Differential effects of everolimus and cyclosporine A on intimal alpha-actin-positive cell dynamics of carotid allografts in mice.

BACKGROUND: We recently demonstrated that donor medial cells are replaced by smooth muscle alpha-actin (SMA)-expressing host cells in murine carotid allografts. In this study, we investigated neointimal cell dynamics including effects of cyclosporine A (CsA) and everolimus (SDZ RAD, Certican). To obtain a functional readout, we measured the effects of these treatments on cardiac allografts. METHODS: Heterotopic heart allotransplantation was performed between C57BL/6 (B6) and BALB/c (B/c) mice. Orthotopic carotid artery allotransplantation was performed between B6 and B/c mice expressing green fluorescent protein (GFP). Both strains served as donors and recipients. Groups of mice were treated for 4 and 8 weeks (heart and carotid recipients, respectively) with placebo, CsA 20 mg/kg per day, or everolimus 1.0 mg/kg per day using Alzet minipumps. At 2, 4, and 8 weeks, carotid grafts were harvested for histology. RESULTS: In the GFP-B/c to B6 strain combination, everolimus but not CsA significantly reduced neointima formation and accumulation of SMA-positive host (non-GFP) cells at doses that did not fully suppress heart rejection. In the B6 to GFP-B/c strain combination, everolimus and CsA strongly prevented heart rejection under treatment, partly suppressed neointima formation, and completely prevented SMA-positive host (GFP) cell accumulation. CONCLUSIONS: Donor-derived cells expressing SMA never appear in the neointima, and such cells are completely recipient derived. Adequate immunosuppression delays or prevents the disappearance of donor cells and the appearance of host cells in the graft, a phenomenon apparently associated with the neointima formation. Even immunosuppression sufficient to prevent heart allograft rejection under treatment completely, diminishes neointima formation only by approximately 50%.

Differential effect of cyclosporine A and SDZ RAD on neointima formation of carotid allografts in apolipoprotein E-deficient mice.

BACKGROUND: Apolipoprotein E-deficient (apoE-/-) mice spontaneously develop hypercholesterolemia and atherosclerotic lesions. This may be an appropriate background for the development of graft vessel disease. The authors therefore investigated the effects of cyclosporine A (CsA) and SDZ RAD (RAD, everolimus, Certican) on neointima formation of carotid allografts in apoE-/- mice. To ascertain that equipotent immunosuppressive doses were used, the authors also investigated their effects on cardiac allografts using the same wild-type strain combination. METHODS: Heterotopic heart allotransplantation was performed in the BALB/c to C57BL/6 strain combination. Graft survival was monitored daily. Orthotopic carotid artery allotransplantation was performed from BALB/c to apoE-/- (C57BL/6) mice. Groups of mice were treated for 8 weeks with placebo; CsA 10, 20, and 30 mg/kg/d; or RAD 0.3, 1.0, and 3.0 mg/kg/d using ALZET minipumps. Body weight, CsA blood levels, serum lipids, and histology were examined 8 weeks after transplantation or on the day of rejection. RESULTS: Compared with the placebo group, CsA or RAD did not affect body weight. Both CsA and RAD prolonged the survival of cardiac grafts in a dose-dependent manner. CsA blood levels were not different between wild-type and apoE-/- recipients. CsA increased total cholesterol and low-density lipoprotein, but not high-density lipoprotein dose-dependently and significantly, but RAD did not affect the lipids. RAD but not CsA significantly attenuated neointima formation. CONCLUSIONS: These results suggest that RAD at a dose preventing organ rejection may also prevent transplant vasculopathy even in the presence of hyperlipidemia.

Dynamics of medial smooth muscle changes after carotid artery transplantation in transgenic mice expressing green fluorescent protein.

BACKGROUND: Recent observations have demonstrated the importance of host cells in neointima formation after transplantation. Because little is known regarding the dynamics of host-derived cells in the graft media, we investigated this question in a mouse carotid artery transplantation model. METHODS: C57BL/6 carotid arteries were orthotopically transplanted into BALB/c mice ubiquitously expressing enhanced green fluorescent protein. Grafts were harvested at 1, 2, 4, and 8 weeks after transplantation for histologic examination. No immunosuppression was used. RESULTS: Immunostaining and semiquantitative analysis of cross sections showed that donor medial smooth muscle cells decreased over time in the graft media, whereas green fluorescent protein-positive/smooth muscle alpha-actin-positive cells (i.e., cells of host origin) increased over time. Interestingly, host cells were located only in the inner media and the neointima at 2 weeks and thereafter also in the outer media, indicating that the host-derived cells entered the media from the luminal side rather than from the adventitia. In longitudinal sections, there were no differences in the accumulation of donor- and host-derived cells between the end and middle regions of the graft media at 8 weeks. CONCLUSIONS: After transplantation, medial cells were replaced by alpha-actin-expressing host cells that were probably derived from circulating precursor cells. Our observations differ from the traditional view of a major contribution of donor medial smooth muscle cells to the neointima formation. Thus, circulating progenitor cells may be important for graft vessel disease.

Efficacy of mycophenolate sodium as monotherapy and in combination with FTY720 in a DA-to-Lewis-rat heart-transplantation model.

BACKGROUND: Mycophenolate sodium (MPS), which is in clinical development as an enteric-coated tablet (Myfortic), has not yet been characterized as a combination partner of FTY720. We therefore report here the effects of MPS and FTY720 mono and combination therapy in a rat heart allograft model. METHODS: Heterotopic heart allotransplantation was performed in the DA-to-Lewis-strain combinations. Groups of six rats were treated with placebo, MPS monotherapy, FTY720 monotherapy, and their combination. To circumvent pharmacokinetic problems, MPS was administered by Alza minipumps, while FTY720 was administered by gavage, both for 4 weeks. Graft survival was monitored daily, followed up by histology. Body weight and hematological parameters were determined at 1 and 4 weeks. RESULTS: The median survival time (MST) was 6 days for placebo, 6, 14.5, and more than 56 days for doses 3, 10, and 30 mg/kg per day MPS, with severe side effects (diarrhea, weight loss, lymphopenia) at the highest dose, and 7 and 8 days for the 0.03 and 0.1 mg/kg per day FTY720. The combination of 3 mg/kg per day MPS with these two FTY720 doses still resulted in rejection under treatment. However, 10 mg/kg per day MPS in combination with 0.03 or 0.1 mg/kg per day FTY720 yielded MSTs of 41 and 43.5 days, respectively, well beyond the 4-week treatment period. Both combination regimens were well tolerated. Both FTY720 doses combined with the highest MPS dose again caused severe side effects. CONCLUSIONS: MPS in monotherapy and combined with FTY720 resulted in steep dose-response curves. However, well tolerated combination regimens could be defined that prevented rejection.