Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury

Abstract

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

Figure 1

HO-1–inducing agents decrease resistance to portal vein blood flow. Livers harvested from obese Zucker rats were perfused for 2 hours on the isolated perfusion rat liver apparatus after 6 hours of cold ischemia. Pretreatment with CoPP or Ad-HO-1 gene transfer (day –1) significantly improved portal venous blood flow compared with untreated, ZnPP, or Ad-βGal pretreated controls throughout the reperfusion period. These data represent the mean ± SE of 4–10 independent perfusions for each group. *P = 0.0001 versus untreated/ZnPP-treated controls.

Figure 2

Bile production in fatty livers perfused for 2 hours on the isolated perfusion rat liver apparatus after 6 hours of cold ischemia. Animals were pretreated with metalloporphyrins, or with Ad-HO-1 gene transfer, or left untreated. Bile production at 30-minute intervals throughout the reperfusion period was significantly higher in the CoPP/Ad-HO-1 groups (*P < 0.05) as compared with untreated, ZnPP-, or Ad-βGal–pretreated controls. These data represent the mean ± SE of 4–10 independent perfusions for each group. *P < 0.05 versus untreated/ZnPP-treated controls.

Figure 3

Effects of I/R injury on sGOT release from fatty livers cold stored for 6 hours followed by 2 hours of ex vivo perfusion. Levels (IU/L) were measured in blood samples taken at 30-minute intervals during the perfusion. Levels of sGOT were significantly lower at 30, 60, and 90 minutes during the perfusion of livers pretreated with CoPP or Ad-HO-1 as compared with untreated controls or those pretreated with ZnPP or Ad-βGal. These data represent the mean ± SE of 4–10 measurements for each group. *P < 0.05 versus untreated/ZnPP-treated control.

Figure 4

Photomicrographs of representative rat fatty livers after 6 hours of cold ischemia and 2 hours of reperfusion on the isolated perfusion rat liver apparatus. (a) Control untreated group with severe lobular distortion, zone 3 ballooning, and hepatocyte necrosis (Banff’s score = 3.0 ± 0.63); (b) ZnPP-pretreated group with marked sinusoidal and vascular congestion (arrow) and profound zone 3 ballooning change (score = 2.86 ± 0.12); (c) CoPP-pretreated and (d) Ad-HO-1–pretreated groups with minimal vacuolar degeneration and almost complete preservation of lobular architecture (scores = 1.21 ± 0.39 and 1.68 ± 0.51, respectively); (e) Ad-HO-1 plus ZnPP–treated group, similar to untreated controls, with profound zone 3 ballooning change accompanied by confluent hepatocyte necrosis (arrow; score = 2.74 ± 0.26); (f) Ad-βGal–pretreated group, similar to untreated controls, showing profound zone 3 ballooning change accompanied by severe vascular congestion and confluent hepatocyte necrosis (arrow; score = 3.0 ± 1.41). ×100, H&E stain; n = 2–3/group.

Figure 5

Western blot analysis of HO-1 protein expression in fatty Zucker livers stored for 6 hours at 4°C, followed by 2 hours of perfusion on the isolated perfusion rat liver apparatus. HO-1 expression was detected by using a polyclonal rabbit anti-rat HO-1 Ab. Lane 1, CoPP treatment; lane 2, Ad-HO-1 gene transfer; lane 3, Ad-HO-1 gene transfer plus ZnPP treatment; lane 4, ZnPP treatment; lane 5, untreated control. HO-1 migrates as a 32-kDa protein. The relative HO-1 expression levels analyzed by densitometer were (in absorbance units): 2.46, 2.12, 1.18, 0.12, and 0.11 for lanes 1, 2, 3, 4, and 5, respectively. Results shown are representative of 2–3 independent experiments.

Figure 6

Prolongation of liver isograft survival. Lean Zucker rats served as recipients of liver transplants from obese Zucker donors. Donor rats were either pretreated with CoPP or Ad-HO-1 or remained untreated before liver procurement followed by 4 hours of cold ischemia. Control animal survival at 14 days was 40% versus 80% and 81.8% in the CoPP and the Ad-HO-1 group, respectively (n = 10–11 rats/group).

Figure 7

Photomicrographs of representative liver isografts transplanted from obese Zucker donors into lean Zucker recipients and harvested at day 1. (a) Untreated group with significant edema and pallor around periportal hepatocytes and severe disruption of lobular architecture with zone 3 necrosis (arrow; Suzuki score = 3.33 ± 0.58); (b) CoPP- and (c) Ad-HO-1–treated groups with minimal pallor, no edema, and complete preservation of lobular architecture (score = 1.33 ± 0.70 and 1.5 ± 0.5, respectively). ×100, H&E stain; n = 2–3/group.

Figure 8

Immunohistochemical staining for infiltrating T cells and macrophages in steatotic rat livers at day 1 and 100 following transplantation into lean Zucker recipients. Sections of liver isografts from untreated or CoPP-pretreated obese Zucker donors were stained with primary mouse mAb against rat T cells (R73) and monocytes/macrophages (ED1). (a) Control day 1 (T cells); (b) CoPP day 1 (T cells); (c) control day 1 (macrophages); (d) CoPP day 1 (macrophages); (e) control day 100 (T cells); (f) CoPP day 100 (T cells); (g) control day 100 (macrophages); (h) CoPP day 100 (macrophages). Original ×272.

Figure 9

Western blot analysis of HO-1 protein expression in fatty livers with or without CoPP pretreatment. Livers were stored for 4 hours at 4°C, transplanted into lean Zucker recipients, and then harvested at 1, 7, 14, and 100 days. The expression of HO-1 was detected by a polyclonal rabbit anti-rat HO-1 Ab. Lane 1, CoPP day 1; lane 2, CoPP day 7; lane 3, CoPP day 14; lane 4, CoPP day 100; lane 5, untreated day 1; lane 6, untreated day 7; lane 7, untreated day 14; lane 8, untreated day 100. HO-1 migrates as a 32-kDa protein. The relative HO-1 expression levels analyzed by densitometer were (in absorbance units): 1.21, 1.27, 1.63, and 2.14 for CoPP-treated livers in lanes 1, 2, 3, and 4, respectively. The HO-1 expression for untreated controls in lanes 5, 6, 7, and 8 were (in absorbance units): 0.09, 0.17, 0.85, and 0.75, respectively. Data shown are representative of 3 independent experiments.