Slit2/Robo Signaling Restores Diabetic Erectile Function via Neurovascular Remodeling

BACKGROUND: Diabetes mellitus-induced erectile dysfunction (DMED) is a common and debilitating vascular-neurogenic complication of diabetes. The poor responsiveness of DMED patients to phosphodiesterase type-5 inhibitors underscores the need for therapies capable of restoring both endothelial integrity and cavernous nerve function. The Slit2/Roundabout (Robo) pathway, a conserved regulator of axon guidance and vascular remodeling, is well positioned to coordinate neurovascular repair; however, its involvement in DMED remains largely undefined. METHODS: Single-cell RNA sequencing from embryonic genital tubercle to postnatal stages was used to map fibroblast-endothelial-neuronal signaling and identify Slit2 as a key secreted ligand. Diabetic and control mouse corpus cavernosum tissues were analyzed for endothelial and neuronal Slit2-Robo1/4 activity. In vitro, recombinant Slit2-N was applied to cells under high glucose to assess proliferation, migration, tube/sprout formation, and neurite outgrowth; Robo1/4 knockdown tested receptor dependency. In vivo, AAV-Slit2 was delivered intracavernously to diabetic mice, with erectile function assessed by sodium-nitroprusside perfusion and electrostimulation, and structural remodeling evaluated by endothelial, neural, and fibrosis markers. RESULTS: Single-cell profiling identified Slit2 as a fibroblast-derived developmental cue for corpus cavernosum morphogenesis. Diabetic mice exhibited markedly reduced endothelial and neuronal Slit2-Robo signaling. Slit2-N restored angiogenic and neuroregenerative functions in vitro under high glucose, enhancing endothelial activity and neurite growth; all effects were lost after Robo1/4 silencing. In vivo, AAV-Slit2 re-established local Slit2 signaling, improved penile perfusion and erectile responses, enhanced endothelial proliferation and nerve regeneration, and reduced fibrosis, indicating substantial neurovascular recovery. CONCLUSIONS: Slit2/Robo signaling is a key regulatory axis disrupted in DMED. Local restoration of Slit2 via AAV delivery drives synchronized neurovascular regeneration and effectively rescues erectile function in diabetic mice. These findings establish Slit2 as a promising therapeutic target for reversing the underlying pathophysiology of DMED rather than merely providing symptomatic relief.