M. Sherman1, M. V. Suresh1, D. Machado-Aranda1, B. Thomas1, N. Talarico1, Y. Shah2, K. Raghavendran1 1University Of Michigan,Acute Care Surgery/General Surgery,Ann Arbor, MI, USA 2University Of Michigan,Molecular Biology And Integrative Physiology,Ann Arbor, MI, USA
Introduction: Lung contusion (LC) due to major thoracic trauma is a significant risk factor for the development of Acute Respiratory Distress Syndrome. The main pathological consequence of LC is hypoxia. The key mediator of adaptation to hypoxia is hypoxia-inducible factor (HIF)-1. We have recently published that a key driver of acute inflammation following LC is HIF-1α. This is based on significant reductions in lung injury and inflammation observed in type II alveolar epithelial cells (AEC) of HIF-1α conditional knockout mice. To better understand the regulation of hypoxic AEC, the current project evaluated the fate of hypoxic AEC following LC.
Methods: LC was induced using a cortical contusion impactor in WT C57Bl6 and ODD-Luc (oxygen dependent domain of HIF linked to luciferase) mice. In the WT mice, global hypoxia was confirmed by pimonidazole staining after LC. In the ODD-Luc mice, subsequent IVIS imaging was performed to assess hypoxia. To evaluate the fate of hypoxic AEC (high luciferase activity), dual staining with caspase-3, TUNEL staining (apoptosis), and Ki67 (proliferation marker) were performed. Finally, laser capture microdissection (LCM) was used to isolate hypoxic and normoxic cells after LC at 24 h, yielding adequate quantities of RNA that were subjected to real-time PCR.
Results: Using Hypoxyprobe (pimonidazole), the presence of global hypoxia with LC was confirmed in the WT mice. Lung, liver, and spleen samples ODD-Luc mice showed increased luminescence at all-time points after LC. Presence of hypoxia in AEC was not uniform. Most of the AEC that were hypoxic following LC showed evidence of caspase-3 activation. Similar results were observed with TUNEL staining. Also, the hypoxic AEC did not show a significant increase in proliferation (ki67). Using directly conjugated fluorescent-tagged luciferase antibody, hypoxic cells were visualized and isolated by LCM. Figure 1 shows the representative immuno-fluorescent staining for luciferase positive hypoxic cells (red circles, Luc+) and the adjacent normoxic cells (blue circles, Luc-) in lung samples in ODD-Luc mice 24 h after LC as well as before and after LCM. Isolated RNA also showed significant increases in VEGFa and caspase-1 in hypoxic AEC compared to normoxic AEC.
Conclusion: Hypoxic AEC following LC specifically undergo apoptosis. Moreover, the hypoxic AEC have increased VEGFa and caspase-1 activation, which confirms our previous finding that HIF-1α regulation of AEC is likely mediated through inflammasome activation.
