Oral Platelet-Derived Growth Factor and Vascular Endothelial Growth Factor Inhibitor Sunitinib Prevents Chronic Allograft Injury in Experimental Kidney Transplantation Model.

BACKGROUND: Expression of both platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) is increased during the development of chronic rejection which remains the major reason for late allograft loss in clinical kidney transplantation. Sunitinib is a tyrosine kinase inhibitor which inhibits both VEGF and PDGF receptors. Here we investigated its effect on the development of chronic rejection. METHODS: Rat aortic denudation model was used to define sunitinib dose. In vitro studies were done to investigate the effect of sunitinib on smooth muscle cell proliferation and migration. Kidney transplantations were performed from dark agouti rat strain (DA) to Wistar furth rat strain rats and syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed either with cyclosporine or with cyclosporine and sunitinib. Grafts were harvested at 5 and 90 days for histology and immunohistochemistry. Serum creatinine levels were measured weekly to monitor graft function. RESULTS: Sunitinib decreased neointimal formation and smooth muscle cell proliferation and migration in a dose-dependent manner. Sunitinib was well tolerated and almost completely prevented chronic rejection changes and preserved significantly better renal graft function after transplantation. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expressions. CONCLUSIONS: These results demonstrate that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation which indicates that sunitinib could be a potential intervention also in clinical kidney transplantation.

Different effects of tacrolimus and cyclosporine on PDGF induction and chronic allograft injury: evidence for improved kidney graft outcome.

INTRODUCTION: Chronic allograft injury is the most frequent cause of long-term kidney allograft loss. Cyclosporine (CsA) nephrotoxicity is an important factor contributing to graft loss. Increased fibrosis has been reported in renal transplants under CsA as compared with tacrolimus (Tac). Platelet-derived growth factor (PDGF) is a well-characterized fibrogenic growth factor in renal disease. OBJECTIVE: Here we investigated the effect of Tac on kidney grafts and PDGF expression both early after transplantation as well as during long-term follow-up of rat renal allografts, and compared it to CsA. Tac and CsA were also studied on PDGF secretion in macrophages in vitro. MATERIALS AND METHODS: Kidney allografts were immunosuppressed with Tac or CsA. Grafts were analyzed by histology and immunohistochemistry. ELISA was used to measure PDGF-levels in Tac and CsA-treated macrophage cultures. RESULTS: Tac ameliorated markedly both acute and chronic changes, whereas moderate to intense histological changes were seen in CsA-treated allografts. Tac also significantly inhibited PDGF expression compared to CsA. At clinically adjusted concentration CsA induced PDGF in macrophages whereas Tac did not. DISCUSSION: Tac may be less fibrogenic than CsA by decreasing PDGF expression in kidney grafts as well as in macrophages. Data presented here suggests that decreased PDGF induction by Tac may be beneficial for later outcome of the kidney graft.

Epidermal growth factor inhibition, a novel pathway to prevent chronic allograft injury.

BACKGROUND: Chronic allograft injury remains a major problem in clinical kidney transplantation and different growth factors participate in its development. Epidermal growth factor (EGF) affects cell proliferation and mitogenesis through its tyrosine kinase receptor. Erlotinib is an orally administered tyrosine kinase inhibitor used in clinical oncology to inhibit EGF signaling. We investigated its effect on the development of chronic allograft injury in an experimental kidney transplantation model. METHODS: Kidney transplantations were performed between Dark Agouti and Wistar Furth rats. Recipients were immunosuppressed either with cyclosporine A (CsA, 1.5 mg/kg/day subcutaneously) or with CsA and erlotinib (10 mg/kg/day orally). Kidney grafts were harvested after 5 and 90 days for histology and immunohistochemistry. Aorta denudation model was used for the erlotinib dose response study to define the optimal dose for the transplantation study. RESULTS: Epidermal growth factor expression was increased in CsA-treated allografts which developed intense chronic changes on day 90. Erlotinib ameliorated neointimal formation in the dose response study. In addition, erlotinib decreased chronic rejection changes and maintained better graft function in kidney transplantation model. Late posttransplant EGF and EGF receptor levels were reduced with erlotinib. CONCLUSION: Based on these findings, EGF mediates in part the development of chronic allograft injury. Its inhibition with erlotinib prevents chronic rejection and maintains better allograft function. Therefore, EGF blocking by erlotinib provides a novel pathway to prevent chronic allograft injury.

Early short-term imatinib treatment is sufficient to prevent the development of chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN), now defined as interstitial fibrosis and tubular atrophy not otherwise specified, is a near universal finding in kidney grafts by the end of the first decade posttransplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. Here, we investigated whether early short-term PDGF inhibition with imatinib could prevent CAN. METHODS: Kidney transplantations were performed from Dark-Agouti (DA) to Wistar-Furth (WF) rats and syngenic controls were done between DA rats. Allografts were immunosuppressed with cyclosporine. One group was also treated with imatinib for the first 30 days after transplantation. Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation. RESULTS: In control allografts, moderate to intense chronic changes were seen, whereas in syngenic grafts, no changes were seen. The early imatinib treatment prevented the development of CAN significantly compared to control allografts. Only few histological changes were seen. Fibrogenic growth factor ligand and receptor induction as well as inflammatory cell response was significantly inhibited by imatinib. Creatinine values of imatinib-treated allografts were also significantly lower compared to controls. CONCLUSIONS: We show that short-term imatinib treatment is sufficient to prevent CAN significantly, indicating that early PDGF induction has an important role in the pathogenesis of CAN. Here, we provide preclinical work that will need to be confirmed in patients with CAN.

FK778 ameliorates post-transplant expression of fibrogenic growth factors and development of chronic rejection changes in rat kidney allografts.

BACKGROUND: Acute rejection is the major risk factor for the development of subsequent chronic allograft nephropathy (CAN), which is the primary reason for late allograft loss in kidney transplantation. Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are the main mitogens mediating mesenchymal cell proliferation. Their early post-transplant induction may start cascades leading to the development of CAN. An immunosuppressive drug, FK778, inhibits de novo pyrimidine biosynthesis and several receptor tyrosine kinases (RTKs). Here we investigated its effects on acute and chronic rejection as well as post-transplant PDGF and TGF-beta expression in combination therapy with calcineurin inhibitors (CNIs). METHODS: Kidney transplantations were performed from DA to WF rats. Syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed with a combination of FK778 (10 mg/kg/day p.o.) and CsA (1.5 mg/kg/day s.c.) or tacrolimus (Tac) (1.5 mg/kg/day p.o.). Grafts were harvested 5 and 90 days after transplantation for histology and immunohistochemistry (PDGF-A, PDGF-B, PDGFR-alpha, PDGFR-beta, TGF-beta, TGF-betaR). The dose response of FK778 on acute rejection was studied with monotherapy of 5, 10 and 20 mg/kg/day. Chronic changes were scored according to the Chronic Allograft Damage Index (CADI). RESULTS: FK778 ameliorated the early post-transplant inflammatory response dose dependently. Additive effects were seen with FK778 and CNIs. Significantly lower CADI scores were seen in combination therapy of FK778 and CNIs compared with CNI monotherapies. FK778 also significantly reduced both early and late PDGF and TGF-beta expression when combined with CNIs. CONCLUSIONS: These results indicate that FK778 could prevent the development of CAN and be a promising therapy also in clinical kidney transplantation.

Immunological and metabolic effects of cis-9, trans-11-conjugated linoleic acid in subjects with birch pollen allergy.

Animal studies suggest that conjugated linoleic acid (CLA) may modulate the immune response, while studies in healthy human subjects have shown little effect and results are controversial. However, the effects of CLA may be more prominent in situations of immune imbalance, such as allergy. We studied the effects of the natural CLA isomer, cis-9, trans-11-CLA, on allergy symptoms and immunological parameters in subjects with birch pollen allergy. In a randomised, placebo-controlled study, forty subjects (20-46 years) with diagnosed birch pollen allergy received 2 g CLA/d in capsules, which contained 65.3 % cis-9, trans-11-CLA and 8.5 % trans-10, cis-12-CLA (n 20), or placebo (high-oleic acid sunflower-seed oil) (n 20) for 12 weeks. The supplementation began 8 weeks before the birch pollen season and continued throughout the season. Allergy symptoms and use of medication were recorded daily. Lymphocyte subsets, cytokine production, immunoglobulins, C-reactive protein, lipid and glucose metabolism and lipid peroxidation were assessed before and after supplementation. The CLA group reported a better overall feeling of wellbeing (P < 0.05) and less sneezing (P < 0.05) during the pollen season. CLA supplementation decreased the in vitro production of TNF-alpha (P < 0.01), interferon-gamma (P < 0.05) and IL-5 (P < 0.05). Total plasma IgE and birch-specific IgE concentrations did not differ between groups, whereas plasma IgA (P < 0.05), granulocyte macrophage colony-stimulating factor (P < 0.05) and eosinophil-derived neurotoxin (P < 0.05) concentrations were lower after CLA supplementation. Urinary excretion of 8-iso-PGF2alpha, a major F2-isoprostane (P < 0.01), and 15-keto-dihydro-PGF2alpha, a primary PGF2alpha metabolite (P < 0.05), increased in the CLA group. The results suggest that cis-9, trans-11-CLA has modest anti-inflammatory effects in allergic subjects.

Primary intraocular lymphoma: improving the diagnostic procedure.

OBJECTIVE: To analyze the clinical features of primary intraocular lymphoma (PIOL) and to describe cytochemical and immunocytochemical findings of the vitreous specimens as well as the reasons for delayed diagnosis of PIOL. DESIGN: Prospective noncomparative study. PARTICIPANTS: Eleven patients referred to the uveitis or medical retina units, Department of Ophthalmology, University of Helsinki, were diagnosed as having PIOL between 2000 and 2005. The median follow-up of the patients was 32 months. METHODS: Clinical features and diagnostic workup of uveitis were described. Twelve vitrectomies were performed on 9 patients. The first 5 biopsies were fixed in an equal volume of 50% alcohol. The specimens of the next 7 vitrectomies were handled without alcohol, and tissue culture medium was added to the samples. MAIN OUTCOME MEASURES: Clinical features of PIOL, intervals from ocular symptoms and from first ophthalmological examination to diagnosis, and the role of a proper handling of the vitreous sample in the diagnosis of PIOL. RESULTS: Six females (54%) and 5 males (46%) (median age, 61 years) were included. Ten patients had ocular symptoms for 1 to 30 months (median, 8) before the first contact with an ophthalmologist. Uveitis was bilateral in 9 patients. Vitreitis was seen in all patients, and it was severe in 8. Fundus lesions dominated in 3 patients. Six patients lost useful vision in one eye before the diagnosis of PIOL. Cytologic and immunohistochemical stainings prepared of the unfixed vitreous specimens showed PIOL in 6 patients. The samples fixed in alcohol were nondiagnostic in 4 patients, and in them, verification of diagnosis was based on brain biopsy (3) or cerebrospinal fluid (1) findings. Seven patients died due to primary nervous system lymphoma. CONCLUSIONS: Diagnosis of PIOL is difficult but can be improved. Severe bilateral vitreitis in an elderly patient is a characteristic finding of PIOL. Alcohol fixation may jeopardize the identification of PIOL cells in the vitreous sample. Optimal handling of the vitreous specimens and examination of the slides by an experienced cytopathologist are critical in the diagnostic workup of PIOL.

Early short-term platelet-derived growth factor inhibition prevents the development of chronic allograft nephropathy in experimental rat kidney transplantation.

Chronic allograft nephropathy (CAN) remains the primary reason for late allograft loss in kidney transplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. When administered continuously the PDGF receptor tyrosine kinase inhibitor imatinib prevents the development of CAN and restores kidney function in experimental kidney transplantation. Herein we investigated whether early short-term imatinib treatment prevented CAN. Kidney transplantations were performed from DA to WF rats and syngenic controls were done between DA rats. Allograft recipients were immunosuppressed with cyclosporine (CsA; 1.5 mg/kg/d sc). One group of allografts was also treated with imatinib (10 mg/kg/d po). Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation for histology and immunohistochemistry (PDGF-AA, -BB, PDGFR-alpha, -beta). Histological changes were scored according to the Chronic Allograft Damage Index (CADI). Among syngenic grafts, no signs of CAN were observed, namely, CADI 0.3 +/- 0.2 (mean +/- SEM). Control allografts showed moderate to intense chronic changes, CADI 6.5 +/- 1.3. Early short-term imatinib treatment significantly prevented the development of CAN compared with control allografts. Only a few histological changes were seen, namely, CADI 3.3 +/- 1.4. Compared with control allografts PDGF ligand and receptor induction was significantly inhibited by imatinib to nearly the same level as in syngenic grafts. Creatinine values of imatinib-treated allografts were also lower than control allografts. Our results demonstrated that early short-term imatinib treatment significantly prevented CAN. This indicated that early PDGF induction has an important role in the pathogenesis of CAN.

Decreased expression of vascular endothelial growth factor in idiopathic membranous glomerulonephritis: relationships to clinical course.

BACKGROUND: Membranous glomerulonephritis (MGN) has a variable clinical course, and factors that determine the prognosis are unknown. Our previous study suggested that urinary excretion of vascular endothelial growth factor (VEGF) is decreased in active MGN, but normalizes in remission. In the present study, VEGF protein and messenger RNA (mRNA) expression were investigated in this disease. METHODS: Twelve patients with clinically active and/or progressive MGN were studied by using urinary assays for VEGF and soluble VEGF receptor-1 (sVEGF-R1), semiquantitative scoring of serial renal biopsy specimens by using immunohistochemical staining for VEGF protein, and in situ hybridization for VEGF mRNA. Results were compared with healthy controls and normal parts of nephrectomized kidneys. RESULTS: Urinary VEGF excretion was decreased significantly (P < 0.001 versus controls) in MGN, but there was no difference in sVEGF-R1 excretion compared with healthy subjects. VEGF protein expression was diminished significantly in glomerular podocytes (P = 0.008), as well as in extraglomerular small arteries (P = 0.03) and arterioles (P = 0.008) in MGN. Total kidney VEGF score (the sum of scores of individual kidney compartments) also was decreased in MGN (P < 0.05) and remained low in repeated biopsies. Expression of VEGF mRNA localized predominantly to podocytes in normal kidneys was greatly reduced in MGN. CONCLUSION: Clinically active MGN is associated with diminished expression of VEGF protein and mRNA, mainly in podocytes, and expression remains depressed in persistently active and/or progressive disease. This is reflected by decreased urinary VEGF excretion. These findings point to potentially reversible podocyte injury and, together with our previous study, suggest that VEGF may have a protective role during the evolution of MGN.

Leflunomide analogue FK778 is vasculoprotective independent of its immunosuppressive effect: potential applications for restenosis and chronic rejection.

BACKGROUND: Leflunomide (LFM) inhibits experimentally both acute and chronic allograft rejection. The inhibition of dihydroorotate dehydrogenase (DHODH) in pyrimidine synthesis is suggested to be the major immunosuppressive mechanism. The mechanism of its vasculoprotective effect is not known, although it may be linked to inhibition of receptor tyrosine kinases (RTK). Here, we have investigated whether sufficient vasculoprotective effect could be obtained upon administration of FK778, a LFM analogue with shorter half-life, and compared the dose response with that of a known platelet-derived growth factor RTK inhibitor, imatinib, after endothelial injury in vivo. METHODS AND RESULTS: Wistar rats were used for aorta denudations. The rats remained untreated or received either FK778 or imatinib (STI571) at decreasing oral doses from 10 mg/kg per day. Half of the animals in both treatment groups also received uridine to reverse DHODH activity. Morphometric analysis was done after 14 day follow-up. In the untreated group, moderate neointima formation was detected. FK778 almost completely inhibited intimal formation, with or without uridine addition (P<0.05). Imatinib also inhibited neointima formation (P<0.05), whereas exogenous uridine reversed its effect. CONCLUSIONS: Our results demonstrate that FK778 inhibits neointima formation by way of a mechanism that is independent of DHODH inhibitory activity on vascular smooth muscle cell. Interestingly, the effect of imatinib was inhibited by uridine, suggesting that part of its action on vascular stenosis could be mediated through inhibition of pyrimidine synthesis.

Chronic allograft nephropathy is prevented by inhibition of platelet-derived growth factor receptor: tyrosine kinase inhibitors as a potential therapy.

BACKGROUND: Chronic allograft nephropathy (CAN) is the primary reason for late allograft loss in kidney transplantation, and currently there is no treatment available for it. Platelet-derived growth factor (PDGF) is suggested to be a major mitogen mediating mesenchymal cell proliferation in CAN. It has been shown that PDGF is already induced at acute renal allograft rejection, indicating a link between acute rejection and subsequent development of CAN. However, the definite effect of PDGF on the pathogenesis of CAN is still unknown. We investigated the role of PDGF in CAN by inhibiting PDGF by imatinib (STI571), a selective PDGF receptor tyrosine kinase inhibitor. METHODS: Kidney transplantations were performed from Dark Agouti (DA) to Wistar-Furth rats, and syngenic control transplantations were performed from DA to DA rats. All allograft recipients were immunosuppressed with cyclosporine A (1.5 mg/kg/day subcutaneously). One group of the animals was also treated with imatinib (10 mg/kg/day orally). Serum creatinine levels and cyclosporine A concentrations were measured once per week until the animals were killed. Grafts were harvested 5 and 90 days after transplantation for histology and immunohistochemistry. RESULTS: Only very few histologic chronic changes, similar to syngenic grafts, were seen in imatinib-treated allografts compared with control allografts. Creatinine values of imatinib-treated allograft recipients and infiltration of inflammatory cells, PDGF ligand, and receptor induction were also at the same level as in syngenic grafts. CONCLUSIONS: Our results demonstrate that imatinib prevents CAN almost completely, indicating that PDGF plays an important role in its pathogenesis. On the basis of our findings, imatinib could be a potential intervention in preventing CAN in clinical kidney transplantation.

Expression of insulin-like growth factors IGF-I and IGF-II, and their receptors during the growth and megakaryocytic differentiation of K562 cells.

Insulin-like growth factors (IGFs) I and II are critical regulators of cell proliferation and differentiation and most of the growth promoting properties of both ligands are mediated by IGF-I receptor (IGF-IR). In the present study we have investigated the role of IGFs in K562 cell line during normal growth and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced megakaryocytic differentiation. Abundant expression of IGF-I, IGF-II and IGF-IR was demonstrated in resting cells and exogenous IGF-I and IGF-II increased 3H-thymidine incorporation in a dose dependent manner. In contrast, we found that basal growth of the cells was inhibited by using anti-IGF-IR mAb. Furthermore, also IGF-I and IGF-II induced DNA synthesis was significantly suppressed by anti-IGF-IR mAb. During megakaryocytic differentiation, expression of IGF-IR increased during first 12h, but after that the expression started to decrease together with IGF-I. Taken together, our data suggest that autocrine production of IGF-I and IGF-II may via IGF-IR play a significant role in the growth and megakaryocytic differentiation of K562 cells.

The use of fine-needle aspiration biopsy in detection of acute rejection in children after liver transplantation.

Diagnosis of acute rejection after liver transplantation is based mainly on clinical signs and the liver core biopsy findings. In this study we retrospectively analyzed our data on the routine use of fine-needle aspiration biopsy (FNAB) after 63 pediatric liver transplantations. A total of 824 FNABs was taken during the postoperative hospitalization, with a mean of 13 biopsies per patient. Forty-nine acute rejection episodes were diagnosed and treated after 39 transplantations (62%). The FNAB analysis detected rejections often before clinical signs. At the time of rejection diagnosis, fever was present in 38% of the patients, and serum bilirubin and alanine aminotransferase were elevated in only 19% and 13%, respectively. The rejections responded well to oral methylprednisolone, and lymphoglobulins were needed in only two episodes (4%). The results indicate that FNAB is a safe and sensitive method for the diagnosis and follow up of acute cellular rejection in pediatric liver recipients.

The effect of acute rejection and cyclosporin A-treatment on induction of platelet-derived growth factor and its receptors during the development of chronic rat renal allograft rejection.

BACKGROUND: In the development of chronic kidney allograft rejection acute rejection (AR) is the single most important risk factor. Although Cyclosporin A (CsA) medication has decreased the incidence of AR, chronic rejection (CR) is still the major reason for late allograft loss. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CR. We have investigated the impact of AR and different doses of CsA on the expression of PDGF ligands and receptors in the development of CR. METHODS: Kidney transplantations were performed from DA to WF rats and syngenic controls were done from DA to DA rats. Two groups of allografts were treated daily with CsA either at low dose (1.5 mg/kg) or high dose (5 mg/kg). Third group of allografts was treated with CsA 5 mg/kg/day for 1 week and then left untreated until the development of AR. AR episodes were treated with CsA 5 mg/kg/day. Grafts were harvested 3 months after transplantation for histology and immunohistochemistry (PDGF-AA, -BB and PDGFR-alpha, -beta). RESULTS: In syngenic grafts no histological signs of CR were seen and the expression of PDGF ligands and receptors remained almost nonexistent. AR episodes increased the chronic rejection changes. High-dose CsA-treatment ameliorated inflammation compared to low-dose CsA-treatment, although it failed to inhibit the development of chronic changes. More fibrosis was even seen in high-dose than in low-dose CsA-treated grafts. CR in each allograft group was associated with induction of all PDGF ligands and receptors (P<0.05 compared with syngenic controls) in interstitial inflammatory cells, capillary endothelium, and arterial smooth muscle cells. In the group with AR episodes the expression was further increased. CONCLUSIONS: Our results demonstrate that CsA treatment cannot inhibit the expression of PDGF ligands and receptors in the development of chronic kidney allograft rejection and that AR episodes induce even more PDGF and its receptors in the graft indicating a link between AR and subsequent development of CR.