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.; David, Larry L.; Appukuttan, Binoy; Wilmarth, Phillip A.

doi:10.1016/j.ajo.2018.03.020

Cited by 23 publications

(25 citation statements)

References 137 publications

(146 reference statements)

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“…As an important part of the blood–tissue barrier, vascular endothelial cells play a critical role in posterior-segment eye diseases. Hyperglycemia, oxidative stress, inflammation, and hypoxia can injure vascular endothelial cells through a variety of complex signaling pathways [ 2 ]. Despite some breakthroughs in understanding these diseases, the common mechanism is unclear.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial DNA drives noncanonical inflammation activation via cGAS–STING signaling pathway in retinal microvascular endothelial cells

Guo

Gan

et al. 2020

Cell Commun Signal

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Background Pathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood–retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli. Methods RMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H2O2), or 25 mM d-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA. Results Pathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation. Conclusions Pathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING–TBK1 signaling pathway.

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Section: Introductionmentioning

confidence: 99%

Mitochondrial DNA drives noncanonical inflammation activation via cGAS–STING signaling pathway in retinal microvascular endothelial cells

Guo

Gan

et al. 2020

Cell Commun Signal

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“…Thus, regulation of βcatenin accumulation and localization to the E-Cadherin/β-catenin complex might be a mechanism whereby Anillin controls not only cell adhesion dynamics, but also Wnt signalling pathway activity 157 . It is also intriguing to note that high levels of anillin expression where found in human choroidal endothelial cells, leading to the obvious hypothesis that Anillin is a potential regulator of choroidal angiogenesis 162,163 . Finally, we have applied weighted gene co-expression network analysis to explore gene coexpression relationships and identify co-expression modules potentially involved in atoh7 function.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome analysis of the zebrafishatoh7−/−mutant,lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases

Covello

Rossello

Filosi

et al. 2020

Preprint

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Short Running Title: Atoh7 genetic networks and human eye diseases. Nonstandard Abbreviations: RPCs: retinal progenitor cells NCRNA: retinal non-attachment ONH: optic nerve hypoplasia ONA: optic nerve aplasia PHPV: persistent hyperplastic primary vitreous RGCs: retinal ganglion cells EFTFs: eye field transcription factors ABSTRACT: Expression of the bHLH transcription protein Atoh7 is a crucial factor conferring competence to retinal progenitor cells for the development of retinal ganglion cells. A number of studies have emerged establishing ATOH7 as a retinal disease gene. Remarkably, such studies uncovered ATOH7 variants associated with global eye defects including optic nerve hypoplasia, microphthalmia, retinal vascular disorders and glaucoma. The complex genetic networks and cellular decisions arising downstream of atoh7 expression, and how their dysregulation cause development of such disease traits remains unknown. To begin to understand such Atoh7-dependent events in vivo we performed transcriptome analysis of wild type and atoh7 mutant (lakritz) zebrafish embryos at the onset of retinal ganglion cell differentiation. We investigated in silico interplays of atoh7 and other disease-related genes and pathways. By network reconstruction analysis of differentially expressed genes we identified gene clusters enriched in retinal development, cell cycle, chromatin remodelling, stress response and Wnt pathways. By weighted gene coexpression network we identified coexpression modules affected by the mutation and enriched in retina development genes tightly connected to atoh7. We established the groundwork whereby Atoh7-linked cellular and molecular processes can be investigated in the dynamic multitissue environment of the developing normal and diseased vertebrate eye. 3 KEY WORDS: Atoh7, Ath5, retinal ganglion cells, transcriptome analysis; inherited eye diseases; human retina; zebrafish 6 anesthetized for 5-10 min in Ethyl 3-aminobenzoate methanesulfonate (MS-222) (Sigma-Aldrich, Saint Louis, MO, USA) in E3 medium. The corresponding body of each embryo was collected and used immediately to perform genotyping analysis to identify the corresponding to lakritz and wild type eyes. All embryos were collected from the same batches of fish stock to maintain a uniform genetic background. DNA extraction from zebrafish body biopsies and genotyping Genomic DNA extraction from each single body was performed in 100 μl of lysis buffer containing Proteinase K-20mg/ml (EuroClone S.p.A. Milan, Italy), 2 M Tris-HCl ph 8.0, 0.5 M EDTA ph 8.0 and 5 M NaCl, 20 % SDS in a final volume of 50 μl ultra H20. After 3 hours of incubation at 65°C, the gDNA was purified with an Ethanol precipitation step and resuspended in 50 μl of Dnase/Rnase H2O. The genotyping was performed by Restriction Fragment Length Polymorphism (RFLP) assay as previously described 6 . An ∼ 300 bp fragment of atoh7 was PCR amplified with 1 U Taq DNA polymerase (Applied Biosystems by Life Technologies, Carlsbad, CA, USA) according to manufacturer protocols in a 30μl PC...

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“…Interestingly, the differences between the two cell-types were reported to be more considerable than inter-donor variations, which denote the heterogeneity of the two vascular beds. Besides, a gene ontology study showed that approximately 9% of differentially expressed genes in REC and CEC belong to genes involved in cell proliferation, implying angiogenesis in the particular case of ECs [18]. Moreover, REC exhibited higher levels of immunologic response or inflammation related genes [107].…”

Section: Gene and Protein Expression In Rec Versus Cecmentioning

confidence: 99%

“…Research focused on comparative gene and protein phenotypes of REC and CEC observed that most proteins were expressed in the same pattern between REC and CEC, in accordance with their similar microvascular phenotype. On the other hand, it has been suggested that angiogenesis inside the retina might be organized by different contrivances to those controlling choroidal neovascularization [17,18]. As a result, choroidal and retinal ECs may have dissimilar expression profiles upon stimulation with oxygen deprivation, thus the understanding of their regulation mechanism renders the development of successful discriminant therapies against PDR and nAMD applicable in the clinic [6].…”

Section: Introductionmentioning

confidence: 99%

The Different Facades of Retinal and Choroidal Endothelial Cells in Response to Hypoxia

Mammadzada

André

2018

IJMS

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Ocular angiogenic diseases, such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, are associated with severe loss of vision. These pathologies originate from different vascular beds, retinal and choroidal microvasculatures, respectively. The activation of endothelial cells (EC) plays pivotal roles in angiogenesis, often triggered by oxygen deficiency. Hypoxia-inducible factors in ECs mediate the transcription of multiple angiogenic genes, including the canonical vascular endothelial growth factors. ECs show notable heterogeneity in function, structure, and disease, therefore the understanding of retinal/choroidal ECs (REC; CEC) biochemical and molecular responses to hypoxia may offer key insights into tissue-specific vascular targeting treatments. The aim of this review is to discuss the differences spanning between REC and CEC, with focus on their response to hypoxia, which could provide innovative and sustainable strategies for site specific targeting of ocular neovascularization.

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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

Cited by 23 publications

References 137 publications

Mitochondrial DNA drives noncanonical inflammation activation via cGAS–STING signaling pathway in retinal microvascular endothelial cells

Mitochondrial DNA drives noncanonical inflammation activation via cGAS–STING signaling pathway in retinal microvascular endothelial cells

Transcriptome analysis of the zebrafishatoh7−/−mutant,lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases

The Different Facades of Retinal and Choroidal Endothelial Cells in Response to Hypoxia

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