Anti-Fibrotic Properties of Mesenchymal Stem Cells in Partial Bladder Outlet Obstruction

• Mesenchymal stem cells inhibit inflammatory and fibrotic signaling of the partial bladder outlet obstruction and preserves normal bladder histology and function

• Author / Creator

  Wiafe, Bridget

• Partial Bladder Outlet Obstruction (pBOO) is characterized by an initial inflammatory response which progresses to smooth muscle hypertrophy, and fibrosis. The resulting high urine storage pressure significantly damages the bladder wall and poses a risk of renal failure. pBOO is characterized by exaggerated stretch, hydrodynamic pressure, and inflammation which cause significant damage to the bladder wall. Several studies have implicated hypoxia in its pathophysiology. However, the isolated progressive effect of hypoxia on bladder cells is not yet defined. As well, current treatment modalities of pBOO are crude and carry high risk of morbidity. Mesenchymal stem cells (MSCs) are undifferentiated multipotent adult cells with reparative, immunomodulatory, anti-inflammatory, and anti-fibrotic capacities. MSCs therapy is an emerging paradigm with several reported experimental successes. However its mechanism of action is not well understood. The experiments of this thesis were designed to investigate the single stress effects of hypoxia on smooth muscle cells of the bladder and the anti-fibrotic effects of bone marrow-derived MSCs on hypoxia-induced pathways. The in-vitro mechanisms were confirmed in whole animal models. Sub-confluent normal human bladder smooth muscle cells (hbSMC) were cultured in 3% O2 tension for 2, 24, 48, and 72 h. Then, this experiment was repeated 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, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respectively. Twenty Sprague Dawley rats were randomly assigned into 5 groups: unobstructed controls, pBOO for 2 weeks, pBOO for 4 weeks, pBOO + MSCs for 2 weeks, and pBOO + MSCs for 4 weeks. pBOO was surgically induced followed by intravenous injection of MSCs. Endpoint urodynamics was performed and bladder tissues harvested for analysis. RT-PCR and immunohistochemistry were performed to study gene and protein expression of major inflammatory and pro-fibrotic markers. Transcription of hypoxia-inducible factor (HIF) 1α and HIF2α were transiently induced after 2 h of hypoxia (p < 0.05), whereas HIF3 was upregulated after 72 h (p < 0.005). HIF1 and HIF3α proteins were significantly induced after 2 and 72 h, respectively. VEGF mRNA increased significantly after 24 and 72 h (p < 0.005). The inflammatory cytokines, TGFβ1 (protein and mRNA), IL 1β, 1L6, and TNFα (mRNA) demonstrated a time-dependent increased expression. Furthermore, the anti-inflammatory cytokine IL-10 was downregulated after 72 h (p < 0.05). Evidence of smooth muscle cell dedifferentiation included increased αSMA, VIMENTIN, and DESMIN. Evidence of pro-fibrotic changes included increased CTGF, SMAD 2, and SMAD 3 as well as COLLAGENS I, II, III, and IV, FIBRONECTIN, AGGRECAN, and TIMP 1 transcripts (p < 0.05). Total Collagen proteins also increased time-dependently (p < 0.05). Both the 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). The induction of pBOO resulted in an upregulation of TGFβ1, SMAD2/3, HIF1α, HIF3α, VEGF, TNFα, mTOR, p70S6K, COL I, and COL III expression in a time dependent manner. This was coupled with a downregulation of IL-10 expression. Increased bladder collagen deposition was directly related to the duration of pBOO and there was an associated high urine storage pressure. Two weeks after therapy, MSCs immunomodulatory effect (defined by reduced TNFα, increased IL-10 and VEGF) was most predominant. Significant downregulation of the pro-fibrotic genes occurred 4 weeks after therapy. End filling pressure, hypertrophy and fibrosis were significantly reduced after MSCs therapy (p<0.05). Together, the studies described in this thesis demonstrate that hypoxia induced significant inflammatory and fibrotic effects in bladder smooth muscle cells similar to pBOO. Furthermore, this work has elucidated the ability of MSCs to target inflammatory and pro-fibrotic pathways to prevent bladder deterioration due to pBOO. The outcomes of this study are promising and lay an important foundation for the prevention and potential treatment of end stage fibrotic bladder with mesenchymal stem cells.

• Subjects / Keywords

  • Hypoxia

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3DV1D43S

• License

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