Human Tissue Kallikrein 1 Protects Against The Development Of Erectile Dysfunction In A Rat Model Of Hyperhomocysteinemia

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The effect of h KLK1 on erectile function in a rat model of hyperhomocysteinaemiaObjective: To identify the existence and expression of Human tissue Kallikrein 1(h KLK1)gene in transgenic rat(TGR).Moreover,we establish the rat model of hyperhomocysteinaemia(HHcy)and determine the erectile function to evaluate the effect of h KLK1.Methods: We obtained the TGR harboring the h KLK1 gene as a generous gift from the Max Delbrück Center for Molecular Medicine(Berlin,Germany).The TGRs were generated by microinjecting a 5.6-kb DNA fragment containing the entire h KLK1 gene,under the control of heavy metal-responsive mouse metallothionein promoter,into the oocytes of SD rats.The presence of the transgene in genomic DNA was verified by Southern blot.Offspring with the homozygous h KLK1 gene were selected for further experiments.We established a rat model of HHcy through a methionine(Met)-rich diet.30 rats were randomly divided into three groups as follows: Control(n = 10)group,the low-dose(4%Met,n = 10)group,and the high-dose(7%Met,n = 10)group.Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7%Met group.Body weight and the level of Hcy were tested during the experiment.One month later,electrostimulation on the rat cavernous nerve with different voltages(2.5 V and 5 V)was performed in all rats.The ratios of maximum(max)ICP and the area under the ICP curve(AUC)to MAP were calculated to evaluate penile erectile function.After the measurement,the penile tissues were harvested for subsequent experiments.Results: Only the TGRs,not the wild type rats(WTRs),contained and expressed the h KLK1 gene through the identification at the levels of genomic DNA,m RNA,and protein.There was no obvious difference in initial body weight and plasma t Hcy levels among all four groups,while the final body weight of 4%Met,7%Met,and TGR+7%Met groups were sharply reduced compared with the Control group.The final t Hcy levels in the 4%Met,7%Met,and TGR+7%Met groups were significantly higher than that in the Control group.Moreover,t Hcy levels in the 7%Met and TGR+7% Met groups were higher than that in the 4%Met group.In addition,there were no obvious differences for t Hcy levels between 7%Met and TGR+7%Met groups.Max ICP/MAP ratios in the 4%Met and 7%Met groups were greatly attenuated compared with that in the Control group,and the value for the 7%Met group was also lower than that for the 4%Met group.However,the Max ICP/MAP ratio was partly,but signigicantly improved in the TGR+7%Met group,though it was still lower than that in the Control group.The ratios of AUC/MAP with 2.5 V and 5 V in all four groups showed the same trend as did the Max ICP/MAP values.There were no differences among the four groups for the MAP.Conclusion: Only the TGRs contained and expressed the h KLK1 gene.The rat model of HHcy was successfully established through the Met-rich diet and HHcy might led to the weight-loss in rats.Moreover,HHcy might impair erectile function in rats,and h KLK1 protected against the development of ED in this rat model.Part ? The effect of h KLK1 on corpus cavernosum endothelial cells in a rat model of hyperhomocysteinaemiaObjective: To evaluate the effect of h KLK1 on corpus cavernosum endothelial cells in a rat model of HHcy and investigate the underlying mechanismMethods: We harvested and stored the penile tissues after measuring the erectile function.Proteins were extracted for western blot and ELISA methods to test the expression of some NADPH oxidase subunits including p22 phox,p47phox,and gp91 phox.Then the levels of MDAand SOD were detected for further investigation.Moreover,the endothelial cells junction proteins(VE-cadherin,occludin,and claudin-5)expression and the Akt/ e NOS pathway activity were also determined.In addition,NO and c GMP levels were also analyzed.Meanwhile,immunofluorescence(IF)staining was also used for determination of PECAM-1,e NOS and n NOS expression levels.Results: Compared to the Control group,the 4%Met and 7%Met groups both showed the higher expression levels of p22 phox,p47phox,and gp91 phox,the higher penile MDA levels and the lower SOD activities,the lower expression levels of endothelial cells junction proteins(VE-cadherin,occludin,and claudin-5),the lower expression levels of p-Akt,e NOS,p-e NOS,n NOS and PECAM-1,and the lower NO and c GMP levels.Moreover,the 7%Met group showed a bigger change than 4%Met group on these targets above.However,the h KLK1 in the TGR+7%Met group could reduce the pathological changes in the penis induced by HHcy,and especially for the SOD activity and the expression levels of Occludin and p-Akt,there was no significant difference for them between TGR+7%Met and Control groups.In addition,there was no obvious difference for Akt expression among all four groups.Conclusions: Oxidative stress,impaired endothelial intercellular junction and endothelial cells content,and cavernous nerve injury were all involved in HHcy-induced ED,while h KLK1 could inhibit these pathological changes to protect the erectile function.Part ? The effect of h KLK1 on corpus cavernosum smooth muscle in a rat model of hyperhomocysteinaemiaObjective: To evaluate the effect of h KLK1 on corpus cavernosum smooth muscle in a rat model of HHcyMethods: We harvested and stored the penile tissues after measuring the erectile function.Proteins were extracted for western blot to test the expression of ?-SMA,TGF-?1,Collagen I,Collagen IV,the Rho A/ROCK pathway and the apoptosis related proteins Bcl-2 and Bax.TUNEL method was also used to study the apoptosis level in the penis.Meanwhile,Masson's trichrome staining were used to evaluate the fibrosis in the penis,and immunohistochemical and immunofluorescence staining were also used for determination of ?-SMA expression.In addition,primary culture and purificantion of corpus cavernosum smooth muscle cells and endothelial cells were performed from rat penis and aorta,respectively.After identification with desmin and PECAM-1,these two cells were co-cultured.LMWK was added to the endothelial cells to test the change of c GMP levels in the corpus cavernosum smooth muscle cells in the co-culture system.Then the co-culture system was cultured with different concentration of Hcy(5?M,30?M and 100?M),and treated with LMWK for 48 hours,followed by the deteremination of c GMP levels,oxidative stress,Rho A/ROCK pathway and apoptosis level in the corpus cavernosum smooth muscle cells.Results: Compared to the Control group,the 4%Met and 7%Met groups both showed the lower ratio of SMC to collagen,the lower ?-SMA expression level,the higher TGF-?1,Collagen I and Collagen IV expression levels,the over-activated Rho A/ROCK pathway and the higher ratio of Bax/Bcl-2.Moreover,the 7%Met group showed a more severe change than 4%Met group.However,the h KLK1 in the TGR+7%Met group could reduce the pathological changes in the penis induced by HHcy.Corpus cavernosum smooth muscle cells and endothelial cells were successfully isolated and co-cultured.The c GMP level in the WTR+LMWK group and TGR+LMWK group were higher than WTR group and TGR group,respectively,after treated with LMWK.In addition,compared to the WTR+LMWK(5?M)group,the WTR+LMWK(30?M)and WTR+LMWK(100?M)groups both showed the lower c GMP levels,the over-activated Rho A/ROCK pathway,the higher gp91 phox expression level and Bax/Bcl-2 ratio.While the h KLK1 in the TGR+LMWK(100?M)group could reduce the pathological changes induced by the high Hcy level in the co-culture system.Conclusions: In vivo and in vitro experiments suggested corpus cavernosum smooth muscle dysfunction and increased apoptosis level were induced by HHcy to impair normal erectile function,while h KLK1 could inhibit these pathological changes to play the protective role.