TGFbeta induced myofibroblast differentiation of rabbit keratocytes requires synergistic TGFbeta,PDGF and integrin signaling

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.     

Influence on cultured human keratocytes by liposome

AIM: To observe the effects on human keratocytes by cationic liposome Lipofectamine^TM2000 (LF2000), to investigate the efficiency and safe range applied in human keratocytes, and establish basis for gene therapy of human keratocytes. METHODS: Human keratocytes cultured in vivo within 3 to 5 passages were used in experiment after being identified. The effects on proliferation of cultured human keratocytes by LF2000 with different concentrations and time were evaluated By MTT; the effects of LF2000 on the survival rate and its relation with 5,10,20,40,80mg/L concentration and time were detected by trypan blue staining. RESULTS: LF2000's effects on human keratocytes were related with concentration and time. The cellular proliferation and survival rate declined when concentration of LF2000 was above certain level, and this effect increased as time became longer. LF2000 had no effect with concentration under 40mg/L for 24 hours. CONCLUSION：LF2000 did not cause cytotoxicity during a concentration range“tested”, and it is hoped to play an important role in gene therapy of human keratocytes.     

脂质体对人角膜基质细胞影响的实验研究

目的:观察脂质体LipofectamineTM2000对人角膜基质细胞的影响,探索其应用于人角膜基质细胞的可行性及安全范围。方法:体外培养人角膜基质细胞,取其第3-5代细胞鉴定后用于实验。采用MTT法检测不同浓度和时间脂质体对人角膜基质细胞增殖率的影响;采用台盼蓝染色法检测对存活率的影响。结果:脂质体对人角膜基质细胞的影响与浓度和作用时间有关。浓度高于一定水平时可引起细胞增殖率和存活率的下降,浓度相同时作用时间越长下降越明显。浓度低于40mg/L作用24h不会对细胞增殖率和存活率产生明显影响。结论:LipofectamineTM2000在一定范围内不引起细胞毒性,有望在角膜基质细胞的基因治疗中发挥重要作用。     

TGF-beta1, TGF-beta receptor II and ED-A fibronectin expression in myofibroblast of vitreoretinopathy

PURPOSE: Formation of scarlike epiretinal membranes (ERMs) constitutes potentially the end stage of evolution of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Among various cellular populations, ERMs contain cells with contractile features typical of myofibroblasts. The current study was conducted to investigate the presence of transforming growth factor (TGF)-beta1, TGF-beta receptor II (RII) and ED-A fibronectin (FN), the main inducers of myofibroblastic differentiation in ERMs in PDR and PVR. METHODS: Samples of ERM were obtained from 23 patients during microsurgery for PVR or PDR. Electron microscopy, immunohistochemistry, and confocal microscopy with antibodies recognizing beta-smooth muscle (SM) actin, desmin, TGF-beta1, TGF-beta receptors I and II, and ED-A FN were performed. RESULTS: alpha-SM actin was detected in all ERMs, whereas desmin was present in 50% of the cases. ED-A FN was expressed in all ERMs in close relation with alpha-SM actin-positive myofibroblasts. In addition, TGF-beta1 and TGF-beta R II were always present, TGF-beta RII being expressed in both alpha-SM actin-positive and negative fibroblastic cells. CONCLUSIONS: Myofibroblast accumulation is a key event in ERM-associated traction retinal detachment occurring during PVR and PDR. The current results suggest that the presence of alpha-SM actin-positive myofibroblasts is probably dependent on the concomitant neoexpression of TGF-beta1, TGF-beta RII, and ED-A FN. The results furnish new data on the mechanism of alpha-SM actin stimulation in fibroblasts in a human pathologic setting.     

CD-34 stromal expression pattern in normal and altered human corneas

OBJECTIVE: To test CD-34 immunoreactivity in stromal cornea cells in normal and pathologic samples obtained from penetrating keratoplasty. DESIGN: Prospective, consecutive histopathologic human tissue study. PARTICIPANTS AND CONTROLS: One hundred two cornea buttons from patients with different diseases, submitted for cornea transplant, were examined. Controls were expired corneas from healthy donor patients who died (n = 4), and globes enucleated for primitive intraocular neoplasias, that is, retinoblastomas (n = 8), and malignant choroidal melanomas (n = 2). METHODS: The expression of CD-34 in stromal cornea cells was examined by immunohistochemistry analysis. Other immunohistochemical stains included an endothelial cell marker (CD-31), common leukocyte antigen, and alpha-smooth muscle actin. MAIN OUTCOME MEASURES: Different diseases that may cause blindness and require penetrating keratoplasty have been tested for CD-34 immunoreactivity. RESULTS: In control corneas, keratocytes present strong and consistent CD-34 immunoreactivity. Diseases leading to the loss of transparency and penetrating keratoplasty, such as keratoconus, herpes keratitis, trauma, and heredofamilial dystrophies, are associated with focal or diffuse loss of CD-34 expression, whereas pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy show normal CD-34 immunoreactivity in most cases and patchy unstained stromal areas in a few cases. CONCLUSIONS: Scar tissue formation in the cornea, as in herpes keratitis and trauma, is always associated with loss of CD-34 immunoreactivity, which may otherwise be a primary event in keratoconus and heredofamilial dystrophies. Both in the pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy, CD-34 immunoreactivity may be normal or lost, hence these two diseases may be considered as one and part of the same group with regard to CD-34 expression, as revealed by immunohistochemistry analysis.     

IGF-II and collagen expression by keratocytes during postnatal development

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

体外培养人角膜基质细胞ICAM-1的表达及调节

目的 探讨细胞间附分子－１（ＩＣＡＭ－１）在角膜基质细胞中表达,以及炎症介质对其表达的调节作用。方法 采用细胞培养、免疫细胞化学和流式细胞技术、观察人角膜基质细胞ＩＣＡＭ－１的表达及脂多糖（ＬＰＳ）和干扰素－γ（ＩＦＮ－γ）对此表达的影响。结果 体外２的基质中基础表达一定量的ＩＣＡＭ－１、ＬＰＳ和ＩＦＮ－γ可上调其表达水平至基础表达的１．４－４．６倍。结论角膜炎症反应中,炎症介质促进基质细胞表达Ｉ     

In vivo and in vitro demonstration of epithelial cell-induced myofibroblast differentiation of keratocytes and an inhibitory effect by amniotic membrane

PURPOSE: To examine the role of epithelial cells in inducing the differentiation of keratocytes into myofibroblasts and to determine whether this effect may be inhibited by amniotic membrane matrix. METHODS: In vivo, a 9-mm diameter, partial-thickness corneal flap was created in 12 rabbit eyes (6 rabbits), which were equally subdivided into three groups. The first group was implanted with one layer of a 6-mm diameter human amniotic membrane, from which the epithelium had been removed by dispase. The second group received an implantation of dispase-treated amniotic membrane with cultured rabbit corneal epithelial cells. The third group received the same implantation as the second group except that the cultured corneal epithelial cells were sandwiched between two layers of membrane. All corneas were removed 2 weeks later and were subjected to Masson trichrome staining and immunofluorescence staining with monoclonal antibodies to alpha-smooth muscle (alpha-SM) actin for myofibroblasts and cytokeratins for epithelial cells. In vitro collagen gels impregnated with different types of human ocular surface fibroblasts were seeded with or without rabbit corneal epithelial cells before testing for gel contraction. RESULTS: Positive staining of alpha-SM actin was noted only in keratocytes adjacent to corneal epithelial cells at the incision site and those grown on the basement membrane side of the amniotic membrane. Negative staining was noted when epithelial cells were removed by dispase or when cultured corneal epithelial cells were sandwiched between two layers of membrane. Gel contraction by fibroblasts was significantly promoted when epithelial cells were seeded on the gel. In the latter situation, positive staining of alpha-SM actin was noted in fibroblasts subjacent to epithelial cells but not in those impregnated in the gel. CONCLUSION: Epithelial cells are capable of inducing the differentiation of adjacent fibroblasts into myofibroblasts; such an induction requires a close epithelial-mesenchymal contact. Amniotic membrane alone does not induce this effect and can help block such induction by epithelial cells.     

Induction of cellular toxicity in cultured porcine corneal keratocytes by endothelin-1.

The effect of endothelin-1 (ET-1) on corneal cells is not well understood. We investigated the biochemical changes of cultured porcine corneal keratocytes under exposure to ET-1. The results indicate that ET-1 has remarkable effects to inhibit corneal keratocytes on 3H-thymidine, 3H-leucine, 3H-uridine uptakes and cellular migration. It is in a dose-dependent manner at concentrations ranging from 10(-7) M to 10(-9) M. The 50% inhibitory dose (ID50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake and 3H-leucine uptake, were 10(-7) M, 10(-0.52) M and 10(-11.8) M, respectively. The dead and living cells were estimated with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of ET-1, the cellular MTT values were also decreased. The ID50 for ET-1 with cell migration assay and MTT assay were measured at 10(-7.86) M and 10(-5.1) M. Endothelin-1 (10(-6) M) promptly changed cellular morphology and attenuated adhesion observed with laser scanning cytometer. Endothelin-1-induced characteristic apoptosis cells were observed using a TUNEL assay that detected fragmented DNA of apoptosis. Western blot assay revealed that endothelin-1 induced proteolysis and decreased in fibronectin protein. These findings indicate that endothelin-1 may lead keratocytes to death resulting from induction of apoptosis and functional loss.     

Involvement of CTGF in TGF-beta1-stimulation of myofibroblast differentiation and collagen matrix contraction in the presence of mechanical stress

PURPOSE: This study was undertaken to investigate the role of connective tissue growth factor (CTGF) in fibroblast-to-myofibroblast differentiation and fibroblast-mediated collagen matrix contraction in the presence of mechanical stress. METHODS: An in vitro three-dimensional contraction model of human corneal-fibroblast-seeded collagen lattices (FSCLs) in the presence of mechanical stress generated by attaching the lattices to the culture well was used to measure FSCL contraction. FSCLs were treated with CTGF; TGF-beta1; serum-free (SF) control medium; or TGF-beta1 plus antisense oligodeoxynucleotides to CTGF; TGF-beta1 plus scrambled-sequence oligodeoxynucleotide to CTGF; or TGF-beta antibody. Expression of alpha-smooth muscle actin (alpha-SMA) by fibroblasts in FSCLs was detected by immunostaining and confocal microscopy, whereas ELISA was used for the fibroblasts cultured on plastic. The conditioned media were analyzed by ELISA for CTGF production. RESULTS: Exogenous CTGF stimulated significantly less collagen matrix contraction and myofibroblast differentiation than TGF-beta1, but similar to that stimulated by SF. TGF-beta1 stimulated fibroblasts to express CTGF. CTGF antisense oligodeoxynucleotide inhibited TGF-beta1-stimulated myofibroblast differentiation and FSCL contraction. Exogenous CTGF circumvented the inhibitory effects of CTGF antisense on FSCL contraction. TGF-beta antibody significantly inhibited FSCL contraction and myofibroblast differentiation under mechanical stress and SF control conditions. CONCLUSIONS: In the presence of mechanical stress, CTGF is necessary for TGF-beta1-stimulation of myofibroblast differentiation and subsequent collagen matrix contraction, but CTGF alone is not sufficient to induce myofibroblast differentiation and collagen matrix contraction. Thus, TGF-beta1 appears to regulate multiple genes that are essential for fibroblast-mediated contraction of stressed matrix, one of which is CTGF.     

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields α-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had −9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at −80 °C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery.     

Differential expression and regulation of TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII and TGF-betaRIII in cultured human corneal, limbal, and conjunctival fibroblasts.

PURPOSE. We have reported that three patterns of cytokine expression are potentially involved between epithelia and fibroblasts of the human ocular surface. The TGF-beta family is a prototypical fibrogenic cytokine responsible for fibroblast activation in wound healing. We investigated how the TGF-beta family is differentially expressed and regulated in cultured human corneal, limbal and conjunctival fibroblasts. METHODS. Human corneal (HCF), limbal (HLF) and conjunctival fibroblast (HJF) were cultured in DMEM-10% FBS until confluence and switched to serum-free DMEM-ITS for 48 h before adding 10 ng/ml of each of eight cytokines for 4 h in three separate experiments. Total RNA was isolated and subjected to Northern hybridization with GAPDH as a control. ELISA was used to determine TGF-beta1 and TGF-beta2 proteins in the media. RESULTS. All three isoforms of TGF-beta and three types of TGF-betaR were expressed by HCF, HLF and HJF. Expression of TGF-beta1 mRNA was strongest and upregulated by the three TGF-betas in all three types of fibroblast. PDGF-BB and TGF-alpha slightly increased TGF-beta1 mRNA. TGF-betas also upregulated TGF-beta3 mRNA in HJF. TGF-betaRI mRNA was the only receptor upregulated by TGF-betas. TGF-betaRII and TGF-betaRIII mRNA were not regulated by all cytokines tested. CONCLUSIONS. TGF-betas auto-induction is the major mechanism upregulating TGF-beta1 expression. Promotion of TGF-beta3 by the TGF-betas may have a special role in HJF. Differential expression and regulation of TGF-betas and TGF-betaRs suggest that each TGF-beta isoform may have specific functions in different ocular surface fibroblasts. No cytokine tested can downregulate TGF-beta1 and the TGF-betaRs.     

Matrix metalloproteinase secretion is stimulated by TGF-beta in cultured lens epithelial cells.

PURPOSE. To determine if TGF-beta regulates the expression of metalloproteinases in chick lens annular pad cells. METHODS. The activity of secreted matrix metalloproteinases was examined with gelatin zymography in primary cultures exposed to TGF-beta. RESULTS. Metalloproteinases with electrophoretic mobilities corresponding to MMP2 and MMP9 were tentatively identified. Activated, processed forms of the two metalloproteinases were also observed. Plasminogen activators potentially capable of initiating metalloproteinase cascades were concomitantly elicited. Metalloproteinase secretion was shown to be specific for TGF-beta stimulation and independent of substrate composition. CONCLUSIONS. These results indicate that TGF-beta-mediated processes could be responsible for localized lens capsular heterogeneity, establishing a substrate suitable for cell migration or the release of matrix-bound factors which influence the terminal differentiation of lens cells.     

cDNA microarray analysis of gene expression changes induced by dexamethasone in cultured human trabecular meshwork cells

PURPOSE: To profile gene expression changes induced by dexamethasone in cultured human trabecular meshwork (TM) cells and identify genes related to the occurrence of steroid-induced glaucoma. METHODS: At confluence, dexamethasone (final concentration 10(-7) M in 0.1% ethanol) or vehicle alone (control, 0.1% ethanol) was applied to cultured human TM cells from eyes of four normal subjects. After 7 days of application, a labeled cDNA probe was synthesized from extracted total RNA and hybridized to a human cDNA microarray containing 2400 genes. After hybridization, the tyramide signal was amplified, and the fluorescent signals on each microarray were scanned and analyzed. RESULTS: In dexamethasone-treated TM cells, simultaneous analysis of 2400 human genes indicated a more than twofold increase in 30 genes. Five of them, myocilin (MYOC), decorin, insulin-like growth factor binding protein 2, ferritin L chain, and fibulin-1C, were the most upregulated genes with higher-than-control expression levels in all four experiments. Their upregulation was further confirmed by semiquantitative RT-PCR. Downregulation, with fluorescent signals decreased to less than a half, was found in 34 genes. The dexamethasone-induced expression changes in most of these TM cell genes have not been reported previously. CONCLUSIONS: cDNA microarray is a useful tool for gene expression analysis that confirms previous reports of upregulated mRNA expression of MYOC after treatment with dexamethasone in human TM cells. Changes in other genes subsequent to the treatment with dexamethasone may also reduce outflow facility, providing new insights into the pathogenesis of steroid-induced glaucoma.