-1?/COX-2 PATHWAY IN AGED TRANSGENIC RATS

normalized to KCl). Data are presented as means SEM. Data were statistically analyzed using a one way ANOVA followed by Tukeys test(GraphPad Prism software). RESULTS: Vaginal wet weight was decreased (P<0.05) in OVXVV animals compared to SHVV, an effect reversed by local estrogen delivery. Qualitative analysis of vaginal cross sections indicated reversal of ovariectomy induced atrophy of the vaginal muscularis with estrogen treatment. In vitro contractility studies with carbachol demonstrated a trend of a lower EC50 (increased sensitivity) of vaginal strips obtained from OVXVV animals compared to SHVV and OVXVE. The amplitude of contraction to 10 uM carbachol was greater of proximal strips from OVXVV animals compared to SHVV and OVXVE (P<0.05). CONCLUSIONS: Our results indicate that vaginal estrogen is effective at reversing not only OVX induced atrophy of the epithelium but also the vaginal muscularis. We report an increased contractile response to carbachol in OVXVV animals, an effect also reversed by vaginal estrogen. Interestingly, previous studies have shown an increase in vaginal sensory innervation in ovariectomized rodents. More studies are needed to evaluate changes in autonomic innervation with this animal model to identify new therapeutic uses of vaginal estrogen treatment.

Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats

Our previous studies had reported that Human Tissue Kallikrein 1 (hKLK1) preserved erectile function in aged transgenic rats, while the detailed mechanism of hKLK1 protecting erectile function in aged rats through activation of cGMP and cAMP was not mentioned. To explore the latent mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 18 months old and divided into four groups: young WTR (yWTR) as the control, aged WTR (aWTR), aged TGR (aTGR) and aged TGRs with HOE140 (aTGRH). Erectile function of all rats was evaluated by cavernous nerve electrostimulation method and measured by the ratio of intracavernous pressure/ mean arterial pressure (ICP/MAP) in rats. Expression levels of cAMP and cGMP were assessed, and related signaling pathways were detected by western blot, immunohistochemistry and RT-PCR. Our experiment results showed erectile function of the aWTR group and aTGRH group was lower compared with those of other two groups. Also, expression levels of cAMP and cGMP were significantly lower than those of other two groups. Moreover, expressions of related signaling pathways including DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP were also downregulated in the corpus cavernosum of rats in aWTR group. Our finding revealed hKLK1 played a protective role in age-related ED. The DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP pathways that were linked to the mechanism hKLK1 could increase the levels of cGMP and cAMP, which might provide novel therapy targets for age-related ED.

AB155. High-throughput sequencing of small RNA component of penile in a post-radical prostatectomy model of erectile dysfunction.

Objective The introduction of nerve-sparing radical prostatectomy represents a milestone in the treatment of prostate cancer. However, a certain percentage of cancer survivors still suffer from erectile dysfunction. Recent research has stated that using PDE 5-inhibitors after radical prostatectomy may lead to biochemical recurrence. This study was performed to identify the expression profile of small RNA in rats with neurogenic erectile dysfunction, and to investigate possible genes and signaling pathways involving in the disease. Methods Neurogenic erectile dysfunction (ED) was induced in male rats by bilateral cavernous nerve crushing injury (BCNI). After 28 days, RNA was isolated from the corpus cavernosum (CC) of both control rats and neurogenic ED rats. Small RNA sequencing was conducted using an Illumina Hiseq 2500/2000 platform. Candidate small RNAs were validated by rael-time polymerase chain reaction. Results Intracavernous pressure (ICP) was significantly decreased in BCNI group compared with SHAM group. Real time PCR validated that miR-9a-5p, miR-203a-5p, miR-378a-3p and miR-3557-5p were upregulated, and meanwhile miR-3084a-3p was downregulated. Conclusion Small RNA, including microRNA, may play an important role in the regulation of genes in CC and some certain miRs may participate in post-prostatectomy ED. Further studies will be designed to investigate the specific mechanisms of these changes.

MP52-10 GENOME-WIDE PROFILING OF LONGNONCODING RIBONUCLEIC ACID EXPRESSION PATTERNS IN CORPUS CAVERNOSUM OF DIABETIC RATS WITH ERECTILE DYSFUNCTION BY MICROARRAY

paraffin after evaluation of erectile function by cavernous nerve electrostimulation. The protein expression of endothelial nitric oxide synthase (eNOS), phospho-eNOS(Ser1177), phospho-eNOS(Thr495), inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), large conductance calcium activated potassium channel (BKCa), L-type calcium channel (ICa-L), RhoA and ROCK1 in CC was detected by western blot. We assayed nitric oxide (NO) using the Griess reagent and nitric oxide synthase (NOS) activity was also analyzed in each group. The concentration of cyclic guanosine monophosphate (cGMP) in CC was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, the transforming growth factor b1 (TGFb1) mRNA and protein expression were analyzed to evaluate the degree of fibrosis in CC tissue. RESULTS: The eNOS and phospho-eNOS(Ser1177) protein expression was significantly decreased while phospho-eNOS(Thr495), ICa-L, RhoA and ROCK1 protein were increased in aWTR group compared to control group, but these results were obviously improved in aTGR group than aWTR group. No significant difference of the iNOS, nNOS and BKCa protein content in CC was found between four groups. The NO level and NOS activity in aTGR was markedly higher than that in aWTR, although it was still lower than yWTR. In accord with the above results, the cGMP concentration was restored dramatically in CC of aTGR. Moreover, hKLK1 could prevent the increase of TGFb1 mRNA and protein expression in CC of aWTR. CONCLUSIONS: These results suggest that hKLK1 may improve erectile function in aged rat via the activation of NO/cGMP and inhibition of RhoA/ROCK signaling pathways as well as anti-tissue fibrosis effect.

Small cell carcinoma of the urinary bladder without gross hematuria: a case report

Small cell carcinoma of the urinary bladder (SCCB) is a rare and aggressive form of bladder cancer with poor prognosis. Hematuria is the main symptom of this malignancy, and most patients have a history of smoking. The disease incidence of malignant bladder tumors in China is approximately 0.74%. Early and accurate diagnosis of SCCB can ensure timely and appropriate treatment of this malignant disease. Oncologic surgery is the standard treatment; however, it may not be a curative approach. Chemotherapy and/or radiotherapy should be performed following surgical removal. This case report describes a patient with a single neoplasm diagnosed as SCCB that arose because of recurrence of bladder cancer after bladder tumor resection. In contrast to previously reported cases, this patient had no gross hematuria and no history of smoking.