- Email: [email protected]
Effect of Mycophenolate Mofetil on Atherosclerosis in a Rabbit Model: Initial Histologic and Immunohistochemical Analyses 1
PHARMACOLOGICAL AGENTS
Effect of Mycophenolate Mofetil on Atherosclerosis in a Rabbit Model: Initial Histologic and Immunohistochemical Analyses T.C. Schreiber, S.M. Greenstein, D.Y. Kim, T.M. Calderon, S. Sun, R.S. Schechner, V.A. Tellis, and J.W. Berman
T
HE CELLULAR responses that define an inflammatory-fibroproliferative response to injury are implicated in the mechanism of atherosclerotic plaque formation. Similar inflammatory responses occur in chronic rejection. Mycophenolate mofetil (MMF) has been shown experimentally to inhibit the vascular changes seen with chronic allograft rejection1,2 as well as to have mixed effects on atherogenesis in animal models.3–5 The purpose of this study was to determine the effect of MMF on atherosclerotic plaque formation. MATERIALS AND METHODS Thirteen male New Zealand White rabbits, weighing 2.8 to 3.4 kg, were used in this study. All animals were fed a high-cholesterol diet for 4 weeks consisting of 1% (wt/wt) cholesterol in 8% (wt/wt) peanut oil mixed with standard rabbit chow.1 MMF was donated by Roche Bioscience (Palo Alto, Calif). The experimental group (n 5 7) was given MMF 80 mg/kg per day subcutaneously,6 whereas the control group (n 5 6) received an equal volume of 5% dextrose as vehicle. At the end of 4 weeks, the aortas were harvested and sectioned for histologic and immunohistochemical analyses. Plaque size was assessed on a scale from 0 to 111. Two observers, one of whom was blinded to the study, analyzed the samples.
RESULTS
The animals tolerated the diet and medication without adverse effects. Weight gain over the 4-week study period was no different between the MMF and control groups. Total cholesterol, HDL, LDL, and triglyceride levels were statistically significantly increased compared with baseline, but there was no statistically significant difference between the two groups. On immunohistochemical examination, there was a trend toward decreased amount of early atherosclerotic plaque (MMF group: 11, 1, 1, 1, 1, 1, 0 vs control group: 111, 11, 11, 1, 1, 1), foamy macrophages, and macrophage migration into the media of vessels in the MMF group as compared with control. Macrophages and monocytes present in the plaques as well as in the © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 961–962 (1998)
subendothelial spaces and media of vessels were identified by monoclonal antibodies against rabbit monocyte/macrophages (RAM 11) and monocyte chemoattractant protein (MCP-1). These changes were observed mainly in the arch. Analysis using monoclonal antibody against a-smooth-muscle actin demonstrated heavy staining in both groups. DISCUSSION
The pathophysiology of atherosclerotic plaque formation is not fully understood. The New Zealand white rabbit develops arterial intimal lesions that resemble human fatty streaks; that is, they consist largely of lipid-laden foam cells, making them good models to study human atherosclerosis.6 The present study confirmed, through immunohistochemistry, the previous reports that foam cells that form these early lesions are largely derived from monocytes.6 Our initial results also suggest that MMF may have an antiatherosclerotic effect in aortas of cholesterol-fed rabbits. A possible mechanism for this effect is by decreasing the severity of the monocyte-macrophage infiltration into the plaque. This may occur by inhibiting monocyte adhesion to endothelial cells as has been previously reported from cell culture experiments.7 The trend toward reduced atherosclerotic plaque in the MMF-treated group did not reach statistical significance because of the high interanimal variability in plaque formation in this animal model. Further studies with greater numbers of animals will be conducted in our laboratory to determine whether these trends will achieve statistical significance. From the Departments of Surgery (T.C.S., S.M.G., D.Y.K., S.S., R.S.S., V.A.T.) and Pathology (T.M.C., J.W.B.), Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, New York, USA. Supported by Roche, Inc (Nutley, NJ). Address reprint requests to Dr S.M. Greenstein, Department of Surgery, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467. 0041-1345/98/$19.00 PII S0041-1345(98)00111-0 961
962
REFERENCES 1. Drew AF, Tipping PG: Atherosclerosis 116:181, 1995 2. Ferns G, Reidy M, Ross R: Am J Pathol 142:403, 1990 3. Jonasson L, Holm J, Hansson GK: Proc Natl Acad Sci USA 85:2303, 1988 4. Emeson EE, Shen ML: Am J Pathol 142:1906, 1993
SCHREIBER, GREENSTEIN, KIM ET AL 5. Roselaar SE, Schonfeld G, Daugherty A: J Clin Invest 96:1389, 1995 6. Hannson GK, Seifert PS, Olsson G, et al: Atheroscler Thromb 11:745, 1991 7. Laurent AF, Dumont S, Poindron P, et al: Exp Hematol 24:59, 1996
Effect of Mycophenolate Mofetil on Atherosclerosis in a Rabbit Model: Initial Histologic and Immunohistochemical Analyses T.C. Schreiber, S.M. Greenstein, D.Y. Kim, T.M. Calderon, S. Sun, R.S. Schechner, V.A. Tellis, and J.W. Berman
T
HE CELLULAR responses that define an inflammatory-fibroproliferative response to injury are implicated in the mechanism of atherosclerotic plaque formation. Similar inflammatory responses occur in chronic rejection. Mycophenolate mofetil (MMF) has been shown experimentally to inhibit the vascular changes seen with chronic allograft rejection1,2 as well as to have mixed effects on atherogenesis in animal models.3–5 The purpose of this study was to determine the effect of MMF on atherosclerotic plaque formation. MATERIALS AND METHODS Thirteen male New Zealand White rabbits, weighing 2.8 to 3.4 kg, were used in this study. All animals were fed a high-cholesterol diet for 4 weeks consisting of 1% (wt/wt) cholesterol in 8% (wt/wt) peanut oil mixed with standard rabbit chow.1 MMF was donated by Roche Bioscience (Palo Alto, Calif). The experimental group (n 5 7) was given MMF 80 mg/kg per day subcutaneously,6 whereas the control group (n 5 6) received an equal volume of 5% dextrose as vehicle. At the end of 4 weeks, the aortas were harvested and sectioned for histologic and immunohistochemical analyses. Plaque size was assessed on a scale from 0 to 111. Two observers, one of whom was blinded to the study, analyzed the samples.
RESULTS
The animals tolerated the diet and medication without adverse effects. Weight gain over the 4-week study period was no different between the MMF and control groups. Total cholesterol, HDL, LDL, and triglyceride levels were statistically significantly increased compared with baseline, but there was no statistically significant difference between the two groups. On immunohistochemical examination, there was a trend toward decreased amount of early atherosclerotic plaque (MMF group: 11, 1, 1, 1, 1, 1, 0 vs control group: 111, 11, 11, 1, 1, 1), foamy macrophages, and macrophage migration into the media of vessels in the MMF group as compared with control. Macrophages and monocytes present in the plaques as well as in the © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 961–962 (1998)
subendothelial spaces and media of vessels were identified by monoclonal antibodies against rabbit monocyte/macrophages (RAM 11) and monocyte chemoattractant protein (MCP-1). These changes were observed mainly in the arch. Analysis using monoclonal antibody against a-smooth-muscle actin demonstrated heavy staining in both groups. DISCUSSION
The pathophysiology of atherosclerotic plaque formation is not fully understood. The New Zealand white rabbit develops arterial intimal lesions that resemble human fatty streaks; that is, they consist largely of lipid-laden foam cells, making them good models to study human atherosclerosis.6 The present study confirmed, through immunohistochemistry, the previous reports that foam cells that form these early lesions are largely derived from monocytes.6 Our initial results also suggest that MMF may have an antiatherosclerotic effect in aortas of cholesterol-fed rabbits. A possible mechanism for this effect is by decreasing the severity of the monocyte-macrophage infiltration into the plaque. This may occur by inhibiting monocyte adhesion to endothelial cells as has been previously reported from cell culture experiments.7 The trend toward reduced atherosclerotic plaque in the MMF-treated group did not reach statistical significance because of the high interanimal variability in plaque formation in this animal model. Further studies with greater numbers of animals will be conducted in our laboratory to determine whether these trends will achieve statistical significance. From the Departments of Surgery (T.C.S., S.M.G., D.Y.K., S.S., R.S.S., V.A.T.) and Pathology (T.M.C., J.W.B.), Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, New York, USA. Supported by Roche, Inc (Nutley, NJ). Address reprint requests to Dr S.M. Greenstein, Department of Surgery, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467. 0041-1345/98/$19.00 PII S0041-1345(98)00111-0 961
962
REFERENCES 1. Drew AF, Tipping PG: Atherosclerosis 116:181, 1995 2. Ferns G, Reidy M, Ross R: Am J Pathol 142:403, 1990 3. Jonasson L, Holm J, Hansson GK: Proc Natl Acad Sci USA 85:2303, 1988 4. Emeson EE, Shen ML: Am J Pathol 142:1906, 1993
SCHREIBER, GREENSTEIN, KIM ET AL 5. Roselaar SE, Schonfeld G, Daugherty A: J Clin Invest 96:1389, 1995 6. Hannson GK, Seifert PS, Olsson G, et al: Atheroscler Thromb 11:745, 1991 7. Laurent AF, Dumont S, Poindron P, et al: Exp Hematol 24:59, 1996