Mesenchymal stem cells inhibit hypoxia-induced inflammatory and fibrotic pathways in bladder smooth muscle cells.

Abstract

Partial bladder outlet obstruction is a multifactorial urological condition in which hypoxia plays a significant role. We recently investigated hypoxia's role as a single stressor and found that hypoxia induced an intense inflammatory and profibrotic switch in bladder smooth muscle cells (bSMCs). With the immunomodulatory capacity of mesenchymal stem cells (MSCs), we aimed to investigate if the hypoxia-signaling pathways can be mitigated using MSCs. Bladder smooth muscle cells were cultured in 3% oxygen tension for 72h with either the direct or indirect co-culture with bone marrow derived MSCs. High pore density transwells were used for indirect co-cultures. Total RNA was extracted for gene expression analysis and the Mesoscale multiplex assay was used for secreted cytokines and growth factor measurements. Total collagen contents were determined using the Sirius Red collagen assay. Hypoxia induced increase of HIF3α, VEGF, TGFβ1, TNFα, IL-1β, IL-6, αSMA, and total collagen expression and decreased IL-10 levels in bSMCs. Both direct and indirect MSCs co-cultures inhibited>50% of hypoxia-induced TGFβ1 and IL-6 expression (p<0.005) in a HIF-independent manner. Also, both MSCs co-culture techniques induced>200% increase in IL-10 protein (p<0.005) and inhibited hypoxia-induced αSMA, collagen I and III transcripts as well as total collagen proteins (p<0.0001). Contrastingly, the hypoxia-induced IL-1β and TNFα were inhibited by only the direct co-cultures (p<0.05). MSCs co-culture with bSMCs potently mitigates hypoxia-induced inflammatory and profibrotic pathways. This work has elucidated the role of cell-cell contact and paracrine immunomodulatory mechanisms of MSCs action and opened avenues for therapeutic intervention.