Elucidation of inflammatory-fibrotic microenvironment in human urethral stricture disease

Abstract (248 words) Background: Urethral stricture disease (USD) is marked by inflammation with subsequent tissue fibrosis that disrupts urinary flow. Recent studies suggest lower urinary tract biome dysbiosis increases proinflammatory cytokines, impeding tissue repair as well as promoting fibrotic scarring of the urethra. Despite these insights, the comprehensive molecular mechanisms underlying the formation, progression, and recurrence of the USD remain unclear. We aim to identify novel molecular targets and designate pathways contributing to USD, thereby identifying potential targets for future therapeutic interventions. Methods: We performed a comprehensive multi-modal analysis in USD patient urine and scar tissues. Urinary cytokines were assayed, transcriptome profiling and pathway enrichment were performed, and network mapping identified fibrotic hubs. Biomechanical tissue stiffness was quantified by atomic force microscopy (AFM), and protein and histological validation employed both immunostaining and Western blotting. Results: IL-1β, IL-6, IL-8, TNF-β, IL-10 were significantly elevated in USD. Transcriptomic profiling revealed 745 differentially expressed genes, with enrichment in ECM organization, cytokine storm, TGF-β, integrin, IL-17A, WNT/β-catenin. ECM organization and cytokine storm are identified as central interactive hubs, dominated by collagen and fibrogenic regulators such as SMAD3 and TGF-β1. Tissue stiffness increased threefold in USD tissues compared to controls, highlighting ECM remodeling. Immunostaining demonstrated enhanced vimentin, elastin, COL1, and TGF-β expression, along with Western blot studies validating elevated protein levels. Conclusion: The integrative approach evaluating pathway enrichment with transcriptome profiling reveals novel interconnections between mechanisms of inflammation, matrix remodeling, and mechanotransduction driving USD. The findings provide a mechanistic foundation for individualized models and therapeutic targets. Graphical abstract