Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1β Production : Transplantation

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Experimental Transplantation

Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1β Production

Cooke, David Tom1,2,3; Hoyt, E Grant1; Robbins, Robert C.1

Author Information

1 Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, CA.

2 Department of General Surgery, Massachusetts General Hospital, Boston, MA.

This research was supported in part by the American Society of Transplantation Council’s Fellowship Grant and the Stanford University Department of Cardiothoracic Surgery Falk Research Fund.

3 Address correspondence to: David Tom Cooke, M.D., c/o Robert C. Robbins, M.D., Stanford University Medical Center, 300 Pasteur Drive, Falk Cardiovascular Research Building, Stanford, CA 94305. E-mail: [email protected].

Received 26 May 2004. Revision requested 29 June 2004. Accepted 15 October 2004.

Transplantation 79(7):p 762-767, April 15, 2005. | DOI: 10.1097/01.TP.0000153368.08861.15

Abstract

Background. 

A significant cause of primary graft failure in lung transplantation is ischemia-reperfusion (I/R). I/R injury generates proinflammatory cytokines, such as interleukin (IL)-1β, and activates the caspase-mediated pathways of alveolar epithelial apoptosis. The authors investigated whether gene transfer of the human antiapoptotic protein Bcl-2 by means of intratracheal adenoviral administration would preserve posttransplant lung function and reduce intragraft activated caspase activity and IL-1β production in syngeneic rat donor lung grafts.

Methods. 

First, 1.0×109 plaque-forming units of AdvBcl-2 in phosphate-buffered saline (PBS), AdvNull empty vector in PBS, or PBS alone was administered intratracheally to ACI (RT1a) rats. Then, the left lungs were procured after 24 hr of in vivo incubation and orthotopically transplanted after 1 hr of cold ischemia into syngeneic recipients. After 2 hr of reperfusion, peak inspiratory pressures (PIP) and donor pulmonary vein PaO2 was measured in all grafts; grafts were then excised and protein extracts were analyzed by enzyme-linked immunosorbent assay (ELISA) and activated caspase-3 and caspase-9 assays.

Results. 

Human Bcl-2 transgene overexpression in donor lung grafts was demonstrated by ELISA of tissue homogenates. Pretreatment of donor lungs with AdvBcl-2 resulted in reduced PIP and increased lung isograft pulmonary vein PaO2 compared with AdvNull or PBS-alone treated controls. In addition, AdvBcl-2 pretreatment led to diminished cytochrome c release into cytosolic extracts and reduced intragraft IL-1β production and inhibited intragraft caspase-3 and caspase-9 activity.

Conclusions. 

Adenoviral overexpression of human Bcl-2 protein limits I/R injury in rat lung isografts. These data suggest that the use of Bcl-2 gene transfer to human lung donors may reduce the oxidative stress of pulmonary grafts after transplantation in clinical lung transplantation.

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Terminal lung diseases such as end-stage emphysema, cystic fibrosis, and idiopathic pulmonary fibrosis have been treated with single- or double-lung transplantation. Despite advancements in organ preservation techniques, ischemia-reperfusion (IR) still serves as a significant cause of posttransplant complications, including primary graft failure and acute rejection (1–3). Post-lung transplant I/R injury leads to the production of highly reactive oxygen species such as O2−, OH−, and H2O2. The resulting oxidative stress promotes apoptosis of pulmonary epithelium, activates transcription regulators such as nuclear factor (NF)-κB, and generates proinflammatory cytokines and chemokines such as interleukin (IL)-1β (1, 4–6). IL-1β is an especially potent cytokine that is released from apoptotic cells and damaged lung epithelium and has been implicated as a contributor to acute lung injury (7–9).

Prior studies in this laboratory demonstrated that increase of native cardiac Bcl-2 reduces I/R injury in a nonfunctional allogeneic heterotopic rat heart transplant model. In addition, the Bcl-2 increase leads to the diminishment of chronic graft coronary artery disease (10, 11). Also, we have previously shown that gene transfer of human Bcl-2 into donor rat hearts by means of antegrade intracoronary injection of an adenovirus overexpressing the human Bcl-2 gene limits apoptosis, free radical release, and tumor necrosis factor-α production postoperatively (12).

Bcl-2 is a well-studied antiapoptotic protein first isolated from a B-cell lymphoma line, and was found to cause oncogenesis by inhibiting apoptosis. Bcl-2 is thought to inhibit apoptosis by stabilizing the outer mitochondrial membrane and preventing release of cytochrome c into the cytosol (7, 13). In the cytosol, free cytochrome c activates and then combines with caspase-9 and apoptosis protease-activating factor-1 to form the apoptosome. The apoptosome then activates procaspase-3 to caspase-3, which then activates other caspases and also serves as a terminal effector, along with caspase-7, of apoptosis by cleaving DNA (14, 15).

In vivo intratracheal administration of adenovirus has been used for gene therapy in rat orthotopic lung transplantation (16, 17). In this study, we tested the hypothesis that human Bcl-2 overexpression by means of intratracheal administration of adenovirus containing the human Bcl-2 gene may limit I/R injury in rat lung transplantation by inhibiting apoptosis and inflammatory cytokine production.

MATERIALS AND METHODS

Animals

Male ACI (RTla) rats weighing 250 to 300 g were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All rats were housed under conventional conditions at the animal care facilities of the Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, California. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985).

Adenoviral Vectors

The adenovirus AdCMVhBcl-2 (AdvBcl-2) was a generous gift from David T. Curiel, M.D. (University of Alabama at Birmingham, Gene Therapy Center, Birmingham, AL). AdvBcl-2 and the empty vector Ad5BglII (AdvNull) were amplified by the University of Iowa Gene Transfer Vector Core (Iowa City, IA). AdvBcl-2 overexpresses the human Bcl-2 gene and has been used in previous in vivo experiments (12, 18).

Intratracheal Adenoviral Administration

ACI rats were anesthetized with inhaled 3% isoflurane followed by an intraperitoneal injection of a Xylazine (5 mg/kg)-ketamine (75 mg/kg) cocktail-combination. The animals were then intubated with a 16-gauge intravenous cannula after direct visualization of the glottis and mechanically ventilated (Harvard Rodent Ventilator, South Natick, MA). The animals were ventilated with a tidal volume of 10 mL/kg at 60 breaths/min and a fraction of inspired oxygen of 1.0. AdvBcl-2 or AdvNull virus in 300 μL of phosphate-buffered saline (PBS) or PBS alone was injected intratracheally through the cannula (n=5 in each group). The animals were then weaned from the ventilator and extubated for 24 hr.

Orthotopic Lung Transplantation

After 24 hr of in vivo adenoviral incubation, donor ACI rats were anesthetized, re-intubated, and mechanically ventilated. Donor animals were anticoagulated with heparin, and after median sternotomy, the lungs were flushed by means of the pulmonary artery with 25 mL (20 mm Hg) of low-potassium dextran glucose perfusate (Perfadex; BioPhausia, Uppsala, Sweden). The left lung was procured and stored in cold (4°C) low-potassium dextran glucose for a total cold ischemia time of 1 hr and then transplanted orthotopically into syngeneic ACI recipients as previously described (19). After 2 hr of warm perfusion, peak inspiratory pressures (PIP) were measured in millimeters of mercury with a manometer connected to the endotracheal tube. The right hilum was tied off with a suture to eliminate contribution from the native lung, and 0.5 mL of blood was drawn from the left donor pulmonary vein with a heparinized needle and the PaO2 was measured using the i-STAT portable clinical analyzer (i-STAT Corporation, East Windsor, NJ). The lung was then maximally inflated, flushed with 25 mL of cold normal saline (20 mm Hg), and excised. The top third of lung tissue was used for caspase analysis, the middle third was used for cytosolic fractionation, and the inferior third was used for enzyme-linked immunosorbent assay (ELISA).

Protein Extraction

The donor lung grafts were divided into thirds, with each third snap-frozen in liquid nitrogen. The superior third was homogenized in caspase lysis buffer, and the inferior third was homogenized in PBS and centrifuged at 12,000g at 4°C for 20 min. The supernatants were assayed for total protein concentration using the bichinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL).

Cytosol Fractionation

Cytosol fractionation was performed using a commercially available kit (BioVision, Mountain View, CA). Briefly, the middle third of donor lung tissue was homogenized in cytosol extraction buffer. Homogenates were then centrifuged at 700g for 10 min at 4°C. The pellet was discarded and the supernatant was centrifuged at 10,000g for 30 min at 4°C. The supernatant was collected as the cytosolic fraction and total protein was measured as mentioned above.

Measurement of Intragraft Human Bcl-2, IL-1β, and Cytochrome c Levels by ELISA

For human Bcl-2 (Oncogene, San Diego, CA), rat IL-1β (BioSource International, Camarillo, CA), and rat cytochrome c (R&D Systems, Minneapolis, MN) protein measurements, ELISA kits were used, and the assays were performed according to the manufacturers’ instructions. Absorbance was read with a microtiter plate reader at 450 nm. Results are presented as picograms per milligram of total protein for IL-1β, units per milligram for Bcl-2, and nanograms per milligram for cytochrome c.

Measurement of Caspase-3 and Caspase-9 Activity

Caspase-3 (Chemicon International, Temecula, CA) and caspase-9 activity was measured using commercial caspase colorimetric protease assay kits (R&D Systems). The superior third of donor lung tissue was homogenized in the appropriate lysis buffer at 4°C. Lysates were centrifuged and the protein content of supernatants was quantified; 150 μg of total protein was used per assay. Caspase activity was determined by a microtiter plate reader at an absorbance of 405 nm of chromophore p-nitroanilide cleaved from the conjugated substrate. Results are presented as micromoles of activity per milligram of total protein.

Human Bcl-2 Immunohistochemistry

To determine the transfection efficiency of adenovirus containing the human Bcl-2 gene, representative ACI recipients underwent intratracheal administration of AdvBcl-2, AdvNull, or PBS alone and allowed to incubate in vivo for 24 hr. The entire left lung was then procured as described above, fixed in 10% formalin, and embedded in paraffin. Human Bcl-2 staining was performed by deparaffinizing imbedded lung tissue. Antigen retrieval was performed by incubating sections in citrate buffer in a microwave for 5 min. The sections were then blocked in normal goat sera at 1:10 dilution for 30 min. Sections were incubated with a 1:250 dilution of rabbit anti-human Bcl-2 (Becton-Dickinson, San Jose, CA) for 2 hr and then 1:500 biotinylated goat anti-rabbit secondary antibody (Jackson Immunologicals, West Grove, PA) for 30 min. Sections were then incubated with streptavidin-horseradish peroxidase at 1:500 dilution for 30 min, exposed to DAB for brown stain, and counterstained with hematoxylin.

Statistical Analysis

Statistical analyses were performed using GraphPad InStat version 3.01 for Windows (GraphPad Software, San Diego, CA). All data are reported as mean±SEM. Repeated-measures analysis of variance with Bonferroni corrections were used to determine significance between various groups. Values of P≤0.05 were considered statistically significant.

RESULTS

Expression of Human Bcl-2 in Donor Lung Grafts

After 24 hr of in vivo incubation, human Bcl-2 overexpression in the AdvBcl-2–pretreated lungs was confirmed by analyzing tissue homogenates with ELISA specific for human Bcl-2. As seen in Figure 1, AdvBcl-2 pretreatment resulted in marked transgene expression when compared with AdvNull pretreatment or PBS alone (595.45±173.03 U/mg total protein vs. 12.30±3.55 U/mg, P=0.028; and 4.50±2.59 U/mg, P=0.027) at the time of transplantation. There is minimal background cross-reactivity to rat Bcl-2. In addition, human Bcl-2 transgene expression occurred in the alveolar epithelium and was expressed in approximately 75% of each grafts as determined on immunohistochemistry with antibody specific for human Bcl-2 (Fig. 2).

F1-4
FIGURE 1. Human Bcl-2 expression after 24 hr of in vivo incubation. *:
P <0.01; ** P <0.05.
F2-4
FIGURE 2. Bcl-2 immunohistochemistry. The brown stain corresponds to human Bcl-2 protein. (A) Positive human Bcl-2 staining in the AdvBcl-2–pretreated lungs after 24 hr compared with no staining in the AdvNull control (A and B, magnification ×200). (C) The human Bcl-2 localized within the alveolar epithelium (magnification ×400). (Color photomicrographs are available as online supplemental material.)

Posttransplant Lung Function

Table 1 describes posttransplant function after 2 hr of reperfusion for each treatment group. Donor lungs pretreated with AdvBcl-2 demonstrated reduced PIP compared with donor lungs pretreated with AdvNull or PBS alone (8.9±0.4 mm Hg vs. 11.0±0.6 mm Hg, P=0.039; and 11.1±0.4 mm Hg, P=0.011). In addition, donor lungs pretreated with AdvBcl-2 resulted in increased PaO2 compared with donor lungs pretreated with AdvNull (542.2±46.2 mm Hg vs. 335.0±60.9 mm Hg, P=0.052) or PBS alone (321.4±80.2 mm Hg, P=0.031).

T1-4
TABLE 1

Cytosolic Cytochrome c Level and Intragraft IL-1β Production

To determine whether the transfected human Bcl-2 protein was performing its physiologic role, cytochrome c release into the cytosol after reperfusion was measured by quantifying the level of cytochrome c in the cytosolic fraction of donor lung grafts. AdvBcl-2 pretreatment resulted in reduced release of cytochrome c into the cytosol (Fig. 3) compared with controls (38.0±4.0 ng/mg vs. 51.3±3.1 ng/mg of total protein in the AdvNull pretreated group, P=0.034; and 53.9±4.8 ng/mg of total protein in the PBS-alone group, P=0.044).

F3-4
FIGURE 3. Cytosolic fraction of cytochrome:
c. AdvBcl-2 pretreatment resulted in reduced release of cytochrome c into the cytosol compared with controls. * P <0.05.

As mentioned earlier, pulmonary epithelial cells have been shown to release IL-1β when injured (7). In this experiment (Fig. 4), we found that AdvBcl-2 pretreatment led to significant reduction of intragraft IL-1β production compared with AdvNull pretreatment (588.4±58.8 pg/mg vs. 828.5±64.7 pg/mg, P=0.025) and PBS-alone pretreatment (913.9±35.4 pg/mg, P=0.002).

F4-4
FIGURE 4. Intragraft IL-1β cytokine production. AdvBcl-2 pretreatment led to reduced intragraft IL-1β levels compared with AdvNull pretreated and PBS-alone pretreated lungs. *:
P <0.05.

Caspase-3 and Caspase-9 Activity

Prior studies in this laboratory examining I/R injury in rat heterotopic heart transplantation have used assays for activated caspase-3 activity as a marker for apoptosis (10). In this study, we examined two important regulators of apoptosis, caspase-3 and caspase-9, which are activated downstream of the Bcl-2 point of intervention. Caspase-3 is a terminal effector of apoptosis and caspase-9 is activated after oxidative stress and associates with the released cytochrome c and Apaf-1 to form the apoptosome. As shown in Figure 5, we found that pretreatment of donor syngeneic lung grafts with AdvBcl-2 led to significant reduction in both caspase-3 activity (24.3±11.8 μM/mg in the AdvBcl-2 group vs. 222.6±34.1 μM/mg in the AdvNull group, P=0.003; and vs. 243.7±61.2 μM/mg in the PBS-alone group, P=0.001) and caspase-9 activity (6.6±3.6 μM/mg in the AdvBcl-2 group vs. 228.4±35.8 μM/mg in the AdvNull group, P=0.003; and vs. 197.4±46.9 μM/mg in the PBS-alone group, P=0.042).

F5-4
FIGURE 5. Intragraft caspase-3 and caspase-9 activity. Pretreatment of donor syngeneic lung grafts with AdvBcl-2 led to reduction in both caspase-3 activity and caspase-9 activity. *:
P <0.05; ** P <0.01.

DISCUSSION

I/R injury remains a major impediment to successful lung transplantation (1, 3, 20–22). Reperfusion leads to the production of unstable oxygen free radicals, which results in the activation of cell-signaling pathways such as those mediated by mitogen-activated protein kinases and critical gene regulators such as NF-κB (1, 4, 23). NF-κB activation further leads to the production of IL-1β, which is a potent mediator of acute lung injury (8). In addition, oxidative stress leads to caspase-mediated apoptosis, and studies by numerous groups confirm that pulmonary epithelial cell apoptosis is deleterious to lung function (24–26).

In this study, we showed that gene transfer of human Bcl-2 before transplantation ameliorates I/R injury in syngeneic lung transplants. These results support prior studies demonstrating that Bcl-2 overexpression in donor grafts is cytoprotective (10–12). However, this is the first study that demonstrates that human Bcl-2 overexpression in lung transplantation improves posttransplant graft function, as illustrated by improved PIP and donor pulmonary vein PaO2 in the AdvBcl-2–pretreated group. Syngeneic grafts were used to clearly delineate improvements mediated by human Bcl-2 on I/R injury without potential masking of positive results by an alloreactive immune response.

The physiologic mechanism of Bcl-2 inhibition of apoptosis is inhibition of cytochrome c egress from the outer mitochondrial membrane (13, 27, 28), thus preventing formation of the apoptosome. Human Bcl-2 is significantly overexpressed in the donor lung isografts in this study; in addition, the exogenously administered Bcl-2 appears to be performing its mechanistic function as evidenced by the diminished cytochrome c protein levels in the cytosolic fraction of AdvBcl-2–pretreated donor lungs compared with controls.

Human Bcl-2 overexpression in this study not only reduced cytochrome c release in the cytosolic fraction of donor lung isografts but also led to an amelioration of apoptosis as indirectly measured by the caspase-3 and caspase-9 activity. The reduced apoptosis mirrored improvements in posttransplant lung function, suggesting that posttransplant apoptosis has deleterious effects on lung transplantation, and in turn, cytoprotection of lung epithelium preserves lung function.

IL-1β is an important cytokine in lung disease in both the transplant setting and in pulmonary infectious disease (7, 8). IL-1β serves as a proinflammatory cytokine that activates cells of innate immunity; promotes the acute-phase response; and causes endothelial cells to generate prostaglandins, platelet-activating factor, and plasminogen activator (29). In addition, IL-1β plays an integral role in cellular apoptosis, in which apoptotic cells release IL-1β (9, 30). Although reduced intragraft production of IL-1β in this study may have been an indirect result of reduced I/R inflammation, significantly diminished IL-1β levels may be a direct result of reduced pulmonary epithelial cell apoptosis. Although we saw trends toward reduction in myeloperoxidase activity (an indirect measure of neutrophil infiltration, as well as cytokines tumor necrosis factor-α, MCP-1, and the rat equivalent of IL-8 [Gro/Cinc-1] in the AdvBc-2–pretreated lungs [data not shown]), these results were not significant, suggesting that reduced apoptosis is the cause of the reduced IL-1β seen in this model.

This study supports the potential use of intratracheal gene transfer of an antiapoptotic protein in inhibiting I/R injury in lung transplantation. However, there are established limitations to adenovirus vector-mediated gene therapy in lung transplantation. The transfection efficiency of adenovirus vectors in the lung is less than in other organ systems. Adenovirus vector transfection requires the presence of coxsackie-adenovirus receptors (CAR) on the target cell for virus attachment (31) in addition to αvβ3 and αvβ5 integrins on the cell surface to mediate virus entry into the cytosol by means of coated pits (32, 33). Lung epithelium has fewer CAR and αv subunits compared with other organ tissue such as the liver and kidney, respectively (34). Also, the CAR and αvβ integrins are expressed on the basolateral surface of bronchial and alveolar epithelium, requiring virus particles to pass through tight junctions and mucosal barriers (35). Moreover, the gene transfer and expression by nonreplicating adenovirus vectors are self-limiting, secondary to the host’s competent immune system (36). However, the results of our experiment serve as a proof of concept. Despite the fact that the transfection efficiency in our system is not optimal because of the points mentioned above, we do find high levels of Bcl-2 transgene products expressed in the alveolar epithelium, and this expression leads to significant improvement in posttransplant lung function and reduction in I/R-mediated apoptosis.

CONCLUSION

This is a unique study illustrating that a physiologic inhibitor of apoptosis such as Bcl-2 can be overexpressed and used as gene therapy to improve acute outcomes of lung transplantation in an animal model. Clinically, other less immunogenic vectors could be used such as DNA-liposome complexes (37) and cell-permeable peptides (38) that could transfer the Bcl-2 gene or intact protein as a therapeutic cargo. Future studies will evaluate allogeneic combinations and determine whether the beneficial effect of human Bcl-2 overexpression is preserved.

ACKNOWLEDGMENTS

The authors thank Anthony Caffarelli, M.D., for expert review of this article and Pauline Chu for assistance with histology.

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Keywords:

Lung transplantation; Ischemia-reperfusion; Apoptosis; Bcl-2; Rat

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