Enhancement of angiogenic potential of endothelial cells by contact with retinal pigment epithelial cells in a model simulating pathological conditions.
OBJECTIVE
Choroidal neovascularization (CNV) is the leading cause of vision loss in chorioretinal diseases involving contact between retinal pigment epithelial (RPE) and endothelial cells (ECs). The aim of this study was to investigate changes in the angiogenic potential of ECs induced by RPE-EC interaction in two models of RPE-EC coculture.


METHODS
RPE and ECs were grown in contact or noncontact coculture. Selection of ECs was achieved using magnetic beads coated with antibodies specific for EC surface proteins. Angiogenesis was assessed by analyzing the expression of EC genes involved in angiogenesis by RT-PCR. Tube formation on Matrigel was used as a functional angiogenesis assay. Expression and activity of matrix metalloproteases (MMPs) were examined by RT-PCR and zymography, respectively.


RESULTS
Coculture of ECs with RPE in the contact model under normoxic conditions induced markedly upregulated EC mRNA expression of 16 genes involved in positive regulation of angiogenesis. Solo ECs subjected to hypoxia demonstrated upregulated expression of the same 16 genes, including VEGF and HIF1. The EC VEGF level was not affected by coculture with RPE in the noncontact model. ECs demonstrated enhanced tube formation on Matrigel after contact coculture with RPE. EC MMP2 mRNA and activity levels were elevated in contact, but not in noncontact, coculture.


CONCLUSIONS
Coculture of ECs with RPE under conditions enabling direct EC-RPE contact enhances the proangiogenic potential of ECs under normoxia, to an extent similar to that induced by hypoxia, suggesting that ECs in direct contact with RPE cells might be more prone to pathologic angiogenesis involved in CNV formation.
Dardik
Rima
R
Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel.
Livnat
Tami
T
Nisgav
Yael
Y
Weinberger
Dov
D
D002450:Cell Communication; D002460:Cell Line; D018920:Coculture Techniques; D003094:Collagen; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D007797:Laminin; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009414:Nerve Growth Factors / Q000378:metabolism; D011509:Proteoglycans; D012333:RNA, Messenger / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000378:metabolism; D012867:Skin / Q000098:blood supply; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D003094:Collagen; D004338:Drug Combinations; D005136:Eye Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007797:Laminin; D020782:Matrix Metalloproteinases; D009414:Nerve Growth Factors; D011509:Proteoglycans; D012333:RNA, Messenger; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C497442:HIF1A protein, human; C467484:VEGFA protein, human; C059485:matrigel