Mechanisms Of Extracellular Matrix Stiffness Affecting Urethral Stricture Progression By Regulating Igfbp3

PurposeUrethral stricture refers to irreversible fibrosis of the urethra,and its incidence is increasing,which seriously threatens national health.The treatment of urethral stricture is still a major problem for urologists,and its easy recurrence makes the prognosis of urethral stricture even less optimistic.The process of urethral stricture is a process of increased stiffness of the extracellular matrix.With the increase of extracellular matrix stiffness,fibroblasts are activated and promote fibrosis,however,there is no clear researches about the mechanisms of extracellular matrix stiffness effecting urethral fibroblasts.This study aimed to investigate the effects of extracellular matrix stiffness on urethral fibroblasts and their effects on urethral strictures,found out possible pathways and make contributions to the treatment of urethral stricture.Method1.Polyacrylamide hydrogels with different matrix stiffness were used to culture the rat urethral primary fibroblasts(2 k Pa,16 k Pa and 32 k Pa)for 48 h to explore the effects of fibroblast to myofibroblast transition(FMT)and fibrosis indexes of rat urethral primary fibroblasts.2.The rat urethral stricture model was established by injuring the rat urethra,and whether the rat urethral stricture model was successfully established was detected by B-mode ultrasonography,urethrography,hematoxylin-eosin staining(H&E)and Masson staining.Atomic force microscope(AFM)was used to quantify the matrix stiffness of normal urethra and stricture urethra tissue in rats,and measure the matrix stiffness of normal urethra and stricture urethra tissue in humans.3.The polyacrylamide hydrogels corresponding to the matrix stiffness of normal urethra and stricture urethra in rats were made,and the extracted rat urethral primary fibroblasts were cultured on the hydrogels for transcriptomic analysis.4.To explore whether the extracellular matrix stiffness causes FMT and fibrosis progress of urethral fibroblasts through the Igfbp3/smad pathway,and further verify the effect of Igfbp3/smad pathway of matrix stiffness affecting urethral fibroblasts by constructing Igfbp3 overexpressing lentivirus and si RNA.5.The expression of Igfbp3/smad pathways protein in rat and human normal urethral tissue and stricture urethral tissue was further detected by immunohistochemical method.Results1.Rat urethral primary fibroblasts were cultured on polyacrylamide hydrogels with different matrix stiffness for 48 h.With the increase of extracellular matrix stiffness,fibroblasts stretched more and protruded pseudopodia,their fibrosis index α-smooth muscle actin(α-SMA)and Collagen I increased,and the migration ability was enhanced.2.Compared with normal urethra,B-mode ultrasonography showed that the urethral stricture tissue with lower echo than normal urethra,and urethrography showed contrast agent defect in urethral stricture.H&E staining showed that the epithelium of the urethra in the control group was intact and the cells were neatly arranged.In the urethral stricture group,the urethral structure was disordered,and inflammatory cell infiltration,connective tissue deposition and fibrous tissue proliferation were also observed.Masson staining showed that the urethral structure was disordered and collagen deposition in the stricture group,indicating that the model of rat urethral stricture was successful.The matrix stiffness of normal urethra measured by AFM was 4.29 k Pa,17.87 k Pa for 2weeks after urethral stricture modeling,and 32.94 k Pa for 4 weeks after modeling in rats.The matrix stiffness of normal human urethra was 5.23 k Pa,and the matrix stiffness of stricture urethra was 41.58 k Pa.3.A total of 246 differential m RNAs were detected ty transcriptomics analysis,and we found the dissonance of the insulin-like growth factor(IGF)system and Igfbp3 molecules may play a role in extracellular matrix stiffness-induced urethral fibrosis.4.With the increase of extracellular matrix stiffness,Igfbp3,p-smad2 and p-smad3 of rat urethral primary fibroblasts increased,smad2 and smad3 remained basically unchanged,fibrosis indexes α-SMA and Collagen I increased,and migration ability strengthened.The above effects can be enhanced by overexpression of Igfbp3 and attenuated by knockdown Igfbp3.5.In normal urethra and urethral stricture tissue of rats and humans,immunohistochemical staining showed that Igfbp3,p-smad2 and p-smad3 increased in urethral stricture tissue compared with normal urethra,while smad2 and smad3 were not significantly changed.ConclusionIncreased extracellular matrix stiffness activates the Igfbp3/smad pathway to promote the FMT process of urethral fibroblasts,increase the expression of fibrosis-related markers,and promote the progression of urethral fibrosis.The progressive fibrosis process in turn increases the matrix stiffness,thereby further promoting the fibrosis process.