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ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

ABSTRACT: The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

SUBMITTER: Meshko B

PROVIDER: S-EPMC10341195 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

Meshko Berbang B Volatier Thomas L A TLA Hadrian Karina K Deng Shuya S Hou Yanhong Y Kluth Mark Andreas MA Ganss Christoph C Frank Markus H MH Frank Natasha Y NY Ksander Bruce B Cursiefen Claus C Notara Maria M

Cells 20230627 13

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) ...[more]

PMID: 37443766

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Project description:PurposeTo assess whether topical application of VEGFR1R2 Trap after corneal transplantation can impair corneal (lymph)angiogenesis and promote murine corneal allograft survival in eyes at high risk of rejection.MethodsWe used the murine model of suture-induced neovascularization and subsequent keratoplasty in eyes at high risk of rejection, which is an established model for local drug application. After transplantation, the mice were treated with either VEGFR1R2 Trap (aflibercept) or human IgG Fc as eye drops for 2 weeks (three times/d). Deposition of VEGFR1R2 Trap in corneal tissue was detected by immunohistochemistry. Two and 8 weeks after transplantation, corneal (lymph)angiogenesis was assessed morphometrically. Dendritic cells (DCs) and regulatory T cells (Tregs) in the draining lymph nodes (dLNs) were examined by flow cytometry. Allograft survival was determined by corneal graft opacity scores.ResultsTopically applied VEGFR1R2 Trap penetrated into corneal host and graft stroma after keratoplasty in eyes at high risk of rejection. Additional postsurgical corneal hemangiogenesis (P < 0.0001) and lymphangiogenesis (P < 0.01) as well as infiltrating CD45+ leukocytes (P < 0.001) and macrophages (P < 0.01) were significantly reduced in the VEGFR1R2 Trap group compared to controls. VEGFR1R2 Trap eye drops significantly decreased the frequency of total CD11c+ DCs (P < 0.01), as well as activated CD11c+MHC II+ DCs (P < 0.01) and CD11c+CD40+ DCs (P < 0.05). In contrast, the frequency of CD200R+ regulatory DCs (P < 0.05) and Tregs in dLNs (P < 0.01) was enhanced. Moreover, long-term allograft survival was also improved (P < 0.05).ConclusionsTemporary, topical application of VEGFR1R2 Trap after corneal transplantation can achieve sufficient anti-VEGF activity, inhibit additional (lymph)angiogenesis, and significantly improve corneal allograft survival in eyes at high risk of rejection.Translational relevanceVEGFR1R2 Trap eye drops after transplantation present a new therapeutic option for patients undergoing corneal transplantation and are at high risk of graft rejection.

Project description:BackgroundDifferentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications.MethodsHuman PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10-11 and 24-25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1.ResultsThe day 24-25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10-11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10-11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs.ConclusionsClinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD.

Dataset Information

ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

Publications

ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

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