Clinical features of 26 cases of COVID-19-associated conjunctivitis.

PURPOSE: To explore the clinical features of COVID-19-associated conjunctivitis with the objective of preventing the spread of infection. STUDY DESIGN: Retrospective cohort study. METHODS: From March 2020 to March 2021, we retrospectively reviewed 26 (9.8%) consecutive COVID-19 patients with conjunctivitis among 282 COVID-19 cases admitted to our hospital. Clinical symptoms, onset date of conjunctivitis, time to patient recovery, and eye drop intervention were investigated. In addition, risk factors for developing conjunctivitis were statistically examined among 206 inpatients available for within 5 days of the onset. A multivariate analysis of conjunctivitis risk factors was performed. RESULTS: Among the 282 COVID-19 patients, 4 (1.4%) had conjunctival hyperemia as the primary symptom. The median time of onset was 4 days after the COVID-19 onset. Hyperemia was observed in all cases, but other ocular symptoms were rare. The median duration of hyperemia was 3 days. A multiple logistic regression analysis revealed that a young age (p=0.005) and current smoking habit (p=0.027) were independent risk factors for conjunctivitis after COVID-19. CONCLUSIONS: COVID-19-associated conjunctivitis is rare in the elderly and strongly associated with a history of smoking. It often occurs in the early stages of infection, and while hyperemia is recognized as a clinical symptom, other ocular symptoms are rare or non-existent. Many cases recover within a short time.

[Combination Therapy of Bevacizumab plus Paclitaxel Was Effective for a Metastatic Choroidal Tumor Secondary to Breast Cancer in a Patient with Ocular Symptoms].

A 44-year-old woman experienced loss of vision and distorted vision in the right eye. After she visited our hospital, she was diagnosed with a right metastatic choroidal tumor. At the age of 35 years, she had undergone surgery for left breast cancer; as recurrence of the breast cancer was suspected, the patient was referred to our department. A CT scan revealed left axillary lymph node swelling, liver metastasis, and lung metastasis. Lymph node needle biopsy was performed under ultrasound guidance, and the pathological findings revealed recurrence of breast cancer. Combination chemotherapy of bevacizumab( BV)plus paclitaxel(PTX)was administered. After chemotherapy, the metastatic lesion had remarkably shrunk, as observed on a CT scan. Optical coherence tomography(OCT)revealed that the tumor was flattened in her right eye. Choroidal metastasis of breast cancer is rare. BV plus PTX therapy was effective for treating choroidal metastasis of breast cancer, and it should be followed by ophthalmological examination over time.

Increased vitreous concentrations of MCP-1 and IL-6 after vitrectomy in patients with proliferative diabetic retinopathy: possible association with postoperative macular oedema.

PURPOSE: To determine whether vitreal concentrations of MCP-1, IL-6 and IL-8 are altered after vitrectomy in patients with proliferative diabetic retinopathy (PDR) and to investigate whether the altered levels of these cytokines are associated with postoperative macular oedema. METHODS: Vitreous samples were collected from 36 eyes of 33 patients with PDR before pars plana vitrectomy without intraocular lens (IOL) implantation, and also from the same 36 eyes during IOL implantation surgery approximately 7 months after the initial vitrectomy. Levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor were measured by flow cytometry using cytometric bead array (CBA) technology. RESULTS: The mean vitreous levels of MCP-1, IL-6 and IL-8 in the samples collected before vitrectomy were significantly higher in patients with PDR than in control patients (p<0.0001). The levels of MCP-1 and IL-6 in the samples collected at the time of IOL implantation were significantly higher than those collected before vitrectomy (p<0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p<0.05). The levels of IL-6 and IL-8, but not MCP-1, in the vitreous from eyes with PDR were inversely correlated with the interval between the initial vitrectomy and the time of implantation surgery. Among the vitrectomised patients, the mean vitreous level of MCP-1 in eyes with diabetic macular oedema (DME) was significantly higher than in those without DME (p=0.028). CONCLUSIONS: The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME.

Increased expression of M-CSF and IL-13 in vitreous of patients with proliferative diabetic retinopathy: implications for M2 macrophage-involving fibrovascular membrane formation.

PURPOSE: We recently demonstrated that M2 macrophages were involved in the development of fibrovascular membranes (FVM) associated with proliferative diabetic retinopathy (PDR) possibly through the induction of periostin. The purpose of this study was to determine whether macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-13, inducers of the M2 polarisation of macrophages from monocytes, are elevated in the vitreous of patients with PDR, and whether M2-polarised macrophages induce periostin production. METHODS: We measured the levels of M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-13, soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples collected from 61 eyes of 47 patients with PDR, and 39 eyes of 36 patients with non-diabetic ocular diseases (control group). Human monocytes were polarised in vitro with GM-CSF, interferon-γ, and lipopolysaccharide for M1 macrophages, and M-CSF, IL-4, and IL-13 for M2 macrophages. Quantitative real-time PCR was used to determine the mRNA level of periostin. RESULTS: The concentrations of M-CSF and IL-13 in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong positive correlation between the vitreous concentrations of M-CSF and sCD163 and periostin. The mean vitreous level of IL-13 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). In vitro studies showed that M2-polarlised macrophages significantly increased the expression of the mRNA of periostin. CONCLUSIONS: These findings indicate that the M2 polarisation of macrophages is induced by M-CSF and IL-13 in diabetic retinas. The presence of M-CSF and IL-13 would then promote FVM formation by periostin production.

Overexpression of CD163 in vitreous and fibrovascular membranes of patients with proliferative diabetic retinopathy: possible involvement of periostin.

AIM: To determine whether CD163, a specific marker for M2 macrophages, is involved in the formation of preretinal fibrovascular membranes (FVMs) present in eyes with proliferative diabetic retinopathy (PDR). METHODS: We measured the levels of soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples from 74 eyes of 62 patients with PDR, 20 eyes of 18 patients with proliferative vitreoretinopathy, and 56 eyes of 54 patients with non-diabetic ocular diseases (control group). Immunohistochemical analyses were performed to determine the expressions of CD68, CD163 and periostin in the surgically resected FVMs and idiopathic epiretinal membranes (ERMs). RESULTS: The concentrations of sCD163 and periostin in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong correlation between the vitreous concentrations of sCD163 and periostin. The mean vitreous level of sCD163 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). The number and percentage of CD163+ macrophages were significantly higher in the FVMs than in the idiopathic ERMs. Immunohistochemical analysis showed co-localisation of CD163 and periostin in FVM cells. CONCLUSIONS: These findings indicate that the overexpression of CD163 by macrophages may be involved in the development of FVMs partly through periostin production.

Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up-regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αV-mediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFß2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.

Comparison of gene expression profile of epiretinal membranes obtained from eyes with proliferative vitreoretinopathy to that of secondary epiretinal membranes.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs. METHODOLOGY/PRINCIPAL FINDINGS: A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 5' end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesion-related genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR. CONCLUSIONS/SIGNIFICANCE: Our results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets.

Antiangiogenic shift in vitreous after vitrectomy in patients with proliferative diabetic retinopathy.

PURPOSE: We determined whether the concentrations of VEGF, erythropoietin, and endostatin in the vitreous are altered after vitrectomy in patient with proliferative diabetic retinopathy (PDR). METHODS: We measured the levels of VEGF, erythropoietin, and endostatin by sandwich ELISA in vitreous samples collected from 38 eyes of 33 patients with PDR before pars plana vitrectomy (without IOL implantation) and the same 38 eyes during IOL implantation 3.1 to 25.7 (mean 6.7) months after the initial vitrectomy. RESULTS: The mean vitreous levels of VEGF (964.5 pg/mL) and erythropoietin (1359.5 pg/mL) in the samples collected before vitrectomy were significantly higher in patients with PDR than in the control patients (0.68 and 70.7 pg/mL, respectively; P < 0.01). The levels of VEGF (292.5 pg/mL) and erythropoietin (557.9 pg/mL) in the samples from eyes with PDR collected at the time of IOL implantation were significantly lower than those collected before vitrectomy (P < 0.01). In contrast, the changes in the level of endostatin were not significant after vitrectomy. The VEGF and erythropoietin levels in the vitreous fluid from patients with PDR were correlated inversely with the interval between the initial vitrectomy and the time of the IOL implantation. CONCLUSIONS: The significant decrease in the intravitreal concentration of VEGF and erythropoietin, and an absence of a significant change in the endostatin indicated a shift in the antiangiogenic balance in the vitreous of patients with PDR after successful vitrectomy.

Involvement of periostin in regression of hyaloidvascular system during ocular development.

PURPOSE: A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression. METHODS: We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay. RESULTS: The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin. CONCLUSIONS: These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels.

Bone marrow-derived monocyte lineage cells recruited by MIP-1ß promote physiological revascularization in mouse model of oxygen-induced retinopathy.

Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1ß (MIP-1ß) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1ß in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1ß was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1ß reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1ß-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1ß is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1ß induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.

Role of tumour necrosis factor-α (TNFα) in the functional properties of hyalocytes.

BACKGROUND/AIM: Tumour necrosis factor-α (TNFα) is an inflammatory cytokine that is upregulated in various vitreoretinal diseases including uveitis and diabetic retinopathy. Recently, our studies have indicated that hyalocytes contribute to the pathogenesis of these diseases. However, the impact of TNFα on the functional properties of hyalocytes is unknown. METHODS: Hyalocytes were isolated from bovine eyes. Cellular proliferation, migration and gel contraction in response to TNFα and the other inflammatory cytokines were analysed by thymidine uptake, Boyden's chamber assay and collagen gel contraction assay, respectively. Furthermore, we estimated the effect of dexamethasone on these properties of hyalocytes. RESULTS: TNFα promoted proliferation, migration and gel contraction by hyalocytes. Dexamethasone inhibited TNFα-induced proliferation but not migration. Dexamethasone did not inhibit TNFα-induced gel contraction but further increased contraction. Furthermore, dexamethasone inhibited TNFα-induced extracellular signal-related kinase (ERK)1/2 phosphorylation in hyalocytes. CONCLUSION: This study indicates that TNFα in vitreous and retina causes activation of hyalocytes, and the activated hyalocytes contribute to the pathogenesis of inflammatory vitreoretinal diseases. Steroid treatment appears to inhibit the activation of hyalocytes in the early stages of the diseases, but might have adverse effects in the late stage through membrane contraction.

Glypican 4, a membrane binding protein for bactericidal/permeability-increasing protein signaling pathways in retinal pigment epithelial cells.

PURPOSE: Originally identified as a lipopolysaccharide binding protein with Gram-negative bactericidal activity in the leukocytes, bactericidal/permeability-increasing protein (BPI) has been shown to induce various effects in retinal cells in vivo and in vitro. METHODS: The authors recently reported that BPI can induce ERK1/2 and Akt activity and that it increases DNA synthesis in the bovine retinal pigment epithelial (RPE) and pericyte cells. The authors have extended the characterization of BPI interaction with membrane proteins from bovine RPE. Crude membrane pools from RPE were isolated, solubilized, and bound to rBPI(21) affinity column. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue, which showed an intense band at 36 kDa consistently displaced by rBPI(21). RESULTS: Tandem mass spectrometry of the 36-kDa band suggested that cell surface protein glypican 4 (GPC4) serves as a putative BPI-binding protein. Heparitinase, phosphatidylinositol-specific phospholipase C, and anti-GPC4 antibody suppressed BPI-induced ERK and Akt phosphorylation in bovine RPE. Moreover, heparitinase also inhibited BPI actions on VEGF and PDGF-B mRNA expression induced by H(2)O(2). CONCLUSIONS: These new findings suggest that GPC4 is a specific binding protein for BPI on RPE to mediate the activation of ERK1/2, Akt, and the mRNA expressions of PDGF-B and VEGF.

Interleukin-18 regulates pathological intraocular neovascularization.

Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Preclinical investigation of fluorometholone acetate as a potential new adjuvant during vitreous surgery.

OBJECTIVE: To examine the effects of intravitreal fluorometholone acetate (FMT) on the morphology and function of the retina and to investigate its possible use for vitreous surgery. METHODS: Brown Norway rat eyes (n = 6, 12 groups) were injected with 0.05 ml of SF6 gas for vitrectomization. Four weeks later, FMT solution was injected into the vitreous cavity/subretinal space of the vitrectomized eyes at doses of 10, 20, and 40 mg/ml (0.05 ml/eye, n = 12 for each group). The retinal function was evaluated by electroretinography (ERG) at 4 and 8 weeks after FMT injection. Retinal toxicity was also assessed histologically by a light microscopy. Sham-operated eyes (0.05 ml of irrigating solution, n = 12) were used as control animals. FMT-assisted pars plana vitrectomy with internal limiting membrane (ILM) peeling was performed in primate eyes (n = 2). Retinal toxicity was assessed by ophthalmoscope, fluorescein angiography and electron microscopy three months after the vitreous surgery. RESULTS: There was no remarkable reduction in any ERG waves at either time interval at 4 and 8 weeks after the intravitreal/subretinal injection of FMT. No obvious histological change was observed in any of the rat eyes either. Using ophthalmoscope, fluorescein angiography and electron microscopy, the appearance of the primate retinas remained to be in a non-pathological condition. CONCLUSION: FMT appears to be a potentially useful tool in assisting vitreous surgery including safe ILM peeling.

Bactericidal/permeability-increasing protein's signaling pathways and its retinal trophic and anti-angiogenic effects.

Bactericidal/permeability-increasing protein (BPI) was originally identified as a lipopolysaccharide (LPS) binding protein with gram-negative bactericidal activity in the leukocytes. In this study, we characterized the previously unknown effects of BPI in the eye and the molecular mechanisms involved in its action. BPI mRNA was detected in bovine retina; retinal pigment epithelium; and primary cultures of bovine retinal pigment epithelial cells (RPE), pericytes (RPC), and endothelial cells (REC); while BPI protein was measured in human vitreous and plasma. BPI, but not control protein thaumatin, activated extracellular regulated kinase (ERK) and AKT, and increased DNA synthesis in RPE and RPC but not in REC. A human recombinant 21 kDa modified amino-terminal fragment of BPI (rBPI21) reduced H2O2-induced apoptosis in RPE and inhibited vascular endothelial growth factor (VEGF)-stimulated ERK phosphorylation in REC when preincubated with VEGF. Intraperitoneal (i.p.)-injected rBPI21 reduced ischemia-induced retinal neovascularization and diabetes-induced retinal permeability. Since BPI has unusual dual properties of promoting RPC and RPE growth while suppressing VEGF-induced REC growth and vascular permeability, the mechanistic understanding of BPI's action may provide novel therapeutic opportunities for diabetic retinopathy and age-related macular degeneration.

Abnormal retinal vascular development in IL-18 knockout mice.

Recent studies have indicated that interleukin 18 (IL-18) might act as either an angiogenic or an angiostatic factor, but the true function of this protein in vascular development is unclear. We therefore investigated the role of IL-18 in the formation of retinal vessels. Development of the retinal vasculature was compared in IL-18 knockout (KO) and wild-type (WT) mice at several different time points. The formation of vessels was evaluated using angiography of flat-mounted retinal samples after inoculation with fluorescein dextran. Retinal samples from both groups were also evaluated through histological examinations, and the expression of angiogenic factors was examined using the reverse-transcription-polymerase chain reaction. The capillary retinal vessels in both WT and IL-18 KO mice had reached the peripheral retina by postnatal day (P) 7. However, IL-18 KO mice showed angiectasis and vascular leakage at P7, especially in the mid-peripheral retina. These symptoms were not observed in WT mice at any stage. Histopathological analysis confirmed abnormal vascular formation in IL-18 KO mice at P14. Interestingly, these abnormalities regressed over time and had disappeared by P84. Several angiogenesis-associated factors, including vascular endothelial growth factor (VEGF), basic fibroblast-growth factor (bFGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), were overexpressed in the retinas of IL-18 KO mice compared with those of WT mice at P14. Interferon-gamma was detected only in WT mouse retinas at P14. These results provide new evidence for the role of IL-18 in retinal vascular development.

Bifunctional properties of peroxisome proliferator-activated receptor gamma1 in KDR gene regulation mediated via interaction with both Sp1 and Sp3.

Vascular endothelial growth factor receptor 2 (KDR) plays a critical role in mediating a variety of vasculogenic and angiogenic processes, including diabetic retinopathy. We previously demonstrated that the promoter activity of the KDR gene in retinal capillary endothelial cells (RCECs) was regulated in part by the relative concentration of positive/negative transcription factors Sp1/Sp3. We also reported that the peroxisome proliferator-activated receptor (PPAR)gamma ligand could inhibit intraocular angiogenesis. In the present study, the role of PPARgamma1 in KDR gene regulation in RCECs was examined. PPARgamma1 protein physically interacted with both Sp1 and Sp3. Transactivation and electrophoretic mobility shift assays clearly demonstrated novel findings that PPARgamma1 increased KDR promoter activity by enhancing the interaction between Sp1, but not Sp3, and KDR promoter region without its ligand in RCECs. The ligand-binding site but not the DNA binding site of PPARgamma1 enhanced the interaction between Sp1 and KDR promoter region. Conversely, PPARgamma1 ligand 15-deoxy Delta (12,14)-prostaglandin J2 dose-dependently suppressed the binding of KDR promoter region with both Sp1 and Sp3, resulting an inhibition of KDR gene expression. In conclusion, PPARgamma1 has bifunctional properties in the regulation of KDR gene expression mediated via interaction with both Sp1 and Sp3.

Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF.

BACKGROUND: The purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism. METHODS: For in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0-1.5 mg/ml). After 3 days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1alpha was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416). RESULTS: Formation of the tubular structure was enhanced 20 to approximately 50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1alpha protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1 microg/ml, P<0.05) or SU5416 (54.8% inhibition at the concentration of 3 microM, P<0.05). CONCLUSIONS: Extravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression.

Suppression of experimental choroidal neovascularization utilizing KDR selective receptor tyrosine kinase inhibitor.

BACKGROUND: We investigated the role of the VEGF-VEGF receptor 2 (KDR) system in the development of choroidal neovascularization (CNV) and its possibility as a therapeutic target utilizing KDR selective receptor tyrosine kinase (RTK) inhibitor (SU5416) both in vitro and in an experimental CNV model. METHODS: VEGF-induced phosphorylation of KDR and p44/p42 MAPK in cultured bovine choroidal endothelial cells (BCECs) was determined by Western blot analysis. The proliferation and in vitro tube formation were analyzed by [3H]thymidine uptake and three-dimensional collagen gel model. For experimental CNV model, intense fundus laser photocoagulation was performed on pigmented rats. The anti-angiogenic efficacy of intraperitoneally injected SU5416 on experimental CNV was evaluated by fluorescein angiography and histology. The extent of fluorescein leakage on late-phase angiograms was scored, and the thickness of CNV membrane was histologically measured under a light microscope. RESULTS: VEGF-induced KDR phosphorylation in cultured BCECs was inhibited by SU5416 in a dose-dependent manner (0-3 microM) with IC50 of 0.29 +/- 0.071 microM. SU5416 treatment also resulted in a dose-dependent prohibition of VEGF-induced p44/p42 MAPK phosphorylation, [3H]thymidine uptake and in vitro tube formation with corresponding concentrations that inhibited KDR phosphorylation. The leakage score on fluorescein angiography for experimental CNV was significantly lower in the SU5416-treated group than in the control group (P<0.01). Histologically, the CNV membranes in the SU5416-treated group were 31.6% thinner than those in the control group (P<0.01). CONCLUSION: These results strengthen the evidence for a critical role of the VEGF-KDR system in the development of CNV, indicating that KDR selective inhibitor might be beneficial for the treatment of intraocular angiogenic diseases, including age-related macular degeneration.