Cytomegalovirus Infection–Enhanced Cardiac Allograft Vasculopathy Is Abolished by DHPG Prophylaxis in the Rat
Background A wealth of clinical and experimental evidence exists for cytomegalovirus (CMV) infection as an accelerating factor in the development of cardiac allograft vasculopathy. In this study, the impact of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) on rat CMV infection–enhanced cardiac allograft vasculopathy is investigated.
Methods and Results Heterotopic rat cardiac allografts were performed from the DA to the WF rat strain, and the recipients were immunosuppressed with cyclosporine A 2 mg·kg−1·d−1 SC for a period of 90 days until the end of experiment. Two groups of recipients were infected intraperitoneally with 105 plaque-forming units of rat CMV, whereas one group was left noninfected and used as controls. One group of rat CMV–infected rats was treated with DHPG with an initial dose of 20 mg/kg IP and a maintenance dose of 10 mg/kg IP twice a day from 1 day before transplantation to 30 days after transplantation. Compared with noninfected rats, rat CMV infection was associated with a significant increase in intimal thickening, from 0.68±0.10 to 1.30±0.12 score units (P<.01), and double the number of vessels affected (P<.01). DHPG treatment significantly reduced intimal thickening in rat CMV–infected rats, from 1.30±0.12 to 0.68±0.13 score units (P<.01), and halved the number of vessels affected (P<.01).
Conclusions The present results demonstrate that DHPG prophylaxis entirely abolishes the accelerating effect of rat CMV infection on cardiac allograft vasculopathy in immunosuppressed rat recipients, which is consistent with our earlier findings demonstrating a similar effect in nonimmunosuppressed rat aortic allografts. Taken together, these results suggest that DHPG might be useful in the prevention of CMV-accelerated cardiac allograft vasculopathy among heart transplant recipients.
A wealth of clinical and experimental evidence exists that CMV infection accelerates the development of intimal lesions in transplantation-associated arteriosclerosis1 2 3 4 5 6 and possibly in ordinary atherosclerosis.7 8 The drug of choice for the treatment of CMV infection is DHPG (ganciclovir), which is an acyclic analogue of the natural 2′-deoxy-guanosine.9 The mechanism of action of this drug depends on the formation of a 5′-triphosphate derivative that is a selective inhibitor of the viral DNA polymerase.10 Human CMV does not encode the viral thymidine kinase, but it has been shown that the human CMV UL 97 open reading frame encodes a protein that controls the phosphorylation of DHPG in human CMV–infected cells.11 12
We have previously demonstrated with nonimmunosuppressed rats that RCMV infection enhances smooth muscle cell proliferation and intimal thickening in aortic allografts and that these changes can be abolished entirely by DHPG prophylaxis.13 The present study was undertaken to test the effect of DHPG on RCMV-enhanced arteriosclerosis in immunosuppressed rat cardiac allografts.
Heterotopic Rat Cardiac Allograft Model
Inbred DA (AG-B4, RT1a) and WF (AG-B2, RT1u) rat strains (weight, 200 to 300 g) (Laboratory Animal Center, University of Helsinki, Finland) were used, and transplantation procedures were performed as described previously.6
The rats were infected intraperitoneally with 105 plaque-forming units of salivary gland–derived RCMV 1 day after transplantation, as described previously.6
CsA (Sandimmun; Sandoz Pharma AG) 50 mg/mL infusion substance was dissolved in Intralipid 200 mg/mL (KabiVitrum) at a final concentration of 2 mg/mL, and the rats received 2 mg/kg SC for the entire experiment. Radioimmunoassay (Sandoz) was applied to determine whole-blood CsA levels.
DHPG (ganciclovir; Cymevene 500 mg; Syntex) was dissolved in 100 mL of 0.9% NaCl at a final concentration of 5 mg/mL. DHPG was administrated intraperitoneally with an initial dose of 20 mg/kg and a maintenance dose of 10 mg/kg twice a day for 30 days.
At least two midsections of the allografts were fixed in 10% phosphate-buffered formalin and stained with Mayer’s hematoxylin and eosin, Masson’s trichrome, and Weigert–van Gieson. The changes in intimal thickness were scored as mild (score 1; <25% occlusion of the lumen) when the intima was readily discernible, moderate (score 2; 25% to 50% occlusion), and severe (score 3; >50% occlusion) when the lumen was encroached on.6 The assigned score was determined by consensus of two observers.
All data are given as mean±SEM. Total variation between the groups was analyzed by the combination of Kruskal-Wallis one-way analysis (Statview 512+, BrainPower Inc) and Dunn test at the significance level of 5% or 1% (Medstat; Astra Group A/S).14 Values of P<.05 were regarded as statistically significant.
As shown in Fig 1⇓, there was no statistical difference in the whole-blood CsA levels between the groups as measured once a week after transplantation. Approximately 40 arteries or arterioles in a cardiac cross section were analyzed in different treatment categories (Table⇓). In cardiac allografts of noninfected rats, 29±4% of the vessels analyzed were affected by intimal thickening, and the mean score for intimal thickness was 0.68±0.10. RCMV infection induced a significant increase in the percent of diseased vessels (to 58±4%; P<.01), and the mean score of intimal thickening increased to 1.30±0.12 (P<.01 compared with nontreated, noninfected allografts). In the DHPG prophylaxis group, the percent of vessels with transplant vascular changes was reduced to the basal level (29±5%; P<.01), and the mean score of intimal thickness was reduced to 0.68±0.13 (P<.01). The effect of DHPG on RCMV infection–enhanced cardiac allograft vasculopathy in individual recipients is shown in Fig 2⇓.
The introduction of the CMV-specific antiviral drug ganciclovir has revolutionized the treatment of severe CMV infections in organ-transplant recipients and other immunodeficient patients.9 10 15 Recently, ganciclovir prophylaxis was shown to significantly delay the onset of chronic rejection, ie, obliterative bronchiolitis in heart-lung and lung transplant patients.13
Our previous study13 in nonimmunosuppressed rat aortic allografts demonstrated that DHPG prophylaxis completely abolished the enhancing effect of CMV infection on aortic allograft arteriosclerosis, whereas DHPG treatment initiated during the infection only partially inhibited the development of intimal lesions. The present study was begun to test the efficacy of DHPG prophylaxis in the prevention of RCMV infection–enhanced cardiac allograft vasculopathy during CsA immunosuppression. A clear-cut result was obtained: whereas RCMV doubled the intimal thickness and the number of vessels affected, the number of vessels with vascular wall changes and the mean score of intimal thickness in the DHPG prophylaxis group were reduced to the basal levels of noninfected CsA immunosuppressed controls.
The results of the previous study also demonstrated a dose-dependent inhibitory effect of DHPG on smooth muscle cell replication in vitro.16 The distinct inhibitory response of DHPG therapy on smooth muscle cell proliferation suggests that the inhibitory effect of DHPG on virus infection–enhanced allograft arteriosclerosis may be linked not only to the inhibition of the viral replication but also to additional pathways. First, the inhibition of CMV replication by DHPG also abolishes the characteristic inflammatory response (“endothelialitis”) of the vascular wall.17 Inflammatory cytokines are potent stimulants of smooth muscle cell growth factors in the vascular wall and, as shown, CMV infection triples the expression of platelet-derived growth factor,18 known to be linked with smooth muscle cell migration and replication.19 Second, smooth muscle cells migrating to the intima in coronary restenosis express elevated levels of human CMV immediate-early antigen IE84 and tumor suppressor protein p53, suggesting that IE84 inactivates p53 and predisposes smooth muscle cells to increased growth, as in malignant tumors.7
Our observations suggest that DHPG may be important in clinical prophylaxis of CMV infection in heart transplant patients not only because it decreases morbidity in acute infection, but also because it inhibits accelerated allograft arteriosclerosis. We suggest that sufficient preclinical information exists that a randomized, double-blind, multicenter clinical trial with the recent oral formula of DHPG should be conducted to validate these experimental observations in humans.
Selected Abbreviations and Acronyms
This work was supported by grants from the Finnish Foundation for Cardiovascular Research, Helsinki University Central Hospital Research Funds, Technology Development Center, and University of Helsinki, Helsinki, Finland, and Syntex Development Research, a division of Syntex (USA) Inc, Palo Alto, Calif. We would like to thank E. Aaltola, T. Lahtinen, and E. Wasenius, Transplantation Laboratory, and G. Grauls, Medical Microbiology, for their excellent technical assistance.
- Received January 28, 1997.
- Revision received April 16, 1997.
- Accepted April 16, 1997.
- Copyright © 1997 by American Heart Association
Lemström KB, Bruning JH, Bruggeman CA, Lautenschlager IT, Häyry PJ. Cytomegalovirus infection enhances smooth muscle cell proliferation and intimal thickening of rat aortic allografts. J Clin Invest. 1993;92:549-558.
Lemström K, Koskinen P, Krogerus L, Daemen M, Bruggeman C, Häyry P. Cytomegalovirus antigen expression, endothelial cell proliferation, and intimal thickening in rat cardiac allografts after cytomegalovirus infection. Circulation. 1995;92:2594-2604.
Speir E, Modali R, Huang E-S, Leon MB, Shawl F, Finkel T, Epstein SE. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science. 1994;265:391-394.
Melnick JL, Adam E, DeBakey ME. Cytomegalovirus and atherosclerosis. Eur Heart J. 1993;14(suppl K):30-38.
Matthews T, Boehme R. Antiviral activity and mechanism of action of ganciclovir. Rev Infect Dis. 1988;10:S490-S494.
Hollander M, Wolfe DA. Non-parametric Statistical Methods. New York, NY: John Wiley & Sons; 1973.
Merigan TC, Renlund DG, Keay S, Bristow MR, Starnes V, O’Connell JB, Resta S, Dunn D, Gamberg P, Ratkovec RM, Richenbacher WE, Millar RG, DuMond C, DeAmond B, Sullivan V, Cheney T, Buhles W, Stinson EB. A controlled trial of ganciclovir to prevent cytomegalovirus disease after heart transplantation. N Engl J Med. 1992;326:1182-1186.
Lemström KB, Bruning JH, Bruggeman CA, Koskinen PK, Aho PT, Yilmaz S, Lautenschlager IT, Häyry PJ. Cytomegalovirus infection-enhanced allograft arteriosclerosis is prevented by DHPG prophylaxis in the rat. Circulation. 1994;90:1969-1978.
Lemström KB, Aho Päivi, Bruggeman CA, Häyry PJ. Cytomegalovirus infection enhances mRNA expression of platelet-derived growth factor-BB and transforming growth factor-β1 in rat aortic allografts: possible mechanism for cytomegalovirus-enhanced graft arteriosclerosis. Arterioscler Thromb. 1994;14:2043-2052.
Koyama N, Hart CE, Clowes AW. Different functions of the platelet-derived growth factor-α and -β receptors for the migration and proliferation of cultured baboon smooth muscle cells. Circ Res. 1994;75:682-691.