Reduced Expression of the Cell Cycle Inhibitors p27 and p57 and Increased Proliferation in Glaucoma Lamina Cribrosa Cells C. O’Brien1, S. McNally1, R. Kirwan2, K. McAllister3, A. Clark4, D. Wallace1,2 1 Dept. Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland. 2 UCD School of Medicine and Medical Science, University College Dublin, Ireland. 3 Histopathology, Mater Misericordiae University Hospital, Dublin, Ireland. 4 Dept. Cell Biology & Anatomy and the North Texas Eye Research Institute, U. North Texas Health Science Center, Ft. Worth, TX, USA. 05/16/13 Background: In glaucoma, fibrosis is seen as a build-up of extracellular matrix (ECM) material in the trabecular meshwork (TM), and in the lamina cribrosa (LC) in the optic nerve head (ONH). Studies have shown increased collagen and elastin in the optic nerve head indicating ECM remodelling. We have also previously shown increased pro-fibrotic genes in glaucomatous LC cells compared to normal LC cells. Within the body there is a normal homeostatic process of wound healing and scarring; however, when this process is allowed to continue unchecked, fibrosis occurs. In the wound healing process, there are increased numbers of myofibroblast cells, but it is unknown if this is due to increased cell proliferation, or decreased cell apoptosis. Recent GWAS studies show that SNPs in the cell cycle gene CDKN2B-AS are associated with an increased vertical cup to disc ratio in glaucoma. Purpose: Here we explore cell cycle gene expression and cell proliferation in glaucoma lamina cribrosa (LC) cells. Methods: We used human donor LC cells (normal and glaucoma) and used Affymetrix Chip microarray technology to study differential gene expression and performed optic nerve head immuno-histochemistry (IHC) with p27(Kip1) and p57(Kip2) antibodies from Leica. Cell proliferation was studied using the MTS assay. Results: The gene chip data showed a significant signal log ratio (SLR) down-regulation in p27(-0.6) and p57(-0.9) in the glaucoma LC cells and the IHC confirmed the reduced p27 and p57 expression in ONH in glaucoma. After 48 hours in routine culture on non-coated tissue culture plastic, glaucoma LC cells show higher proliferation indices than normal control cells. TGFβ treatment of normal LC cells also reduced the SLR in p27(-0.6) and p57(-0.7). Normal Lamina Cribrosa 40X Glaucoma Lamina Cribrosa Figure 1: Wound Healing. Within the body there is a normal homeostatic process of wound healing and scarring; however, when this process is allowed to continue unchecked, fibrosis occurs . In the wound healing process, there are increased numbers of myofibroblast cells, but it is unknown if this is due to increased cell proliferation, or decreased cell apoptosis. 40X NLC GLC Figure 4: Increased Proliferation of Glaucomatous LC Cells. An MTS assay was used to determine cell proliferation of normal and glaucomatous LC cells. We found increased proliferation of glaucomatous LC cells compared to normal. Phase contrast analysis of human lamina cribrosa cell morphology reveals a loss of symmetry in glaucoma cell architecture. Figure 2: Cell Cycle. P27 and P57 both control cell cycle in the G1 phase. P27 prevents activation of the cyclin E-CDK2 or cyclin D-CDK4 complexes. P57 binds to and inhibits cyclin/CDK complexes. This causes cell cycle arrest at the G1 phase. GLAUCOMA COMPARED TO NORMAL LC CELLS Probe Gene SLR 202246_s_at cyclin-dependent kinase 4 -0.5 205529_s_at core-binding factor, runt domain, alpha subunit 2; translocated to, 1; cyclin D-related 208711_s_at cyclin D1 (PRAD1: parathyroid adenomatosis 1) 209112_at cyclin-dependent kinase inhibitor 1B (p27, Kip1) -0.6 209215_at tetracycline transporter-like protein 0.5 211892_s_at prostaglandin I2 (prostacyclin) synthase -0.7 213348_at cyclin-dependent kinase inhibitor 1C (p57, Kip2) -0.9 TGFβ-TREATED COMPARED TO CONTROL LC CELLS Probe Gene SLR 202246_s_at cyclin-dependent kinase 4 -0.5 205529_s_at core-binding factor, runt domain, alpha subunit 2; translocated to, 1; cyclin D-related -0.9 207530_s_at cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) 1.3 208131_s_at prostaglandin I2 (prostacyclin) synthase -0.6 209112_at cyclin-dependent kinase inhibitor 1B (p27, Kip1) 209644_x_at cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 0.6 213348_at cyclin-dependent kinase inhibitor 1C (p57, Kip2) -0.7 Figure 3: Decreased P27 and P57 in Glaucomatous and TGFβ-treated LC Cells Compared to Normal LC Cells. mRNA from lamina cribrosa cells was used in an Affymatrix Chip microarray to determine gene expression of cyclins. Genes showing a signal log ratio (SLR) of > +/-0.5 are presented in the above tables. P27 and P57 are decreased in glaucomatous LC cells, and in TGFβ-treated LC Cells, compared to NLC cells. Green shading indicates a decrease in gene expression, while blue shading indicates an increase in gene expression. P27 and P57 are highlighted in red text. Figure 5: Decreased Levels of P27 and P57 in Glaucomatous ONH. Immunohistochemistry was conducted on optic nerve head sections from normal and glaucomatous donors for P27 and P57. We found decreased P27 and P57 in the ONH sections from glaucomatous donors when compared to the normal donors. Conclusions: This study demonstrates increased proliferation in glaucoma LC cells associated with reduced expression of the cell cycle inhibitors p27/p57, which may help to explain the activated myofibroblast nature of LC cells in glaucoma.