Allograft vasculopathy
Bcl-2–mediated inhibition of apoptosis in rat cardiac allografts worsens development of graft coronary artery disease

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Abstract

Background:

We hypothesized that adenovirally mediated Bcl-2 transfection of donor hearts would reduce the apoptosis that occurs during acute rejection while worsening the development of chronic graft coronary artery disease (GCAD).

Methods:

PVG donor hearts were treated with either AdvBcl-2 or AdvNull virus before heterotopic transplantation into ACI rats. Bcl-2 expression was assessed on post-operative day 4 (POD) 4 by western blot. Apoptosis was measured using 99mTechnetium-bound–annexin V imaging and caspase 3 activity assay. Allograft survival was determined in a separate cohort of animals. Long-term–treated animals were then assessed for measures of GCAD on POD 90.

Results:

Western blot analysis showed upregulation of Bcl-2 expression in AdvBcl-2–treated hearts. 99mTc–annexin V images demonstrated decreased uptake in the AdvBcl-2 group (1.41 ± 0.33% vs 1.94 ± 0.37%, p = 0.026). Caspase 3 activity was also significantly lower in this treatment group (0.112 ± 0.032 vs 0.204 ± 0.096, p = 0.049). Allograft survival was similar in both groups, respectively (7.7 ± 1.2 vs 6.8 ± 1.5 days, p = 0.340). GCAD, as determined by percent luminal narrowing (5.9 ± 6.1% vs 1.6 ± 1.5%, p = 0.039), intima-to-media ratio (5.1 ± 5.1% vs 1.5 ± 1.7%, p = 0.040) and percent of affected vessels (15.1 ± 9.9% vs 5.3 ± 4.4%, p = 0.009), was higher for the AdvBcl-2 group.

Conclusion:

Treatment of cardiac allografts with AdvBcl-2 resulted in a reduction of apoptosis that did not significantly improve short-term graft survival, but worsened chronic GCAD.

Section snippets

Animals

Adult male ACI (RT-1a) and PVG (RT-1c) rats, weighing between 200 and 250g, were purchased from Harlan Sprague-Dawley (Indianapolis) and housed in the animal care facilities at Stanford University Medical Center (Stanford, CA). They were kept under standard temperature, humidity and timed lighting conditions and were provided rat chow and water ad libitum. Animals were treated in a humane manner, in compliance with the Guide for the Care and Use of Laboratory Animals, prepared by the Institute

Bcl-2 expression

Treatment of donor hearts ex vivo with adenovirus-expressing Bcl-2 resulted in upregulation of this gene product as measured by western blot analysis on POD 4. Expression in the null virus–treated hearts was similar to the baseline levels seen in native, non-transplanted hearts (Figure 1). Densitometric analysis of Bcl-2 bands on western blots demonstrated significant differences in protein expression (AdvBcl-2, 93,765 ± 18,538; AdvNull, 48,802 ± 21,906; native hearts, 42,770 ± 9,159) Units

Discussion

Prevention of apoptosis has emerged as a potential strategy for the treatment of acute allograft rejection. Excessive programmed cell death has been implicated in several disease processes such as ischemia–reperfusion injury, autoimmune disorders and acute rejection of transplanted organs.15 Treatment of these disorders with anti-apoptotic therapy, however, represents something of a “double-edged sword.” For example, interruption of apoptosis may interfere with the normal mechanism by which

Conclusions

AdvBcl-2 treatment of cardiac allografts in this rodent model resulted in a reduction of apoptosis that did not significantly prolong short-term allograft survival. Graft coronary artery disease, however, was exacerbated, possibly due to the lack of vascular smooth muscle cell apoptosis during vessel remodeling. Our study has highlighted an important long-term concern regarding the use of anti-apoptotic therapy as a potential adjunct to current modes of immunosuppression in cardiac

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