HB-EGF protects the lungs after intestinal ischemia/reperfusion injury

Abstract

Background: Acute respiratory distress syndrome continues to be a major source of morbidity and mortality in critically-ill patients. Heparin binding EGF-like growth factor (HB-EGF) is a biologically active protein that acts as an intestinal cytoprotective agent. We have previously demonstrated that HB-EGF protects the intestines from injury in several different animal models of intestinal injury. In the current study, we investigated the ability of HB-EGF to protect the lungs from remote organ injury after intestinal ischemia/reperfusion (I/R).

Methods: Mice were randomly assigned to one of the following groups: (1) sham-operated; (2) sham+HB-EGF (1200 microg/kg in 0.6 mL administered by intra-luminal injection at the jejuno-ileal junction immediately after identification of the superior mesenteric artery); (3) superior mesenteric artery occlusion for 45 min followed by reperfusion for 6 h (I/R); or (4) I/R+HB-EGF (1200 microg/kg in 0.6 mL) administered 15 min after vascular occlusion. The severity of acute lung injury was determined by histology, morphometric analysis and invasive pulmonary function testing. Animal survival was evaluated using Kaplan-Meier analysis.

Results: Mice subjected to intestinal I/R injury showed histologic and functional evidence of acute lung injury and decreased survival compared with sham-operated animals. Compared with mice treated with HB-EGF (I/R+HB-EGF), the I/R group had more severe acute lung injury, and decreased survival.

Conclusion: Our results demonstrate that HB-EGF reduces the severity of acute lung injury after intestinal I/R in mice. These data demonstrate that HB-EGF may be a potential novel systemic anti-inflammatory agent for the prevention of the systemic inflammatory response syndrome (SIRS) after intestinal injury.

Figure 1

Lung histologic injury scoring. Shown are representative photomicrographs of hematoxylin and eosin stained lung sections from: A) a sham operated mouse, B) a sham operated mouse that was treated with HB-EGF, C) a mouse subjected to intestinal I/R, and D) a mouse subjected to intestinal I/R but treated with HB-EGF. Panel E represents quantification of lung injury scores, based on septae congestion, intra-alveolar infiltration and alveolar hemorrhage. Bars represent sham animals (n = 6), sham+HB-EGF animals (n = 5), I/R animals (n = 11), and I/R+HB-EGF animals (n = 11). Scores represent the averaged results of two blinded observers.

Figure 2

Lung morphometric analysis. A) Alvoelar surface area and B) alveolar septae thickness of H & E stained lung tissue were determined using Image Pro digital image analysis software. C) Pulmonary diffusion capacity was calculated as the alveolar surface area divided by the alveolar septae thickness. Bars represent sham animals (n = 4), I/R animals (n = 7), and I/R+HB-EGF animals (n = 7).

Figure 3

Lung inflammatory cell infiltration. Shown are representative photomicrographs of tissue sections immunohistochemically stained for CD11b/c to indicate infiltration of macrophages and polymorphonuclear leukocytes from: A) a sham operated mouse, B) a sham operated mouse that was treated with HB-EGF, C) a mouse subjected to intestinal I/R, and D) a mouse subjected to intestinal I/R but treated with HB-EGF. Arrows indicate positively stained cells. Panel E represents quantification of positively stained cells, represented by the number of inflammatory cells counted in four optical fields viewed at 200× magnification. Bars represent sham animals (n = 6), sham+HB-EGF animals (n = 5), I/R animals (n = 11), and I/R + HB=EGF animals (n = 11). Panel F represents MPO levels where n = 8 animals per group.

Figure 4

Lung apoptosis. TUNEL-positive cells stained in red are indicated by arrows. Nuclei were counterstained with Prolong Gold antifade reagent containing DAPI. Shown are representative images of: A) a sham operated mouse, B) a sham operated mouse that was treated with HB-EGF, C) a mouse subjected to intestinal I/R, and D) a mouse subjected to intestinal I/R but treated with HB-EGF. Panel D represents quantification of TUNEL-positive stained cells. The apoptotic cells in lung were scored randomly in 10 optical fields viewed at 400× magnification. Bars represent sham animals (n = 6), sham+HB-EGF animals (n = 5), I/R animals (n = 10), and I/R + HB-EGF animals (n = 10).

Figure 5

Akt activation. Shown are representative Western blots demonstrating phosphorylated-Akt and total Akt levels in the lungs from animals exposed to: A) intestinal ischemia for 45 min followed by reperfusion for 1 h and B) intestinal ischemia for 45 min followed by reperfusion for 6 h. Panels C and D represent densitometry results for the 1 h ad 6 h reperfusion time points respectively. The densitometry results are expressed as activated Akt normalized to total Akt levels from three independent experiments that used separate groups of mice. There were no statistically significant differences in expression levels of activated Akt in the lungs at either the 1 h or 6 h reperfusion time points.

Figure 6

Pulmonary vascular permeability. Pulmonary vascular permeability was determined using the Evans blue dye assay. Bars represent n = 4 in each group.

Figure 7

Pulmonary function tests. A) Lung resistance and B) lung compliance were measured using the SCIREQ Flexivent system at the 6 h reperfusion time point. Tracheas were cannulated, and mice were ventilated using a computer controlled small animal ventilator. Two sets of measurements of pulmonary resistance and compliance were obtained using a single compartment model (n = 6 animals in each group).

Figure 8

Kaplan-Meier survival analysis. Mice were subjected to 45 min of ischemia (I/R, n=12); 45 min of ischemia with administration of HB-EGF (I/R + HB-EGF, n=10) or sham-surgery (sham, n = 5), and were monitored every 2 h for 6 h, every 12 h for the next 24 h, and every 24 h until the end of the two-week study period.