Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas

PURPOSE: To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). METHODS: Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. RESULTS: All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. CONCLUSION: The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.     

A comparison of ocular melanocyte and uveal melanoma cell invasion and the implication of alpha1beta1, alpha4beta1 and alpha6beta1 integrins

BACKGROUND/AIMS: Posterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The beta1 family of integrins, alphavbeta3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement in uveal melanoma we analysed the expression of the beta1 integrins, alphavbeta3, MMP-2, and MMP-9 in 10 primary posterior uveal melanomas, and correlated expression with invasive potential in vitro. Comparable studies were undertaken on cultures of melanocytes. METHODS: Expression of integrins was studied by immunohistochemistry, secretion of MMP-2 and MMP-9 by zymography, and the invasive potential was assessed using a transwell model. RESULTS: MMP-2 was secreted by all uveal melanomas and seven of 10 secreted MMP-9. Among uveal melanoma, invasion levels of 4-25% were observed and the major integrins expressed were alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1, and avbeta3. Melanocytes did not express alpha1beta1, alpha4beta1, and alpha6beta1. CONCLUSION: The laminin binding alpha6beta1 integrin was not expressed by either melanocytes or tumours with spindle morphology, which are considered to have a better prognosis. It is possible that expression of the alpha6beta1 integrin may prove useful as a prognostic indicator.     

Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa

PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR. Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals. RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis. Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels. In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice. MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria. CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP. Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response.     

Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas

PURPOSE: To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS: RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS: More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression. CONCLUSIONS: Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.     

Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: comparison of an immortalized cell line to primary cells, and their response to retinoic acid.

PURPOSE: The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS: Primary cells, maintained in serum free medium, were exposed to 10(-6) M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (alpha(3), alpha(6), alpha(V), beta(1), beta(2), beta(3) and beta(4)), laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS: Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins alpha(6) (CD49f) and alpha(V) (CD51) were expressed by primary and immortalized cells. Expression of integrin alpha(6) was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin alpha( V) expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin alpha(6) expression by primary cells. Both cell types also expressed integrins beta( 1), beta(2) and beta(3), but beta(4) was detected only in immortalized cells. Lacrimal acinar cells do not express integrin alpha(3). CONCLUSIONS: Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin alpha(V), but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.     

Human diabetic corneas preserve wound healing,basement membrane,integrin and MMP-10 differences from normal corneas in organ culture

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

Expression of membrane-type matrix metalloproteinases 4, 5, and 6 in mouse corneas infected with P. aeruginosa.

PURPOSE: To investigate the expression and regulation of membrane-type matrix metalloproteinases (MT-MMPs) 4, 5, and 6 in the mouse corneas infected with Pseudomonas aeruginosa. METHODS: C57BL/6J mice were intracorneally infected with P. aeruginosa. The expression of MT4-, MT5-, and MT6-MMP was detected at both the mRNA and protein levels by RT-PCR and immunoblot analysis. Immunohistochemical staining was performed to localize the expression of MT4- and MT5-MMP in the mouse corneas. RESULTS: Expression of MT4- and MT5-MMP was detected in the normal (uninfected) cornea by RT-PCR and immunoblot analysis. When infected with P. aeruginosa, the corneas showed significant induction of each MT-MMP. Localization of MT4- and MT5-MMP revealed that the expression of MT5-MMP was restricted to the epithelial tissue in the normal cornea, whereas the induced expression of MT4- and MT5-MMP was predominantly in the substantia propria, which contained most of the infiltrating cells. MT6-MMP expression was not detected in the uninfected cornea but was upregulated in the infected corneas. CONCLUSIONS: Expression of MT4-, MT5-, and MT6-MMP was induced in corneas infected with P. aeruginosa. Immunohistochemistry showed predominant immunoreactivity of MT4- and MT5-MMP in the substantia propria. Previous histologic studies have revealed different patterns of inflammatory cell infiltration with an increased number of polymorphonuclear neutrophils (PMNs) during the early stage of inflammation and increased macrophages during the late stage. These results indicate a good correlation between the overexpression of the MT-MMPs in the infected corneas and the inflammatory response-that is, leukocyte infiltration-indicating that inflammatory cells such as macrophages and PMNs may play a role in the upregulation of MT-MMPs during corneal infection, which in turn can cause the destruction of corneal tissue.     

Expression and distribution of MMPs and TIMPs in human uveal melanoma

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).     

Phenotypic investigation of cell junction-related proteins in gelatinous drop-like corneal dystrophy

PURPOSE: To identify the molecules involved in the pathogenesis of gelatinous drop-like corneal dystrophy (GDLD) by using immunohistochemical analysis of the expression of tight junction (TJ)-, desmosome-, and basement membrane (BM)-related proteins in human corneas with GDLD. METHODS: Mutation analysis was performed on samples from three Japanese women with GDLD. Four corneal buttons from these patients were examined histopathologically by Congo red staining and immunohistochemical analysis for the expression of TJ-related proteins (ZO-1, occludin, and claudin-1), desmosome components (desmoplakin), BM-related proteins (integrins alpha6, beta4, alpha3, and beta1; laminin-5; and collagens IV and VII), amyloid P component, and lactoferrin. RESULTS: Mutation analysis revealed that all three women had the Q118X mutation on M1S1. There were accumulations, primarily beneath the epithelium, of Congo-red-positive deposits with birefringence under polarized light. The BM was abnormally thickened and demonstrated a bandlike area. Immunofluorescence analysis revealed that neither ZO-1 nor occludin was expressed in the TJ areas of surface epithelial cells; there was no expression of claudin-1 or desmoplakin in the epithelial surface layer of GDLD corneas. Integrins alpha6, beta4, alpha3, and beta1 was expressed along the serrated surface of the BM, laminin-5 and collagens IV and VII were widely expressed throughout the BM, and lactoferrin was expressed in the amyloid deposits and the thickened BM. CONCLUSIONS: This is the first demonstration of the unique expression patterns of the major cell-junction-related proteins in GDLD corneas. The results show that in corneas with the Q118X mutation, there is a disturbance in cell-to-cell and cell-to-substrate junctions. These findings are an important step toward elucidating the pathogenesis of GDLD.     

Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.     

The effect of selective cyclooxygenase-2 inhibitor on corneal angiogenesis in the rat.

PURPOSE. Eicosanoids that are present in inflamed tissues are thought to play a significant role in angiogenesis. Cyclooxygenase, a key enzyme in eicosanoid synthesis, has recently been shown to exist in two isoforms: the constitutive COX-1 and the inducible COX-2. This study was undertaken to determine the role of COX-2 in the corneal angiogenic response. METHODS. Angiogenesis in the rat cornea was provoked by chemical cautery. Either NS-398, a selective COX-2 inhibitor, or indomethacin, a non-selective COX inhibitor, was applied topically 3 times daily for 4 days. Neovascularization was quantitated by digital image analysis in corneal flat preparations. To test their inhibitory effects on eicosanoid synthesis, normal or cauterized corneas were incubated in the culture medium with the inhibitor. Prostaglandin E2 in the medium was assayed using an enzyme-linked immunosorbent assay. RESULTS. Both NS-398 and indomethacin significantly inhibited corneal neovascularization with the % inhibition of 36.4 +/- 9.6%, and 38.5 +/- 9.0%, respectively, when applied topically at a concentration of 0.1% (p <.001). Neither reduced the angiogenic response at a concentration of 0.01% or below. PGE(2) production in the cauterized cornea was 2.0 times higher than that in the controls. In normal corneas, indomethacin inhibited PGE(2) synthesis by 80%, whereas NS-398 inhibited it by no more than 20%. In contrast, in injured corneas, both indomethacin and NS-398 inhibited PGE(2) synthesis in a similar fashion, with a maximal inhibition rate of 75 to 80%. CONCLUSIONS. Our results suggest that COX-2 induction in cauterized corneas increases the level of eicosanoids, which result in corneal angiogenesis.     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

The role of integrin alpha5beta1 in the regulation of corneal neovascularization

Integrins are transmembrane receptor proteins critical for growth and stabilization of vessels, but the mechanisms by which integrin activities are involved in neoangiogenesis of the eye remain unclear. Specific inhibitors to fibronectin receptor integrin alpha(5)beta(1) impeded pathological neovascularization in vivo. Our objective was to determine whether alpha(5)beta(1) plays a role in ocular angiogenesis, and whether a novel alpha(5)beta(1)-inhibiting small molecule is able to reduce angiogenesis in a model of inflammatory corneal neovascularization. Corneal neovascularization was induced in C57Bl/6 mice by NaOH-application and debridement of the limbal epithelium. Mice were randomized into six groups receiving either no treatment, or intraperitoneal osmotic pumps delivering three different doses of integrin antagonist or control substance on day 10 after scraping. In order to quantify the neovascular response, flatmounts were stained with FITC-CD31. Integrin alpha(5) expression was determined by immunohistochemistry and quantified by semiquantitative western blot analysis. Influence of integrin antagonist treatment on the mRNA expression of VEGF, bFGF and integrin alpha(5) was quantified by real-time RT-PCR. Vascularized corneas demonstrated a strong up-regulation of integrin alpha(5) within affected areas. Animals treated systemically with alpha(5)beta(1)-inhibiting small molecule showed a significant inhibition and regression of corneal neovascularization. PCR analysis evinced a significant up-regulation of VEGF and integrin alpha(5) mRNA levels in injured animals compared to controls, and a significant reduction of integrin alpha(5) mRNA in substance-treated animals compared to control substance, but no significant differences of bFGF levels in all groups. Western blot analysis of integrin alpha(5)beta(1) protein expression showed a trend towards up-regulation in injured animals, both control substance-treated and those treated with the alpha(5)beta(1)-inhibiting small molecule. Systemic delivery of an alpha(5)beta(1)-inhibiting small molecule inhibits and regresses corneal neovascularization induced by mechanical-alkali burn corneal injury. These results suggest an essential role for the integrin alpha(5)beta(1) in pathological neovascular processes of the cornea. Integrin alpha(5)beta(1) inhibitors could become a new approach for treatment of neovascularization in the eye.     

Insulin-like growth factor-1 induces migration and expression of laminin-5 in cultured human corneal epithelial cells

PURPOSE: The effects of insulin-like growth factor (IGF)-1 on laminin (Ln)-5 and the associated integrins during in vitro HCEC migration were examined. Also investigated were the effects of IGF-1 on the migration of human corneal epithelial cells (HCECs). METHODS: HCEC migration was examined by wound-healing and chemoattraction assays. For migration inhibition assays, HCECs were pretreated with inhibitors of the IGF-1 receptor (alphaIR3), the PI3-K/AKT pathway (LY294002), and the MEK-ERK pathway (PD98059). The expression levels of Ln-5 and fibronectin (Fn) were determined by Western blot analysis, and the expression levels of the beta1 and alpha3 integrins were determined by confocal microscopy and Western blot analysis. The migration inhibition with anti-integrin alpha3 and beta1 antibodies was also determined. RESULTS: HCEC migration was significantly increased in the presence of IGF-1 and Ln-5. IGF-1 enhanced the production of Ln-5 in both a dose- and time-dependent manner, and this upregulation was blocked by pretreatment with alphaIR3 or LY294002. IGF-1 treatment upregulated expression of beta1 integrin, but not alpha3 integrin. The HCEC migration facilitated by IGF-1 was inhibited with the anti-integrin antibody for beta1. However, there was no cross-talk between Ln-5 and integrin beta1 production. CONCLUSIONS: The results reveal that IGF-1 induces HCEC migration through the independent production of Ln-5 and beta1 integrin, which are directed at least in part by activation of the PI3-K/AKT pathway, but are not affected by the MEK-ERK pathway.     

Fibronectin fragments promote human retinal endothelial cell adhesion and proliferation and ERK activation through alpha5beta1 integrin and PI 3-kinase

PURPOSE: Extracellular matrix degradation is associated with neovascularization in diabetic retinas. Fibronectin fragments (Fn-fs) are generated during vascular remodeling. The effects of cellular fibronectin (Fn) and selected Fn-fs on adhesion, proliferation, and signal transduction in human retinal endothelial cells (HRECs) were characterized. METHODS: Relative quantitative RT-PCR, flow cytometry, and immunocytochemistry determined integrin expression on HRECs. Adhesion was evaluated by coating plastic with Fn or Fn-fs of 45, 70, 110, or 120 kDa, and MTT conversion was used to measure proliferation and survival. Peptide inhibitors and blocking antibodies determined adhesive sites and integrins used for adhesion. Pharmacologic inhibitors and Western analyses were used to evaluate intracellular signaling. RESULTS: HRECs produced significant levels of alpha(2), alpha(3), alpha(5), alpha(v), beta(1), beta(3), and beta(5) integrin subunit mRNA. Flow cytometry of surface integrin expression revealed high levels of alpha(3), alpha(5), and beta(1) and lower levels of alpha(1), alpha(v), beta(3), and beta(5). These results were confirmed by immunocytochemistry. For adhesion to Fn and Fn-fs. the alpha(5)beta(1) integrin was essential. Pharmacologic inhibitors of PI 3-kinase blocked adhesion to Fn and Fn-fs, whereas the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 blocked phosphorylation. The 110- and 120-kDa Fn-fs showed a concentration-dependent increase in proliferation, whereas 500 ng of the 70 kDa Fn-f-induced proliferation. Addition of III1-C, a matrix assembly domain, increased the proliferative effect of these Fn-fs. CONCLUSIONS: Fn and its Fn-fs modulate HREC adhesion and proliferation through signal-transduction pathways involving coupling of the alpha(5)beta(1) integrin through PI 3-kinase. Mitogenic signals for endothelial cells from degraded extracellular matrix may contribute to the development of diabetic retinopathy.