Inhibition of human scleral fibroblast cell attachment to collagen type I by TGFBIp.

PURPOSE: Transforming growth factor beta-induced protein (TGFBIp; 68 kDa) is a secreted extracellular matrix (ECM) protein that has been demonstrated to regulate cell attachment in a variety of cell types. The sclera synthesizes and secretes TGFBIp, which may function to facilitate scleral ECM remodeling events associated with myopia development. Here the authors report that human scleral fibroblasts (HSFs) express TGFBI and that its protein product, TGFBIp, mediates an effect on cell attachment. METHODS: TGFBI/TGFBIp expression was evaluated by RT-PCR and immunoblot of HSF lysates and culture supernatants. The effect of rTGFBIp (50 microg/mL) on cell attachment to collagen type I was determined with the use of fluid-phase cell attachment assays in HSFs, human foreskin fibroblasts (HFFs), and human corneal stroma fibroblasts (HCFs). Binding assays using biotinylated rTGFBIp were used to assess TGFBIp binding to the HSF surface. Flow cytometry and immunocytochemistry were used to determine both alphavbeta3 and alphavbeta5 expression and localization to the HSF cell surface. RESULTS: HSFs expressed TGFBI and secreted TGFBIp (approximately 833 ng/h). rTGFBIp significantly decreased (25 microg/mL; P

Microarray analysis of choroid/RPE gene expression in marmoset eyes undergoing changes in ocular growth and refraction.

PURPOSE: Visually guided ocular growth is facilitated by scleral extracellular matrix remodeling at the posterior pole of the eye. Coincident with scleral remodeling, significant changes in choroidal morphology, blood flow, and protein synthesis have been shown to occur in eyes undergoing ocular growth changes. The current study is designed to identify gene expression changes that may occur in the choroid/retinal pigment epithelium (RPE) of marmoset eyes during their compensation for hyperopic defocus as compared to eyes compensating for myopic defocus. METHODS: Total RNA was isolated from choroid/RPE from four common marmosets (Callithrix jacchus) undergoing binocular lens treatment using extended wear soft contact lenses of equal magnitude but opposite sign (+/-5 diopter [D]). After reverse transcription, cDNA was labeled and hybridized to a human oligonucleotide microarray and gene transcript expression profiles were determined. Real-time polymerase chain reaction (PCR) and western blot analysis were used to confirm genes and proteins of interest, respectively. RESULTS: Microarray analyses in choroid/RPE indicated 204 genes were significantly changed in minus lens-treated as compared with plus lens-treated eyes (p<0.05, Student's t-test). Differential choroid/RPE expression of protein tyrosine phosphatase, receptor type, B (PTPRB), transforming growth factor beta-induced (TGFBI), and basic fibroblast growth factor 2 (FGF-2) were confirmed by real-time PCR. TGFBIp was confirmed at the protein level by western blot analysis in marmoset and human cornea, choroid/RPE, and sclera. CONCLUSIONS: The present study demonstrated that significant gene expression changes occur in the marmoset choroid/RPE during visually guided ocular growth. The identification of novel candidate genes in choroid/RPE of marmoset eyes actively accelerating or decelerating their rates of ocular elongation may elucidate the choroidal response during the regulation of postnatal ocular growth and may lead to the identification of choroid/RPE signaling molecules that participate in scleral remodeling.

Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts.

In order to understand the effect of mechanical strain on scleral extracellular matrix remodeling, human scleral fibroblasts were subjected to equibiaxial stretch in vitro and the expression of proteoglycans, metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were evaluated. Isolated human scleral fibroblasts were seeded onto flexible bottom culture plates, and subjected to a cyclic stretch regimen of 15% equibiaxial stretch for 45 s followed by 15s of rest for 6-48 h in the presence of 35SO4. Newly synthesized proteoglycans were measured in the medium by CPC precipitation of radiolabelled glycosaminoglycans. MMP-2 activity and expression levels were measured in the medium by, Western blot, gel zymography and real-time PCR. Steady state levels of TIMP-2 mRNA and membrane-type MMP, MT1-MMP (MMP-14) mRNA were measured in the cell layer using real-time PCR. The predominant gelatinolytic enzyme secreted by scleral fibroblasts was the pro-enzyme form of MMP-2 (ProMMP-2). Mechanical stretch resulted in a significant increase of ProMMP-2 after 12 and 48 h (+76.28%, p<0.05; +19.56%, p<0.01, respectively). Mechanical stretch significantly increased the production of the active form of MMP-2 (ActiveMMP-2) after 48 h (+59.72%, p<0.05) and decreased levels of TIMP-2 mRNA (-22%, p<0.05). The rate of scleral proteoglycan synthesis and the steady state levels of MMP-2 and MMP-14 mRNA were not significantly affected by mechanical stretch. These results suggest that mechanical strain stimulates the activation of MMP-2 by scleral fibroblasts, possibly through increased levels of ProMMP-2 and reduced levels of TIMP-2. Increased levels of ActiveMMP-2 in the sclera would be expected to contribute to scleral extracellular matrix degradation, scleral thinning and possible ocular ectasia.