G. R. Bicknell

Rapamycin reduces expression of fibrosis-associated genes in an experimental model of renal ischaemia reperfusion injury

CHRONIC ALLOGRAFT nephropathy (CAN) remains a major cause of renal transplant failure. It is characterised by a progressive functional deterioration, which is associated histologically with graft fibrosis. Cyclosporin has no impact on the development of CAN, and its nephrotoxicity is a contributor to the functional and histological deterioration found in this condition. The new immunosuppressive agent rapamycin inhibits growth factordriven proliferation in a number of cells crucial to the development of fibrosis including smooth muscle cells, endothelial cells, and fibroblasts. It therefore has a potential role in the therapy of CAN. In this experiment, to further explore this role both cyclosporin and rapamycin were administered to a rodent model of renal ischaemia reperfusion injury, which has previously been shown to be associated with up-regulation of fibrosis-associated genes.

Rapamycin inhibits vascular remodeling in an experimental model of allograft vasculopathy and attenuates associated changes in fibrosis-associated gene expression

BACKGROUND Rapamycin inhibits extracellular matrix (ECM) accumulation (fibrosis) and vascular remodeling in experimental models of chronic allograft dysfunction (CAD) by poorly understood mechanisms. The aim of this study was to assess the effect of rapamycin on the expression of fibrosis-associated genes and correlate this with observed changes in ECM remodeling in an experimental of model allograft vasculopathy. METHODS Vascular remodeling and ECM accumulation (picrosirius red) were measured by computerized histomorphometry of F344-to-Lewis rat aortic allograft sections harvested at serial timepoints. Expression of fibrosis associated genes was studied by means of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS Rapamycin (0.5 mg/kg/day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodeling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intragraft gelatinase, collagen III and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA levels. At a lower dosage (0.25 mg/kg/day), rapamycin inhibited intimal hyperplasia and medial ECM accumulation, but there was a lesser effect on vascular remodeling. Lower dose allografts were also seen to have a more severe inflammatory infiltrate and larger amounts of intragraft matrix metalloproteinase 9 (MMP 9) mRNA than those treated with the higher dose. CONCLUSIONS These data suggest that, in addition to the tissue response to injury, the alloimmune injury itself may contribute directly to the vascular remodeling that occurs in allograft vasculopathy. Rapamycin at higher but not lower doses inhibited both of these pathologic processes.

Differential effects of modern immunosuppressive agents on the development of intimal hyperplasia

Abstract Modern immunosuppressive agents such as tacrolimus and rapamycin are claimed to be associated with a reduction in vascular narrowing, a central feature of chronic rejection. This study assesses the effect of cyclosporine, tacrolimus and rapamycin on the development of intimal thickening, fibrosis‐associated genes and deposition of extracellular matrix (ECM) proteins in a model of intimal hyperplasia. Male Sprague‐Dawley rats received either no treatment or 5 mg/kg cyclosporine, 0.1 mg/kg tacrolimus or 0.05 mg/kg rapamycin. Animals underwent left common carotid balloon angioplasty, and intima medial ratios, pro‐fibrotic gene expression and ECM accumulation were calculated at 14 and 28 days. Cyclosporine was associated with increased intimal thickening compared to controls (P<0.004). Tacrolimus had no effect on intimal thickening, whilst rapamycin significantly inhibited intimal thickening at both 14 and 28 days (P<0.004 and P<0.026, respectively). All groups significantly inhibited matrix metalloproteinase (MMP)‐2, MMP‐9, tissue inhibitor of metalloproteinases (TIMP)‐l, transforming growth factor (TGF)‐β and collagen III expression at 14 days (P<0.001), but increased ECM deposition. However, rapamycin marginally reduced ECM deposition compared to cyclosporine (P<0.06). Treatment with cyclosporine was associated with worsening of vascular narrowing, whilst rapamycin showed a beneficial reduction in intimal thickening. Treatment with all immunosuppressive agents resulted in increased ECM deposition. Rapamycin may halt the progression of vascular narrowing compared to both cyclosporine and tacrolimus.

Fibrosis-associated gene expression in renal transplant glomeruli after acute renal allograft rejection.

Acute allograft rejection is thought to be a risk factor for chronic allograft nephropathy, the cardinal features of which are vasculopathy, interstitial fibrosis and glomerulosclerosis. Fibrosis‐associated genes might act as ad interim surrogate markers for chronic allograft nephropathy. The aim of this study was to determine mRNA expression of fibrosis‐associated genes in glomeruli plucked from protocol renal transplant biopsies, in patients with or without a history of acute rejection.

The effect of combined rapamycin/cyclosporine on the changes in pro-fibrotic gene expression that occur during the development of allograft vasculopathy in rats, compared with cyclosporine or rapamycin in isolation

Abstract Chronic allograft dysfunction, the leading cause of solid‐organ transplant failure, is characterised by histological evidence of extracellular matrix (ECM) accumulation (fibrosis). The aim of this study was to compare the effect of combined rapamycin and cyclosporine therapy on fibrosis‐associated gene expression and ECM turnover during the development of allograft vasculopathy, compared with either agent alone. Lewis recipients of F344 rat thoracic‐to‐abdominal aorta transplants were administered rapamycin, cyclosporine, combined rapamycin and cyclosporine or no treatment. F344‐to‐F344 isografts served as controls. Six grafts in each group were harvested at 16 weeks. Vascular remodelling and ECM accumulation (Sirius red) were measured by computerised histomorphometry of aortic sections. Messenger RNA was extracted from frozen tissue, and expression of fibrosis‐associated genes was studied by means of semiquantitative reverse transcription polymerase chain reaction (RT‐PCR). Rapamycin (0.5 mg/kg per day) or cyclosporine (5 mg/kg per day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodelling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intra‐graft gelatinase, collagen III and tissue inhibitor of metalloproteinase (TIMP)‐1 mRNA levels. Combined rapamycin and cyclosporine inhibited intimal hyperplasia; however, there was a lesser effect on vascular remodelling and medial ECM accumulation. Combined‐treatment aortic allografts were also seen to have a moresevere inflammatory infiltrate and larger amounts of intra‐graft matrix metalloproteinase (MMP)‐9, transforming growth factor (TGF)‐β and TIMP‐1 mRNA than those treated with monotherapy. Rapamycin and cyclosporine act synergistically to inhibit intimal hyperplasia but not the inflammatory infiltrate, allograft fibrosis or vessel remodelling. In the high‐responder F344‐to‐Lewis rat model, effective immunosuppression is required to reduce graft fibrosis.