Normal Conjunctival Fibroblasts Research Articles

Recombinant human heat shock protein 27 can inhibit ultraviolet B‐induced differentiation in pterygial‐derived fibroblast

PurposeTo investigate the effect of recombinant human heat shock protein 27 (rhHSP27) on the differentiation of human pterygial‐derived fibroblast.MethodsHuman conjunctival and pterygial fibroblasts were isolated and cultured from specimens of normal conjunctiva and pterygium head, which were donated during pterygium excision. Cultured conjunctival and pterygial fibroblasts were exposed to 20 mJ/cm2 of ultraviolet‐B (UVB) irradiation with and without 4 ug/mL rhHSP27 pre‐treatment. Western blot analyses of α‐smooth muscle actin (α‐SMA), a marker for myofibroblast, were performed in each group and quantified using Image Gauge 4.0 software. The ratios of α‐SMA / GAPDH and phosphorylated HSP27 / GAPDH were compared.ResultsThe α‐SMA level in Pterygial fibroblasts showed 2.85 ± 0.11 fold greater than that of normal conjunctival fibroblasts (P < 0.001). The level of α‐SMA was increased by 1.92 ± 0.02 times (P < 0.001), when normal conjunctival fibroblasts were exposed to UVB irradiation without rhHSP27 pre‐treatment. However, when normal conjunctival fibroblasts were exposed to UVB irradiation with rhHSP27 pre‐treatment, the fibroblasts demonstrated 0.82 ± 0.04 fold smaller α‐SMA level than control did (P = 0.04).ConclusionsThe rhHSP27 revealed inhibitory effects on the UVB irradiation‐induced differentiation from normal conjunctival fibroblasts to pterygial myofibroblast. The rhHSP27 can be a preventive treatment against pterygium growth.

Ultraviolet-A Irradiation Upregulated Urokinase-Type Plasminogen Activator in Pterygium Fibroblasts through ERK and JNK Pathways

Effects of ultraviolet (UV) B on the pterygium and its cells have been studied previously, whereas little is known on the effects of UVA. Urokinase-type plasminogen activator (uPA) is a protease involved in tissue remodeling and cell migration, and its levels are increased in pterygium. The purpose of our study was to investigate the effects of UVA on the expression of uPA in cultured pterygium fibroblasts. Cultured fibroblasts from early-stage pterygia and normal conjunctiva were irradiated with different dosages of UVA and compared to nonirradiated cells. uPA activities in the medium and uPA mRNA in the cells were measured by casein zymography and RT-PCR, respectively. Total and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), and c-Jun N-terminal kinase (JNK) levels of cells treated with and without UVA were measured by Western blotting. Inhibitors of p38 (SB203580), ERK (UO1026), and JNK (SP600125) were added before the irradiation of UVA to test their effects. UVA irradiation increased the uPA mRNA levels in pterygium fibroblasts and the uPA activities in cultured medium, which was accompanied with an increase in phosphorylated ERK and JNK. The ERK and JNK inhibitor, but not p38 MAPK inhibitors, significantly decreased the UVA-induced expression of uPA by pterygium fibroblasts. Normal conjunctival fibroblasts were less sensitive to UVA irradiation compared to the pterygium fibroblasts. UVA stimulated the production of uPA, a key factor in the modulation of extracellular matrixes, inflammatory processes, and angiogenesis. This may have a role in the development and progression of pterygium.

Profibrotic Phenotype of Conjunctival Fibroblasts from Mucous Membrane Pemphigoid

Ocular mucous membrane pemphigoid is an immunobullous disease in which excessive conjunctival fibrosis causes blindness, and the pathogenesis of scarring is incompletely understood. To establish whether profibrotic fibroblasts with an altered phenotype exist in ocular mucous membrane pemphigoid, we compared the functional characteristics of pemphigoid conjunctival fibroblasts to normal conjunctival fibroblasts with respect to cell division; migration; collagen contraction; matrix metalloproteinase, secretion of collagen and chemokines; and myofibroblast differentiation. We found that pemphigoid fibroblasts showed increased cell division (P = 0.01), increased migration in serum-free medium (72 ± 18 migrated cells versus 33 ± 11, P = 0.04), increased collagen contraction in the presence of 10 ng/ml tumor necrosis factor-α, increased collagen type I secretion (P = 0.03), increased secretion of matrix metalloproteinase-3 (P = 0.03), and increased secretion of eotaxin in response to interleukin-13 (P = 0.04). Differences between pemphigoid and normal conjunctival fibroblasts with respect to collagen contraction and MMP secretion in the presence of interleukin-13 were also observed. Together, these findings indicate that pemphigoid conjunctival fibroblasts have a profibrotic phenotype that is maintained in vitro. No differences between pemphigoid fibroblasts obtained from acutely inflamed versus clinically uninflamed conjunctiva were observed. Developing effective antifibrotic therapies will require understanding of the mechanisms that both induce and maintain the profibrotic phenotype.

Glucose-6-phosphate-dehydrogenase Deficiency as a Risk Factor for Pterygium

Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP), providing reducing power (NADPH) and pentose phosphates. The purpose of this study was to investigate the possible involvement of G6PD deficiency (G6PD-) in the pathogenesis of pterygium. Erythrocyte G6PD activity was evaluated in 123 pterygium patients and in 112 age-matched control patients. Enzyme activity, mRNA, rate of growth, green autofluorescence, response to oxidative stress, and cholesterol metabolism were determined in pterygium fibroblasts (PFs) and in normal conjunctival fibroblasts (NCFs) isolated from G6PD normal (NCFs+ and PFs+) and G6PD- (NCFs- and PFs-) patients. Higher prevalence of G6PD- was found in patients affected by primary pterygium than in control subjects, both men and women, suggesting that this enzymatic defect may be a predisposing factor for pterygium. G6PD activity was significantly lower in NCFs- than in NCFs+, but not in PFs- than in PFs+. In PFs-, G6PD mRNA levels were significantly higher than in PFs+. Growth-stimulated NCFs- grew at half the rate of NCFs+, although PFs- and PFs+ grew at the same rate. Increased green autofluorescence and susceptibility to oxidative stress were observed in PFs (+/-) and in NCFs-, but not in NCFs+. Moreover, ex vivo PFs (+/-) accumulated more lipids than corresponding NCFs. The results of this study, although restricted to a limited group of subjects (i.e., those of Sardinian ancestry), suggest that G6PD- not only does not protect against pterygium, but may even be considered a risk factor for the development of this disorder.

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.

Modulation of Cholesterol Homeostasis by Antiproliferative Drugs in Human Pterygium Fibroblasts

The authors have previously shown that the growth of cultured fibroblasts obtained from primary pterygia was associated with an increase in cholesterol esterification, suggesting that alterations of cholesterol homeostasis may be involved in the development and progression of this disorder. This investigation was conducted to determine whether antiproliferative agents such as pioglitazone (PIO) and everolimus (EVE) may inhibit proteins involved in the cholesterol ester cycle and the proliferation of pterygium fibroblasts (PF). Quiescent normal conjunctival fibroblasts and PFs were treated with or without inhibitors of cell proliferation (PIO and EVE) or with inhibitors of cholesterol esterification-progesterone (Pg) and Sandoz compound (SaH)-and then were stimulated to growth by 10% fetal calf serum (FCS). Cell proliferation was assessed by counting cells. Trypan blue uptake was used to determine cell viability. mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. PIO and EVE significantly abolished the increase in cholesterol esters, acyl-coenzyme A cholesterol acyltransferase (ACAT1), and multidrug resistance protein (MDR1) mRNA observed in growing cells. Each inhibitor upregulated ATP-binding cassette-A1 (ABCA1), neutral cholesterol ester hydrolase (NCEH) mRNA, and caveolin-1 expression in a manner similar to that of specific inhibitors of cholesterol esterification such as Pg and SaH. Intracellular modifications of cholesterol homeostasis may be relevant to pterygium development. Moreover, antiproliferative agents such as PIO and EVE may represent a potential topical medication in the prevention and inhibition of pterygium growth at an early stage, probably by modulation of cholesterol ester metabolism.

Pterygial Derived Fibroblasts Express Functionally Active Histamine and Epidermal Growth Factor Receptors

Pterygia are characterised by a fleshy outgrowth of altered conjuctival tissue over the cornea and are most common in tropical regions. Pterygial fibroblasts are characteristically distinct from normal conjunctival fibroblasts, and therefore the aim of this study was to determine the presence and functional significance of histamine and epidermal growth factor (EGF) receptors in these cells. Pterygial specimens were cultured in vitro and cellular outgrowths were phenotypically characterised as fibroblasts using vimentin and cytokeratin staining. Intracellular calcium mobilization was used to characterise the functional activity of histamine receptors on these cells. Maximal response was obtained with 100μM histamine. However, lower concentrations of histamine also caused mobilization of calcium that were totally abolished by pre-incubation with H1 but not H2 or H3 receptor antagonists. EGF receptor was diffusely expressed over the cell surfaces. EGF stimulated receptor internalization, ERK protein phosphorylation and intracellular calcium mobilization. Therefore, fibroblasts derived from human pterygia express functionally active histamine and epidermal growth factor receptors. Controlled modification of either the receptors or the appropriate ligands could have beneficial effects in pterygia treatment.

Mechanism of Abnormal Elastin Gene Expression in the Pinguecular Part of Pterygia

Elastodysplasia and elastodystrophy are two known manifestations in the conjunctival, ie, pinguecular, part of pterygia. But the mechanisms are still not understood. The purpose of this study is to investigate the mechanism of enhanced elastin gene expression in fibroblasts from the pinguecular part of pterygia, which is related to abnormal elastic fiber expression in the pinguecular part of pterygia. Elastin in surgical specimens of normal conjunctiva and the pinguecular part of pterygia from age-matched patients was detected by immunohistochemical staining. Northern hybridization and quantification of radiolabeled tropoelastin were performed in conjunctival fibroblasts cultured under different doses of ultraviolet (UV) B irradiation, and in cultured pinguecular fibroblasts from pterygia. In vitro translation was also performed to analyze the tropoelastin production in rabbit reticulocyte lysate. The level of tropoelastin in reticulolysates from UV-treated conjunctival and pinguecular fibroblasts of pterygia was higher than in normal conjunctival fibroblasts. The coding sequence and 3'- untranslated region of tropoelastin mRNAs were amplified by reverse transcription-polymerase chain reaction, and mutations were checked by DNA sequencing. Immunohistochemical staining revealed elastin in pinguecular subepithelial connective tissues of pterygia, but not in normal conjunctiva. Tropoelastin mRNA levels were not elevated in cultured pinguecular or conjunctival fibroblasts with or without ultraviolet B irradiation. However, tropoelastin synthesis was enhanced in culture medium of pinguecular and UV-irradiated conjunctival fibroblasts, but not in normal conjunctival fibroblasts. Direct DNA sequencing revealed mutations in the 3'-untranslated region but not in the coding sequence of tropoelastin mRNA, in both pinguecular and UV-irradiated conjunctival fibroblasts. The increased expression of tropoelastin in pinguecular and UV-irradiated fibroblasts is not a result of increased levels of steady-state mRNA, but is a result of posttranscriptional modification of tropoelastin.

Suppression of TGF-� signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane

Suppression of TGF-� signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane

Suppression of TGF-ß signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane

Purpose. When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFß signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF). Methods. HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-ß1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-ß1, ß2 and ß3; TGF-ß receptors (TGF-ßR) type I, II and III; a-smooth muscle actin (a-SM), ß1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor ß (PDGFR-ß); and GAPDH as a loading control. MTT assay was used for cell proliferation. Results. Amniotic membrane markedly suppressed the transcript expression of TGF-ß2, ß3 and all three types of TGF-ß receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of ß1 integrin, a-SM actin, and FGFR1/ flg was mildly suppressed. In contrast, expression of TGF-ß1 and PDGFR-ß remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed. Conclusions. Amniotic membrane matrix uniquely suppresses TGF-ß signaling in both types of fibroblasts. It may also suppress signaling via CD44, ß1 integrin and FGFR1/ flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.

Growth factors in cultured pterygium fibroblasts: immunohistochemical and ELISA analysis.

In order to study growth factors in the pathogenesis and recurrence of pterygium, we grew pterygium tissues in culture and compared fibroblasts from primary and from recurrent pterygia with reference to the fibroangiogenic growth factors basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha). We used indirect immunohistochemical procedures against human b-FGF, PDGF, TGF-beta and TNF-alpha. As controls, we used cultured normal human conjunctival fibroblasts. A serum-free conditioned medium (CM) from confluent fibroblasts derived from primary and recurrent explants was assessed by enzyme-linked immunosorbent assay to determine the level of the above-mentioned growth factors. Immunoreactivity of b-FGF was stronger in recurrent than in primary pterygium fibroblasts. PDGF immunolabeling was stronger in primary than in recurrent pterygium fibroblasts. TGF-beta and TNF-alpha immunolabeling was weak in both pterygia. All these growth factors were very sparse in normal conjunctival fibroblasts. Basic-FGF and TGF-beta 1 were found in the CM from both primary and recurrent pterygium, while PDGF and TNF-alpha were not detectable. The strong immunoreactivity and the release of b-FGF in cultured fibroblasts of recurrent pterygia suggest that fibroblasts may play an important role in the recurrence of pterygium.

TNP-470 (a fungus-derived inhibitor of angiogenesis) reduces proliferation of cultured fibroblasts isolated from primary pterygia: a possible drug therapy for pterygia

PURPOSE. To study drug therapy for pterygium, especially the effect of a fumagillin analog, TNP-470, a potent anti-angiogenic compound, on the growth of cultured fibroblasts obtained from primary pterygia and normal human conjunctiva. METHODS. Cultured pterygium fibroblasts (PF) were exposed to different concentrations of TNP-470 every other day for 7 days (Treatment A) and to a single dose before 4 days of culture (Treatment B). Human normal conjunctival fibroblasts (HCF) were treated with TNP-470 every other day for 7 days. The cells were observed daily by phase contrast microscopy. Cell proliferation was assessed by counting cells with a hemocytometer. Trypan blue uptake was used to determine cell viability at harvest. RESULTS. TNP-470 induced a significant inhibition of PF and HCF proliferation in a dose-dependent manner (P <. 0001). At the lowest dose of TNP-470 (100 pg/ml), the cumulative inhibitory effect of TNP-470 was more potent than the sustained inhibitory effect observed by treatment B in one high dose. Nevertheless, the cytotoxic effect was dose-dependent and more marked after treatment A than after treatment B. After washing out of the drug, partial reversibility was observed at doses lower than 5 mg/ml with a significant increase of viability. HCF were less sensitive to TNP-470 and doses less than 5 mg/ml were not cytotoxic. CONCLUSION. TNP-470 appears to have a marked inhibitory effect on PF proliferation, and it may be of considerable value in the prevention of pterygium growth and recurrence.