The effects of 5-fluorouracil on ocular tissues in vitro and in vivo after controlled release from a multifunctional implant.

PURPOSE: To evaluate the effects of 5-fluorouracil (5-FU) on ocular cells in vitro and the effects of degradable 5-FU-loaded poly(DL-lactide-co-glycolide; PDLGA) 50:50 implant in the rabbit eye in vivo. METHODS: Cytotoxicity was assessed with a tetrazolium salt WST-1 cell proliferation/viability test and a lactate dehydrogenase (LDH) leakage test in rabbit corneal stromal fibroblasts (SIRCs), bovine corneal endothelial cells (BCECs), human conjunctival epithelial cells (IOBA-NHCs), human retinal pigment epithelial cells (ARPE-19), and human corneal epithelial cells (HCECs). The 5-FU-loaded PDLGA implants were surgically placed in rabbit eyes with a deep sclerectomy technique and the histopathology of the eyes was examined. RESULTS: In vitro, 5-FU affected cell proliferation and survival in a time- and dose-dependent manner. In the WST-1 test, adverse effects in serum-free conditions started from 0.0005 mg/mL 5-FU in SIRCS and HCECs, whereas in other cell types, 0.005 mg/mL 5-FU hindered cell proliferation. In serum-free conditions 72-hour 5 mg/mL 5-FU treatment decreased cell viability to 40% in BCECs and to 10% to 15% in other cell types. 5-FU had no or very minor effects on LDH leakage. In vivo, the 5-FU implant showed no signs of toxicity in cornea and retina, whereas in the conjunctival stroma near the implantation site, some inflammatory cells and a marked subepithelial condensation of stromal connective tissue was observed during the postoperative period of 4 weeks. CONCLUSIONS: 5-FU had a broad therapeutic range, and the 5-FU implant showed only minor tissue reactions in conjunctiva at the surgical site. 5-FU is a possible candidate for controlled drug release.

Histopathology of the three implanted degradable biopolymers in rabbit eye.

New straightforward applications of new biopolymers are needed in glaucoma surgery. The aim of this study was to compare biocompatibility of three biomaterials in rabbit eyes after deep sclerectomy; a collagen implant (AquaFlow) represented the "gold standard". A blend of 85:15 poly(L-lactide-co-glycolide) and 70:30 poly(L-lactide-co-1,3-trimethylene carbonate) copolymers in a molar ratio of 70:30 (Bio-1 = Inion GTR membrane) and poly(DL-lactide-co-glycolide with molar compositions of 50:50 (Bio-2) and 85:15 (Bio-3) were inserted into rabbits eyes. Bio-1, Bio-2 or Bio-3 caused very mild eye irritation or tissue response which was comparable to that of the collagen implant. The biodegradation time of Bio-1, Bio-2, and Bio-3 implants was over one year, 3 months and 6 months, respectively. Implant mapping by Fourier transform infrared (FTIR) microscopy revealed a heterogeneous distribution of degradation products throughout Bio-1, Bio-2, and Bio-3. All implants were surrounded by a very fine tissue capsule which was not visible after total degradation of the implants. The FTIR spectrum of tissue capsule around Bio-1 was almost identical to that around Bio-2 whereas significant differences were observed in the spectrum of the tissue capsule around Bio-3. Despite some differences in tissue response, all tested implants represent biologically acceptable materials for drainage devices in glaucoma surgery.

VEGFR-1 and -2 regulate inflammation, myocardial angiogenesis, and arteriosclerosis in chronically rejecting cardiac allografts.

OBJECTIVE: Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. METHODS AND RESULTS: Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow- or non-bone marrow-derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. CONCLUSIONS: These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

Angiogenic growth factors in cardiac allograft rejection.

Normal adult vasculature is in a quiescent state. In transplanted hearts, peri- and postoperative ischemic and alloimmune stimuli may be interpreted as inadequate tissue perfusion leading to activation of angiogenic signaling. Although this may have protective functions, improper activation of cardiac allograft endothelial cells and smooth muscle cells may actually result in impaired survival of cardiac allografts. In this paper, we review the current knowledge on angiogenic growth factors, vascular endothelial growth factor, angiopoietins, and platelet-derived growth factor in cardiac allografts. We also discuss the potential for therapies aimed at angiogenic growth factors in preventing and treating cardiac allograft rejection and transplant coronary artery disease.

Inhibition of tumor necrosis factor-alpha attenuates myocardial remodeling in rat cardiac allografts.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) elicits a wide range of pro-inflammatory activities on target cells and mediates diverse cardiovascular processes ranging from heart failure to atherosclerosis. Recently, we demonstrated that TNF-alpha regulates the platelelet-derived growth factor (PDGF)-A/PDGF-Ralpha activation pathway in rat cardiac allograft arteriosclerosis. The aim of this study was to determine the kinetics and biologic role of TNF-alpha and its receptors, TNF-R1 and TNF-R2, in rat cardiac allografts. METHODS: Heterotopic heart transplantations were performed from Dark Agouti to Wistar-Furth rats. In the acute rejection model, recipients were given no immunosuppression and grafts were removed 5 days after transplantation. In the chronic rejection model, cyclosporine (CsA) was administered and grafts were removed at 60 days. To investigate the functional role of TNF-alpha in chronic rejection, recipients received recombinant human soluble TNF receptor p80/IgG1 Fc fusion protein (rhu TNF-R2:Fc). RESULTS: During acute and chronic rejection, an increase in intragraft TNF-alpha and TNF-R2 mRNA expression was recorded, but not TNF-R1 mRNA expression. Prominent induction of TNF-alpha and TNF-R2 immunoreactivity was localized to medial cells of coronary arteries and interstitial inflammatory cells, whereas cardiomyocytes showed moderate immunoreactivity to TNF-alpha and its receptors. Inhibition of the TNF-alpha-mediated pathway by TNF-R2:Fc did not affect the incidence or intensity of arteriosclerotic lesions in rat cardiac allografts; however, it significantly inhibited myocardial remodeling with a concomitant decrease in myocardial TNF-alpha expression but not intragraft PDGF immunoreactivity. CONCLUSIONS: We conclude that inhibition of TNF-alpha attenuates myocardial remodeling but is not rate-limiting for arteriosclerotic lesion formation.

Combined vascular endothelial growth factor and platelet-derived growth factor inhibition in rat cardiac allografts: beneficial effects on inflammation and smooth muscle cell proliferation.

BACKGROUND: Perivascular inflammation and subsequent smooth muscle cell (SMC) proliferation are central in the development of cardiac allograft arteriosclerosis. We examined the effect of combined inhibition of proinflammatory vascular endothelial growth factor (VEGF) and SMC mitogen platelet-derived growth factor (PDGF) in rat cardiac allografts. METHODS: Heterotopic cardiac transplantations were performed between fully major histocompatibility mismatched rat strains receiving cyclosporine A immunosuppression. In situ hybridization and immunohistochemistry were performed to examine VEGF and PDGF ligand and receptor (R) expression. Protein tyrosine kinase inhibitors PTK787 and imatinib were used to inhibit VEGFR and PDGFR activity, respectively. Rat coronary artery SMC migration and proliferation assays were used to examine the effect of VEGF and PDGF and tyrosine kinase inhibitors in vitro. RESULTS: Both ligand and receptor expression of VEGF and PDGF were detected in chronically rejecting allografts. In vitro, PDGF-BB mediated rat coronary artery SMC migration and proliferation was completely inhibited with imatinib and partially with PTK787. In vivo, combined treatment with PTK787 and imatinib significantly reduced the formation of neointimal lesions in arteries of cardiac allografts at 8 weeks, producing a greater effect than either drug alone. PTK787, in contrast with imatinib, reduced the number of ED1 macrophages and PDGF-B immunoreactivity in the allografts at 4 weeks. CONCLUSIONS: Blocking VEGF and PDGF receptor signaling in cardiac allografts has distinctive effects on inflammation and SMC proliferation, suggesting that targeting both inflammation and pathologic vascular remodeling may be needed to inhibit cardiac allograft arteriosclerosis.

Role of angiogenic growth factors in transplant coronary artery disease.

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long-term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR-2 /CD34+ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet-derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.

Platelet-derived growth factor receptor inhibition reduces allograft arteriosclerosis of heart and aorta in cholesterol-fed rabbits.

BACKGROUND: Crosstalk between pro-inflammatory cytokines and platelet-derived growth factor (PDGF) regulates smooth-muscle-cell proliferation in cardiac-allograft arteriosclerosis. In this study, we tested the effect of STI 571, a novel orally active protein tyrosine kinase (PTK) inhibitor selective for PDGF receptor (PDGF-R) on transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. METHODS: Cardiac allografts were transplanted heterotopically from Dutch Belted to New Zealand White rabbits. A 0.5% cholesterol diet was begun 4 days before transplantation. Recipients received STI 571 5 mg/kg per day or vehicle intraperitoneally throughout the study period of 6 weeks. Cyclosporine A was given as background immunosuppression. RESULTS: In cardiac allografts of vehicle-treated rabbits, 76.2+/-2.1% of medium-sized arteries were affected by intimal thickening, and the percentage of arterial occlusion was 45.0+/-5.0%. Treatment with STI 571 reduced the incidence of affected medium-sized arteries to 41.2+/-8.1% (P <0.05) and the arterial occlusion to 27.6+/-5.0% ( P<0.05). In addition, we observed that STI 571 treatment reduced intimal lesion formation in proximal ascending aorta of transplanted hearts from 72.3+/-19.9 to 12.7+/-1.9 microm ( P<0.05). Our results also show that STI 571 significantly inhibited accelerated arteriosclerosis in medium-sized arteries of recipients' own hearts. CONCLUSIONS: The results of the present study suggest that PDGF-R activation may regulate the development of transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Thus, PTK inhibitors may provide new strategies for prevention of these fibroproliferative vascular disorders.

Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis.

BACKGROUND: Cardiac allograft arteriosclerosis is a complex process of alloimmune response, chronic inflammation, and smooth muscle cell proliferation that includes cross talk between cytokines and growth factors. METHODS AND RESULTS: Our results in rat cardiac allografts established alloimmune response as an alternative stimulus capable of inducing vascular endothelial growth factor (VEGF) mRNA and protein expression in cardiomyocytes and graft-infiltrating mononuclear inflammatory cells, which suggests that these cells may function as a source of VEGF to the cells of coronary arteries. Linear regression analysis of these allografts with different stages of arteriosclerotic lesions revealed a strong correlation between intragraft VEGF protein expression and the development of intimal thickening, whereas blockade of signaling downstream of VEGF receptor significantly reduced arteriosclerotic lesions. In addition, in cholesterol-fed rabbits, intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector that encoded mouse VEGF(164) enhanced the formation of arteriosclerotic lesions, possibly secondary to increased intragraft influx of macrophages and neovascularization in the intimal lesions. CONCLUSIONS: Our findings suggest a positive regulatory role between VEGF and coronary arteriosclerotic lesion formation in the allograft cytokine microenvironment.

Crosstalk of endothelin-1 and platelet-derived growth factor in cardiac allograft arteriosclerosis.

OBJECTIVES: In this study, we investigated the crosstalk of endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) in coronary artery smooth muscle cell (SMC) proliferation in the rat cardiac allograft model. BACKGROUND: Previous studies have suggested an independent role of ET-1 and PDGF in the development of cardiac allograft arteriosclerosis (i.e., chronic rejection). METHODS: Heterotopic heart transplantations were performed from Dark Agouti to Wistar Furth rats. Grafts were harvested after five days in an acute rejection model and after 60 days in a chronic rejection model. In the in vitro part of the study, SMC proliferation and migration were quantitated, as well as messenger ribonucleic acid (mRNA) levels of ET-1 and PDGF ligands and receptors after growth factor stimulation. RESULTS: Acute rejection induced both ET-1 receptors in the arterial wall. On linear regression analysis of chronically rejecting cardiac allografts, a strong correlation between intimal thickening and immunoreactivity of ET-1 and ET receptors A and B (ET(A) and ET(B)) in the arterial walls was observed. Treatment with Bosentan, a mixed ET-1 receptor antagonist, significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac allografts, as well as total intragraft ET(A) and ET(B) mRNA expression and intimal cell ET-1 and receptor immunoreactivity. This was associated with significantly reduced intragraft PDGF beta-receptor (PDGF-Rbeta) mRNA expression. In contrast, CGP 53716, a protein tyrosine kinase inhibitor selective for the PDGF receptor, did not reduce intragraft ET-1, ET(A) or ET(B) mRNA expression. In rat coronary artery SMC cultures, ET-1 stimulation significantly upregulated PDGF-Ralpha and -Rbeta mRNA expression and augmented PDGF-BB-mediated SMC proliferation as well as PDGF-AB- and PDGF-BB-mediated SMC migration. CONCLUSIONS: Our results suggest that the ET-1/PDGF-Rbeta/PDGF-BB axis may operate in SMC migration and proliferation in cardiac allograft arteriosclerosis, thus explaining the marked beneficial effects of blocking the signaling downstream of ET-1 receptors.