Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury.

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1ß mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.

HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury.

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1ß, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1ß mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Trauma-hemorrhagic shock-induced pulmonary epithelial and endothelial cell injury utilizes different programmed cell death signaling pathways.

Intestinal ischemia after trauma-hemorrhagic shock (T/HS) results in gut barrier dysfunction and the production/release of biologically active and tissue injurious factors in the mesenteric lymph, which, in turn, causes acute lung injury and a systemic inflammatory state. Since T/HS-induced lung injury is associated with pulmonary endothelial and epithelial cell programmed cell death (PCD) and was abrogated by mesenteric lymph duct ligation, we sought to investigate the cellular pathways involved. Compared with trauma-sham shock (T/SS) rats, a significant increase in caspase-3 and M30 expression was detected in the pulmonary epithelial cells undergoing PCD, whereas apoptosis-inducing factor (AIF), but not caspase-3, was detected in endothelial cells undergoing PCD. This AIF-mediated pulmonary endothelial PCD response was validated in an in situ femoral vein assay where endothelial cells were found to express AIF but not caspase-3. To complement these studies, human umbilical vein endothelial cell (HUVEC), human lung microvascular endothelial cell (HLMEC), and human alveolar type II epithelial cell (A549) lines were used as in vitro models. T/HS lymph induced the nuclear translocation of AIF in HUVEC and HLMEC, and caspase inhibition in these cells did not afford any cytoprotection. For proof of principle, AIF silencing in HUVEC reversed the cytotoxic effects of T/HS on cell viability and DNA fragmentation. In A549 cells, T/HS lymph activated caspase-3-mediated apoptosis, which was partially abrogated by N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). Additionally, T/HS lymph did not cause the nuclear translocation of AIF in A549 cells. Collectively, T/HS-induced pulmonary endothelial PCD occurs via an AIF-dependent caspase-independent pathway, whereas epithelial cells undergo apoptosis by a caspase-dependent pathway.

Intravenous injection of trauma-hemorrhagic shock mesenteric lymph causes lung injury that is dependent upon activation of the inducible nitric oxide synthase pathway.

OBJECTIVE: To test the hypothesis that gut-derived factors carried in trauma-hemorrhagic shock (T/HS) lymph is sufficient to induce lung injury. Additionally, because our previous studies showed that T/HS-induced nitric oxide production was associated with lung injury, we examined whether T/HS lymph-induced lung injury occurs via an inducible nitric oxide synthase (iNOS)-dependent pathway. BACKGROUND: We have previously shown that T/HS-induced lung injury is mediated by gut-derived humoral factors carried in the mesenteric lymph. However, it remains unclear whether T/HS lymph itself is sufficient to induce lung injury, or requires the activation of other factors during the T/HS period to exert its effect. METHODS: Mesenteric lymph collected from T/HS or trauma-sham shock (T/SS) animals was injected intravenously into male rats at a rate of 1 mL/h for 3 hours. At the end of infusion, lung injury was assessed by lung permeability and lung histology. The effect of iNOS inhibition on T/HS lymph-induced lung injury was studied and this was further confirmed in iNOS knockout mice. Finally, iNOS immunohistochemistry was performed to identify the cells of origin of iNOS. RESULTS: The injection of T/HS lymph, but not sham shock lymph, caused lung injury. This was associated with increased plasma nitrite/nitrate levels as well as induction of iNOS protein in the lung, liver, and gut. Treatment with the selective iNOS inhibitor aminoguanidine prevented T/HS lymph-induced lung injury. iNOS knockout mice, but not their wild-type controls, were resistant to T/HS lymph-induced lung injury. By immunohistochemistry, neutrophils and macrophages, rather than parenchymal cells, were the source of T/HS lymph-induced lung iNOS. CONCLUSIONS: These results indicate that T/HS lymph is sufficient to induce acute lung injury and that lymph-induced lung injury occurs via an iNOS-dependent pathway.

Molecular signatures of trauma-hemorrhagic shock-induced lung injury: hemorrhage- and injury-associated genes.

The etiology of trauma-hemorrhagic shock (T/HS)-induced acute lung injury has been difficult to elucidate because of, at least in part, the inability of in vivo studies to separate the noninjurious pulmonary effects of trauma-hemorrhage from the tissue-injurious ones. To circumvent this in vivo limitation, we used a model of T/HS in which T/HS lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the noninjurious systemic response to T/HS by comparing the pulmonary molecular responses of rats subjected to T/HS, which did and did not develop lung injury, with those of nonshocked rats. Using high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 h after the end of the shock or sham-shock period, 139 of 8,799 assessed genes were identified by significant analysis of microarrays. Hemorrhage without the secondary effects of lung injury modulated the expression of 21 genes such as interleukin 1beta, metallothionein-2, and myeloctomatosis oncogene (c-myc). In response to injury, 42 genes were identified to be differentially expressed. Upregulated genes included the L1 retroposon and guanine deaminase, whereas downregulated genes included catalase and superoxide dismutase 1. Real-time polymerase chain reaction confirmed the differential expression for selected genes. PathwayAssist analysis identified interleukin 1beta as a central regulator of two subpathways of stress response-related genes (c-myc and superoxide dismutase 1/catalase) as well as several unrelated genes such as lipoprotein lipase. Our model system provided a unique opportunity to distinguish the molecular changes associated with T/HS-induced acute lung injury from the systemic molecular response to T/HS.

Persistent HIF-1alpha activation in gut ischemia/reperfusion injury: potential role of bacteria and lipopolysaccharide.

In both animal models of hemorrhagic shock and clinical settings, shock-induced gut ischemia has been implicated in the development of the systemic inflammatory response syndrome and distant organ injury, yet the factors transducing these events remain to be fully determined. Because hypoxia-inducible factor (HIF-1), a transcription factor composed of oxygen-labile HIF-1alpha and constitutive HIF-1beta subunits, regulates the physiologic/pathophysiologic response to hypoxia and ischemia, we examined the HIF-1 response in two rat models of gut ischemia-reperfusion. We found that ileal nuclear HIF-1alpha protein levels were induced in rats subjected to trauma (laparotomy) plus hemorrhagic shock for 90 min relative to their trauma sham-shock and naïve counterparts and that this trauma hemorrhagic shock-induced mucosal HIF-1alpha protein response persisted after 1 h and 3 h of reperfusion. Likewise, in a model of isolated gut ischemia-reperfusion injury, where the superior mesenteric artery was occluded for 45 min, nuclear HIF-1alpha were induced in the gut mucosa relative to their sham counterparts and persisted after 1 h and 3 h or reperfusion. Similar to the in vivo response, in vitro hypoxia induced HIF-alpha expression in three different enterocyte cell lines (rat IEC-6 and human Caco-2 and HT-29 cell lines). However, in contrast to the in vivo response, HIF-1 expression rapidly disappeared on subsequent reoxygenation. Because in vivo enterocytes are exposed to bacteria, we tested whether the in vitro HIF-1alpha response would persist on reoxygenation if the enterocytes were cocultured with bacteria. P. aeruginosa, an enteric bacterium, markedly induced enterocyte HIF-1alpha protein levels under normoxic conditions. Furthermore, the addition of P. aeruginosa during either the hypoxic or reoxygenation phase prevented the degradation of HIF-1alpha protein levels. Moreover, the observation that lipopolysaccharide induced HIF-1alpha expression in a time-dependent manner in IEC-6 cells indicated that the induction of HIF-1 by exposure to P. aeruginosa is not dependent on bacterial viability. In conclusion, these results suggest that HIF-1alpha activation is an early reperfusion-independent event in models of gut ischemia-reperfusion and that this HIF-1alpha response is potentiated by the presence of P. aeruginosa or lipopolysaccharide.

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms.

OBJECTIVE: To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. SUMMARY BACKGROUND DATA: Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. METHODS: Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. RESULTS: T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. CONCLUSIONS: Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.