Estrogen Receptor Hormone Agonists Limit Trauma Hemorrhage Shock-Induced Gut and Lung Injury in Rats

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) is a major cause of death in trauma patients. Earlier studies in trauma hemorrhagic shock (T/HS) have documented that splanchnic ischemia leading to gut inflammation and loss of barrier function is an initial triggering event that leads to gut-induced ARDS and MODS. Since sex hormones have been shown to modulate the response to T/HS and proestrous (PE) females are more resistant to T/HS-induced gut and distant organ injury, the goal of our study was to determine the contribution of estrogen receptor (ER)alpha and ERbeta in modulating the protective response of female rats to T/HS-induced gut and lung injury.The incidence of gut and lung injury was assessed in PE and ovariectomized (OVX) female rats subjected to T/HS or trauma sham shock (T/SS) as well as OVX rats that were administered estradiol (E2) or agonists for ERalpha or ERbeta immediately prior to resuscitation. Marked gut and lung injury was observed in OVX rats subjected to T/HS as compared to PE rats or E2-treated OVX rats subjected to T/HS. Both ERalpha and ERbeta agonists were equally effective in limiting T/HS-induced morphologic villous injury and bacterial translocation, whereas the ERbeta agonist was more effective than the ERalpha agonist in limiting T/HS-induced lung injury as determined by histology, Evan's blue lung permeability, bronchoalevolar fluid/plasma protein ratio and myeloperoxidase levels. Similarly, treatment with either E2 or the ERbeta agonist attenuated the induction of the intestinal iNOS response in OVX rats subjected to T/HS whereas the ERalpha agonist was only partially protective.Our study demonstrates that estrogen attenuates T/HS-induced gut and lung injury and that its protective effects are mediated by the activation of ERalpha, ERbeta or both receptors

Trauma Hemorrhagic Shock-Induced Lung Injury Involves a Gut-Lymph-Induced TLR4 Pathway in Mice

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation

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

Estradiol and ERβ are more effective than ERα in conferring protection against T/HS induced lung injury.

<p>A) T/HS-induced lung injury, as represented by the percentage of EBD leak within the BALF or B) by the BALF/plasma protein ratio, was increased in the ovariectomized rats and reduced by the administration of estradiol of the estrogen receptor agonists. Data expressed as mean ± SD with 6–8 rats per group. *p<0.01 vs all other groups except the ERα T/HS group. #p<0.01 vs ERα T/SS group. C) T/HS increased the degree of lung neutrophil sequestration, as reflected in MPO levels, in the OVX rats and this was abrogated by the administration of E2 and the ERβ but not the ERα agonist. *p<0.05 vs all other groups.</p

iNOS response is abrogated by both E2 and ERβ.

<p>A) The number of iNOS positive enterocytes was increased in all of the T/HS groups but was highest in the OVX rats and the OVX rats receiving the ERα agonist. Data expressed as mean ± SD with a minimum of 100 villi counted per animal with 6–8 animals per group. * p<0.01 vs all other groups, **p<0.01 vs all other groups, and #p<0.05 vs T/HS groups. B) Representative immunohistochemistry micrographs showing iNOS staining of enterocytes.</p

Estradiol and both estrogen receptor agonists ERα and ERβ are protective against T/HS induced gut injury.

<p>A) The extent of villus injury 3 hours after T/HS was reduced back to the levels observed in the PE females by all three estrogen-based therapies. A minimum of 200 villi per animal was counted with 6–8 animals per group. *p<0.001 vs all other T/HS groups, #p<0.05 vs all T/HS groups. B) The incidence of bacterial translocation was increased only in the OVX group subjected to T/HS. *p<0.01 vs all other groups with 6–8 animals per group.</p

TRIF and Myd88 deficiency confer full and partial protection against T/HS lymph induced microvascular permeability.

<p>A) WT and Myd88^−/− and B) WT and TRIF^mut mice were infused with porcine T/SS and T/HS lymph for 3 hr and lung permeability to EBD was performed. Data expressed as mean ± SE (n = 5–8 mice/ group).</p

TLR2 does not mediate T/HS lymph-induced lung injury.

<p>WT and TLR2^−/− mice infused with porcine T/HS and T/SS lymph for 3 hr. A) Lung permeability to EBD was performed. Data expressed as mean ± SE (n = 4–6 mice/ group). B) MPO levels (U/g) were measured in lung homogenates. Data expressed as mean ± SE (n = 5–6 mice/group).</p

TLR4 deficiency reduces T/HS lymph induced pulmonary iNOS protein levels.

<p>A) and B) Western blot of iNOS in lung WCEs of WT and TLR4^mut mice infused with porcine T/SS and T/HS lymph for 3 hr. B) Densitometry was performed to quantify iNOS and total p42/p44 MAPK expression. Data expressed as mean ± SE (n = 4–7 mice/group).</p

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-1β, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1β 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