Renal heparan sulfate proteoglycans modulate fibroblast growth factor 2 signaling in experimental chronic transplant dysfunction.

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats.

BACKGROUND: Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction. METHODS: Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence. FINDINGS: Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function. INTERPRETATION: Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.

Development of transplant vasculopathy in aortic allografts correlates with neointimal smooth muscle cell proliferative capacity and fibrocyte frequency.

OBJECTIVE: Transplant vasculopathy consists of neointima formation in graft vasculature resulting from vascular smooth muscle cell recruitment and proliferation. Variation in the severity of vasculopathy has been demonstrated. Genetic predisposition is suggested as a putative cause of this variation, although cellular mechanisms are still unknown. Using a rat aorta transplant model we tested the hypothesis that kinetics of development of transplant vasculopathy are related to neointimal smooth muscle cell proliferative capacity and fibrocyte frequency, the latter being putative neointimal smooth muscle ancestral cells. METHODS: Aortic allografts were transplanted in Lewis and Brown Norway, as well as MHC-congenic Lewis.1N and Brown Norway.1L recipients. Severity of transplant vasculopathy was quantified 4, 8, 12 and 24 weeks after transplantation. Host-endothelial chimerism, as a reflection of vascular injury, was determined by specific immunofluorescence. Neointimal smooth muscle cell proliferative capacity was determined in vitro and in situ. Fibrocyte frequency and phenotype were determined after in vitro culture by cell counting, immunofluorescence and in situ zymography. RESULTS: Compared to Lewis, Brown Norway recipients developed accelerated transplant vasculopathy which is dependent on the presence of Brown Norway non-MHC-encoded determinants. Accelerated transplant vasculopathy was associated with increased levels of host-endothelial chimerism and increased neointimal smooth muscle cell proliferation, the latter being accompanied by increased endothelial and smooth muscle cell-derived neuropilin-like protein mRNA expression. Moreover, accelerated transplant vasculopathy was associated with increased frequency of circulating gelatinase-expressing CD45(+)vimentin(+) fibrocytes. CONCLUSION: Susceptibility for transplant vasculopathy appears to be genetically controlled and correlates with neointimal smooth muscle cell proliferative capacity and circulating fibrocyte frequency.

Donor and recipient contribution to transplant vasculopathy in chronic renal transplant dysfunction.

BACKGROUND: Chronic transplant dysfunction is the leading cause of long-term renal allograft loss. One of the histologic hallmarks of chronic transplant dysfunction is transplant vasculopathy characterized by accumulation of smooth muscle cells (SMCs) in the arterial subendothelial space, leading to ischemic graft failure. Currently, no therapy is available for transplant vasculopathy, and knowledge of the origin (donor vs. recipient) of neointimal cells may contribute to develop adequate strategies. METHODS: Origin of neointimal SMCs, endothelial, and tubular cells was determined in four nephrectomy samples from male recipients transplanted with a female kidney. Recipient-derived cells were detected using X- and Y-chromosome-specific fluorescent in situ hybridization combined with immunofluorescent staining. Specificity and sensitivity of fluorescent in situ hybridization were determined with corresponding controls. RESULTS: No Y-chromosome-positive cells were detected in the female to female graft, whereas approximately 31% of nucleated cells in male to male grafts had a detectable Y-chromosome. In female to male grafts, a recipient-derived population of neointimal alpha-smooth muscle actin-positive SMCs were detected (6%, range 3%-11%). Percentages of recipient-derived arterial endothelial cells, glomerular endothelial cells, and tubular epithelial cells were 14% (range 4%-32%), 19% (range 7%-31%) and 3% (range 2%-5%), respectively. CONCLUSIONS: Both donor- and recipient-derived cells contribute to vascular remodeling in clinical renal transplantation. The presence of alpha-smooth muscle actin in donor- and recipient-derived cells supports a constructive role for these cells in neointimal formation. However, the predominance of donor-derived cells in the neointima points to these cells as the likely therapeutic target.

Spironolactone ameliorates transplant vasculopathy in renal chronic transplant dysfunction in rats.

Chronic transplant dysfunction (CTD) is the leading cause of long-term renal allograft loss and is characterized by specific histological lesions including transplant vasculopathy, interstitial fibrosis, and focal glomerulosclerosis. Increasing evidence indicates that aldosterone is a direct mediator of renal damage via the mineralocorticoid receptor (MR). The MR antagonist spironolactone is renoprotective in native chronic kidney disease, but its effects on CTD are unknown. We studied the effects of spironolactone treatment on CTD development in the Dark Agouti-to-Wistar-Furth renal allograft transplant model, by treatment with 20 mg/kg spironolactone or vehicle daily by oral gavage from 2 days before transplantation (donors and recipients) throughout the experiment (12 wk, recipients). Dark Agouti-to-Dark Agouti isografts served as negative controls. Spironolactone significantly ameliorated the development of transplant vasculopathy in allografts by reducing the number of affected intrarenal arteries. In addition, spironolactone treatment showed a trend toward reduced proteinuria and focal glomerulosclerosis, and significantly reduced glomerular macrophage influx. However, spironolactone treatment did not affect interstitial fibrosis, interstitial macrophage influx, creatinine clearance, and systolic blood pressure. We conclude that spironolactone selectively ameliorates transplant vasculopathy and glomerular lesions in renal CTD in rats. These results suggest that spironolactone may have renoprotective potential as an adjunct treatment in renal transplantation to ameliorate CTD.

Dichotomous effects of rosiglitazone in transplantation-induced systemic vasodilator dysfunction in rats.

BACKGROUND: Transplantation-induced systemic endothelial dysfunction causes severe cardiovascular morbidity and mortality after transplantation. Interventions that improve systemic endothelial function after transplantation and furthermore reduce intragraft vascular dysfunction might improve graft and patient survival. Treatment with the PPARgamma agonist rosiglitazone is an intervention that potentially fulfills these criteria. In this study, we determined the effect of rosiglitazone treatment on transplantation-induced endothelial dysfunction and vasomotor activity in an experimental model for chronic transplant dysfunction in rats. METHODS: Lewis abdominal aortic allografts were orthotopically transplanted into Brown Norway recipients that received either regular chow or chow containing rosiglitazone (approximately 4.2 mg/day). Endothelium-dependent (response to metacholine) and total (response to sodium nitrite) vasodilatory responses were determined in autologous thoracic aortic rings using an ex vivo organ bath setup. Measurements were performed 8 weeks after transplantation. RESULTS: Aortic allografting induced systemic endothelial dysfunction as measured by reduced endothelium-dependent vasodilation in the recipient's vascular system. Rosiglitazone treatment restored endothelium-dependent vasodilatory responses to pretransplantation levels. However, rosiglitazone treatment reduced the total dilatory response despite normalized endothelial function, indicating impairment of vascular smooth muscle cell vasomotor activity. CONCLUSIONS: Rosiglitazone treatment after allogeneic transplantation restores endothelial function but impairs vascular smooth muscle cell vasomotor activity. This dichotomous effect of rosiglitazone might impede use of rosiglitazone after organ transplantation since this potentially increases cardiovascular risk despite improved endothelial cell function.

The source of neointimal cells in vein grafts: does the origin matter?

The focus of this Commentary is the existing plasticity in the origin of neointimal vascular smooth muscle cells and endothelial cells after vein grafting.

Rosiglitazone attenuates transplant arteriosclerosis after allogeneic aorta transplantation in rats.

BACKGROUND: Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts. METHODS: Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either approximately 5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro. RESULTS: Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P<0.01 vs. nontreated). Rosiglitazone reduced T cell alloreactivity which was not mediated through modulation of CD4+CD25+FoxP3+ regulatory T cells. Reduced development of transplant arteriosclerosis coincided with reduced intragraft expression of stromal-derived factor-1alpha and platelet-derived growth factor receptor-beta. Finally, rosiglitazone reduced growth-factor-driven proliferation of both medial and neointimal vascular smooth muscle cells in vitro, which was not mediated through PPARgamma. CONCLUSION: PPARgamma agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.