The Effect And Underlying Mechanisms Of S100A1 In Restoring Erectile Function In Diabetic Rat Model With Erectile Dysfunction

Part I Establishment of a diabetic rat model with erectile dysfunction and identification of angiogenic genes involved in the pathogenesis of diabetic erectile dysfunctionObjective:To establish a diabetic rat model with erectile dysfunction(ED),and find out angiogenic genes involved in the pathogenesis of diabetic ED.Methods: A streptozotocin-induced type I diabetes rat model was established.At 8 weeks after diabetes was induced,erectile function was evaluated by observing apomorphine(APO)-induced erection in all surviving rats.Rats that presented an erectile response were marked as APO-positive;otherwise,the result was APO-negative.Subsequently,intracavernosal pressure(ICP)and mean arterial pressure(MAP)was measured by electrical stimulation of the cavernous nerve.Penis samples were harvested to detect the expression of relevant proteins with immunohistochemistry,western blot and immunofluorescence.Global m RNA expression in corpus cavernosum from normal control rats,APO-positive rats and APO-negative rats were measured by using Affymetrix Rat Gene 2.0 ST array.Angiogenic genes were further sifted and expression data was validated in both m RNA and protein levels.Results: Compared with normal control group,the max ICP/MAP in the APO-positive group displayed a slight decrease,while APO-negative group showed a remarkable decrease.The relative expression levels of advanced glycation end products(AGEs),receptor of AGEs(RAGE)and malondialdehyde(MDA)were significantly increased and superoxide dismutase(SOD)was decreased in the diabetic groups,and there were significant differences in these levels between APO-positive and APO-negative groups.When compared with APO-positive rats,the protein levels of endothelial nitric oxide synthease(e NOS)and p-e NOSSer1177 were significantly decreased,down stream release of nitric oxide(NO)and cyclic guanosine monophosphate(c GMP)were also declined in APO-negative rats.The ratio of TUNEL-positive cells to total cells in APO-negative rats was increased compared with APO-positive rats.Conversely,the endothelium content was obviously decreased.Global gene expression alterations were analyzed in the penis in normal control,APO-positive and APO-negative rats.We further considered significant changes in expression as a 1.5-fold difference.As a result,8 genes ultimately were gradually upregulated or downregulated within the groups.Of these genes,1 angiogenic gene(IGFBP-3)was upregulated,and 7 genes(GREM1、CXCL13、S100A1、SMOC1、PDGFD、CREB3L1、F13A1)were downregulated.The differential expression of S100A1 was further validated by q RT-PCR,immunohistochemistry and western blot.In line with the microarray findings,the m RNA and protein levels of S100A1 were gradually downregulated within the groups.Ex vivo study showed that high glucose treatment significantly decreased the m RNA and protein levels of S100A1 in rat aortic endothelial cells.Conclusions: APO testing is necessary to be conducted to confirm ED after the establishment of a diabetic rat model.The decreased expression of S100A1 during hyperglycemia might be important in the development of diabetic ED.Part II The effect and underlying mechanisms of S100A1 in restoring erectile function in diabetic rat model with erectile dysfunctionObjective: To explore whether overexpression of S100A1 could improve erectile function in diabetic rat model with ED.Methods: To overexpress S100A1 ex vivo,we construct a recombinant adenovirus(ad)vector expressing S100A1.The ad-S100A1/ad-GFP was transfected in to rat aortic endothelial cells(RAECs),and cells were assigned to either normal(NG,5.5 mmol/L)or high glucose(HG,30 mmom/L)treatment.After incubation of 2 days,RAECs were treated with dynasore,an inhibitor of endocytosis.After incubation for indicated times,RAECs were collected.A streptozotocin-induced type I diabetes rat model was established.At 8 weeks after diabetes was induced,erectile function was evaluated by observing APO-induced erection in all surviving rats.Only APO-negative rats were used for further experiments.To overexpress S100A1 in vivo,we construct a recombinant adeno-associated virus(AAV)vector expressing S100A1.A 25-gauge insulin syringe was used to administer a single injection of AAV-S100A1/AAV-GFP(1010 parts/70μL)into corpus cavernosum of diabetic ED rats.At 2 weeks after gene therapy,ICP and MAP was measured by electrical stimulation of the cavernous nerve.Subsequently,the corpus cavernosum were collected.Relevant protein expression was detected by inmmunofluorescence and western blot.Results: The upregulation of S100A1 in ad-S100A1 group was validated by q RT-PCR and western blot.In cells treated with ad-S100A1,vascular endothelial growth factor receptor 2(VEGFR2)accumulated and formed many large bright clusters in RAECs which were colocalized with early endosome antigen 1(EEA1).Upregulation of S100A1 induced dramatically increase the phosphorylation levels of VEGFR2(Tyr 1175),protein kinase B(Akt,Ser 473)and endothelial nitric oxide synthase(e NOS,Ser 1177),but had little effect on the total VEGFR2,Akt and e NOS expression.Importantly,the phenomenon including the accumulation of VEGFR2 and colocalization with EEA1,VEGFR2-Akt signaling pathway activation were abrogated in the presence of dynasore,but not e NOS.S100A1 colocalized with e NOS throughout the RAECs,indicating S100A1 might directly interact with e NOS.Additionally,exposure of RAECs to HG increased pro-apoptotic protein expression in HG-treated cells compared with NG-treated cells,and these differences were reduced by ad-S100A1 treatment.In vivo experiments indicated that a single injection of AAV-S100A1 significantly restored erectile function in diabetic rats,compared with an injection of AAV-GFP.S100A1 overexpression significantly promoted VEGFR2 internalization and subsequently triggered the Akt-e NOS pathway in diabetic erectile tissues.Marked increases in nitric oxide,endothelium content,and cyclic guanosine monophosphate were noted in AAV-S100A1-treated diabetic ED rats.Conclusions: S100A1 may play a potential role in restoring erectile function in diabetic rats with ED through(i)enhancing endothelial function by modulating VEGFR2-Akt-e NOS pathway or directly interacting with e NOS,and(ii)promoting endothelial cell survival.