Indiana University-Purdue University Indianapolis (IUPUI) / Rationale: CD55 down-regulation on airway epithelium correlates with local complement activation observed in hypoxia-associated pulmonary diseases. Therefore, we hypothesized that induction of hypoxia inducible factor 1 alpha (HIF-1α) in hypoxic airway epithelium, mediates CD55 down-regulation.
Methods: Chetomin and HIF-1α siRNA inhibited HIF-1α in hypoxic SAECs (1% O2), and mice lungs (10% O2). DMOG mediated HIF-1α stabilization in normoxic SAECs and mice lungs (21% O2). Transduction of SAECs with AdCA5 also stabilized HIF-1α. CD55 and CA9 transcripts were measured by RT-PCR. CD55 and HIF-1α protein expression was assessed by western blots. In vivo, immunohistochemistry (IHC) confirmed CD55 and HIF-1α expression. C3a and C5a levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA.
Results: HIF-1α was induced in 6 hour hypoxic SAECs (p<0.05), but CD55 transcripts were repressed (p<0.05). CD55 protein was down-regulated by 72 hours (p<0.05). CA9 transcripts were elevated by 48 -72 hours (p<0.05 and p<0.01, respectively). In vivo, CD55 transcripts and protein were down- regulated by 24 hours post-hypoxia (p<0.01) which corresponded to complement activation (p<0.05) in BALF. However, CA9 was increased (p<0.01). Chetomin (100nM) treatment in 6 hour hypoxic SAECs, recovered CD55 transcripts (p<0.01) and protein (p<0.05), but down-regulated CA9 (p<0.05). Similarly, in vivo chetomin (1mg/ml) treatment recovered CD55 protein (p<0.01) and down-regulated CA9 (p<0.01). Silencing HIF-1α (50nM) in hypoxic SAECs restored CD55 transcripts by 6 hours (p<0.05), and protein expression by 24 hours (p<0.05). However, CA9 was repressed (p<0.01). In vivo silencing of HIF-1α (50µg) restored CD55 protein expression (p<0.05) but down-regulated CA9 (p<0.05). Stabilizing HIF-1α in normoxic SAECs via DMOG (1µM), down-regulated CD55 transcripts and protein (p<0.01), but increased CA9 within 6-24 hours (p<0.05 and p<0.01, respectively). HIF-1α induction by DMOG (1mg/ml) in normoxic mice lungs down-regulated CD55 transcripts (p<0.01) and protein (p<0.01), but increased CA9 (p<0.05). Induction of HIF-1α in AdCA5 (50 PFUs/cell) transduced normoxic SAECs, resulted in CD55 protein down-regulation (p<0.05), but increased CA9 (p<0.001).
Conclusions: HIF-1α down-regulates CD55 on airway epithelium. Targeting this mechanism may be a potential therapeutic intervention for attenuating complement activation in hypoxic pulmonary diseases.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/8002 |
| Date | 08 June 2015 |
| Creators | Pandya, Pankita Hemant |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
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