A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration-Associated
Markus A Loeven1, Jasper J van Gemst1, Carolien M S Schophuizen2
1Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Insights
A new conditionally immortalized choroidal endothelial cell (ciChEnC) line was developed for studying age-related macular degeneration (AMD). This cell line mimics choriocapillary cells and shows altered complement factor H binding, aiding AMD research.
Area of Science:
- Ophthalmology
- Cell Biology
- Immunology
Background:
- Choroidal endothelial cells are crucial in age-related macular degeneration (AMD) pathogenesis.
- Existing protocols for primary choroidal endothelial cell isolation rely on human donor eyes, posing a significant limitation.
- A novel cell line is needed to overcome these limitations and facilitate AMD research.
Purpose of the Study:
- To establish and characterize a conditionally immortalized choroidal endothelial cell (ciChEnC) line.
- To enable in vitro studies of AMD pathogenesis within the choriocapillary microenvironment.
- To investigate the binding characteristics of complement factor H variants to these cells.
Main Methods:
- Choroidal endothelial cells were isolated using magnetic-activated cell sorting.
- Cells were conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase.
- Characterization involved assessing endothelial marker expression, functional responses, barrier function, and complement factor H binding via ELISA.
Main Results:
- The ciChEnC line maintained endothelial morphology and key protein expression (von Willebrand factor, vascular endothelial cadherin) for over 27 passages.
- Cells exhibited functional characteristics including LDL uptake, tube formation on Matrigel, and increased ICAM-1 expression upon TNF-α stimulation.
- The ciChEnC line displayed choriocapillary cell markers (e.g., PVAS, HLA ABC, CA IV) and synthesized chondroitin sulfate and heparan sulfate, with decreased binding of the AMD-associated Factor H 402H variant.
Conclusions:
- A novel ciChEnC cell line with choriocapillary characteristics has been successfully established.
- This cell line provides a valuable tool for investigating AMD pathogenesis in the choriocapillary microenvironment.
- The observed differences in Factor H binding highlight potential mechanisms in AMD development.
Purpose:
Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established.
Methods:
Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA.
Results:
ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant.
Conclusions:
A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.
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