Purchase this article with an account.
Parviz Mammadzada, Johann Gudmundsson, Anders P Kvanta, Helder Andre; Comparative analysis of ocular endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3629.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal and choroidal endothelial cells (REC and CEC) are the main cellular components of angiogenesis in the eye. Considering the possible heterogeneity of EC from different vascular beds, comparative study of REC and CEC can lead to deeper understanding of the various mechanisms through which EC become activated in ocular neovascular conditions.
The posterior segment of human consented-donor eyes (24 years old) was microdissected into separate tissues (retina and choroid). Isolation of REC and CEC from these tissues was achieved by CD31-positive selection. Characterization was performed by immunostaining for EC specific markers (CD31, isolectin, and acetylated-LDL uptake).<br /> Total RNA was extracted from normoxic (21% O2) or hypoxic (1% O2, 6 and 12 hours) cells. qPCR arrays were used to investigate a multitude of genes relevant for EC biology and angiogenesis. Gene expression differences were calculated based on ΔΔCt method from triplicates (fold changes > ±2 with p < 0.05 were considered).
Positive staining was confirmed to 100% of the cultured REC and CEC. No phenotypical differences were mapped between REC and CEC. qPCR analysis displayed that, in normoxia, REC showed different basal level of gene expression for angiopoietin-like 4 (10 fold up), monocyte chemotactic protein 1(also known as CCL2) (3 fold down), endothelin 1 (12 fold up), factor 3 (6 fold up) and CD31 (4 fold up), when compared to CEC. At hypoxia these genes were upregulated in CEC, with the exception of CCL2 which was downregulated. In hypoxic REC only CCL2 gene presented a similar expression change, while all other referred genes did not show observable changes. In addition, insulin-like growth factor 1 (IGF1) and placental growth factor (PGF) were upregulated in REC under hypoxia, while no similar changes were observed in CEC.
Despite REC and CEC exhibiting similar morphologies, differences in gene expression under normoxia and hypoxia clearly confirmed heterogeneity of these two EC lines. Hypoxia activated a series of angiogenesis-related genes in CEC, but not in REC. Interestingly, the genes upregulated in REC in response to hypoxia (IGF and PGF) have been shown to be involved in promoting angiogenesis by synergism with VEGF secreted from ischemic retinal tissue. This data indicates that growth factors involved in ocular neovascular conditions, among others in nAMD and PDR, can be different.
This PDF is available to Subscribers Only