Insulin like growth factor 1 (IGF 1) is a growth factor involved in control of proliferation of human retinal pigment epithelial (hRPE) cells (Spraul et al. 2000). Abnormal proliferation of hRPE cells has been shown to be present in the development of epiretinal membranes found in proliferative eye disease (Nagineni et al 2005). Lambooij et al. (2003) has shown that in eyes with choroidal neovascularization (CNV), IGF mRNA in the retina has the same distribution pattern as in normal retina. Further, IGF-1 and its receptor were co-localized in the normal human eye and in eyes with neovascular age-related macular degeneration (AMD), a disease that causes blindness. This suggests an autocrine function of IGF-I in the normal human retina as well as a possible role of abnormal regulation in the pathogenesis of neovascular AMD (Slomiany and Rosenzweig 2004). Abnormal IGF 1 synthesis, proliferation of hRPE cells, and hRPE cell migration to extracellular matrix (ECM) has also been implicated in the etiology of membrane formation in age related macular degeneration.

In addition, high concentrations of connective tissue growth factor (CTGF) correlate with increased intraocular fibrosis (Kuiper et al. 2006). CTGF was first identified as a 38-kDa cysteine-rich protein isolated from fibroblast conditioned culture media from human umbilical vein endothelial cells (Bradham et al. 1991). It is secreted by human vascular endothelial cells and is related to the SRC-induced immediate early gene product CEF-10. It was later found to stimulate the synthesis of extra-cellular matrix components and play a role in fibrosis.

Hyperglycemia is an independent risk factor that may contribute to the development of neovascularization via various signaling pathways (Khan and Chakrabarti 2007; Rosenthal et al. 2004). To investigate the role of CTGF in fibrotic peri-retinal membrane formation in patients with AMD-related proliferative vitreoretinopathy, we examined whether CTGF was regulated by high glucose and IGF-1 in in vitro cultured hRPE cells. We also exposed hRPE cells from an AMD patient and nonAMD patients to elevated glucose and IGF-1 in presence and absence of the ERK kinase inhibitor PD98059 (Zelivianski et al. 2003) to determine if the ERK kinase signaling pathway is involved in any changes detected.

48.2 Methods

48.2.1 Chemicals

IGF1 was purchased from Sigma Chemicals, St. Louis, MO. Anti-CTGF was purchased from R & D Systems, Minneapolis, MN. 3H-thymidine and 14C-Methionine were purchased from Amersham Corporation, Arlington Heights, IL. Ham’s F-12 nutrient medium, Dulbecco’s minimum essential media (DMEM), Hank’s balanced salt solution, fetal bovine serum (FBS), penicillin, streptomycin and trypsin were purchased from GIBCO BRL, Gaithersburg, MD. PD98059 was purchased from Cell Signaling Technology, Beverly, MA.

48.2.2 Establishment and Maintenance of hRPE Cell Cultures

Primary cultures of hRPE cells were established from human eyes obtained from a patient with AMD and four without AMD as described previously (Kusaka et al. 1998; Kothary et al. 2001). Briefly, the anterior segment, vitreous and the retina of human eyes were surgically removed. The posterior segments were then washed with balanced salt solution, filled with papain (0.623 mg/ml in cystein/EDTA) and incubated for 1 h at 37◦C. The Papain was aspirated and replaced with Ham’s F-12 nutrient medium containing 15% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.075% (wt/vol) sodium bicarbonate (medium-1). The now loosely adherent hRPE cells were detached by gentle brushing and hydrostatic pressure with a sterile fire polished Pasteur pipet. The cells were plated in 16-mm Primaria plates and incubated at 37◦C in a 95% air/5% CO2 incubator. The medium was changed every 3 days until the cells were confluent. Primary cultures were then washed with Hank’s balanced salt solution and subcultured by trypsinization with 0.5 g/100 ml trypsin and 0.2 g/100 ml EDTA in Hank’s normal salt solution (Sigma T-3924) at 37◦C for 10 min. The cell suspension was centrifuged at 500×g and replated. The morphology of cells was examined daily by phase-contrast microscopy. For maintenance of cell lines, cells were plated in 75-mm flasks at density of 50,000 cells/flask. The medium was changed every three days until the cells were ready for trypsinization. Cells were counted by hemocytometer and viability was assessed by trypan blue exclusion.

48.2.3 Cellular Proliferation

Proliferation of cultured hRPE cells was determined by tritiated thymidine incorporation (3H-thy) and viable cell count by the trypan blue exclusion method as described previously (Kothary and Del Monte 2008). It showed that IGF-1 and fetal bovine serum are mitogenic and stimulate 3H-thy incorporation as well as increase in viable cell number of cultured hRPE cells obtained from the non-AMD control eyes as well as eyes from an AMD patient (Data not shown). This demonstrates that we have cell culture system that responds to biological stimuli and is suitable for intracellular signaling studies.

48.2.4 Immunoprecipitation Assay

To measure intracellular CTGF synthesis, hRPE cells were labeled by 14-C- Methionine and then treated with elevated glucose (hyperglycemia) and IGF-1 in the presence and absence of the ERK kinase inhibitor, PD98059, using the method described previously (Bitar et al. 1996; Kothary et al. 2006). hRPE cells were then lysed with zwittergent 3–12 and precipitated with antibody specific for CTGF.