Conjunctival Fibroblasts Research Articles

Expression profiles of collagens, HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA. This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.

Tear and mucus eotaxin-1 and eotaxin-2 in allergic keratoconjunctivitis

Objective Eotaxin-1 and eotaxin-2 are potent eosinophil chemotactic and activating peptides that may be implicated in the pathogenesis of the chronic allergic eye diseases vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). The purpose of this study was to measure these chemokines in tear and mucus samples of active-disease patients and in vitro cultured conjunctival epithelial cells and fibroblasts. Design Comparative, observational case series and in vitro study. Participants Sixteen patients with clinically active and untreated VKC or AKC, six age-matched control patients, and five nonactive seasonal allergic conjunctivitis patients. Methods Tears were collected from the active VKC and AKC patients, and from the normal patients. Mucus was collected from six of these VKC patients. Tears were also collected from an additional five allergic patients after obtaining a positive reaction to conjunctival allergen challenge. Conjunctival epithelial cell and conjunctival fibroblast cultures were exposed to interleukin (IL)-4, IL-13, and tumor necrosis factor-α (TNF-α), or to combinations of these cytokines. Main outcome measures Levels of eotaxin-1 and eotaxin-2 in tears, mucus, and culture medium. Results High levels of eotaxin-1 and eotaxin-2 were found in mucus of VKC patients, whereas only eotaxin-2 was found to have increased significantly in tears of VKC and AKC patients compared with those of normal patients. Mucus contained higher levels of chemokines than did tears. Both tear eotaxin-1 and eotaxin-2 were correlated significantly with the percent of eosinophils in tear fluid. Eotaxin-1 also was correlated significantly with the sum clinical score and corneal involvement in VKC patients. Conjunctival epithelial cells in culture did not produce eotaxin-1 or eotaxin-2, either at baseline or after cytokine exposure. Conjunctival fibroblasts produced eotaxin-1 only after exposure to IL-4, TNF-α, and the combination of IL-4 plus TNF-α or IL-13 plus TNF-α. Conclusions Eotaxin-1 and eotaxin-2 are implicated in eosinophil recruitment and in the pathogenesis of VKC and AKC. Cytokine-stimulated conjunctival fibroblasts may be one source of eotaxin-1 in severely allergic tissues.

Overexpression of Insulin-like growth factor-binding protein-2 in pterygium body fibroblasts.

To examine the expression pattern of insulin-like growth factor-binding protein (IGFBP)-2 in cultured primary pterygium fibroblasts and compare it with expression in normal conjunctival fibroblasts. Profile of gene expression by normal conjunctival and primary pterygium fibroblasts was performed by using a cDNA microarray. The overexpression of IGFBP-2 thus identified was further confirmed by RT-PCR and Western blot analysis of cultured cells and by immunohistochemistry on primary pterygium and normal conjunctival tissue sections. A dramatically increased expression of IGFBP-2 mRNA was demonstrated in cDNA microarray membranes from two different pterygium fibroblasts. This finding was confirmed by RT-PCR in four additional different pterygium fibroblasts and by Western blot analysis of their culture supernatants. Immunohistochemistry of frozen sections from primary pterygium demonstrated increased staining in extracellular matrix of the stroma, compared with that of the normal conjunctiva. IGFBP-2 was also found in goblet cells of both normal conjunctival and pterygium epithelia. The increased expression of IGFBP-2 mRNA and protein in pterygium fibroblasts is further strong evidence to support the transformed phenotype of these cells and helps explain why there is increased growth of fibrovascular tissue. This phenotype may be used as a marker to assess the malignant nature of pterygium growth and recurrence.

Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts.

To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-gamma elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-alpha increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-gamma and TNF-alpha.

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.

Gene transfer of soluble TGF-beta type II receptor inhibits experimental proliferative vitreoretinopathy.

This study was conducted to investigate a method of gene therapy for proliferative vitreoretinopathy (PVR) by inhibiting type beta transforming growth factor (TGF-beta). PVR was induced in pigmented rabbits by intravitreal injection of 50 000 rabbit conjunctival fibroblasts after vitrectomy. Subsequently, the eyes received an intravitreal application of adenovirus vector encoding a soluble type II TGF-beta receptor (AdTbeta-ExR, n = 10) or adenoviral vector expressing beta-galactosidase (AdLacZ) (n = 10) or balanced salt solution (BSS) (n = 6). The eyes were examined ophthalmoscopically for 28 days after surgery, and the clinical stage of PVR was evaluated on a scale of zero to five. Histological examinations were performed on the treated eyes on day 28. All control eyes injected with AdLacZ or BSS developed PVR, characterized by retinal detachment and the formation of intravitreal membranes within 7 days. The eyes injected with AdTbeta-ExR also developed features of PVR, but the average severity from day 5 to day 28 was significant lower than in the control eyes (P < 0.05). TGF-beta plays an important role in PVR progression in a PVR model, and prevention of TGF-beta signaling could be therapeutically useful.

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.

Histamine-induced cytokine production and ICAM-1 expression in human conjunctival fibroblasts

Purpose. Conjunctival fibroblasts stimulated with histamine (H) may be directly involved in the inflammatory and remodeling processes of chronic allergic conjunctival diseases. Methods. Proinflammatory cytokine and growth factor production, and the expression of intercellular adhesion molecule-1 (ICAM-1) were studied in conjunctival fibroblast cultures challenged with different concentrations of H (from 10 -9 M to 10 -4 M). Interleukin (IL)-1, IL-4, IL-6, IL-8, tumor necrosis factor-alfa (TNF-a), fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor-beta (TGFß-1) were measured in supernatants. ICAM-1 expression was evaluated by a fluorescence activated cell sorter (FACS). Inhibitory effects of the H-1 antagonists (antiH): emedastine, levocabastine, and azelastine, and of the antiH-2, cimetidine, on H-stimulated fibroblasts were evaluated by measuring both cytokines in supernatants and the cellular expression of ICAM-1. Results. Histamine increased the production of IL-1, IL-6 and IL-8, and ICAM-1 expression. TNF-a, IL-4 and growth factor production were not modified by histamine. The antiH-1, emedastine, significantly reduced H-induced production of IL-1, IL-6 and IL-8, while azelastine reduced only IL-1. Levocabastine and cimetidine were less effective. The histamine-induced increase in ICAM-1 expression was inhibited by emedastine but not by azelastine and levocabastine. Conclusions. Histamine has pro-inflammatory effects on conjunctival fibroblasts, inducing the production of cytokines and the expression of ICAM-1. Emedastine significantly reduced cytokine and ICAM-1 expression from H-stimulated fibroblasts. Conjunctival fibroblasts may contribute to the maintenance of inflammation in chronic allergic diseases.

Apoptosis in the cornea in response to epithelial injury: significance to wound healing and dry eye.

Chronic ocular surface injury is a hallmark of dry eye disease. The extent of objective surface abnormality is highly variable between patients, and roughly correlates with the severity of the disease. These surface changes extend from mild conjunctival rose bengal staining to diffuse conjunctival and corneal rose bengal and fluorescein staining. Previous studies have demonstrated that corneal surface injury triggers keratocyte apoptosis and a subsequent wound healing response in the stroma. However, the majority of patients with mild to moderate surface abnormalities from dry eye have no evidence of stromal haze, unstable refractive error, or other changes that would be expected if significant ongoing stromal wound healing were triggered by the surface changes. Superficial injury to the corneal or conjunctival epithelium probably does not induce stromal wound healing because of the intact barrier function of the epithelium and lack of penetration of proapoptotic cytokines into the corneal stroma and subconjunctival tissues. New data suggest in response to IL-1 or tumor necrosis factor (TNF) alpha is monocyte chemotactic and activating factor (MCAF)18 (also Hong, Liu, and Wilson, unpublished data, 2000). This upregulation of MCAF in response to IL-1 or TNF alpha stimulation was confirmed by RNAse protection assay (mRNA) and western blotting (protein). MCAF has chemotactic effects on monocyte and macrophage cells. This observation has led us to formulate a model to explain factors attracting inflammatory cells into corneal stroma or subconjunctival connective tissue in response to epithelial injury. This hypothesis holds that: 1) Significant injury to the epithelium (mechanical or other) results in the release of IL-1 and TNF alpha from the epithelial cells. 2) If the injury to the epithelium is sufficient to break down the epithelial barrier function, then IL-1 and TNF alpha penetrate into the stroma or subconjunctival tissues. Some types of injury might directly trigger release of IL-1 or TNF alpha from the basal epithelial cells. 3) IL-1 or TNF alpha that penetrates into the subepithelial tissues binds to receptors expressed by keratocytes or conjunctival fibroblasts, and triggers MCAF production. 4) MCAF attracts and activates macrophages. Work is in progress to characterize the effects of MCAF. We also hope to identify other cytokines released by keratocytes or conjunctival fibroblasts that attract T cells and polymorphonuclear cells in response to epithelial injury or other pathophysiological triggers.

Effects of cyclosporin A on human conjunctival fibroblasts.

To evaluate the effects of cyclosporin A (CsA) on cytokine and/or collagen production, cell growth, and apoptosis in conjunctival fibroblast cultures. Fibroblast cultures derived from normal subjects and patients with vernal keratoconjunctivitis and pemphigoid were exposed to different concentrations of CsA for either 24 hours or 30 days. The effects were evaluated by the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test to assess cell proliferation, and by the measurement of procollagen I (PIP) and procollagen III (PIIIP) cytokines and total protein in culture medium. CsA-induced apoptosis was assessed by fluorescence-activated cell sorter analysis. After 24 hours of exposure to doses of CsA of more than 10 microg/mL, cell proliferation and migration were significantly reduced. Cyclosporin A reduced PIP and interleukin 1 (IL-1) production in a dose-dependent manner. Interleukin 6 and IL-8 were increased by 10 microg/mL of CsA, whereas transforming growth factor beta, PIIIP, and total protein were unaffected. Cyclosporin A exposure induced apoptosis in a time- and dose-dependent manner. Long-term exposure to CsA reduced IL-6 but did not modify PIIIP production. Exposure to CsA directly modified fibroblast behavior. Cyclosporin A ability to accelerate apoptosis in clinically fibrotic tissues may prove to be therapeutic and useful in hyperproliferative conjunctival disorders.

Accumulation of latent transforming growth factor-beta binding protein-1 and TGF beta 1 in extracellular matrix of filtering bleb and of cultured human subconjunctival fibroblasts.

To examine immunohistochemically whether extracellular matrix (ECM) of the filtering bleb and of cultured human subconjunctival fibroblasts contains latent TGF beta binding protein-1 (LTBP-1) and TGF beta. An enucleated human eye that had undergone trabeculectomy and cultured human subconjunctival fibroblasts were processed for light microscopic immunohistochemistry. Antibodies against LTBP-1, collagen types, fibrillin-1 and TGF beta s were used. TGF beta 1 was located by detecting beta 1-latency associated peptide (LAP). LTBP-1, beta 1-LAP and fibrillin-1 were all located in the subepithelial ECM as well as in the basal epithelial cells of the conjunctiva over the filtering bleb. TGF beta 2 and beta 3 were immunolocated to epithelium and/or fibroblasts/keratocytes. ECM deposited in confluent fibroblast cultures was positive for beta 1-LAP, LTBP-1 and fibrillin-1, whereas sparse cells were negative. LTBP-1, beta 1-LAP and fibrillin-1 are co-localized to the ECM of the filtering bleb and of cultured conjunctival fibroblasts. Both conjunctival epithelium and fibroblasts are considered to be the source of TGF beta in healing bleb. ECM secreted by in vivo and in vitro subconjunctival fibroblasts may works as a scavenger or repository of TGF beta.

APOPTOSIS IN THE CORNEA IN RESPONSE TO EPITHELIAL INJURY: SIGNIFICANCE TO WOUND HEALING AND DRY EYE.

Chronic ocular surface injury is a hallmark of dry eye disease. The extent of objective surface abnormality is highly variable between patients, and roughly correlates with the severity of the disease. These surface changes extend from mild conjunctival rose bengal staining to diffuse conjunctival and corneal rose bengal and fluorescein staining. Previous studies have demonstrated that corneal surface injury triggers keratocyte apoptosis and a subsequent wound healing response in the stroma. However, the majority of patients with mild to moderate surface abnormalities from dry eye have no evidence of stromal haze, unstable refractive error, or other changes that would be expected if significant ongoing stromal wound healing were triggered by the surface changes. Superficial injury to the corneal or conjunctival epithelium probably does not induce stromal wound healing because of the intact barrier function of the epithelium and lack of penetration of proapoptotic cytokines into the corneal stroma and subconjunctival tissues. New data suggest in response to IL-1 or tumor necrosis factor (TNF) alpha is monocyte chemotactic and activating factor (MCAF)18 (also Hong, Liu, and Wilson, unpublished data, 2000). This upregulation of MCAF in response to IL-1 or TNF alpha stimulation was confirmed by RNAse protection assay (mRNA) and western blotting (protein). MCAF has chemotactic effects on monocyte and macrophage cells. This observation has led us to formulate a model to explain factors attracting inflammatory cells into corneal stroma or subconjunctival connective tissue in response to epithelial injury. This hypothesis holds that: 1) Significant injury to the epithelium (mechanical or other) results in the release of IL-1 and TNF alpha from the epithelial cells. 2) If the injury to the epithelium is sufficient to break down the epithelial barrier function, then IL-1 and TNF alpha penetrate into the stroma or subconjunctival tissues. Some types of injury might directly trigger release of IL-1 or TNF alpha from the basal epithelial cells. 3) IL-1 or TNF alpha that penetrates into the subepithelial tissues binds to receptors expressed by keratocytes or conjunctival fibroblasts, and triggers MCAF production. 4) MCAF attracts and activates macrophages. Work is in progress to characterize the effects of MCAF. We also hope to identify other cytokines released by keratocytes or conjunctival fibroblasts that attract T cells and polymorphonuclear cells in response to epithelial injury or other pathophysiological triggers.

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.

Role of histamine in allergic conjunctivitis.

Histamine remains the main mediator released by both specific allergic and non-specific mast cell activation. Histamine is the classic mediator of itching, flare and redness. The effects of histamine in allergic conjunctivitis are mediated by H1-receptor activation on blood vessels and nociceptive nerves. Histamine effects may be prolonged and exaggerated since a defect in the histaminase enzymes has been recently demonstrated in VKC. The effects of histamine on conjunctival tissues may be more complex than those manifested by the simple symptom of itching. In fact, proinflammatory effects of histamine on conjunctival epithelial cells and fibroblasts have been demonstrated. Preliminary results showed that the H1 antagonist, emedastine, reduces significantly cytokine production from histamine-stimulated fibroblasts.

The potential of active metabolites of antihistamines in the management of allergic disease.

Since the discovery of histamine in the ®rst decade of the 20th century, there has been increasing evidence that this biogenic amine, synthesized predominantly in mast cells and basophils, is released in in ammatory processes and plays a key role in the pathogenesis of urticaria and allergic airway diseases, including asthma, rhinitis, and urticaria (1±3). Studies have demonstrated that histamine levels are signi®cantly increased in the bronchoalveolar lavage (BAL) of symptomatic asthmatics, compared with asymptomatic asthmatics (4, 5), and that allergen challenge leads to a marked increase in the levels of histamine in asthmatics (6, 7). Similarly, studies have demonstrated that allergen challenge also leads to signi®cant increases in the levels of histamine in the nasal secretions of patients with allergic rhinitis (8±10). A comparison of patients with chronic urticaria and healthy subjects has also demonstrated that the former release greater amounts of histamine, both spontaneously and after antigen provocation (11±13). In addition, some studies have demonstrated that the responsiveness of the skin to histamine is slightly increased in patients with urticaria (11). Mechanistic studies have demonstrated that the action of histamine arises from its interaction with one of at least three speci®c histamine receptors, H1, H2, and H3, located on various histamine-responsive target tissues. Interaction of histamine with the H1 receptor leads primarily to smooth-muscle contraction, vasodilatation and hypotension, increased vascular permeability with formation of tissue oedema, increased respiratory mucus secretion, and parasympathetic nerve stimulation (14, 15). These effects can subsequently lead to bronchial obstruction in asthma; nasal blockage, sneezing, itching, and discharge in rhinitis; and itchy skin wheals/ ares in urticaria. At the cellular level, histamine may also act on in ammatory cells and lead to release of mediators, such as leukotrienes and cytokines, and increased expression of class II antigens (HLA-DR) and ICAM-1 on epithelial cells (16, 17), effects which could, in turn, exacerbate the pathology and symptoms of allergic disease. In contrast, effects attributable to H2 receptors include augmentation of gastric acid secretion and increases in gastric and respiratory mucus secretion (14). H3 receptors have been implicated in autocrine regulation of histamine synthesis and release from nerve tissue (14).

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.

Histamine Effects on Conjunctival Fibroblasts from Patients with Vernal Conjunctivitis

Histamine, an important mast cell mediator in allergic disorders, may affect extracellular matrix production and cell growth in vernal keratoconjunctivitis (VKC). In the present study, the histamine reactivity of conjunctival fibroblasts derived from VKC patients was investigated in vitro. Conjunctival fibroblast cultures were derived from biopses of 8 tarsal VKC patients and 5 normal subjects. These cells were maintained in vitro and stimulated with different concentrations of histamine with and without H1 (clorpheniramine) and H2 (cimetidine) receptor antagonists. Comparisons were made to fibroblasts grown in the same media without histamine and to fibroblasts stimulated with just antihistamine. The effects of histamine were evaluated by: (1) the MTT test to assess cell proliferation; (2) an in vitro wound model for cell migration and (3) the measurement of procollagen I (PIP) and procollagen III (PIIIP) in supernatants for collagen production. Results showed: (1) While VKC-derived fibroblasts proliferated at a faster rate than normal cells in unstimulated media, after histamine stimulation, VKC and normal cells grew at a similar rate. Both H1 and H2 antagonists significantly inhibited (P<0.05) histamine-induced cell proliferation. (2) Histamine enhanced cell migration after wounding; this effect was inhibited only by H2 antagonism. (3) When stimulated with histamine, VKC fibroblasts produced significantly more PIP than those in control media. Furthermore, VKC-derived fibroblasts were more sensitive to histamine challenge, producing significantly more PIP than normal fibroblasts. H1 and H2 antagonists did not modify histamine-stimulated PIP production. The enhanced proliferative and productive capacity of VKC fibroblasts may be the result of a selective overgrowth of one or more fibroblast subpopulations in a chronically inflamed tissue. Histamine increased proliferation, migration and collagen production in both normal and VKC fibroblasts. Since H2 antagonism modulated both cell growth and migration, but not histamine-induced collagen production, the latter may be mediated by a different receptor. These results showed that histamine is at least partially responsible for fibroblast stimulation.

Functionally Adapted Surfaces on a Silicone Keratoprosthesis

Silicone intraocular lenses as well as silicone sponges and encircling bands on the bulbar surface are widely used and are well tolerated. The aim of this project is a new one-piece silicone keratoprosthesis with enhanced cell adhesion in the haptic region to optimize the keratoprosthesis stability. These investigations show how enhanced profileration of conjunctival fibroblasts and, therefore, improved tissue compatibility can be achieved by hydrophilizing and by protein immobilisation on a hydrophobic silicone surface. This allows a combination of desired chemical and mechanical properties of the silicone bulk material with surfaces of improved tissue compatibility. Silicone foils with surface modifications of different kinds were tested. Experiments were done using cell cultures with murine fibroblasts L-929 and human conjuctival fibroblasts. Cytotoxicity assays were carried out with cells grown on the material in direct contact, as well as in indirect contact, with extracts (EN 30993-5). Viability stains by means of fluoresceindiacetate and ethidiumbromide together with morphology analyses by hemalaun-staining were performed. For the unmodified and modified foils themselves and their extracts any negative influence on cell cultures of murine and human cells could be excluded. There was a gradual improvement of cell morphology, spreading and proliferation dependent on the degree of surface modification. Covalently immobilised fibronectin showed the best results in contrast to adsorptive binding. Silicone surfaces can be modified chemically with bioactive proteins. These modifications are cell compatible and do not result in toxic reactions. The degree and type of silicone hydrophilization results in improved development of cell morphology, spreading and proliferation. Even better results are obtained after covalent binding of bioactive proteins like fibronectin. Improved biocompatibility with enhanced cellular overgrowth has been demonstrated in vitro for the modified silicone of the haptic region. We believe that this type of modification will help in reducing extrusion problems observed with former keratoprostheses.

The lack of extracellular laminin ß2 chain deposition correlates to the loss of conjunctival epithelial keratin K4 localization in culture

Purpose. To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics. Methods. Keratin K4 and laminin ß2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin ß2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts. The synthesis and secretion of laminin ß2 chain by conjunctival fibroblasts in culture was determined by western blot analysis and immunoprecipitation. The ability of conjunctival epithelium to respond to exogenous laminin ß2 chain was assayed by culturing epithelial cells on a laminin matrix isolated from human placenta. Results. In culture, conjunctival fibroblasts synthesize and secrete laminin ß2 chain but do not deposit this chain into an extracellular matrix substrate or basement membrane-like structure. The lack of extracellular deposition of this chain correlates to the gradual loss of keratin K4 protein in conjunctival epithelial cell culture. Conjunctival epithelium remains responsive to laminin ß2 chain in vitro because keratin K4 localization can be rescued in these cells by culture on a substrate of exogenous placental laminin. Conclusions. In vitro, alterations in native conjunctival epithelial-mesenchymal interactions results in aberrant basement membrane laminin isoform composition. This, in turn, leads to the loss of adult epithelial cell phenotype characteristics, suggesting that at least some aspects of conjunctival epithelial cell differentiation are regulated by the extracellular matrix.