| Part I Novel noninvasive quantification of 2-D Shear Wave? Elastography for Penile Corpus Cavernosum lesions in Hyperlipidemia Induced Erectile Dysfunction Rabbits.Objective: Biopsy is currently the only method to reveal the histological lesions of penile corpus cavernosum(CC)in the clinical diagnosis of erectile dysfunction(ED).Our study evaluated the effectiveness of noninvasive two-dimensional Shear Wave? Elastography(2-D SWE)in quantifying penile CC lesions in hyperlipidemia induced ED rabbits.Materials and methods: A total of 12 New Zealand White rabbits were divided into two groups randomly.Six of them were fed a high-cholesterol diet containing 2% cholesterol and 8.5% lard and 7.5% yolk powder for 10 weeks and others were fed normal diet as control.Ultrafast ultrasound device-Aixplorer? was used to measure the shear wave elastic quantitative(SWQ)value of penile CC.Erectile function was investigated by papaverine intracavernous injection and immunohistochemical staining and Western Blot assessment were performed to determine the smooth muscle cells(SMCs)phenotypic transition and extracellular matrix(ECM)accumulation.Results: After 10 weeks,high cholesterol diet fed rabbits showed worse lipid profiles compared to control group.The SWQ values obtained from penile CC in hyperlipidemia group were remarkably higher than that of control group and the ?ICP/MAP was decreased dramatically.In accordance,histological examination showed numerous empty cellular structures like adipocytes in the penile CC of hyperlipidemia group.Immuohistochemical staining and Westerm blot assessments demonstrated ECM accumulation in CC SMCs.Also,the SMC phenotypic transition was affected as indicated by the reduction in ?-SMA,calponin-1 and increase in p-MLC20,osteopontin expression.Conclusion: Hyperlipidemia resulted in ECM accumulation accompanied with SMCs phenotypic transition in penile CC and impaired the erectile function eventually.These might in turn lead to variations in the SWQ values.These suggested that 2-D SWE is a novel,noninvasive and effective approach that distinguishes hyperlipidemia induced ED penile CC pathologies from normal.Part II Fasudil,a Rho A/Rho kinase inhibitor,attenuates Penile Corpus Cavernosum lesions and improves erectile function in Hyperlipidemic Rabbits.Objective: Hyperlipidemia can cause ED by impairing the SMCs of penile CC.We use fasudil,a Rho A/Rho kinase inhibitor,to treat the hyperlipidemia induced ED rabbits and evaluate its effectiveness in improving erectile function.Materials and methods: A total of 27 New Zealand White rabbits aged 14~16 weeks were divided into three groups randomly.The control group(CO)were fed with normal diet.Hyperlipidemia group(HL)and Fasudil treatment group(FS)were fed a highcholesterol diet containing 2% cholesterol,8.5% lard and 7.5% yolk powder for 10 weeks.At the end of the 8th week,FS group were administered with fasudil hydrochloride injection intravenously once daily for 2 weeks(Dosage: 1mg/kg,diluted with 100 ml 0.9%Na Cl).At the end of 10 th weeks,penile CC strips were obtained from three rabbits of each group.The relaxation responses were investigated in an organ bath.Erectile function was assessed by papaverine intracavernous injection in the remain and Immuohistochemical staining and Western Blot assessment were performed to determine the activity of Rho A/Rho kinase pathway,SMCs apoptosis,SMCs phenotypic transition and ECM accumulation.Results: One rabbit of FS group died during the high cholesterol diet fed and the data was eliminated.At the end of 10 th week,HL(n=9)and FS(n=8)groups showed worse lipid profiles compared to CO group(n=9)and there was no difference between the HL and FS group.The relaxation responses to gradient concentration of fasudil(0.01,0.1,1,10,100?mol/L)were compared among the three groups.There was no difference between HL(n=3)and FS(n=3)groups(-0.01±0.03% Vs 0.03±0.04%,-0.06±0.05% Vs 0.00±0.00%,-0.04±0.06% Vs 0.02±0.05%,0.16±0.02% Vs 0.22±0.04%,0.45±0.04% Vs 0.64±0.08%,respectively,p>0.05).However,the relaxation responses of both groups were worse than that of the CO group(0.04±0.03%?0.09±0.02%?0.25±0.05%?0.34±0.06%?0.66±0.01%).ICP measurement showed that the ?ICP/MAP of HL(n=6)decreased dramatically compared to the CO group(n=6)(0.17±0.02 Vs 0.40±0.04,p<0.01).Erectile function restored in FS(n=5),that ?ICP/MAP was significant higher than that of HL(?ICP/MAP:0.31±0.30 Vs 0.17±0.02,p<0.01).Immuohistochemical staining and Western Blot assessments demonstrated ROCK-2 protein expression were remarkably higher in HL than that of FS and CO(p<0.01),indicating the activation of Rho A/Rho kinase pathway in hyperlipidemic rabbits' penile CC without Fasudil inhibition.Compared to the CO group,the increase of Caspase-3 and PARP protein expression demonstrated significant SMCs apoptosis in HL(p<0.01)and FS(p<0.05).As to the ECM accumulation,TGF-? protein expression and Collagen?/? ratio of HL increased dramatically compared to the CO(p<0.01).TGF-? protein expression decreased in FS(p<0.01),but Collagen?/? ratio was still higher than CO(p<0.01)with no statistical differences compared to the HL,although the values decreased.Furthermore,SMCs phenotypic transition was affected as indicated by the reduction in ?-SMA,calponin-1 and increase in p-MLC20,osteopontin expression in HL compared to the CO(p<0.01)and Fasudil ameliorated this lesion(p<0.01).Conclusion: Hyperlipidemia resulted in Rho A/Rho kinase pathway activation in penile CC.Lesions of SMCs apoptosis,ECM accumulation and SMCs phenotypic transition in penile CC can impair the erectile function and lead to ED eventually.Chronical inhibition by fasudil administration attenuates these lesions and improves erectile function,indicating that Rho A/Rho kinase pathway might play an important role in the pathological process of ED.Part III Rho A/Rho kinase activity affects hypoxia induces autophagy in penile corpus cavernosum smooth muscle cells.Objective: Hypoxia causes ED through multiple factors such as neuropathy,endocrine hormones and cell metabolism disorder,etc,of which the lesions of penile CC SMCs are essential.Autophagy is a dynamic,self-catabolic process that has been implicated in a wide range of physiological processes and the pathogenesis of diverse diseases.We conducted this study to investigate the role of Rho A/Rho kinase pathway in hypoxia induced autophagy in penile CC SMCs.Materials and methods: A 6 months-old,sexual mature male New Zealand White rabbit was sacrificed and the SMCs from penile CC were obtained for culture in vitro.The primary cultured cells were divided into 5 groups: 1)Control group,incubated in normal oxygen concentration(21%O2+5%CO2+74%N2,37?);2)Hypoxia group,incubated under hypoxia condition(1%O2+5%CO2+94%N2,37?);3)Hypoxia/Vector group,cells transfected with plasmid;4)Hypoxia/Rho A+ group,cells transfected with Rho A overexpression plasmid;5)Hypoxia/Rho A-group,cells transfected with Rho A knockdown plasmid.Group 3~5 were all incubated in the same hypoxia condition as group 2 when plasmids successfully transfected.After 48 hours incubation,all SMCs were harvested for assessment of autophagy-associated markers by RT-PCR and Western Blot assessment.Results: Hypoxia induced an activation of Rho A/Rho kinase pathway in penile CC SMCs in vitro.RT-PCR and Western Blot assessment demonstrated the Rho A expression were significantly higher in group 2)and group 3)than that of group 1),both in m RNA and protein level(p<0.05).The Rho A regulated plasma transfection successfully interfere the Rho A/Rho kinase pathway activity.The Rho A m RNA and protein expression increased dramatically in group 4)compared to the other 4 groups(p<0.01),while decreased remarkably in group 5)(p<0.01).Furthermore,autophagy occurred under hypoxia conditions as m RNA of Beclin-1,Atg5 and Atg7 in group 2)~5)were significant higher that group 1(p<0.05).Up-regulation of Rho A could decrease the m RNA expression of Beclin-1 compared to other 4 groups(p<0.01),while inhibition of Rho A increased Beclin-1 and decreased Atg5 and Atg7 m RNA expression(p<0.05).It could also be demonstrated in Western Blot analysis that compared to group 1),hypoxia increased the protein expression of LC3?/?,Beclin-1,Atg5 and Atg7(p<0.05).Up-regulation of Rho A increased and deceased Beclin-1 and Agt7 protein expression(p<0.05),while inhibition of Rho A showed opposite results and incresed LC3 ?/? ratio(p<0.05).Conclusion: Hypoxia induced autophagy in penile CC SMCs,which could be affected by the regulation of Rho A/Rho kinase pathway activity.These results provide a novel insight into the pathogenesis of penile CC SMCs and give evidence for the occurrence of Rho A/Rho kinase mediated autophagy in ED. |
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