J. Luclano1, B. Kautza1, P. Waltz1, B. Zuckerbraun1,2 1University Of Pittsburg,Pittsburgh, PA, USA 2VA Pittsburgh Healthcare System,Pittsburgh, PA, USA
Introduction: Tissue and cellular hypoxia are a component of many disease processes, including shock states and ischemia/reperfusion insults. Heme oxygenase (HO) enzymes, which are the rate limiting enzymes in the breakdown of heme to free iron, biliverdin, and carbon monoxide (CO), are critical to the maintenance of cellular homeostasis. HO enzymes have been implicated in the sensing of oxygen levels and the regulation of oxygen consumption. Additionally, mitochondrial responses are critical to changes in oxygen sensing. Mitochondria act as critical rheostats within a cell to orchestrate cellular responses to various stimuli, including hypoxia. The purpose of these investigations was to test the hypothesis that HO-2 protects againts hemorrhagic shock or hypoxia induced injury. Furthermore that HO-2 serves as a critical regulator of hypoxic responses in hepatocytes via modulation of mitochondrial signaling.
Methods: C57BL/6 male mice were hemorrahged to a MAP of 20 mmg for 30 minutes and then resuscitated with 2X the maximum shed blood volume with lactated Ringers. Some mice were pretreated with control siRNA or HO-2 specific siRNA 72 hours prior to hemorrhage and resuscitation. Primary hepatocytes were harvested and cultured from C57BL/6 or gp91phox-/- mice. Cells were exposed to standard culture conditions (5%CO2, and 21% O2) or hypoxia (5%CO2, 1%O2) for 0-12 hours. In some experiments hepatocytes were pretreated with control siRNA, HO-1 siRNA, or HO-2 siRNA. Additionally, tin protoporphyrin (SnPP) was utilized as a pharmacological inhibitor of HO. Western blots or immunohistochemistry for HO-1, HO-2, hypoxic signaling proteins, mitochondrial fusion pathways, and oxidative phosphorylation proteins were performed. Mitochondrial reactive oxygen species were measured by mitosox fluorescence. ANOVA was used for statistical analysis and significance wsa assumed a s P<0.05.
Results: HO-2 siRNA pretreatment effectively limited expression of HO-2 in the liver and exacerbated organ injury ( serum ALT increased 2.5+/-0.9 fold over control siRNA, shocked mice, P<0.05). Additionally, there were significant increases in serum markers of inflammation (IL-6 and TNF-alpha).HO-2 but not HO-1 was expressed in hepatocytes at baseline and was localized within mitochondria. SnPP or HO-2 siRNA, but not HO-1 siRNA inhibited hypoxia induced mitochondrial ROS. Furthermore, hypoxia increased HIF1a protein levels, and this was inhibited by HO-2 siRNA or SnPP. Furthermore, 12 hours of hypoxia exposure led to increased expression of proteins of oxidative phosphorylation and this was inhibited by HO-2 siRNA.
Conclusion: HO-2 limits organ injury and inflammation in hemorrhagic shock and resuscitation. This may be through the influence on hypoxia induced mitocohndrial signaling. Understanding critical adaptive responses in hypoxic type of insults are critical to impriving care and the development of future therapeutics.