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Hypoxia-increased expression of genes involved in inflammation, dedifferentiation, pro-fibrosis, and extracellular matrix remodeling of human bladder smooth muscle cells

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Abstract

Partial bladder outlet obstruction (pBOO) is characterized by exaggerated stretch, hydrodynamic pressure, and inflammation which cause significant damage and fibrosis to the bladder wall. Several studies have implicated hypoxia in its pathophysiology. However, the isolated progressive effects of hypoxia on bladder cells are not yet defined. Sub-confluent normal human bladder smooth muscle cells (hbSMC) were cultured in 3% O2 tension for 2, 24, 48, and 72 h. RNA, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respectively. 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, TGFB (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 1, 2, 3, and 4, fibronectin, aggrecan, and TIMP 1 transcripts (p < 0.05). Total collagen proteins also increased time-dependently (p < 0.05). Together, these results show that exposure of hbSMC to low oxygen tension results in intense hypoxic cascade, including inflammation, de-differentiation, pro-fibrotic changes, and increased extracellular matrix expression. This elucidates mechanisms of hypoxia-driven bladder deterioration in bladder cells, which is important in tailoring in vivo experiments and may ultimately translate into improved clinical outcomes.

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Acknowledgments

We acknowledge the Northern Alberta Urology Foundation for funding this project.

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Authors and Affiliations

  1. Department of Surgery, University of Alberta, Edmonton, Alberta, Canada

    Bridget Wiafe, Adetola Adesida, Thomas Churchill, Esther Ekpe Adewuyi, Zack Li & Peter Metcalfe

  2. Department of Surgery, Division of Pediatric Urology, University of Alberta Hospital, 2C3.79 WMC 8440-112 Street NW, Edmonton, Alberta, T6G 2B7, Canada

    Peter Metcalfe

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  1. Bridget Wiafe
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  2. Adetola Adesida
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  3. Thomas Churchill
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  4. Esther Ekpe Adewuyi
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  5. Zack Li
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  6. Peter Metcalfe
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Correspondence to Peter Metcalfe.

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Editor: Tetsuji Okamoto

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Wiafe, B., Adesida, A., Churchill, T. et al. Hypoxia-increased expression of genes involved in inflammation, dedifferentiation, pro-fibrosis, and extracellular matrix remodeling of human bladder smooth muscle cells. In Vitro Cell.Dev.Biol.-Animal 53, 58–66 (2017). https://doi.org/10.1007/s11626-016-0085-2

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  • DOI: https://doi.org/10.1007/s11626-016-0085-2

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