Abstract
Introduction: :
Choroidal neovascularization (CNV) is the most devastating complication of age–related macular degeneration. Circulating endothelial progenitor cells could be involved in formation of newly formed capillaries and promote neovascularization during this process.
Purpose: :
To evaluate functional and gene expression differences between OEC from aged donors and three endothelial cell lines: human umbilical vein endothelial cells (HUVEC), human cardiac microvascular endothelial cells (HMVEC–C) and human dermal microvascular endothelial cells (HMVEC–D).
Methods: :
Peripheral blood mononuclear cells (PBMNC) were collected from six aged patients and isolated cells were grown in EGM–2 medium on fibronectin–coated plates. OEC were characterized by FACS and immunocytochemistry. Differences in gene expression patterns between OEC, mature endothelial and retinal pigment epithelial (RPE) cell lines were quantitatively and qualitatively examined using Human Genome 133 plus 2 GeneChips (Affymetrix) using BRB tool package and verified by FACS, Western blot and ELISA. Differences in representation of functional gene groups between cell lines were analyzed by GoMiner tool (NCI). Functional differences between cell lines were confirmed by FACS, immunocytochemistry, cell–to–cell adhesion assays, and protein quantification of secreted proteins.
Results: :
Microarray analysis demonstrated that large group of genes were differentially expressed (1896 genes, p value 0.001) between the cell lines. Hierarchical clustering and GoMiner analysis revealed that significant upregulation of cell adhesion, cell migration, blood vessel formation and pro–angiogenic transcripts were overrepresented in OEC compared to the three standard endothelial cell lines and RPE cells. High level of protein expression and/or secretion of VE–Cadherin, ICAM1, CXCR4, E–Selectin, PDGF, IL–8 by OEC was confirmed by FACS, ELISA and WB. Cell to cell adhesion of OEC to the HUVEC’s demonstrated significant differences in adhesion properties of OEC compared to the three other standard endothelial cell lines.
Conclusions: :
OEC derived from aged patients compared to other standard endothelial cell lines demonstrated significant differences in gene expression, protein expression and/or secretion of gene products involved in cell adhesion, cell migration, blood vessel formation and angiogenesis affecting their functional properties and therefore substantiating their pathologic function in CNV formation.
Keywords: choroid: neovascularization • gene microarray • cytokines/chemokines