Hypoxia-increased expression of genes involved in inflammation, dedifferentiation, pro-fibrosis, and extracellular matrix remodeling of human bladder smooth muscle cells Bridget Wiafe 1 & Adetola Adesida 1 & Thomas Churchill 1 & Esther Ekpe Adewuyi 1 & Zack Li 1 & Peter Metcalfe 1,2 Received: 26 February 2016 /Accepted: 3 August 2016 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2016 Abstract Partial bladder outlet obstruction (pBOO) is char- acterized 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% O 2 tension for 2, 24, 48, and 72 h. RNA, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respective- ly. 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 in- duced 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 ex- pression. 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 in- cluded 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 ex- pression. This elucidates mechanisms of hypoxia-driven blad- der deterioration in bladder cells, which is important in tailor- ing in vivo experiments and may ultimately translate into im- proved clinical outcomes. Keywords Hypoxia . Inflammation . Cytokines . Dedifferentiation . Fibrotic Introduction Partial bladder outlet obstruction (pBOO) occurs as a sequela of benign prostate hyperplasia, posterior urethral valve dis- ease, urethral stricture disease, cancer, and neural tube defects. The resistance to voiding initially results in a compensatory response, with bladder smooth muscle hypertrophy. However, excessive, prolonged exposure can lead to decompensation (Levin et al. 1990). A myriad of factors including stretch, hypoxia, hydrodynamic pressure, and/or inflammation are in- volved in the etiology of bladder damage. Also, in the aged population, hypoxia has been known to correlate with in- creased urological disorders (Pinggera et al. 2008). In the early development of pBOO, there is a sequential process of inflammation, hypertrophy, and eventually culmi- nating in fibrosis (Metcalfe et al. 2010). Further in its devel- opment, the obstructed hypercontractile bladder is exposed to more frequent yet prolonged cycles of ischemia during the filling and voiding stages (Schröder et al. 2001). Even though many studies have identified hypoxia in the muscle layer of the obstructed bladder, the effects of hypoxia as a sole stressor on bladder injury remain unknown. * Peter Metcalfe pmetcalf@ualberta.ca 1 Department of Surgery, University of Alberta, Edmonton, Alberta, Canada 2 Department of Surgery, Division of Pediatric Urology, University of Alberta Hospital, 2C3.79 WMC 8440-112 Street NW, Edmonton, Alberta T6G 2B7, Canada In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-016-0085-2