Department of Urology, San Diego VA Health Care System and University of California, San Diego, California.
Department of Surgery, San Diego VA Health Care System and University of California, San Diego, California.
Neurourol Urodyn. 2020 Feb;39(2):625-632. doi: 10.1002/nau.24281. Epub 2020 Jan 21.
To elucidate the precise cellular and molecular mechanisms that underlie urethral fibrogenesis.
Endoluminal electrocautery injury (using Karl Storz 10 Fr. Pediatric urethroscope) was employed in male rabbits (n = 6) to create mucosal injury. Retrograde urethrogram (RUG) and endoluminal ultrasound techniques were used to assess severity and changes in luminal cross-sectional area. Six control rabbits were subjected to sham injury, in which the electrocautery was inserted but not powered. Urethral tissues were harvested 30 days postinjury and subjected to RNA sequencing and quantitative polymerase chain reaction (qPCR) to determine changes in gene expression. Histological, immunostaining, and Western blot studies were used to determine changes in protein expression of known markers of fibrosis (eg, collagen, Integrinαv, GIV/Girdin, transforming growth factor-β (TGF-β), and pSMAD1,2,3).
Trichrome staining confirmed increased connective tissue in urethral scar tissues. Immunostaining revealed a potential role for epithelial to mesenchymal cell transition (EMT) and positive labeling for all fibrotic markers (eg, collagen-1, Integrin αv, GIV/Girdin, transforming growth factor-β (TGF-β), and SMAD1,2,3). Western blot analysis confirmed increased protein levels of these fibrotic markers.
Our RNA sequencing and qPCR studies, in conjunction with our protein data, suggest that urethral mucosal fibrogenesis may be mediated by novel fibrogenic signaling pathways involving Wnt-β catenin, TGF-β, GIV/Girdin, and EMT which lead to increased collagen deposition. Therapeutic strategies targeting these pathways may be beneficial in attenuating fibrogenesis and stricture progression.
阐明尿道纤维化形成的确切细胞和分子机制。
采用腔内电灼损伤（使用 Karl Storz 10Fr. 小儿尿道镜）对雄性兔（n=6）进行尿道黏膜损伤。逆行尿道造影（RUG）和腔内超声技术用于评估严重程度和管腔横截面积的变化。6 只对照兔接受假损伤，电灼器插入但未通电。损伤后 30 天采集尿道组织，进行 RNA 测序和定量聚合酶链反应（qPCR），以确定基因表达的变化。组织学、免疫染色和 Western blot 研究用于确定纤维化已知标志物（如胶原蛋白、整合素αv、GIV/Girdin、转化生长因子-β（TGF-β）和 pSMAD1、2、3）的蛋白表达变化。
三色染色证实尿道瘢痕组织中结缔组织增加。免疫染色显示上皮间质细胞转化（EMT）和所有纤维化标志物（如胶原蛋白-1、整合素αv、GIV/Girdin、转化生长因子-β（TGF-β）和 SMAD1、2、3）的阳性标记具有潜在作用。Western blot 分析证实这些纤维化标志物的蛋白水平增加。
我们的 RNA 测序和 qPCR 研究结合我们的蛋白数据表明，尿道黏膜纤维化可能是由涉及 Wnt-β 连环蛋白、TGF-β、GIV/Girdin 和 EMT 的新型纤维化信号通路介导的，导致胶原沉积增加。针对这些途径的治疗策略可能有益于减轻纤维化和狭窄进展。
