Functional Characteristics of Connective Tissue Growth Factor on Vitreoretinal Cells
-
首席医学网
2008年05月25日 16:34:31 Sunday
-
作者：Takeshi Kita, Yasuaki Hata, Muneki Miura, Shuhei Kawahara, Shintaro Nakao, and Tatsuro Ishibashi 作者单位：the Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
加入收藏夹
【摘要】 Connective tissue growth factor (CTGF) level is elevated in eyes with proliferative vitreoretinal diseases, such as proliferative diabetic retinopathy and proliferative vitreoretinopathy (PVR), as we previously reported, but its functional characteristics on vitreoretinal cells are yet to be clarified. In this study, we demonstrated a growth-promoting effect of CTGF on cultured hyalocytes and bovine retinal pigment epithelial cells (BRPEs) with the induction of p44/p42 mitogen-activated protein kinase phosphorylation and [3H]thymidine incorporation. CTGF also stimulated the synthesis of fibronectin by hyalocytes and BRPEs without significant effect on collagen gel contraction by these cells. On the other hand, CTGF had no direct effects on the proliferation, migration, or in vitro tube formation by vascular endothelial cells. Nevertheless, CTGF promoted vascular endothelial growth factor (VEGF) gene expression by hyalocytes and BRPEs. Although the concentrations of both CTGF and VEGF in the human vitreous samples with proliferative vitreoretinal diseases were elevated, there was no significant correlation between these concentrations. These findings indicate that CTGF appears to be involved in the formation of proliferative membranes without direct regulation of their cicatricial contraction in the pathogenesis of proliferative vitreoretinal diseases. Whereas CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, it is possible that CTGF has indirect effects by modulating the expression of VEGF.
【关键词】 BAEC, bovine aortic endothelial cell; BREC, bovine retinal capillary endothelial cell; BRPE, bovine retinal pigment epithelial cell; CTGF, connective tissue growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; MAPK, mitogen-a
Proliferative vitreoretinal diseases, such as proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), still remain common causes of severe vision loss or blindness despite the dramatic development of vitreoretinal surgery (1,2). Better understanding of the pathogenesis of proliferative vitreoretinal diseases is thus needed for improved management of the diseases.
PVR and PDR cause tractional retinal detachment due to the formation of contractile preretinal fibrous membranes as a result of excessive wound healing in the pathological conditions. The membrane formation is characterized by the proliferation and migration of the cells and the excessive synthesis and deposition of extracellular matrix proteins (3). Previous studies including our own revealed that various types of ocular cells, such as pigment epithelial cells, hyalocytes, glial cells, vascular endothelial cells, and fibroblast-like cells, appear to play a crucial role in the formation of preretinal fibrous membrane (3–5). Another important pathological complication of PDR is retinal neovascularization, which often leads to vitreous hemorrhage and causes blindness. Retinal neovascularization due to PDR is known to be associated with retinal capillary nonperfusion and to be characterized by the proliferation and migration of retinal vascular endothelial cells and the development of new vessels, frequently followed by the formation of fibrovascular membrane together with the contractile proliferative membrane (6).
The tissue repair process is regulated by a number of polypeptides including cytokines and growth factors. Connective tissue growth factor (CTGF) is a 38-kDa cysteine-rich polypeptide that was originally identified from conditioned medium of human umbilical vein endothelial cells (HUVECs) (7). CTGF, considered to be a downstream mediator of transforming growth factor- (TGF-) (8,9), is indicated to induce the production of extracellular matrix, such as collagen and fibronectin, and to cause fibrosis (10). Recent studies have shown that CTGF is overexpressed in the membranes of eyes with PVR (11) or PDR (12), suggesting that CTGF might be involved in the pathogenesis of PVR and PDR. In addition, our previous study revealed that CTGF is overexpressed also in the vitreous with PVR and PDR and additionally demonstrated that various types of vitreoretinal cells could be the sources of CTGF (13). However, little is known about the cellular effects of CTGF in the pathogenesis of proliferative vitreoretinal diseases. We hypothesized that CTGF would have some effects on vitreoretinal cells in an autocrine and/or a paracrine manner and play a role in the progression of proliferative vitreoretinal diseases, such as PVR and PDR.
Furthermore, CTGF has been recently indicated to be one of the regulators of angiogenesis. In vitro, CTGF has been demonstrated to have proangiogenic effects on HUVECs (14) and bovine aortic endothelial cells (BAECs) (15), and in vivo, CTGF has been indicated to induce angiogenesis in rat corneal pocket implants (16) and to be involved in tumor angiogenesis (17) and choroidal neovascularization (18,19). However, to the best of our knowledge, the effects of CTGF on retinal neovascularization and retinal vascular endothelial cell functions have yet to be investigated.
In the present study, we investigated cellular effects of CTGF using hyalocytes and RPEs to address the effect of CTGF on the formation of contractile proliferative membrane and also using retinal vascular endothelial cells to address the effect of CTGF on retinal neovascularization. Furthermore, we addressed the expressional interaction between CTGF and vascular endothelial growth factor (VEGF), a potent angiogenic molecule in vitro and in vivo.
RESEARCH DESIGN AND METHODS
Reagents.
Recombinant human CTGF was purchased from PeproTech (London), and recombinant human TGF-2 and VEGF were from Sigma-Aldrich (Tokyo). Rabbit polyclonal antibodies against phospho-p44/p42 mitogen-activated protein kinase (MAPK) and p44/p42 MAPK were obtained from Cell Signaling Technology (Beverly, MA), and mouse monoclonal antibody against fibronectin was from Santa Cruz Biotechnology (Santa Cruz, CA). In addition, horseradish peroxidase–conjugated secondary antibody was obtained from Bio-Rad (Hercules, CA).
Cell culture.
Bovine eyes were obtained from a local abattoir. The posterior part of the vitreous body was extracted and washed once in Dulbecco's modified Eagle's medium (DMEM; Sigma). The vitreous was chopped into several pieces and incubated in type I collagen–coated dishes filled with DMEM containing 10% fetal bovine serum (FBS; Invitrogen-Gibco, San Diego, CA) for 1 week. The cells that proliferated on the dishes were then subcultured in type I collagen–coated dishes in DMEM supplemented with 10% FBS as well. Cultured hyalocytes obtained between passages 4 to 7, which had shown no obvious morphological changes, were used in the following experiment. Isolated cells were immnocytochemically identified as hyalocytes expressing S-100 protein, but expressed neither glial fibrillary acidic protein nor cytokeratin, as previously described (20). Bovine retinal pigment epithelial cells (BRPEs) and bovine retinal capillary endothelial cells (BRECs) were also isolated from bovine eyes and cultured as previously reported (21,22).
Phosphorylation of p44/p42 MAPK.
Hyalocytes, BRPEs, and BRECs were starved with 1% calf serum and untreated or treated with CTGF (100 ng/ml) for the indicated time (2, 5, 15, 30, and 60 min) with the same media, and the cells were lysed with 1x Laemmli buffer (50 mmol/l Tris [pH 6.8], 2% SDS, and 10% glycerol) containing protease inhibitor and phosphatase inhibitors (1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l NaF, and 0.5 mmol/l Na3VO4). The lysed protein samples were separated by SDS-PAGE, followed by electrophoretic transfer to nitrocellulose membranes (New England Biolabs, Beverly, MA). The blots were blocked with skim milk and incubated overnight at 4°C with antibody against phospho-p44/p42 MAPK (1:1,000). Washed with Tris-buffered saline with Tween (20 mmol/l Tris [pH 7.5], 500 mmol/l NaCl, and 0.1% Tween-20) three times for 10 min each, the membranes had been incubated with horseradish peroxidase–labeled goat anti-rabbit IgG (Bio-Rad) (1:4,000) for 30 min at room temperature. Visualization was performed with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) according to the manufacturer's protocol. Lane-loading differences were normalized by reblotting the membranes with antibodies against p44/p42 MAPK (1:1,000).
[3H]thymidine incorporation.
The hyalocytes, BRPEs, and BRECs were respectively seeded into 24-well plates at a density of 1 x 104 cells/well. The media were replaced by DMEM with 1% calf serum the next day. After 24 h, the cells were unstimulated or stimulated with CTGF (1, 10, and 100 ng/ml) for 18 h with the same media. [3H]thymidine was then added (0.25 μCi/well) for an additional 6 h, and after that, the cells were washed, fixed, and lysed. Incorporated [3H]thymidine was determined by scintillation counting, as previously described (23).
Collagen gel contraction assay.
The contraction assay was performed as we previously described (24). Hyalocytes and BRPEs were collected by the treatment of cultures with trypsin-EDTA for 3 min, washed with DMEM, and resuspended in DMEM at a density of 2.2 x 106 cells/ml. Type I collagen (Koken, Tokyo, Japan), two kinds of reconstitution buffer, hyalocytes or BRPEs suspension, and distilled water were mixed on ice at a ratio of 7:1:1:1:1 (final concentration of type I collagen gel 1.9 mg/ml; final cell density 2 x 105 cells/ml). The resultant mixture (0.5 ml) was added to a 24-multiwell plate (Nunc, Roskilde, Denmark), and the formation of collagen gel was induced by incubation at 37°C for 60 min. After gels formed, 0.5 ml DMEM containing 10% FBS was added to each well. After 48 h of incubation, the cells were starved with DMEM containing 1% calf serum. The gels were freed from the walls of the culture wells with a microspatula and used for the experiments. The diameter of the collagen gel was measured with a ruler at indicated time points after stimulation. For quantitative purposes, contraction data are presented as the reduction in diameter of the collagen gels.
Northern blot analysis.
Northern blot analysis was performed as previously described (21). Subconfluent hyalocytes, BRPEs, and BRECs under normal growth medium conditions were collected, and total RNA was extracted by the acid guanidine thiocyanate-phenol-chloroform extraction method. Radioactive fibronectin or VEGF cDNA probe was generated using a labeling kit (Multiprime; Amersham) and [32P]dCTP (NEN, Boston, MA). Quantitation of Northern blot analysis was performed using a computing phosphorescence imager (PhosphorImager with ImageQuant software analysis; Molecular Dynamics, Sunnyvale, CA). Lane-loading differences were normalized by rehybridization with radiolabeled 36B4 cDNA probe as an internal control gene.
Scratch wound assays.
The migration of BRECs was assessed in scratch wound assays. After the cells in six-well plates were confluent and starved with DMEM containing 1% calf serum, monolayers in each well were scratched using a micropipette tip (1-ml blue tip) and rinsed gently with PBS before stimulating the cells with or without VEGF (25 ng/ml) and CTGF (100 or 300 ng/ml). Wound closure was monitored for 48 h and photographed using a digital camera, and the number of cells that migrated from initial borders per same unit area was counted. These experiments were repeated three times with similar results.
In vitro tube formation assay.
The effect of CTGF on capillary formation was evaluated using an angiogenesis assay kit (KZ-1000; Kurabo, Tokyo, Japan) according to the provided instructions. Briefly, at day 1, HUVECs cocultured with human fibroblasts were incubated in optimized medium that was contained in the kit (KZ-1500; Kurabo), and the cells were stimulated with or without CTGF (100 or 300 ng/ml). Recombinant human VEGF (25 ng/ml) was added to the medium as a positive control. The media were replaced at days 4, 7, and 9. At day 11, the HUVECs were fixed and stained by an anti-human CD31 antibody (Kurabo). Total areas of tube-like structures were quantified by image analysis using angiogenesis quantitation software (KSW-5000U; Kurabo) and statistically analyzed.
Enzyme-linked immunosorbent assay.
This study was carried out with approval from the institutional review board and performed in accordance with the ethical standards of the 1989 Declaration of Helsinki. We obtained written informed consent to participate from the patients. Vitreous samples were collected from patients who underwent pars plana vitrectomy because of macular hole, rhegmatogenous retinal detachment (RRD), PDR, and PVR, which are the same samples in which we investigated the concentrations of TGF-2 and CTGF in our previous study (13). Concentrations of VEGF in the vitreous were measured by human VEGF immunoassay kit (R&D Systems) according to the manufacturer's protocol. Undetectable concentrations of VEGF were determined <31.2 pg/ml. The concentrations of CTGF had been analyzed already in our previous study (13). We investigated whether there is a correlation between the concentrations of VEGF and CTGF in the vitreous with PDR and PVR.
Statistical analysis.
The experimental data are expressed as means ± SD. Statistical significance was assumed when P < 0.05 using the Student's t test in normally distributed populations. The correlation between the differences in CTGF and the differences in VEGF was statistically analyzed using StatView version 5.0 (SAS Institute, Cary, NC), and statistical significance was assumed at P < 0.05 as well.
RESULTS
Growth-promoting effect of CTGF on hyalocytes and BRPEs.
We addressed the phosphorylation of p44/p42 MAPK and [3H]thymidine incorporation in the presence of CTGF to investigate whether CTGF stimulates cell growth activity. As shown in Fig. 1A and C, whereas quiescent hyalocytes and BRPEs both showed a slight phosphorylation state of p44/p42 MAPK, CTGF stimulated the phosphorylation state of p44/p42 MAPK within 5 min, and maximum activation was achieved after 15 min and maintained at least up to 60 min by both types of cells. Furthermore, CTGF significantly promoted [3H]thymidine incorporation by hyalocytes and BRPEs in a dose-dependent manner (0–100 ng/ml) (Fig. 1B and D). It suggests that CTGF might have growth-promoting effect on hyalocytes and BRPEs.
Collagen gel contraction by TGF-2 and CTGF.
Next, to examine the contractile property of hyalocytes and BRPEs in the presence of CTGF, hyalocytes, and BRPEs, respectively, embedded in type I collagen gels were starved and untreated or treated with TGF-2 (0.3 nmol/l) or CTGF (100 ng/ml) for 5 days. Whereas TGF-2 significantly decreased the diameter of the collagen gel (hyalocytes, 34.9%, and BRPEs, 36.3% reduction in diameter compared with control, P < 0.01), the gels treated with CTGF showed no significant contraction up to 5 days (Fig. 2A and B). In addition, protein expressions of fibronectin, one of the extracellular matrix proteins, of those kinds of cells embedded in collagen gels were both enhanced in the presence of CTGF compared with control, but the expression was less promoted than that in the presence of TGF-2 (Fig. 2C).
CTGF-induced expression of fibronectin mRNA by hyalocytes and BRPEs.
To confirm the effect of CTGF on extracellular matrix synthesis, we investigated changes in mRNA expression of fibronectin by hyalocytes and BRPEs in the presence of CTGF. The cells were starved and left unstimulated or stimulated with CTGF (100 ng/ml) for 1, 4, 10, and 24 h, and then total RNA was extracted for Northern blot analysis. As shown in Fig. 3A and B, fibronectin mRNA expression by both hyalocytes and BRPEs was significantly promoted by CTGF in a time-dependent manner up to 24 h (2.07-fold by hyalocytes and 1.75-fold by BRPEs, respectively, compared with control, P < 0.01), demonstrating that CTGF was associated with the synthesis of the extracellular matrix by hyalocytes and BRPEs.
CTGF does not induce proliferation and migration of BRECs.
We also investigated the proliferative effect of CTGF on BRECs by the examination of phosphorylation of p44/p42 MAPK and [3H]thymidine incorporation. Whereas phosphorylation state of p44/p42 MAPK and thymidine incorporation by hyalocytes and BRPEs in the presence of CTGF were stimulated, neither of them by BRECs were stimulated in the presence of CTGF (100 ng/ml) (Fig. 4A and B). As shown in Fig. 5A and B, compared with the positive control VEGF (2.37-fold compared with control, P < 0.01), CTGF at concentration of 100 or 300 ng/ml did not significantly promote migration of BRECs in scratch wound assays.
CTGF did not promote in vitro tube formation.
In the presence of VEGF, HUVECs generated tube-like structures, and the area of the structures was significantly greater than control (1.95-fold compared with control, P < 0.01), whereas CTGF did not promote formation of tube-like structures at the concentration of either 100 or 300 ng/ml compared with control (Fig. 6A and B).
CTGF induced VEGF mRNA expression by hyalocytes and BRPEs.
To investigate whether CTGF enhances VEGF expression, hyalocytes, BRPEs, and BRECs were starved and unstimulated or stimulated with CTGF (100 ng/ml) for 4 h. Total RNA was extracted, and Northern blot analysis was performed for the detection of VEGF mRNA. As shown in Fig. 7, VEGF mRNA was enhanced by hyalocytes and BRPEs in the presence of CTGF, whereas CTGF did not promote VEGF mRNA expression by BRECs.
Correlation between CTGF and VEGF in the vitreous in eyes with PDR and PVR.
Average concentrations of CTGF protein in the vitreous were 38.90 ng/ml (macular hole; n = 15), 39.87 ng/ml (RRD; n = 18), 56.63 ng/ml (PDR; n = 41), and 69.47 ng/ml (PVR; n = 10). Concentrations of CTGF in eyes with PDR or PVR were significantly elevated compared with those with macular hole and RRD, respectively (P < 0.01) (13). Average concentrations of VEGF protein in the vitreous were 11.17 pg/ml (macular hole; n = 15), 17.49 pg/ml (RRD; n = 18), 939.2 pg/ml (PDR; n = 41), and 173.3 pg/ml (PVR; n = 10). Concentrations of VEGF in eyes with PDR or PVR were also significantly elevated compared with those with macular hole and RRD, respectively (PDR vs. macular hole and RRD, P < 0.01 and PVR vs. macular hole and RRD, P < 0.05) (data not shown). However, there was no significant correlation between the concentrations of CTGF and VEGF in the vitreous with PDR and PVR (r = 0.195, P = 0.171, Fig. 8).
DISCUSSION
Recent studies, including our previous one, have demonstrated that CTGF levels were elevated in the vitreous and proliferative preretinal membranes in eyes with PDR and PVR (11–13) and thus suggested the involvement of CTGF in the pathogenesis of proliferative vitreoretinal diseases. In the present study, we demonstrated the functional characteristics of CTGF on vitreoretinal cells, such as hyalocytes, BRPEs, and BRECs, for the first time and clarified its functional mechanisms, at least in part. Furthermore, we addressed the expressional interaction between CTGF and VEGF in vitro and in vivo.
CTGF enhanced the phosphorylation state of p44/p42 MAPK and [3H]thymidine incorporation, increased the number of cells significantly (at the concentration of 100 ng/ml; data not shown), and also promoted mRNA and protein expression of fibronectin by both hyalocytes and BRPEs, suggesting that CTGF might stimulate cell growth and extracellular matrix synthesis by hyalocytes and BRPEs. In addition, our previous study revealed that hyalocytes and BRPEs could also be the sources of CTGF (13), and fibronectin is indicated to be a major component of the extracellular matrix proteins observed in epiretinal membranes of PDR and PVR (25). Taken together, CTGF secreted by hyalocytes and RPEs might modulate the formation of proliferative fibrous membrane in an autocrine and paracrine manner in the pathogenesis of proliferative vitreoretinal diseases, such as PDR and PVR. On the other hand, CTGF could not significantly induce the contraction of type I collagen gel embedding hyalocytes and BRPEs in this study, whereas TGF-2 remarkably induced its contraction (26,27). CTGF is indicated to share many biological functions with TGF- in causing fibrosis, and thus it is considered to be one of the downstream mediators of TGF- (8,9). CTGF might not share the contractile property with TGF-, despite enhanced expression of fibronectin by both types of cells. Therefore, it is considered that CTGF might not be involved or have minimal involvement, if any, in the cicatricial contraction of proliferative membranes in eyes with proliferative vitreoretinal diseases.
Whereas CTGF might have a growth-promoting effect on hyalocytes and BRPEs, CTGF enhanced neither phosphorylation state of p44/p42 MAPK nor [3H]thymidine incorporation by BRECs, suggesting that CTGF might not have a direct effect on the proliferation of BRECs. These results indicated that the effect of CTGF on cellular proliferation might be cell-type dependent. In addition, CTGF did not promote the migration by BRECs or in vitro tube-like formation by HUVECs co-incubated with dermal fibroblasts, suggesting that CTGF might not be directly associated with neovascularization. However, those results are more controversial, because this result does not support some of previous findings that examined cellular functions of CTGF on various types of endothelial cells. Regarding cell proliferation, CTGF has been indicated to promote cell proliferation by BAECs (15) but not to directly mediate cell proliferation by HUVECs (14) or choroidal endothelial cells (18). In addition, CTGF has been demonstrated to promote the migration of endothelial cells by Boyden chamber assay and to promote in vitro tube formation by HUVECs, BAECs, and choroidal endothelial cells (14,15,18). This difference from our results may be due to endothelial cell heterogeneity, which has been well documented.
Another reason is presumed that CTGF has been indicated to interact with other angiogenic proteins at many different levels and to use indirect mechanisms to regulate endothelial cell function (28). It has been reported that CTGF modulates the activity of basic fibroblast growth factor, one of the potent angiogenic molecules, by HUVECs (14), and it has recently been revealed that CTGF transcriptionally regulates VEGF by airway smooth muscle cells (29). We also demonstrated that CTGF stimulates VEGF mRNA expression by hyalocytes and BRPEs but not by BRECs. VEGF is widely known as a mitogen of endothelial cells and as a vasopermeability factor (30). Therefore, even though CTGF had no direct effects on the proliferation or migration of BRECs or on tube formation by HUVECs, it is possible that CTGF-induced VEGF secreted by hyalocytes and RPEs might have such effects on retinal vascular endothelial cells in a paracrine manner in vivo. Besides, conversely, VEGF has been reported to enhance CTGF expression by retinal capillary endothelial cells (RECs) (31). It is also possible that VEGF-induced CTGF secreted by RECs might mediate the formation of proliferative fibrous membrane in proliferative vitreoretinal diseases through the proliferation and extracellular matrix synthesis by hyalocytes and RPEs in a paracrine manner.
According to those in vitro analyses, there might be an expressional correlation between CTGF and VEGF in the progression of proliferative vitreoretinal diseases, such as PDR and PVR. In addition, CTGF knockout mice exhibit reduced VEGF expression and vascular defects during embryogenesis and fetal development, despite the absence of the examination of retinal capillary vessels (32). However, we demonstrated that there is no significant correlation between the concentrations of CTGF and VEGF in the vitreous with PDR and PVR, even though concentrations of CTGF and VEGF were both significantly higher in the vitreous with PDR and PVR than those in the vitreous with nonproliferative vitreoretinal diseases (macular hole and RRD), respectively (data not shown), similar to the previous reports (13,33). CTGF expression is known to be strongly enhanced by TGF- and also promoted by hypoxia (34) and high glucose (35), and VEGF expression is also known to be potently enhanced by hypoxia and further promoted by high glucose and many other cytokines and growth factors, such as TGF- and tumor necrosis factor-. Therefore, VEGF-induced CTGF and CTGF-induced VEGF are respectively presumed to be only minor components in entire expressions of CTGF and VEGF in the vitreous with proliferative vitreoretinal diseases. A fairly recent study sustains our results at least partially, which demonstrated that the CTGF level in the vitreous significantly correlates with the severity of fibrosis but not neovascularization activity by multifactorial ANOVA in consideration of sex, age, diabetes, degree of neovascularization, degree of hemorrhage, and fibrosis score (36).
In this study, we demonstrated functional characteristics of CTGF on vitreoretinal cells and indicated that CTGF might play an important role in the formation of proliferative fibrous membranes in proliferative vitreoretinal diseases, such as PDR and PVR, but CTGF might not directly associate with the cicatricial contraction of the membranes. Furthermore, our results also suggested that CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, however, we demonstrated the possibility that CTGF might have indirect effects by modulating the expression of VEGF by neighboring cells. Further investigations in vitro and in vivo are still necessary to clarify the interaction among CTGF, VEGF, and other cytokines or growth factors and their direct or indirect effects on the pathogenesis of proliferative vitreoretinal diseases.
【参考文献】
Fong DS, Aiello L, Gardner TW, King GL, Blankenship G, Cavallerano JD, Ferris FL III, Klein R: Diabetic retinopathy. Diabetes Care 26 (Suppl. 1):S99–S102, 2003
Pastor JC, de la Rua ER, Martin F: Proliferative vitreoretinopathy: risk factors and pathology. Prog Retin Eye Res 21:127–144, 2002
Campochiaro PA: Pathogenic mechanisms in proliferative vitreoretinopathy. Arch Ophthalmol 115:237–241, 1997
Jerdan JA, Pepose JS, Michels RG, Hayashi H, de Bustros S, Sebag M, Glaser BM: Proliferative vitreo retinopathy membranes: an immunohistochemical study. Ophthalmology 96:801–810, 1989
Vinores SA, Campochiaro PA, Conway BP: Ultrastructural and electron-immunocytochemical characterization of cells in epiretinal membranes. Invest Ophthalmol Vis Sci 31:14–28, 1990
Jerdan JA, Michaels RG, Glaser BM: Diabetic preretinal membranes: an immunohistochemical study. Arch Ophthalmol 104:286–290, 1986
Bradham DM, Igarashi A, Potter RL, Grotendorst GR: Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 114:1285–1294, 1991
Grodendorst GR: Connective tissue growth factor: a mediator of TGF- action on fibroblasts. Cytokine Growth Factor Rev 8:171–179, 1997
Blom IE, Goldschmeding R, Leask A: Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy Matrix Biol 21:473–482, 2002
Leask A, Holmes A, Abraham DJ: Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis. Curr Reumatol Rep 4:136–142, 2002
Hinton DR, He S, Jin ML, Barron E, Ryan SJ: Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy. Eye 16:422–428, 2002
Hinton DR, Spee C, He S, Weitz S, Usinger W, LaBree L, Oliver N, Lim JI: Accumulation of NH2-terminal fragment of connective tissue growth factor in the vitreous of patients with proliferative diabetic retinopathy. Diabetes Care 27:758–764, 2004
Kita T, Hata Y, Kano K, Miura M, Nakao S, Noda Y, Shimokawa H, Ishibashi T: Transforming growth factor-2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor. Diabetes 56:231–238, 2007
Kireeva ML, Latinkic BV, Kolesnikova TV, Chen CC, Yang GP, Abler AS, Lau LF: Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development. Exp Cell Res 233:63–77, 1997
Shimo T, Nakanishi T, Kimura Y, Nishida T, Ishizeki K, Matsumura T, Takigawa M: Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. J Biochem (Tokyo) 124:130–140, 1998
Babic AM, Chen CC, Lau LF: Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19:2958–2966, 1999
Shimo T, Nakanishi T, Nishida T, Asano M, Sasaki A, Kanyama M, Kuboki T, Matsumura T, Takigawa M: Involvement of CTGF, a hypertrophic chondrocyte-specific gene product, in tumor angiogenesis. Oncology 61:315–322, 2001
He S, Jin ML, Worpel V, Hinton DR: A role for connective tissue growth factor in the pathogenesis of choroidal neovascularization. Arch Ophthalmol 121:1283–1288, 2003
Watanabe D, Takagi H, Suzuma K, Oh H, Ohashi H, Honda Y: Expression of connective tissue growth factor and its potential role in choroidal neovascularization. Retina 25:911–918, 2005
Sakamoto T, Sakamoto H, Sheu SJ, Gabrielian K, Ryan SJ, Hinton DR: Intercellular gap formation induced by thrombin in confluent cultured bovine retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 35:720–729, 1994
Hata Y, Nakagawa K, Ishibashi T. Inomata H, Ueno H, Sueishi K: Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis. Virchows Arch 426:479–486, 1995
Aiello LP, Robinson GS, Lin YW, Nishio Y, King GL: Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display. Proc Natl Acad Sci U S A 91:6231–6235, 1994
Hata Y, Rock SL, Aiello LP: Basic fibroblast growth factor induces expression VEGF receptor KDR through a protein kinase C and p44/p42 mitogen activated protein kinase-dependent pathway. Diabetes 48:1145–1155, 1999
Noda Y, Hata Y, Hisatomi T, Nakamura Y, Hirayama K, Miura M, Nakao S, Fujisawa K, Sakamoto T, Ishibashi T: Functional properties of hyalocytes under PDGF-rich conditions. Invest Ophthalmol Vis Sci 45:2107–2114, 2004
Ioachim E, Stefaniotou M, Gorezis S, Tsanou E, Psilas K, Agnantis NJ: Immunohistochemical study of extracellular matrix components in epiretinal membranes of vitreoproliferative retinopathy and proliferative diabetic retinopathy. Eur J Ophthalmol 15:384–391, 2005
Hirayama K, Hata Y, Noda Y, Miura M, Yamanaka I, Shimokawa H, Ishibashi T: The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes. Invest Ophthalmol Vis Sci 45:3896–3903, 2004
Miura M, Hata Y, Hirayama K, Kita T, Noda Y, Fujisawa K, Shimokawa H, Ishibashi T: Critical role of the Rho-kinase pathway in TGF-beta2-dependent collagen gel contraction by retinal pigment epithelial cells. Exp Eye Res 82:849–859, 2006
Brigstock DR: Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61). Angiogenesis 5:153–165, 2002
Burgess JK, Ge Q, Poniris MH, Boustany S, Twigg SM, Black JL, Johnson PR: Connective tissue growth factor and vascular endothelial growth factor from airway smooth muscle interact with the extracellular matrix. Am J Physiol Lung Cell Mol Physiol 290:L153–L161, 2006
Miller JW, Adamis JP, Aiello LP: Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy. Diabete Metab Rev 13:37–50, 1997
Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP, King GL: Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathway in retinal vascular cells. J Biol Chem 275:40725–40731, 2000
Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM: Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 2003 130:2779–2791, 2003
Sydorova M, Lee MS: Vascular endothelial growth factor levels in vitreous and serum of patients with either proliferative diabetic retinopathy or proliferative vitreoretinopathy. Ophthalmic Res 37:188–190, 2005
Shimo T, Kubota S, Kondo S, Nakanishi T, Sasaki A, Mese H, Matsumura T, Takigawa M: Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line. Cancer Lett 174:57–64, 2001
Murphy M, Godson C, Cannon S, Kato S, Mackenzie HS, Martin F, Brady HR: Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 274:5830–5834, 1999
Kuiper EJ, de Smet MD, van Meurs JC, Tan HS, Tanck MW, Oliver N, van Nieuwenhoven FA, Goldschmeding R, Schlingemann RO: Association of connective tissue growth factor with fibrosis in vitreoretinal disorders in the human eye. Arch Ophthalmol 124:1457–1462, 2006
订阅登记：
请您在下面输入常用的Email地址、职业以便我们定期通过邮箱发送给您最新的相关医学信息，感谢您浏览首席医学网！