Physiologic Stimulation of Bladder Smooth Muscle Cell Growth is Mediated by Mammalial Target of Rapamycin (Mtor)

Abstract

Purpose Maladaptive bladder muscle overgrowth in human bladder dysfunction is instigated by coordinate responses to mechanical strain, tissue hypoxia, and extracellular matrix (ECM) remodeling. Though mTOR's role in vascular smooth muscle cell (SMC) growth and cardiomyocyte hypertrophy is known, mTOR's role in bladder SMC (BSMC) hypertrophy and hyperplasia is unknown. Rapamycin, a specific inhibitor of mTOR, induces differentiation of vascular SMC, and reverses overgrowth of SMC in vitro and in vivo. Our purpose was to study rapamycin's ability to reverse the de-differentiated phenotype of BSMC during injury. Material and Methods BSMCs were slowly ramped up to 5% mechanical strain on flexible collagen membranes to mimic bladder distension in vivo. A humidified hypoxia chamber was used for BSMC culture at 3%O2 to simulate local tissue hypoxia. To simulate altered ECM induced by stretch and hypoxia, denatured collagen (DNC) was used as substrate and stimulus and compared to native fibrillar collagen. Proliferation was assessed by 3H-thymidine incorporation or cell counting. Real-time PCR and immunofluorescent staining for smooth muscle actin (SMA) assessed BSMC differentiation. Transfections were performed with HA-S6 K wildtype and constutively active mutants. Westerns for proteins of the mTOR pathway were also performed. Results Rapamycin significantly inhibited BSMC proliferation in response to hypoxia (p = 0.0001), DNC, and strain (p = 0.0001) while maintaining basal growth. Strain induced a significant decrease in SMA mRNA, which was restored by rapamycin. Similarly, rapamycin restored a spindle-like contractile morphology in BSMC on DNC. Many components of the mTOR pathway increased in activity post-strain, including S6 K and S6. Forced expression of S6 K resulted in decreased SMA immunofluorescence, as compared to untransfected neighbouring cells. Conclusions Rapamycin inhibition of BSMC growth induced by three coordinate stimuli, which define hypertrophic bladder dysfunction (stretch, hypoxia and altered ECM), demonstrates the potential of mTOR and S6 K as targets of therapy for maladaptive BSMC hyperplasia and hypertrophy.