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Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs.

ABSTRACT: PURPOSE:Diseases that involve retinal or choroidal vascular endothelial cells are leading causes of vision loss: age-related macular degeneration, retinal ischemic vasculopathies, and noninfectious posterior uveitis. Proteins differentially expressed by these endothelial cell populations are potential drug targets. We used deep proteomic profiling to define the molecular phenotype of human retinal and choroidal endothelial cells at the protein level. METHODS:Retinal and choroidal vascular endothelial cells were separately isolated from 5 human eye pairs by selection on CD31. Total protein was extracted and digested, and peptide fractions were analyzed by reverse-phase liquid chromatography tandem mass spectrometry. Peptide sequences were assigned to fragment ion spectra, and proteins were inferred from openly accessible protein databases. Protein abundance was determined by spectral counting. Publicly available software packages were used to identify proteins that were differentially expressed between human retinal and choroidal endothelial cells, and to classify proteins that were highly abundant in each endothelial cell population. RESULTS:Human retinal and/or choroidal vascular endothelial cells expressed 5042 nonredundant proteins. Setting the differential expression false discovery rate at 0.05, 498 proteins of 3454 quantifiable proteins (14.4%) with minimum mean spectral counts of 2.5 were differentially abundant in the 2 cell populations. Retinal and choroidal endothelial cells were enriched in angiogenic proteins, and retinal endothelial cells were also enriched in immunologic proteins. CONCLUSIONS:This work describes the different protein expression profiles of human retinal and choroidal vascular endothelial cells, and provides multiple candidates for further study as novel treatments or drug targets for posterior eye diseases. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

SUBMITTER: Smith JR

PROVIDER: S-EPMC6109601 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs.

Smith Justine R JR David Larry L LL Appukuttan Binoy B Wilmarth Phillip A PA

American journal of ophthalmology 20180317

<h4>Purpose</h4>Diseases that involve retinal or choroidal vascular endothelial cells are leading causes of vision loss: age-related macular degeneration, retinal ischemic vasculopathies, and noninfectious posterior uveitis. Proteins differentially expressed by these endothelial cell populations are potential drug targets. We used deep proteomic profiling to define the molecular phenotype of human retinal and choroidal endothelial cells at the protein level.<h4>Methods</h4>Retinal and choroidal ...[more]

PMID: 29559410

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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.

Dataset Information

Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs.

Publications

Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs.

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