Dataset Information

Molecular, Cellular, and Functional Heterogeneity of Retinal and Choroidal Endothelial Cells.

ABSTRACT:

Purpose

To investigate the endothelial heterogeneity across distinct vascular beds in the inner and outer blood-retinal barriers.

Methods

We evaluated the molecular, cellular, and functional differences between primary human retinal endothelial cells (HRECs) and human choroidal endothelial cells (HCECs) in terms of angiogenic and vasculogenic properties, permeability, and transcytosis. Tube formation assay, cell migration assay, in vitro permeability assay, microfluidic sprouting assay, and transcriptome analysis were performed.

Results

HRECs showed higher proliferation and migration activity than did HCECs, whereas the tube formation ability was similar between HRECs and HCECs. Under angiogenic stimuli, HCECs displayed earlier sprouting angiogenesis, but the overall speed was faster and more stable in HRECs. HRECs expressed higher levels of adherens junctional proteins, whereas the tight junctional genes and transcytosis-related genes were more highly expressed in HCECs. Angiopoietin-2 was predominantly expressed in HRECs, but vascular endothelial growth factor (VEGF) receptors were more strongly expressed in HCECs. Platelet-derived growth factor subunit B (PDGFB) was more highly expressed in HRECs, which correlates to the lower degree of pericyte coverage in choroidal blood vessels.

Conclusions

Retinal and choroidal ECs showed significant cellular and molecular heterogeneities that correlated with their functional characteristics. Retinal ECs are vasculogenic with high migratory characteristics and faster angiogenic sprouting, and they are more responsive to VEGF-induced permeability. In contrast, choroidal ECs express high levels of transcytosis genes, and they are vasculogenic, rather proliferative, adept in generating tip cells, and less responsive to VEGF-induced permeability.

SUBMITTER: Kim SJ

PROVIDER: S-EPMC10382998 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Publications

Molecular, Cellular, and Functional Heterogeneity of Retinal and Choroidal Endothelial Cells.

Kim Soo Jin SJ Lim Joon Seo JS Park Jun Hyeong JH Lee Junyeop J

Investigative ophthalmology & visual science 20230701 10

<h4>Purpose</h4>To investigate the endothelial heterogeneity across distinct vascular beds in the inner and outer blood-retinal barriers.<h4>Methods</h4>We evaluated the molecular, cellular, and functional differences between primary human retinal endothelial cells (HRECs) and human choroidal endothelial cells (HCECs) in terms of angiogenic and vasculogenic properties, permeability, and transcytosis. Tube formation assay, cell migration assay, in vitro permeability assay, microfluidic sprouting ...[more]

PMID: 37504962

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Project description:Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, we hypothesized that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from 6 human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasite:cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8747 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false discovery rate set at 5 %. For selected genes, differences in level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphical descriptive analysis demonstrated strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 of 8,747 transcripts (8.9 %) were differentially represented. Notably, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. Our findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte. Experiment Overall Design: Retinal and choroidal vascular endothelial cells were isolated from both eyes of 6 human cadaver donors. Endothelial cells of each subtype from each donor were separately pooled and cultured. In a first series of experiments (Experiment 1), donor-matched cultures of retinal and choroidal endothelial cells derived from 3 donors were incubated with either T. gondii tachyzoites or medium alone. In a second set of experiments (Experiment 2), cultured retinal and choroidal endothelial cells derived from the other 3 donors were stimulated with LPS or medium alone. Whenever possible, 2 replicate dishes of endothelial cells were subjected to each set of conditions. Subsequent to incubations, mRNA was extracted from a cell lysate prepared from each dish of cells, and cRNA derived from each mRNA preparation was separately hybridized to one oligonucleotide expression array.

Project description:BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Dataset Information

Molecular, Cellular, and Functional Heterogeneity of Retinal and Choroidal Endothelial Cells.

Purpose

Methods

Results

Conclusions

Publications

Molecular, Cellular, and Functional Heterogeneity of Retinal and Choroidal Endothelial Cells.

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