Isolated Hypoxia is Sufficient to Induce an Inflammatory Response in Human Bladder Smooth Muscle Cells and is Reversed with Mesenchymal Stem Cells.
Peter Metcalfe, MD, MSc, FRCSC, Bridget Waife, BSc, MSc, Tom Churchill, BSC. PhD., Adetola Adesida, BSc, PhD..
University of Alberta, Edmonton, AB, Canada.
Introduction: Detrusor decompensation after a partial bladder outlet obstruction (pBOO) is initiated with an inflammatory response. Although stretch has historically been implicated, hypoxia has recently come to the forefront as a significant contributor to the pathophysiology. However, it has been difficult to separate the effect of tissue stress from stretch from the hypoxia secondary to impaired vascular flow.
Furthermore, mesenchymal stem cells (MSC) have demonstrated excellent therapeutic potential in preventing bladder decompensation after pBOO. They have demonstrated potent paracrine anti-inflammatory properties in several in-vivo models.
Therefore we hypothesized that human bladder smooth muscle cells (hbSMC) cultured in hypoxic conditions would display a hypoxic and inflammatory cascade similar to pBOO and that co-culture with MSC would inhibit this response.
Materials and Methods: hbSMC were commercially acquired and maintained in normoxic culture conditions as per manufacturer recommendation. Cells were then moved to hypoxic (3% O2) conditions for 2, 24, 48, or 72 hours. RT-PCR was performed for hypoxic, inflammatory, and fibrotic mediators as well as indicators of epithelial mesenchymal transformation (EMT). ELISA and Western blot were performed to confirm protein levels in accordance with mRNA.
The experiments were then repeated with either a direct or indirect co-culture with MSC. High pore density trans-well culture plates (0.4 um) that prevented direct MSC contact with bladder cells were used as indirect co-culture technique to determine if the inhibition was secondary to direct trophic stimulation or by paracrine extracellular communication.
Results: After hypoxia, there was an immediate, but transient increase in HIF-1 and 2a, but HIF-3a levels rose only after 72 hours of hypoxia. VEGF levels mirrored this with a bi-modal increase in transcription. mRNA for TNF-a, TGF-B, IL-1B, and IL-6 all increased significantly after hypoxia, with the greatest levels seen after 72 hours. aSMA, Vimentin, desmin, and SMAD 2+3 also increased, indicating activation of EMT. Finally, mediators of collagen and fibrosis, such as collagen 1,2,3, 4, TIMP, fibronectin and aggrecan were also significantly increased. ELISA and Western blot confirmed elevation of proteins concordant with mRNA levels.
Both direct and indirect co-culture with MSC were effective in reducing the inflammatory, EMT, and pro-fibrotic gene cascades, whereby almost none of the gene expressions were elevated significantly above controls, and were significantly lower than hbSMC alone. However, HIF-1a and VEGF levels were not significantly affected, and IL-10 was significantly higher than hypoxic hbSMC cultured alone. Indirect co-culture demonstrated similar results.
Conclusions: Isolated hypoxia, without tissue stretch, was sufficient to initiate the hypoxic, inflammatory, EMT, and pre-fibrotic cascades that have been implicated in pBOO. We are the first to report the presence of HIF-3a in the bladder, and its variable response may provide clues to an alternative pathologic pathway. MSCs were very effective in eliminating the inflammatory response, and the effect was not dependent on direct cell contact. This effect was independent of persistent elevations of HIF and VEGF. These experiments provide further insight into the pathophysiology of pBOO and will translate into further experiments with our animal model and progress to human trials.
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