Complement-mediated release of fibroblast growth factor 2 from human RPE cells

Ping Yang; Samantha E Neal; Kristen L Buehne; Grace M Tewkesbury; Mikael Klingeborn; Yae-Young Yang; Peter Baciu; Glenn J Jaffe

doi:10.1016/j.exer.2021.108471

Complement-mediated release of fibroblast growth factor 2 from human RPE cells

Exp Eye Res. 2021 Mar:204:108471. doi: 10.1016/j.exer.2021.108471. Epub 2021 Jan 28.

Authors

Ping Yang¹, Samantha E Neal¹, Kristen L Buehne¹, Grace M Tewkesbury¹, Mikael Klingeborn¹, Yae-Young Yang², Peter Baciu², Glenn J Jaffe³

Affiliations

¹ Department of Ophthalmology, Duke University Medical Center, Durham, NC, 27710, USA.
² Department of Biology, Allergan, Inc, Irvine, CA, 92612, USA.
³ Department of Ophthalmology, Duke University Medical Center, Durham, NC, 27710, USA. Electronic address: jaffe001@mc.duke.edu.

PMID: 33516764
DOI: 10.1016/j.exer.2021.108471

Abstract

Purpose: Complement activation is associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Fibroblast growth factor 2 (FGF2) and membrane attack complex (MAC) are present in eyes of patients with CNV. Herein, we investigated the effect of complement activation on FGF2 release in human retinal pigment epithelial (RPE) cells.

Methods: Cultured human RPE cells were primed with an anti-RPE antibody and then treated with C1q-depleted human serum in the presence or absence of Tec kinases inhibitor (LFM-A13). 38 cytokines/chemokines levels were measured by Luminex technology. Secretion of FGF2 and interleukin (IL)-6 was assessed by ELISA. Tec protein was measured by Western blot. mRNA expression of FGF2, chemokine (C-X-C motif) ligand 1 (CXCL-1), and family members of Tec kinases was evaluated by qPCR. Cell viability and MAC deposition were determined by WST-1 assay and flow cytometry, respectively.

Results: Complement activation caused increased FGF2 and IL-6 release. FGF2 was released when C6-depleted human serum was reconstituted with C6. Anti-C5 antibody significantly attenuated complement-mediated FGF2 release, but not IL-6. FGF2 mRNA levels were not affected, while CXCL-1 mRNA levels were increased by complement activation. FGF2-containing extracellular vesicles were detected in response to complement challenge. Tec mRNA and protein were expressed in RPE cells. In the presence of LFM-A13, secretion of FGF2, but not IL-6, and MAC deposition were significantly decreased and cell viability was significantly increased in complement-treated cells when compared to controls.

Conclusions: Complement plays an important role to release FGF2 from RPE cells. Tec kinase is involved in MAC formation and complement-mediated FGF2 release. This information suggests a role for complement activation to mediate neovascularization in conditions such as AMD, and may elucidate potential therapeutic targets.

Keywords: Age-related macular degeneration; Complement; Fibroblast growth factor 2; Retinal pigment epithelial.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Blotting, Western
Cells, Cultured
Choroidal Neovascularization / metabolism
Complement Activation / physiology*
Complement Membrane Attack Complex / metabolism
Complement System Proteins / metabolism*
Enzyme-Linked Immunosorbent Assay
Female
Fibroblast Growth Factor 2 / genetics
Fibroblast Growth Factor 2 / metabolism*
Flow Cytometry
Humans
Interleukin-6 / metabolism
Macular Degeneration / metabolism
Male
Middle Aged
RNA, Messenger / genetics
Real-Time Polymerase Chain Reaction
Retinal Pigment Epithelium / metabolism*

Substances

Complement Membrane Attack Complex
Interleukin-6
RNA, Messenger
Fibroblast Growth Factor 2
Complement System Proteins

Grants and funding

P30 EY005722/EY/NEI NIH HHS/United States