Conjunctival Fibroblast Proliferation Research Articles

MicroRNA (miR)-29 Modified Bone Marrow Stem Cells (BMSCs) Regulating Wnt-β-Catenin-p53 Pathway to Inhibit the Migration and Proliferation of Conjunctival Fibroblasts

To explore the mechanism of miR-29 modified bone marrow stem cells (BMSCs) in regulating the Wnt/β-Catenin-p53 pathway to inhibit the migration and proliferation of conjunctival fibroblasts. New Zealand rabbit BMSCs were randomly divided into control group, miR-29NC group, and miR-29mimic group. MTT assay, cell scratch test, and Western blot were used to detect the proliferation, migration, and protein expressions in constituent fibroblast. Cell proliferation in the miR-29mimic group was attenuated at 24 h and 48 h (P <0.05). The expression of miR-29 in miR-29 mimic group was upregulated, while Wnt/β-Catenin-p53 protein was decreased (P < 0.05). MiR-29 modified BMSC can inhibit the expression of Wnt/β-Catenin-p53 pathway and suppress the proliferation and migration of conjunctival fibroblasts.

Anti-scarring effects of butaprost on human subconjunctival Tenon's fibroblasts.

To investigate the toxicity of the E-prostanoid 2 (EP2) receptor agonist, butaprost against human subconjunctival (Tenon's capsule) fibroblasts, and to determine the underlying mechanism. We isolated Tenon's fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate (cAMP) levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon's tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the cAMP cascade, which might be an important mechanism underlying this effect. Butaprost effectively reduces the subconjunctival scarring response. Given the significance of wound healing modulation in blebs, butaprost's inhibitory effect on subconjunctival Tenon's fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery.

Evaluation of Chitosan/Aptamer Targeting TGF-β Receptor II Thermo-Sensitive Gel for Scarring in Rat Glaucoma Filtration Surgery.

This study was designed to develop a chitosan (CS) thermo-sensitive gel combined with aptamer S58 targeting transforming growth factor-beta receptor II (TGF-β RII) and to investigate the antifibrotic effects of CS/S58 gel in a rat glaucoma filtration surgery (GFS) model. In vitro aptamer S58 release rate from the CS/S58 gel were detected, and the effect of mitomycin-C (MMC), TGF-β2, CS, or CS/S58 gel on wound healing were investigated in a rat GFS model by detecting scar-related factors and the involved inflammatory response. The levels of collagen I and α-smooth muscle actin (α-SMA) were detected by immunohistochemistry and Western blotting. The control and TGF-β2 eyes exhibited densely packed collagen fibers with no evidence of filtration after day 7. The pronounced increase in filtration efficiency was associated with thinner fibers, and a loosely organized subconjunctival matrix was observed in CS/S58 gel-treated eyes. The levels of collagen I and α-SMA were downregulated in CS/S58 gel-treated eyes. Conjunctival fibroblast proliferation and the inflammation response were also suppressed in the CS/S58 gel-treated group. This study presents evidence that the antifibrotic effect of chitosan in combination with aptamer S58 is superior to chitosan alone in a rat GFS model. Chitosan/S58 gel may be considered to be a promising antifibrotic agent for a local drug therapy.

Validation of the Glaucoma Filtration Surgical Mouse Model for Antifibrotic Drug Evaluation

Glaucoma is a progressive optic neuropathy, which, if left untreated, leads to blindness. The most common and most modifiable risk factor in glaucoma is elevated intraocular pressure (IOP), which can be managed surgically by filtration surgery. The postoperative subconjunctival scarring response, however, remains the major obstacle to achieving long-term surgical success. Antiproliferatives such as mitomycin C are commonly used to prevent postoperative scarring. Efficacy of these agents has been tested extensively on monkey and rabbit models of glaucoma filtration surgery. As these models have inherent limitations, we have developed a model of glaucoma filtration surgery in the mouse. We show, for the first time, that the mouse model typically scarred within 14 d, but when augmented with mitomycin C, more animals maintained lower intraocular pressures for a longer period of time concomitant with prolonged bleb survival to beyond 28 d. The morphology of the blebs following mitomycin C treatment also resembled well-documented clinical observations, thus confirming the validity and clinical relevance of this model. We demonstrate that the antiscarring response to mitomycin C is likely to be due to its effects on conjunctival fibroblast proliferation, apoptosis and collagen deposition and the suppression of inflammation. Indeed, we verified some of these properties on mouse conjunctival fibroblasts cultured in vitro. These data support the suitability of this mouse model for studying the wound healing response in glaucoma filtration surgery, and as a potentially useful tool for the in vivo evaluation of antifibrotic therapeutics in the eye.

Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

Inhibition of Cell Proliferation by Mitomycin C Incorporated into P(HEMA) Hydrogels

The technique of mitomycin C (MMC) drug delivery and its application in glaucoma surgery are not standardized with resultant inconsistencies in the results. Also, one time application of MMC does not seem to have the same efficacy after glaucoma drainage device surgeries compared with trabeculectomies. This preliminary study examined the efficacy of a slow release form of MMC for its ability to inhibit cell proliferation in vitro. MMC was incorporated into 1% P(HEMA) hydrogels using a redox polymerization method. For some experiments, unreacted low molecular weight components were removed from the hydrogels before the MMC was incorporated. Sterile disks (8 mm) of each polymer sample were affixed to 60 mm tissue culture dishes, and the dishes were inoculated with COS-1 cells or early passage human conjunctival fibroblasts. After 7 days in culture, the number of cells in each dish was determined. Cell morphology was assessed in replicate cultures after fixation and staining. Hydrogels with unreacted low molecular weight components slowed cell proliferation and induced morphologic changes. Early passage human conjunctival fibroblasts were more sensitive than COS-1 cells both to intrinsic contaminants in the hydrogels and to incorporated MMC. Once contaminants had been removed, MMC-loaded hydrogels inhibited conjunctival fibroblast proliferation in a dose-dependent fashion, with an IC50 of approximately 0.15 mg/g polymer. This study demonstrates that a slow release form of MMC can inhibit cell proliferation in vitro. Future experiments will focus upon the efficacy of this polymer-bound form during in vivo wound healing.

Enhancement of the Mechanical and Biological Properties of a Biomembrane for Tissue Engineering the Ocular Surface

In this study, we have developed and optimised a novel gelatin-chitosan (GC) substrate for use as a cellular carrier for tissue-engineered conjunctival epithelium. The substrate was fabricated by casting and the mechanical properties of the substrate, including tensile strength and elongation, were measured. Using the MTT, cell proliferation assay with rabbit conjunctival fibroblasts, we optimised the G:C ratio to enhance cytocompatibility. Rabbit conjunctival epithelial cells were immunostained using monoclonal antibodies for keratin 4 and pancytokeratin to investigate the biological effects of the GC substrate on the proliferation and differentiation of epithelial cells. We found that increasing the amount of gelatin resulted in an increase in elasticity (from 1:9 to 1:1 ratio), reaching a maximum (101.89% +/- 7.13%) at a ratio of 1:1. The MTT assay showed that the proliferation of conjunctival fibroblasts significantly increased from 0.068 +/- 0.017 to 0.177 +/- 0.011 (P = 0.014) as the gelatin was increased from 20% (1:4) to 50% (1:1). Additional studies using tissue-cultured conjunctiva explants showed that these explants grew well on the substrate, forming a multilayered epithelium. Cell morphology on this substrate was similar to that of cells grown on culture dishes alone. Positive staining of keratin 4 and pancytokeratin indicated that the substrate supported normal differentiation of conjunctival epithelial cells. By enhancing the proportion of gelatin, both the mechanical and biological properties of the chitosan substrate were improved. The results also suggest that this GC biomembrane may be a useful candidate for reconstructive tissue engineering of the conjunctiva.

Tryptase Increases Proliferative Activity of Human Conjunctival Fibroblasts through Protease-Activated Receptor-2

Tryptase that is released by mast cell degranulation has recently been thought to play a key role in wound healing in allergic bronchitis. Conjunctival fibroblasts secrete mediators and extracellular matrices that could exacerbate inflammation and papillary formation in allergic conjunctivitis. This study was conducted to investigate the effect of tryptase on the proliferation of conjunctival fibroblasts and studied whether this effect was mediated by protease-activated receptor (PAR)-2. Conjunctival fibroblasts were cultured with or without tryptase (0.1 ng/mL to 1.0 microg/mL), and the proliferation rate was assessed after 48 hours. The effects of tryptase inhibitors (leupeptin, benzamidine) and a PAR-2 agonist (SLIGKV) were examined. The existence of PAR-2 mRNA and protein in conjunctival fibroblasts was examined by RT-PCR and Western blot analysis, respectively. The existence of PAR-2 in cultured conjunctival fibroblasts and conjunctival papillae from patients with vernal keratoconjunctivitis, as well as conjunctival tissue from normal subjects was examined by immunohistochemistry. Conjunctival fibroblast proliferation was upregulated by tryptase in a dose-dependent manner (P < 0.001). Leupeptin and benzamidine inhibited tryptase-induced fibroblast proliferation (P < 0.05), and SLIGKV mimicked tryptase's effect. PAR-2 mRNA and protein were detected in cultured conjunctival fibroblasts using RT-PCR and Western blot analysis. PAR-2 immunoreactivity in both the cultured conjunctival fibroblasts and in stromal cells in excised conjunctival tissues was observed. Tryptase increased conjunctival fibroblast proliferation and this response appeared to be mediated by PAR-2. Mast cells are the most likely source of tryptase in the conjunctiva and may play an important role in chronic exacerbations with conjunctival papillary formation in allergic conjunctivitis.

Protection of Human Conjunctival Fibroblasts from NO-Induced Apoptosis by Interleukin-4 or Interleukin-13

To examine the possible roles of T helper type 2 (Th2) cell-derived cytokines in formation of the giant papillae characteristic in individuals with vernal keratoconjunctivitis, the effects of these cytokines on the proliferation and apoptosis of cultured human conjunctival fibroblasts were investigated. Apoptosis was induced by the NO donor sodium nitroprusside. Cell viability was determined by measurement of mitochondrial metabolic activity, and apoptotic cells were identified on the basis either of nuclear morphology after staining with 4',6-diamidino-2-phenylindole or of TUNEL staining. The activation of antiapoptotic signaling mediated by the protein kinase Akt was assessed by immunoblot analysis and by an in vitro kinase assay. Expression of interleukin (IL)-4 and IL-13 receptor subunits was examined by reverse transcription and polymerase chain reaction analysis and by flow cytometry. IL-4 and IL-13, but not IL-5, IL-9, or IL-10, induced the proliferation of conjunctival fibroblasts as well as protecting these cells from NO-induced apoptosis. Both IL-4 and IL-13 induced the phosphorylation of Akt and increased the kinase activity of this enzyme in a manner that was sensitive to the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. These inhibitors also blocked the antiapoptotic effects of IL-4 and IL-13. Transcripts encoding IL-4 and IL-13 receptor components were detected in conjunctival fibroblasts, and the proteins were expressed at the cell surface. Among the various Th2 cytokines tested, only IL-4 and IL-13 induced the proliferation of human conjunctival fibroblasts and protected these cells from apoptosis. These effects may contribute to the formation of giant papillae in individuals with vernal keratoconjunctivitis.

IL-4-induced cell proliferation and production of extracellular matrix proteins in human conjunctival fibroblasts

Giant papillae, characteristic lesions of vernal keratoconjunctivitis, are formed as a result of the proliferation of conjunctival fibroblasts, the deposition of extracellular matrix, and the infiltration of inflammatory cells. The concentration of interleukin (IL)-4 is also increased in the tear fluid of individuals with ocular allergic diseases. The possible role of IL-4 in the development of giant papillae was investigated by examining the effects of this cytokine on cultured human conjunctival fibroblasts. Reverse transcription and polymerase chain reaction analysis revealed the presence of transcripts encoding the IL-4 receptor α chain in these cells, and flow cytometry demonstrated the expression of this protein on the cell surface. IL-4 induced the proliferation of conjunctival fibroblasts in a concentration-dependent manner, and this effect was inhibited by neutralizing antibodies to the IL-4 receptor. Enzyme immunoassays revealed that IL-4 also increased in a concentration-dependent manner the amounts of procollagen type I C-peptide and fibronectin released into the culture supernatant by conjunctival fibroblasts. A whole-cell enzyme-linked immunosorbent assay showed that IL-4 increased the deposition of collagen type III by conjunctival fibroblasts. Furthermore, reverse transcription combined with real-time polymerase chain reaction analysis revealed that IL-4 increased the abundance of collagen type III mRNA in these cells. These results demonstrate that human conjunctival fibroblasts express receptors for IL-4, and that IL-4 stimulates both the proliferation of and the production of extracellular matrix proteins by these cells. These effects of IL-4 might contribute to the formation of giant papillae in individuals with vernal keratoconjunctivitis.

The inhibition of vitamin K3 on rabbit fibroblast proliferation in vitro.

Vitamin K3 that is commonly used to treat hemorrhagic diseases can inhibit human lymphatic neoplasms in vitro. This study was undertaken to investigate the effects of vitamin K3 on rabbit conjunctival fibroblast proliferation in cell culture. Vitamin K3 was added to cultured rabbit conjunctival fibroblasts. Remarkable inhibition was observed at a concentration of 4 mg/l. Because of its lower toxicity, vitamin K3 may prove to be of significant value in the treatment of intraocular proliferative disorders.

Biological activity of N-(4-hydroxyphenyl) retinamide-O-glucuronide in corneal and conjunctival cells of rabbits and humans.

Previous studies of topical retinoic acid for treatment of ocular surface disease met with limited success due to instability and irritancy of the retinoid and lack of efficacy in keratoconjunctivitis sicca. There has, however, been continued interest in the treatment of mucin deficiency and cicatrizing conjunctival diseases, such as ocular cicatricial pemphigoid (OCP), topically with retinoids. In this study the biological activity of stable, water-soluble, synthetic retinoid, N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) was investigated in vivo and in vitro using conjunctival and corneal epithelium and fibroblasts. Vitamin A-deficient rabbits with stage 3-4 corneal xerosis and squamous metaplasia confirmed by conjunctival impression cytology were treated with topical 0.1% 4-HPROG in an artificial tear vehicle for 3 weeks. Impression cytology was repeated at 2 and 3 weeks and at 3 weeks conjunctival biopsies were fixed for histology. Growth curves were generated using conjunctival fibroblasts of rabbits and humans (normals and patients with cicatrizing conjunctival disease including OCP and Stevens-Johnson syndrome) cultured in the 10(-8)-10(-6) M 4-HPROG. In vivo, corneal xerosis cleared in three days. A normal conjunctival epithelium was restored by 2 weeks and goblet cells were present by 3 wk, with no change in vehicle-treated controls. No ocular irritation occurred. In vitro, 10(-6) M 4-HPROG inhibits growth of rabbit conjunctival fibroblasts. The retinoid had no effect on proliferation of conjunctival fibroblasts from normal humans but the doubling time of cells from patients with OCP increased significantly, from 50.9 +/- 10.01 h (control) to 61.5 +/- 8.95 h (retinoid). Proliferation of conjunctival fibroblasts from a patient with Stevens-Johnson syndrome was also inhibited. N-(4-hydroxyphenyl) retinamide-O-glucuronide is biologically active and merits further study to determine its efficacy in controlling conjunctival fibrosis and treating ocular surface squamous metaplasia.

The inhibition of fibroblast proliferation by a novel monokine: an in vitro and in vivo study.

We have identified a product of rabbit macrophages that inhibits fibroblast proliferation. Tested in vitro against several fibroblast populations, this monokine inhibited rabbit conjunctival fibroblast proliferation by 85% (p = 0.005), human conjunctival fibroblast proliferation by 88% (p = 0.005), human hypertrophic scar fibroblast proliferation by 85% (p = 0.005), and human keloid fibroblast proliferation by 79% (p = 0.005). Additionally (in an in vivo model), this monokine was injected into healing rabbit wounds and inhibited fibroblast proliferation by 33% after 7 days (p = 0.0001) and by 27% after 2 weeks (p < 0.0001). Preliminary analysis of the active factor demonstrates that it is not species-specific, has a molecular weight less than 3000 d, is resistant to degradation by trypsin and carboxypeptidase A, is heat-stable, and is produced by macrophages largely in the first 3 days of culture.

The effects of 5-fluorouracil and mitomycin C on the corneal endothelium.

5-Fluorouracil (5-FU) and Mitomycin C (MMC) are used as adjunct chemotherapy during glaucoma filtering surgery to suppress conjunctival fibroblast proliferation. Since part of these agents may gain access to the anterior chamber and cause cytotoxicity to the corneal endothelium we set up an in vitro system to establish a dose-response effect. Cytotoxicity of MMC and 5-FU was quantified using Mosmann's colorimetric assay in a bovine endothelial cell culture system. In this assay the respiratory activity of the cells is used as a marker for cell viability. After incubation for 5 minutes the 3.0 mg/ml concentration of MMC showed endothelial cytotoxicity, whereas no endothelial toxicity of 5-FU was noted in concentrations up to 50 mg/ml. After incubation for 30 minutes endothelial cytotoxicity was demonstrated for 50 mg/ml of 5-FU and 1 mg/ml of MMC. After an exposure-time of 60 minutes the toxicity level remained 50 mg/ml for 5-FU but decreased to 0.5 mg/ml for MMC. We conclude that with respect to the clinically used concentrations and methods of application of 5-FU and MMC in vivo endothelial toxicity is not to be expected. However, in cases of accidental access of MMC to the anterior eye chamber and following a reduction of aqueous turnover rate the safety of MMC is unwarranted.

Fluorouracil (5-FU) and cytarabine (ara-C) inhibition of corneal epithelial cell and conjunctival fibroblast proliferation.

We evaluated the antiproliferative properties of fluorouracil (5-FU) and cytarabine (ara-C) in a tissue culture model of rabbit corneal epithelial cells and conjunctival fibroblasts. Cells were treated with fluorouracil or cytarabine for five days. Corneal epithelial cell proliferation was inhibited to 50% of control proliferation by 0.6 mg/L of fluorouracil or 30 micrograms/L of cytarabine. Proliferation of conjunctival fibroblasts was inhibited to 50% of control by 0.5 mg/L of fluorouracil or 50 micrograms/L of cytarabine. The antiproliferative effect of 10 mg/L of fluorouracil was not blocked by thymidine. The inhibition of proliferation produced by 100 or 1,000 micrograms/L of cytarabine was blocked by 2'-deoxycytidine at concentrations of 10(-5) M and 10(-4) M, respectively. The antiproliferative effects of fluorouracil and cytarabine appear similar for both cell types. Blockage of cytarabine-related ocular toxic effects may be possible with 2'-deoxycytidine.

Topical fluorouracil. Pharmacokinetics in normal rabbit eyes.

Postoperative subconjunctival fluorouracil injections may be a useful adjunct to standard glaucoma filtering surgery in eyes that are at high risk of failure. Topical administration would be preferable to subconjunctival administration; however, there are no data on the ocular penetration of topically applied fluorouracil. Consequently, we investigated the pharmacokinetics of topically administered fluorouracil labeled with carbon 14 in normal rabbit eyes. One drop (approximately 2.4 mg) of fluorouracil resulted in the following concentrations at 0.5 and six hours, respectively: 17.3 and 0.9 micrograms/g of conjunctiva; 24.3 and 1.3 micrograms/g of cornea; 14.6 and 0.2 micrograms/mL of aqueous; 0.8 and 0.5 microgram/g of lens; 1.1 and 0.3 microgram/g of vitreous; and 0.2 and less than 0.1 microgram/mL of serum. Three drops (approximately 7.2 mg) of fluorouracil resulted in the following concentrations at 0.5 and eight hours, respectively: 589.8 and 1.3 micrograms/g of conjunctiva; 502.9 and 1.8 micrograms/g of cornea; 199.6 and 0.8 micrograms/mL of aqueous; 6.2 and 0.5 micrograms/g of lens; 6.8 and 0.5 micrograms/g of vitreous; and 1.3 and 0.2 microgram/mL of serum. Since a fluorouracil concentration of 0.2 microgram/mL inhibits rabbit conjunctival fibroblast proliferation in cell culture by 50%, these data suggest that topically applied fluorouracil achieves sufficient levels in the ocular compartments and tissues to have potential therapeutic applications.

Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation.

A variety of antimetabolites, steroids, and nonsteroidal anti-inflammatory agents were tested for their ability to inhibit rabbit dermal and conjunctival fibroblast proliferation in cell culture. Doxorubicin hydrochloride and fluorouracil produced notable inhibition in concentrations of less than 1 mg/L. Meclofenamate sodium and indomethacin produced notable inhibition at concentrations of 11 and 40 mg/L, respectively. Dexamethasone sodium phosphate and triamcinolone acetonide produced inhibition at 200 and 150 mg/L, respectively, but paradoxically increased proliferation almost two-fold at concentrations ranging from 1 to 30 mg/L under identical culture conditions. Methotrexate sodium demonstrated only limited effectiveness. This assay system may be a useful approach to drug selection in the treatment of a variety of ocular proliferative disorders. Fluorouracil may prove to be of significant value in the treatment of intraocular proliferative disorders.