Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model

Purpose: To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. Methods: The effect of silibinin on the expression of α‐smooth muscle actin (α‐SMA) and vimentin in response to transforming growth factor‐β1 (TGF‐β1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin‐modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF‐β receptor–related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. Results: TGF‐β1 elevated the expression of α‐SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF‐β1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF‐β1‐treated HTFs was inhibited by silibinin. The effects of silibinin on TGF‐β1‐stimulated HTFs were mediated via the down‐regulation of TGF‐β receptor–related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α‐SMA at the site of blebs in vivo. Conclusion: Silibinin inhibited the TGF‐β receptor–related signalling pathway in TGF‐β‐treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo.     

Bevacizumab modulates epithelial‐to‐mesenchymal transition in the retinal pigment epithelial cells via connective tissue growth factor up‐regulation

Purpose: To investigate the effect of bevacizumab treatment on connective tissue growth factor (CTGF) expression and the induction of epithelial‐to‐mesenchymal transition in ARPE‐19 cells and human donor retinal pigment epithelium (HRPE) cells in vitro. Methods: We quantitated the protein and gene expression level of CTGF by ELISA. The effect of Fc–Fc receptor (Fc–FcR) interaction on CTGF expression was evaluated by CD64 siRNA silencing. Expression of epithelial‐to‐mesenchymal transition markers, alpha‐smooth muscle actin (α‐SMA) and zona occludens protein (ZO‐1) was evaluated by Western blot. Cell migration and collagen gel contraction assay were examined by light microscopy, and collagen production was measured by ELISA. Results: Bevacizumab stimulation increased CTGF expression in ARPE‐19 and HRPE cells in a dose‐dependent manner. CD64 gene silencing inhibited the effect of bevacizumab‐induced CTGF up‐regulation. Bevacizumab increased the expression of α‐SMA and decreased the expression of ZO‐1 in ARPE‐19 cells. Bevacizumab also caused the release of type‐1 collagen and increased cell migration and contraction of collagen. Conclusions: Bevacizumab exerts pro‐fibrotic effects on human RPE cells at clinical doses by up‐regulation of CTGF expression via an Fc–FcR interaction. This effect of bevacizumab may be one of the underlying mechanisms involved in age‐related macular degeneration therapy or intravitreal bevacizumab‐associated complications.     

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Background: Transforming growth factor β (TGF‐β) plays an important role in diabetic retinopathy. βIG‐H3 is a downstream target molecule of TGF‐β that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. Methods: We observed bovine retinal pericytes apoptosis and the increased expression of TGF‐β and βIG‐H3 induced by high concentrations of glucose in the cell culture media. An anti‐TGF‐β antibody was used to block glucose‐induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild‐type or mutant βIG‐H3 lacking Arg‐Gly‐Asp (RGD) sequences in order to validate the effects of βIG‐H3 and RGD signalling on retinal pericytes apoptosis. Results: A cell death‐detecting enzyme‐linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF‐β levels as compared with 5.5 mM glucose after 5 days, and βIG‐H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF‐β increased cell death and βIG‐H3 levels in a dose‐dependent manner, with a maximal effect observed at 1 ng/mL. An anti‐TGF‐β antibody nearly completely blocked high glucose‐induced cell death. Wild‐type βIG‐H3‐transfected cells showed a significant increase in cell death as compared with mutant βIG‐H3‐transfected (Mycb‐c) cells, untransfected or mock‐transfected cells. Conclusion: These results suggest that hyperglycaemia‐induced expression of TGF‐β and βIG‐H3 contributes to accelerated retinal pericytes apoptosis. βIG‐H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.     

Antioxidants reduce TGF‐beta2‐induced gene expressions in human optic nerve head astrocytes

Purpose: The goal of the present study was to investigate whether the antioxidants vitamin E, vitamin C and vitamin B1 can reduce the transforming growth factor‐beta2 (TGF‐β2)‐induced gene expressions in cultured human optic nerve head (ONH) astrocytes. Methods: Cultured human ONH astrocytes were pretreated with different concentrations of vitamin E, vitamin C and vitamin B1 and then exposed to 1.0 ng/ml TGF‐β2 for 24 hr. Expression of the heat shock proteins Hsp27 and αB‐crystallin, the extracellular matrix (ECM) component fibronectin and the ECM‐modulating protein connective tissue growth factor (CTGF) was detected by immunohistochemistry or real‐time PCR analysis. Results: TGF‐β2 increased the expression of Hsp27, αB‐crystallin, fibronectin and CTGF in human ONH astrocytes. Pretreatment with different concentrations of vitamin E, vitamin C and vitamin B1 reduced the TGF‐β2‐stimulated gene expressions. Conclusion: In cultured human ONH astrocytes, the TGF‐β2‐stimulated gene expressions could be reduced by pretreatment with vitamin E, vitamin C and vitamin B1. Therefore, the use of antioxidants in glaucomatous optic neuropathy might be a promising approach to prevent TGF‐β2‐induced cellular changes in ONH astrocytes.     

Differential effects of TGF‐β and FGF‐2 on in vitro proliferation and migration of primate retinal endothelial and Müller cells

Purpose: During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming growth factor‐β (TGF‐β) and fibroblast growth factor (FGF‐2) to influence this process by regulating proliferation and migration of retinal endothelial and macroglial cells. Methods: We assessed the effects of exogenous TGF‐β and FGF‐2 on the proliferation and migration of cultured endothelial (RF/6A) and Müller cell (MIO‐M1) lines. Cell proliferation was measured using a MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) colorimetric assay over 72 hr. Cell migration was measured using a scratch‐wound assay over 72 hr. Results: Transforming growth factor‐β inhibited the proliferation of endothelial and Müller cells and inhibited the migration of Müller cells, but not endothelial cells, compared to untreated controls. Conversely, FGF‐2 increased endothelial cell proliferation but inhibited endothelial cell migration. Fibroblast growth factor‐2 increased migration of Müller cells but had little effect on proliferation except at higher concentrations (20 ng/ml). Conclusion: Taken together, these observations indicate that TGF‐β and FGF could work in concert to inhibit endothelial cell proliferation and migration, respectively; this may have implications for establishing and maintaining the avascular zone of primate fovea.     

NGF and TGF‐β mRNA expression during pregnancy in a rat corneal wound healing model

Background: Growth factors seem to play a major role in corneal wound healing and TGF‐β seems to be associated with abnormal healing after corneal surgical procedures. Few studies have analysed the role of NGF and TGF‐β on corneal wound healing during pregnancy. The aim of the present study was to create an animal model to evaluate the expression of NGF and TGF‐βs during corneal wound healing in two groups: control and pregnant rats. Methods: Corneal mRNA for NGF and the three isoforms of TGF‐β were analysed by RT‐PCR, in a time‐course experiment on different days after epithelial wounding (2, 7, 14 days) in pregnant and control groups Results: The results show high corneal mRNA expression for NGF and TGF‐β1 without any variation throughout the healing process or pregnancy evolution. However, we detected a different expression of corneal mRNAs for TGF‐β2 and TGF‐β3 in the control group. This data was not detected in the pregnant group. Discussion: Our results suggested that pregnancy could have a relevant role on TGF‐β2 and TGF‐β3 mRNA expression during the corneal wound healing process. Additional research should be performed to corroborate these findings.     

增生性玻璃体视网膜病变增生膜中结缔组织生长因子与转化生长因子β受体和细胞外基质相关性的研究

目的 观察结缔组织生长因子（CTGF）在不同级别增生性玻璃体视网膜病变（PVR）增生膜中的表达及其与转化生长因子β受体（TGF-βR）和细胞外基质（ECM）出现的关系，探讨PVR发病过程中CTGF与TGF-βR和ECM产生的相关性。 方法 采用链霉亲和素生物素复合物（SABC）免疫组织化学方法，检测玻璃体切除手术获得的43例PVR增生膜中CTGF、TGF-βRⅡ、纤维连接蛋白（FN）和Ⅰ型及Ⅲ型胶原蛋白的表达，应用Spearman相关分析判断两样本之间有无相关性。 结果 PVR增生膜中CTGF、TGF-βRⅡ蛋白高表达，阳性表达的细胞主要是上皮样细胞。免疫组织化学显示C级膜中二者阳性表达率分别为70.6%和76.5%，D级膜中分别为73.9%和69.6%。统计学分析结果显示，CTGF、TGF-βRⅡ各级阳性表达率与膜分级间无相关性（P＞0.05）。PVR膜中FN、Ⅰ型和Ⅲ型胶原染色在实质细胞和细胞外间质均有表达;统计学分析结果显示，CTGF与TGF-βRⅡ、FN、Ⅰ型及Ⅲ型胶原的表达呈正相关(P＜0.01)。 结论 PVR增生膜中CTGF、TGF-βRⅡ蛋白表达上调，推测CTGF的产生与TGF-βR的激活有关，CTGF加重增生膜中RPE细胞合成FN和胶原等ECM，参与了PVR增生膜的形成和发展。 (中华眼底病杂志, 2006, 22: 192-195)     

Keratinocyte transglutaminase in proliferative vitreoretinopathy

PURPOSE: Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease. METHODS: Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-beta2, basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-beta2-modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-beta2 treatment. RESULTS: mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-beta2 treatment. Silencing of CTGF expression did not attenuate the TGF-beta2-mediated induction of kTgase. CONCLUSIONS: The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-beta2. TGF-beta2-mediated increase seems to be independent of CTGF.     

Regulation of cell-mediated collagen gel contraction in human retinal pigment epithelium cells by vascular endothelial growth factor compared with transforming growth factor-β2

Background: To explore the potential role of vascular endothelial growth factor compared with transforming growth factor‐β2 in the regulation of human retinal pigment epithelium cell‐mediated collagen gel contraction. Methods: The retinal pigment epithelium cell mediated type I collagen gel contraction assay was performed to evaluate and compare the effect of vascular endothelial growth factor and transforming growth factor‐β2. The number of viable retinal pigment epithelium cells in the gel and the expression of α‐smooth muscle actin were analysed. Results: Both vascular endothelial growth factor and transforming growth factor‐β2 caused a time dependent gel contraction, associated with over expression of α‐smooth muscle actin in retinal pigment epithelium cells undergoing a fibroblast like transformation. The decrease in volume of the collagen gel and increase in α‐smooth muscle actin expression were more significant in the transforming growth factor‐β2‐treated group than in vascular endothelial growth factor‐treated group beginning at day 2, and the growth of retinal pigment epithelium cells was significantly more inhibited in the transforming growth factor‐β2‐treated group compared with the vascular endothelial growth factor‐treated group after day 1 (P < 0.05). Transforming growth factor‐β2 stimulation increased both vascular endothelial growth factor mRNA expression and secretion. The α‐smooth muscle actin expression and the change in volume of collagen gel were significantly positively correlated in both experimental groups. Conclusions: Both vascular endothelial growth factor and transforming growth factor‐β2 can cause induction of retinal pigment epithelium cell‐mediated collagen gel contraction in vitro via partial upregulation of α‐smooth muscle actin expression.     

Toxoplasma gondii protects against H2O2‐induced apoptosis in ARPE‐19 cells through the transcriptional regulation of apoptotic elements and downregulation of the p38 MAPK pathway

Purpose: Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii‐induced suppression of apoptosis in retinal cells have not been analysed in detail. Methods: We investigated the role of T. gondii infection in H₂O₂‐induced apoptosis in human retinal pigment epithelial cells (ARPE‐19) by monitoring the activities of apoptosis‐regulating molecules and mitogen‐activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK. Results: T. gondii infection significantly inhibited the cellular toxicity of H₂O₂ (500 μm ) and increased cell viability in a multiplicity of infection (MOI)‐dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE‐19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of pro‐apoptotic factors, such as Bad and Bax, and the activation of caspase‐3. Infection with T. gondii increased the expression of the anti‐apoptotic factor Bcl‐2 in ARPE‐19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H₂O₂ treatment. Exposure to H₂O₂ increased the expression of heme oxygenase‐1 (HO‐1) in ARPE‐19 cells, and its expression was significantly inhibited in H₂O₂‐treated infected cells. Conclusion: The protective function of T. gondii infection against ROS‐induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stress‐induced intracellular signalling.     

Angiopoietin‐like protein 4 (ANGPTL4) is induced by high glucose in retinal pigment epithelial cells and exhibits potent angiogenic activity on retinal endothelial cells

Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro‐ versus anti‐angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro‐angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE‐19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE‐19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE‐19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE‐19 and RNA interference (RNAi). Results: The conditioned medium from ARPE‐19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE‐19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE‐19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy.     

Stage specificity of novel growth factor expression during development of proliferative vitreoretinopathy

OBJECTIVE: To compare the relative levels of connective tissue growth factor (CTGF), platelet-derived growth factor alpha (PDGF-AA), and hepatocyte growth factor (HGF) in glial and retinal pigment epithelial (RPE) cells of epiretinal membranes from proliferative vitreoretinopathy (PVR). METHODS: A total of 37 PVR membranes, of various stages, underwent fluorescent immunohistochemisty and confocal laser scanning microscopy to localize CTGF, HGF, and PDGF-AA in RPE and glial cells. RESULTS: Numerous RPE, and relatively fewer glial cells, were found in all stages of PVR. CTGF immunoreactivity increased from early to late stage PVR and was principally expressed by RPE cells in early stage, and by glial cells in late stage PVR. HGF, expressed by both RPE and glial cells, was principally expressed in mid-stage PVR. PDGF-AA, expressed by both cell types, demonstrated a uniform level of staining throughout all stages of PVR. CONCLUSIONS: RPE and glial cells contribute to the expression of CTGF, HGF, and PDGF-AA during PVR, but with specific developmental patterns. PDGF-AA is expressed uniformly throughout all stages of PVR, while HGF expression peaks during mid stage, and CTGF expression is highest during late stage PVR. These results allow for the development of stage-specific therapeutics for PVR that may allow targeting of the early proliferative and/or the late tractional stages of PVR.     

Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy

AIMS: To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors.Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes. RESULTS: In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-beta. The TGF-beta is activated, upregulating expression of CTGF. Under the influence of TGF-beta and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury. CONCLUSIONS: HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.     

结缔组织生长因子在兔视网膜增生膜中的表达

目的观察结缔组织生长因子（CTGF）在实验性增生性玻璃体视网膜病变（PVR）增生膜中的表达,探讨CTGF在PVR视网膜增生膜形成过程中的作用。方法采用Nobuyo的方法从兔视网膜中分离视网膜色素上皮（RPE）细胞并进行体外培养和传代。第3代RPE细胞制备密度为1.1×10^5/mL的细胞悬液。32只日本大耳白兔,取8只作为正常对照组,其余24只采用玻璃体腔内注入RPE细胞悬液的方法建立PVR动物模型。动物分别于造模后7、30、60d处死并摘除眼球,每次处死8只动物。光学显微镜下观察视网膜增生膜的组织学改变,免疫组织化学法检测PVR视网膜增生膜中CTGF的表达。结果造模后5d检眼镜下可见视网膜增生组织开始形成,增生膜随时间的推移逐渐增厚。组织学检查显示,造模后7d兔视网膜表面可见红染的条索状和网状胶原纤维,并可见大量的增生细胞分布其中。光学显微镜下可见视网膜内界膜变厚、粗糙、断裂或结构不清,视网膜各层结构欠清。免疫组织化学法检测表明,正常对照组在玻璃体及视网膜内未见CTGF的特异性染色。造模后7d,CTGF主要表达于视网膜表面增生细胞;造模后30d,CTGF主要表达于增生的细胞及增生的胶原纤维组织中;造模后60d,CTGF主要表达于增生的胶原纤维组织中。结论 CTGF在实验性PVR动物模型中呈高表达,提示CTGF参与了PVR增生膜的形成。     

Clearance of dying ARPE‐19 cells by professional and nonprofessional phagocytes in vitro– implications for age‐related macular degeneration (AMD)

Acta Ophthalmol. 2011: 89: e30–e34

Abstract.

Purpose: Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age‐related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE‐19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively. Methods: Death through extracellular matrix detachment using polyHEMA‐coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE‐19 cells. Two‐coloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin‐V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested. Results: The clearance of anoikic and apoptotic cells by nondying ARPE‐19 cells over 8 hr of co‐incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE‐19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages. Conclusions: ARPE‐19 cells are more efficient in clearing anoikic than UV‐induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE‐19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.     

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.     

Expression of advanced glycation end products and related molecules in diabetic fibrovascular epiretinal membranes

Purpose: To investigate associations between expressions of advanced glycation end products (AGEs), transforming growth factor‐β (TGF‐β), tumour necrosis factor‐α (TNF‐α) and integrins and correlations between their expression and level of vascularization and proliferative activity in diabetic fibrovascular epiretinal membranes. Methods: Membranes from eight patients with active proliferative diabetic retinopathy and nine patients with inactive proliferative diabetic retinopathy were studied by immunohistochemistry. Results: Blood vessels expressed AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin in 5, 13, 8 and 8 membranes, respectively. Stromal cells expressed AGEs, TNF‐α and α_vβ₃ integrin in 15, 13 and 3 membranes, respectively. There was no immunoreactivity for α_vβ₅, α₅β₁ and α₂β₁ integrins. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing AGEs (r_s = 0.496; P = 0.043), TGF‐β (r_s = 0.777; P < 0.001) and TNF‐α (r_s = 0.699; P = 0.002). There were significant correlations between number of blood vessels expressing AGEs and number of blood vessels expressing TGF‐β (r_s = 0.532; P = 0.028) and TNF‐α (r_s = 0.626; P = 0.007). The correlation between number of blood vessels expressing TNF‐α and α_vβ₃ integrin was significant (r_s = 0.617; P = 0.008). Number of blood vessels expressing CD34 (P = 0.001), TGF‐β (P = 0.006) and TNF‐α (P = 0.002) and stromal cells expressing AGEs (P = 0.001) and TNF‐α (P = 0.004) were significantly higher in active membranes than in inactive membranes. Conclusion: Interactions of AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin might be involved in pathogenesis of proliferative diabetic retinopathy fibrovascular proliferation.