Human Tenon's Capsule Fibroblasts Research Articles

Changes of type I collagen and fibronectin expressions on cultured human Tenon's capsule fibroblasts transfected with Smad 7 vector

To investigate type I collagen and fibronectin expressions in the cultured human Tenon's capsular fibroblasts (HTFs) transfected with Smad 7 vector and to elucidate the possibility of Smad 7 in blocking tissue fibrosis after filtration surgery. Nucleofector(TM) was used to transfect Smad 7 vector into HTFs. The expressions of alpha2-type I procollagen (COL1A2) mRNA and fibronectin mRNA were detected by reverse transcription real-time quantitative polymerase chain reaction (real-time RT-PCR). The concentration of carboxyterminal propeptide of type I procollagen (PICP) in culture media was detected by radioimmunoassay. The above mentioned experiments were also detected before and after HTFs stimulated by TGF-beta(2) (10 ng/ml). HTFs and vector-HTFs were used as control groups. Smad 7 was successfully transfected into HTFs evidenced by Smad 7 over expression. The expression of COL1A2 mRNA in Smad 7-HTFs was decreased 40.47% and 37.94%, respectively, when compared with the control HTFs and pCMV5-HTFs group. The PICP concentration of Smad 7-HTFs in the culture medium was decreased 52.10% and 57.35%, respectively, comparing to the two control groups. Furthermore, Smad 7-HTFs attenuated the increase of COL1A2 mRNA expression and PICP concentration of HTFs stimulated by TGF-beta(2). There was no difference in the fibronectin mRNA expressions between Smad 7-HTFs and the two control groups. Smad 7 reduces the expression of COL1A2 mRNA and the synthesis of type I collagen, but has no effect on fibronectin mRNA expression, which may imply its ability to inhibit tissue fibrosis after filtration surgery.

Expression of bone morphogenetic proteins (BMPs), their receptors, and activins in normal and scarred conjunctiva: Role of BMP-6 and activin-A in conjunctival scarring?

Bone morphogenetic proteins (BMPs) and activins are multifunctional growth factors, which also affect wound healing and tissue fibrosis. To determine their putative role in conjunctival wound healing and scarring, we investigated the expression of various BMPs, BMP receptors, and activins in normal and scarred human conjunctival tissue and in cultured human Tenon's capsule fibroblasts on the mRNA and protein level. Messenger RNA expression of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, the BMP receptors type I (BMPR-IA, BMPR-IB) and II (BMPR-II), and of activin A and B was investigated by semi-quantitative RT–PCR in normal conjunctival specimens and in scarred filtering blebs as well as in cultured Tenon's capsule fibroblasts obtained from patients with primary open-angle glaucoma (POAG), pseudoexfoliation (PEX) glaucoma and cataract. Immunohistochemistry was used to study the protein expression of BMP-2, BMP-4, BMP-6, BMP-7, and activin A in normal and scarred conjunctival tissue as well as in cultured Tenon's capsule fibroblasts. BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, all BMP receptors, and activin A were expressed on the mRNA and protein level in conjunctival biopsies without showing any differences between groups of patients. The mRNA and protein expression of both BMP-6 and activin A was found to be significantly increased in scar tissue compared with normal conjunctiva and could be immunolocalized to epithelial cells, vascular endothelia, stromal fibroblasts, and macrophage-like cells. However, no significant increase in receptor gene expression was observed in scar tissue. With the exception of BMP-7, all growth factors and receptors were also expressed in cultured Tenon's fibroblasts without showing any differences between cultures derived from normal and scarred conjunctival specimens. These findings suggest various BMPs and activin A as components of the conjunctival cytokine meshwork regulating tissue homeostasis and wound healing and provide evidence that alterations in the expression of BMP-6 and activin A, in particular, are associated with conjunctival scarring. Modulation of BMP/activin activities may, therefore, be explored as new approaches for managing postoperative conjunctival scarring responses in glaucoma patients.

Anti-proliferative effect of herpes simplex virus thymidine kinase gene system on human Tenon capsule fibroblasts in vitro

To observe anti-proliferative effect of herpes simplex virus thymidine kinase gene system (HSV-tk) on human Tenon capsule fibroblasts (HTFs) and it's mechanism. Retroviral vector was constructed containing HSV-tk gene and transfer to packaging cell line PT67. The positive clones was selected with G418 and the supernatant was collected which contains virus particles. The titer of the virus was also calculated. The virus particles were verified by transmission electron microscope (TEM). PCR was carried out to detect the integrity of tk gene into viral DNA genome. HTFs cells were infected with the virus and the positive clones were selected for propagation culture. RT-PCR and Western blot were used to detect tk gene expression. The anti-proliferative effect of tk gene on HTFs cells was determined by MTT. Apoptosis of cells was detected by Hoechst 33258 DNA staining and flow cytometry. The morphologic changes of the cell were observed by TEM. The retroviral vector pL (tk) SN was constructed successfully which was verified by enzyme cutting. The shape of virus particles derived from the package cells appeared to be oval or spherical under TEM with the diameter around 100 nm. The tk gene was detected in the viral DNA genome by PCR. The virus titer was 4 x 10(7) cfu/ml. The expression of tk gene was detected by RT-PCR and Western blot both in mRNA and protein level. The anti-proliferative effect of tk gene on HTFs cells was dose-dependent, all cells were died 5 days after 5 x 10(-3) g/L GCV being added, IC50 = 6 x 10(-4) g/L. Both necrosis and apoptosis were observed in this study and the apoptosis rate was increased with increasing dose of GCV. The proliferation of HTFs cells in vitro could be effectively inhibited by the killing effect of HSV-tk-GCV system through the pathway of necrosis and apoptosis.

Preliminary study of inhibition of human Tenon's capsule fibroblasts in vitro by RNA interference targeting IKK-beta

To determine whether small interference RNA (siRNA) targeting the inhibitor of kappaB kinase-beta (IKK-beta) could be used to suppress the IKK-beta expression, and inhibit the proliferation of human Tenon's capsule fibroblasts in vitro. IKK-beta specific siRNA designed from the human gene sequence was transfected into the cultured human Tenon's capsule fibroblasts, and a non-targeted siRNA was transfected as a negative control. The mRNA of IKK-beta was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression was determined by Western blotting. Cell viability of the cultured human Tenon's capsule fibroblasts with series of RNA interference at 5, 10, 25, 50, 100, and 200 nmol/L was evaluated by MTT assay 48 hours after transfection. The expression of IKK-beta was significantly (P < 0.05) suppressed at both mRNA and protein levels after transfection. The proliferation of the cultured human Tenon's capsule fibroblasts was inhibited at all the transfected concentrations at different rates (10.72%, 23.35%, 30.84%, 51.25%, 50.06% and 49.63% respectively). The highest level of inhibition was observed at 50 nmol/L of siRNA concentration. IKK-beta specific siRNA is effective in suppressing the IKK-beta expression and inhibiting the proliferation of the cultured human Tenon's capsule fibroblasts. RNA interference may offer a new alternative to post-operational management of scar tissue formation.

Mitomycin-C Induces the Apoptosis of Human Tenon’s Capsule Fibroblast by Activation ofc-JunN-Terminal Kinase 1 and Caspase-3 Protease

To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C. Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining. Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C. Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.

Effects of latanoprost eye drops on the proliferation of human Tenon's fibroblasts in tissue culture

To observe whether there is a direct effect of latanoprost eye drops on the proliferation of cultured human Tenon's capsule fibroblasts (HTCFs) in vitro. Human Tenon's capsule fibroblasts were cultured in vitro. Latanoprost eye drops was serially diluted in order to make nine concentrations ranging from 5.00 microg/ml, 0.50 microg/ml, 0.05 microg/ml to 5 x 10(-8) microg/ml. Fibroblasts growth was evaluated with MTT assay. Morphological changes were observed under phase contrast microscope. Majority of cultured human Tenon's capsule fibroblasts was dead at the concentration of Latanoprost (5 microg/ml), the A values were significantly decreased (P < 0.01). There were no significant changes of the A values (P > 0.05) in lower concentration (under 0.05 microg/ml) compared with control. there was no statistical difference in proliferation of HTCFs after latanoprost treatment for 24 hours, 48 hours, 72 hours. Latanoprost has the toxic effect on the fibroblast at higher concentrations, but there is no influence on the proliferation of HTCFs at the lower concentrations.

Experimental study on the effects of interferon-gamma gene on Tenon's capsule fibroblasts in vitro

To investigate the effects of interferon-gamma (IFN-gamma) gene on Tenon's capsule fibroblasts in vitro. Using lipofectAMINE, IFN-gamma gene was transferred to human Tenon's capsule fibroblasts with plasmid pcDNA3IFN-gamma in vitro. Cells treated with plasmid pcDNA3 without IFN-gamma gene were used as the controls. The transfected fibroblasts were screened by G418. The expression of IFN-gamma was determined by RT-PCR, immunohistochemistry and flow cytometry assay. The effects of IFN-gamma on the proliferation of Tenon's capsule fibroblasts were evaluated by flow cytometry and MTT. The Tenon's capsule fibroblasts transferred the IFN-gamma gene could express the IFN-gamma RNA and protein transiently. The proliferation of the fibroblasts transferred the IFN-gamma gene was inhibited. The proliferation of the Tenon's capsule fibroblasts in vitro can be inhibited by transferred IFN-gamma gene.

Tocotrienol inhibits proliferation of human Tenon’s fibroblasts in vitro: a comparative study with vitamin E forms and mitomycin C

To evaluate the potential of the vitamin E compound alpha-tocotrienol as antifibrotic agent in vitro. Using human Tenon's capsule fibroblast cultures, the antiproliferative and cytotoxic effects of the different vitamin E forms alpha-tocopherol, alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocotrienol were compared with those of mitomycin C. To mimic subconjunctival and regular oral application in vivo, exposure time of serum-stimulated and serum-restimulated fibroblasts (SF and RF, respectively) to vitamin E forms was set at 6 days. Cultures were only exposed for 5 min to mitomycin C due to its known acute toxicity and to mimic the short-time intraoperative administration. Proliferation (expressed as % of control) was determined by DNA content quantification on days 2, 4 and 6, whereas cytotoxicity was assessed by cell morphology and glucose 6-phosphate dehydrogenase (G6PD) release after 24 h. alpha-Tocopherol and alpha-tocopheryl acetate stimulated growth of SF, but not RF. Reduction of fibroblast content by alpha-tocopheryl succinate was accompanied by increased G6PD release and necrosis. Contrary to alpha-tocopheryl succinate, 50 microM or repeatedly 20 microM of alpha-tocotrienol significantly inhibited proliferation without causing cellular toxicity (maximal effect: 46.8%). RF were more sensitive to this effect than SF. Mitomycin C 100-400 microg/ml showed a stronger antiproliferative effect than alpha-tocotrienol (maximal effect: 13.8%). Morphologic characteristics of apoptosis were more commonly found under treatment with mitomycin C. Of the vitamin E forms tested, only alpha-tocotrienol significantly inhibited growth at non-toxic concentrations. In this in vitro study, antiproliferative effects of mitomycin C were stronger than those of alpha-tocotrienol.

Effect of diabetes mellitus and hyperglycemia on the proliferation of human Tenon's capsule fibroblasts: Implications for wound healing after glaucoma drainage surgery

Glaucoma drainage surgery in diabetic patients is associated with a relatively poor prognosis due to increased scarring at the site of surgery, secondary to increased proliferation of human Tenon's capsule fibroblasts (hTCF). This is in marked contrast to diabetic wound healing at other sites, where it is generally impaired. The aim of this study was to determine why diabetics show an increased ocular scarring response in comparison to that found at other sites. Under normoglycemic conditions, hTCF isolated from diabetics showed a mean reduction in short-term proliferation (95% CI) of 45 +/- 12% compared with normal controls (p < 0.001). Under hyperglycemic conditions, proliferation of diabetic hTCF was reduced by 21 +/- 11% (p < 0.01) compared with nondiabetic controls. When exposed to transforming growth factor-beta2 (1-10,000 pg/ml) and platelet-derived growth factor-BB (0.5-500 ng/ml) under both normo- and hyperglycemic conditions, there was a dose-related increase in proliferation of both diabetic and nondiabetic controls. There was no significant difference in response to cytokine stimulation between the two groups at any of the cytokine concentrations used. Western blot analysis did not show any apparent difference in the expression of platelet-derived growth factor receptor alpha, mitogen-activated protein kinase/ERK2, or transforming growth factor-beta receptor II to account for the reduced proliferation of diabetic hTCF. These results suggest that hTCF behave in a manner similar to fibroblasts from other nonocular sites and that the increased proliferation and scarring response found in vivo may be secondary to the previously noted elevated cytokine concentrations in the aqueous and vitreous of diabetics.

Comparative effects of TGF-β1 and TGF-β2 on extracellular matrix production, proliferation, migration, and collagen contraction of human Tenon's capsule fibroblasts in pseudoexfoliation and primary open-angle glaucoma

Purpose. To comparatively investigate the effects of TGF-β 1 and TGF-β 2 on extracellular matrix production, proliferation, migration, and collagen contraction of cultured human Tenon's capsule fibroblasts derived from patients with pseudoexfoliation (PEX) syndrome, PEX glaucoma, primary open-angle glaucoma (POAG), and cataract. Methods. Tenon's capsule fibroblasts obtained from four groups of patients were cultured and stimulated with different concentrations (0·1–10 ng ml −1) of TGF-β 1 or TGF-β 2 for up to 14 days. Cell proliferation was determined with the WST-1 colorimetric assay, cell migration by using the Transwell assay system, and collagen contraction by computerised analysis of three-dimensional collagen lattices and immunohistochemistry for α-smooth muscle actin expression. Expression and synthesis of extracellular matrix components (fibronectin, collagen types I and III) was assessed by enzyme-linked immunosorbent assays, by real-time RT-PCR, and by transmission electron microscopy. Results. Both TGF-β 1 and TGF-β 2 in pathophysiological concentrations of 0·1–5 ng ml −1 stimulated cell proliferation, migration, collagen contraction, α-smooth muscle actin expression as well as mRNA expression and secretion of fibronectin, collagen type I, and collagen type III by Tenon's fibroblasts derived from all groups of patients. TGF-β stimulation occurred in a concentration-dependent manner with different peak activities associated with different fibroblast functions. There was some variability among the different groups of patients with an increased response of cells derived from PEX and POAG patients as compared to cataract patients. Although no statistically significant differences were found between both TGF-β isoforms, TGF-β 1 had a more pronounced stimulatory effect on expression and synthesis of extracellular matrix components including the production of elastic microfibrils, particularly in cells derived from patients with PEX syndrome/glaucoma. Conclusions. These findings suggest a significant contribution of TGF-β 1 in addition to TGF-β 2 to the conjunctival scarring process following glaucoma filtration surgery. Due to its pronounced fibrogenic potential, TGF-β 1 may become another focus for targeting drug therapy, particularly in patients with PEX glaucoma.

APC0576 decreases production of pro-inflammatory chemokine and extracellular matrix by human Tenon's capsule fibroblasts

Purpose. To control intraocular pressure in patients treated by trabeculectomy, progressive inflammation, fibroblast proliferation, and the enhanced expression of extracellular matrix (ECM), causes of scar formation at the bleb, must be prevented. Using human Tenon's capsule fibroblasts (TCFs), we examined the effect of APC0576, a suppressor of NF-κB-dependent gene activation of human vascular endothelial cells that does not adversely affect cell viability, on the production of pro-inflammatory chemokine and ECM. Its effect on TCF proliferation was also assessed. Methods. Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines IL-8 and MCP-1 in the supernatant of TCFs stimulated with IL-1α in the presence or absence of APCO576 and ECM proteins, COOH-terminal peptide of type I procollagen (PIP), fibronectin (FN), and laminin (LN) in supernatants and lysates of TCFs stimulated with IL-1α or TGF-β. The proliferative response of IL-1α-stimulated TCFs was examined using the SF formazan solution reaction. Results. APC0576 significantly suppressed the production by TCFs of IL-8 ( p<0·0001), MCP-1 ( p<0·005), PIP (supernatants: p<0·0001, cell lysates: p<0·0001), LN (supernatants: p<0·0001, lysates: p<0·0001), and FN (supernatants: p<0·0001, lysates: p<0·0001) as well as their proliferation ( p<0·0001). Conclusions. APC0576 suppressed pro-inflammatory chemokine and ECM production in TCFs as well as their proliferation. It may represent a novel candidate for the postoperative management of patients treated by trabeculectomy.

The investigation of tranilast on the proliferation and migration of human Tenon's capsule fibroblasts

To investigate the effect of tranilast, N-(3,4-dimethoxycinnamoyl) anthramilic acid, on the proliferation and migration of human Tenon's capsule fibroblasts. fibroblasts were cultured from Tenon's tissue of a glaucoma patient after trabeculectomy. The subcultured cells were incubated with DMEM medium containing different concentrations of tranilast for 72 h. The growth of fibroblasts was measured by methyl thiazlyl tetrazolium (MTT) assay and the cell count, and migration was evaluated by crutch method. The expression level of Protein kinase C (PKC) in fibroblasts was tested by immunohistochemical associated with image biological analysis (IBAS) methods. Treated with tranilast varying from 12.5 mg/L to 100.0 mg/L concentration, the proliferation of fibroblasts declined in a dose dependent manner. The migration of fibroblasts decreased from 40.20 +/- 5.83 to 22.50 +/- 4.21 and 9.80 +/- 2.14 cells/per field (P < 0.05) at 50.0 and 100.0 mg/L, respectively, and PKC expression was suppressed by tranilast from 0.2591 +/- 0.0038 to 0.2375 +/- 0.0106 and 0.1273 +/- 0.0573 Absorption value (P < 0.05) at 50.0 and 100.0 mg/L, respectively. Tranilast inhibited the proliferation as well as migration of fibroblasts in vitro, at least in part, by downregulation of PKC expression.

Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and collagen production in vitro.

To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production. Free-floating fibroblast-populated type I collagen lattices were prepared with human Tenon's capsule fibroblasts. Lattice areas were photographed and digitally analyzed to indicate the degree of lattice contraction. Quantitative competitive reverse transcription-polymerase chain reaction (QCRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify mRNA and protein respectively for MMP-1, -2, and -3 by fibroblasts during lattice contraction. Gelatin zymography demonstrated activity of MMPs released into the conditioned medium of contracting lattices. Concentrations of the broad-spectrum MMP inhibitors ilomastat, CellTech (Slough, UK), and BB-94 were added to the contracting fibroblast-populated collagen lattices. Secreted C-terminal propeptide of type I collagen was measured in conditioned medium of contracting lattices by ELISA. Fibroblast proliferation in the presence of concentrations of ilomastat was measured by using the reagent water-soluble tetrazolium-1 (WST-1). During contraction of type I collagen lattices, Tenon's capsule fibroblasts expressed MMP-1, -2, and -3 mRNA and protein. Zymography demonstrated the release of four gelatinolytic species into the conditioned medium of contracting lattices (57, 72, 91, and 100 kDa). Inclusion of MMP inhibitors in the zymogram-developing buffer reduced the proteolytic activity of the detected bands. MMP inhibition (1-100 microM) significantly reduced fibroblast-mediated collagen lattice contraction (P < 0.05), and this effect was found to be reversible. Ilomastat also significantly inhibited production of collagen in a dose-dependent manner (P < 0.05). No effect on fibroblast proliferation was found in the presence of ilomastat. MMPs are produced during Tenon's capsule fibroblast-mediated collagen lattice contraction. Broad-spectrum MMP inhibition significantly reduced matrix contraction and production without cell toxicity. Future clinical use of MMP inhibitors may be possible, because MMP inhibition significantly reduces fibroblast functions associated with contractile scarring.

Anti-allergy Drugs Inhibit the Proliferation of Human Tenon's Capsule Fibroblasts and Maintain the Experimental Filtering Blebs on Rabbit Eyes

To examine whether tranilast and ketotifen fumarate inhibit the growth of human Tenon's capsule fibroblasts and maintain experimental filtering blebs on rabbit eyes. Human Tenon's capsule fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and direct counting of cell number. Production of collagen and transforming growth factor-beta 1 (TGF-beta 1) was measured with corresponding enzyme-linked immunosorbent assay (ELISA). In experimental filtering surgery performed on rabbit eyes, the effects of the topical drugs on intraocular pressure and on maintenance of the filtering blebs were observed. Both tranilast and ketotifen inhibited the growth of the fibroblasts and half-inhibitory concentrations were 200 microM and 70 microM, respectively. Low concentration (10 microM) of tranilast and ketotifen decreased the synthesis of collagen but neither drug had any obvious effect on TGF-beta 1 production. Both drugs prolonged the life of the experimental filtering bleb significantly [12.5 +/- 3.2 (mean +/- standard deviation) days, 13.9 +/- 3.4 days, respectively; control, 10.5 +/- 2.4 days]. Tranilast and ketotifen inhibited the growth of human Tenon's capsule proliferation and are capable of maintaining experimental filtering blebs in rabbit eyes.

The Effect of Integrin Antibodies on the Attachment and Proliferation of Human Tenon's Capsule Fibroblasts

The integrins are protein heterodimers consisting of noncovalently associated α and β subunits. The adhesive interactions mediated by integrins are necessary for cellular survival and proliferation. In this study we investigated the effects of three different integrin antibodies on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Human Tenon's capsule fibroblasts were cultured into 96 well plates and treated with different concentrations (ranging from 10−6to 1 μg ml−1) of three different integrin antibodies: human integrin α-2 antibody, human integrin α-3 antibody and human integrin α-5/FnR (fibronectin receptor) antibody. Coulter counter, hexosaminidase, and3H-thymidine assays were used to determine the inhibitory effects of these integrin antibodies on ocular fibroblasts on days 0 (attachment), 1,3 and 7 following antibody treatment. The concentration of each antibody required to produce a proliferation 50% less than the control (ID50) was calculated for each assay. With respect to attachment, all three antibodies studied displayed some inhibitory activity. All three antibodies also displayed dose-dependent antiproliferative properties, especially at the highest concentration tested after 7 days of exposure. The integrin α-2 antibody was the most potent of the inhibitors, followed by the integrin α-3 antibody, with the integrin α-5 antibody being the least potent antibody tested. In addition, the anti-proliferative activities of the integrin α-2 and integrin α-3 antibodies increased with increasing incubation time. In conclusion, these integrin antibodies demonstrated some inhibitory effects on the attachment and proliferation of human Tenon's capsule fibroblasts in culture. Further investigation will be required to determine whether integrin antibodies can significantly limit scar formation in vivo without significant toxicity.

Effect of cytokines on regulation of the production of transforming growth factor beta-1 in cultured human Tenon's capsule fibroblasts.

Transforming growth factor-beta1 (TGF-beta1) is thought to play a pivotal role in the regulation of the wound healing process after glaucoma filtering surgery. The aim of the present study was to investigate whether platelet-derived growth factor isoforms (PDGF-AA, PDGF-BB), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) modulate the production of latent and/or active TGF-beta1 by cultured human Tenon's capsule fibroblasts (HTF). Human Tenon's capsule fibroblasts were seeded at two different densities (30 cells/mm2 and 150 cells/mm2) and stimulated for five days with PDGF-AA, PDGF-BB, bFGF, EGF, IL-1beta and TGF-beta1. Control cells were treated with serum-free medium (WM/F12). The concentrations of latent and active TGF-beta1 in the medium were determined using an immunoassay before and after activation of TGF-beta1 by transient acidification. The concentration of latent TGF-beta1 in conditioned media from HTF seeded at high density (150 cells/mm2) significantly increased after stimulation with 5 ng/ml TGF-beta1 (151.5 +/-41.7 pg/ml) or 10 ng/ml IL-1beta (45.7+/-8.1 pg/ml). The concentration of active TGF-beta1 in conditioned media also significantly increased after stimulation of HTF with 5 ng/ml TGF-beta1 (48.4+/-27.5 pg/ml). The present results indicate that TGF-beta1 is the most potent inducer of its own synthesis in HTF. Activation of an autocrine TGF-beta1 loop may play a role in the wound healing response after glaucoma filtering surgery.

Cytokine regulation of hyaluronate production by human Tenon?s capsule fibroblasts

The high-molecular weight glycosaminoglycan hyaluronate (HA), a component of the extracellular matrix, has been shown to play important roles in many biological processes including cell proliferation, migration and differentiation. In the present study, the effect of cytokines on production of hyaluronate (HA) by human tenon's capsule fibroblasts (HTF) was determined. HTF (2(nd) passage) were seeded at a cell density of 30 cells/mm(2) and stimulated by six different cytokines (platelet-derived growth factor (PDGF)-AA, PDGF-BB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)-beta1). Controls were treated with aliquots of serum-free medium only. Concentrations of HA were determined using a radiometric assay based on the specific binding of HA to HA binding proteins. The concentration of HA in conditioned medium of HTF was significantly increased only after stimulation with PDGF-AA [10 and 100ng/ml], IL-1beta [1 and 10ng/ml] and TGF-beta1 [5ng/ml]. Production of HA by HTF is regulated by PDGF-AA, IL-1beta and TGF-beta1 and is speculated to be involved in the wound healing reaction after glaucoma filtration surgery.

Cellular Photoablation to Control Postoperative Fibrosis in Filtration Surgery: in vitro Studies

The purpose of this study was to evaluate the feasibility of cellular photoablation using fluorescence-generated photoreaction products as a method to control postoperative fibrosis in filtration surgery. The fluorescent probe, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) is a cell membrane permeable compound rendered membrane-impermeable and fluorescent upon cleavage by intracellular esterases. Human scleral and Tenon's capsule fibroblasts were cultured and used as the target cells. Uptake and retention of the probe were determined with a fluorescence multi-well plate reader. Fibroblasts with or without intracellular probe were irradiated under conditions of fluorescence microscopy with diffuse blue light (450–490 nm, 1.68×102mW m2-1). The viability of cells was examined by trypan blue exclusion and crystal violet test. To better mimic a wound healing process the effect of cellular photoablation was verified in artificial lesions produced in cultured monolayers loaded with different concentrations of the probe. Uptake and retention of BCECF-AM is dependent on ambient concentration. When incorporated the probe is lethal to those cells exposed to the appropriate photo-irradiation. Cells exposed to BCECF-AM (for 45 min) at a concentration of approximately 10 μm and irradiated for 1 min resulted in 100% cell death. Cellular photoablation in contrast to chemotherapeutic agents acts only on the targeted cells. This method shall be pursued as an alternative therapy to control postoperative fibrosis in filtration surgery.

Effect of Diclofenac Sodium and Dexamethasone on Cultured Human Tenon's Capsule Fibroblasts

To investigate the effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts. Two experiments were conducted. In the first experiment, fibroblasts were treated with either diclofenac sodium or dexamethasone at different concentrations, and the cell growth was quantified by using Coulter counter and hexosaminidase methods at 1, 3, 5, and 7 days after adding the drugs. In the second experiment, the cells were treated with each drug for 24 hours and then the cultures were switched to a drug-free medium. The cell growth was quantified at day 7 after removing the drugs from the medium. In the first experiment, inhibition of fibroblast growth in a dose-dependent manner was observed from days 1 to 7 in the cultures treated with each drug. The inhibitory was more pronounced in the diclofenac treated cultures. The typical spindle-shaped fibroblasts treated with higher concentrations of the drugs became spherical cells. In the second experiment, inhibition was not observed when the cultures were switched to a drug-free medium. The spherical cells recovered to spindle-shaped cells and proliferated as normal cells. Our results have shown that diclofenac sodium and dexamethasone can significantly inhibit human Tenon's capsule fibroblast growth in a cell culture model. The inhibitory effect was not observed when the cultures were switched after 24 hours to a drug-free culture medium.

Ultrastructural changes during contraction of collagen lattices by ocular fibroblasts.

Contraction and scarring of the cornea and conjunctiva following disease or injury are major causes of visual morbidity. The aim of this study was to identify any specific ultrastructural features of ocular fibroblast behavior in different collagen lattices in order to understand some of the mechanisms of cell-mediated contraction. Normal human Tenon's capsule fibroblasts were cultured within both restrained and floating collagen lattices for periods of up to 13 days and then analyzed using transmission electron microscopy. The contractile force of these fibroblasts was also tested using the culture force monitor, an instrument capable of measuring the minute forces exerted by cells within a collagen lattice. The results showed differences in the behavior of fibroblasts cultured in the two gel models. The features seen in restrained gels suggest that fibroblasts were actively migrating across and through the lattice. These migratory features were not seen to the same extent in untethered gels, which lack the inherent tension and support of the tethered model. We hypothesize that contraction of the collagen matrix in tethered lattices is due to cellular migration and that this fact cannot be ascertained from untethered gels. Both lattice models have experimental value, but it is important to appreciate what mechanical signals cells receive from the matrix in order to understand cellular behavior.