Human corneal endothelial cells (HCEC) are inhibited in G1-phase of the cell cycle. This prevents cell division from replacing dead cells and can result in critically low cell density. Our LONG-TERM GOALS are to identify the molecular basis for this inhibition and to develop methods to stimulate proliferation to increase cell density. HCEC at all donor ages possess proliferative capacity; however, there is an intrinsic, age-related decrease in their response to serum, implying increased inhibition with increasing donor age. The GOAL of our studies is to identify the inhibitory mechanisms responsible for this age-related decrease in serum responsiveness. An exciting, novel approach will be used based on studies of replicative senescence. We will test the HYPOTHESIS that the age-related decrease in ability of HCEC to respond to serum is due to increased inhibition by the cyclin-dependent kinase inhibitor, p21CIP1 (p21). Both ex vivo corneas and cultured HCEC from young (<30yo) and older donors (>50yo) will be used in these studies.
In Aim 1, image analysis of Mcm2 expression will permit calculation of the percent of HCEC in each age-group that are competent to replicate in response to serum.
In Aim 2, Western blots, real-time PCR, and mobility shift assays will test if there is an age-related decrease in the activity of key G1-phase regulators that suggests increased inhibition by p21.
Aim 3 will test if p21 siRNA treatment of cultured cells will promote proliferation in HCEC from OLDER donors.
Aim 4 will test if p21 siRNA treatment will promote an increase in endothelial cell density in ex vivo corneas from OLDER donors.
Aims 3 and 4 will use staining for cell cycle markers and direct cell counts to document proliferation.
In Aim 4, image analysis will test if p21 siRNA treatment will promote an increase in cell density in corneas with densities <1500 cells/mm2. If our hypothesis is correct, molecular approaches could tap the residual proliferative capacity of HCEC in older donors by decreasing p21 expression.
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