Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

Abstract

Purpose: Corneal endothelial dystrophies are characterized by endothelial cell loss and dysfunction. Recent evidence suggests that corneal endothelial cells (CECs) can regenerate although they do not do so under normal conditions. This work sought to test whether CECs can be stimulated to proliferate in organ culture by wounding and/or by treatment with the engineered human fibroblast growth factor 1 (FGF1) derivative TTHX1114. Methods: Human donor corneas obtained from eye banks were maintained in organ culture in the presence or absence of TTHX1114. Wounds in the corneas were created by quartering the corneas. The CEC monolayer was identified as a regular layer by Hoechst staining of the nuclear DNA with cell outlines delineated by immunohistochemical identification of ZO-1. Nuclei and nuclei incorporating 5-ethynyl-2¢-deoxyuridine (EdU) were counted using ImageJ. Results: CECs in normal corneas in undisturbed monolayers had low, but measurable, rates of proliferation. CECs at the edge of a wound had higher rates of proliferation, probably due to the release of contact inhibition. TTHX1114 increased proliferation at wound edges. After 7 days of culture, proliferating CECs formed contiguous groups of labeled cells that did not migrate away from one another. TTHX1114-treated cells, including the EdU labeled proliferating cells, retained normal morphology, including cell/cell junction ZO-1 staining. Conclusions: Proliferation of CECs in organ-cultured corneas is low, but can be stimulated by wounding or by the administration of TTHX1114 with the effects of each being additive. The CEC monolayer appears to have a population of progenitor cells that are susceptible to stimulation.

Healing of Ocular Herpetic Disease Following Treatment With an Engineered FGF-1 Is Associated With Increased Corneal Anti-Inflammatory M2 Macrophages

“…Fibroblast growth factor-1 (FGF-1), a naturally occurring protein, promotes tissue repair and regenerates corneal tissue (19). The FGF family consists of a group of homologous growthpromoting polypeptides that increase proliferation, angiogenesis, and wound healing (24)(25)(26).…”

“…TTHX1114 (N-Met C16S/A66C/C117V FGF1) was prepared as described ( 19 ). During primary herpes infection, HSV-1-infected mice received topical eye treatment with 400 ng/ml TTHX1114 (4ul/eye i.e., 1.6 ng/eye) or equivalent amount of vehicle (PBS) (mock-treatment) from day1 to da 14 days PI (two times/day).…”

New developments in the biology of fibroblast growth factors

“…Corneal endothelial dystrophies, such as Fuchs endothelial corneal dystrophy, have a progressive loss of corneal ECs leading to edema, corneal opacity, and loss of vision (Vedana et al, 2016). A stabilized derivative of FGF1 (TTHX1114) was found to be protective in a chemical injury corneal epithelial explant assay and to stimulate proliferation in cultured human corneal ECs and is currently in clinical trials (Eveleth et al, 2018(Eveleth et al, , 2020. In vivo, in cryo-lesioned cornea, TTHX1001 (a similar mutant FGF1), enhanced corneal EC regeneration and showed a significant clearing of opacity relative to untreated controls (Weant et al, 2021).…”

Healing of Primary and Recurrent Mouse Ocular Herpetic Disease Following Topical Ocular Treatment with an Engineered Fibroblast Growth Factor-1 (FGF-1) is Associated with Increased Frequency and Function of Corneal Anti-Inflammatory M2 Macrophages

“…Fibroblast growth factor-1 (FGF-1), a naturally occurring protein, promotes tissue repair and regenerates corneal tissue (23). The FGF family consists of a group of homologous growth-promoting polypeptides that increase proliferation, angiogenesis, and wound healing (24–26).…”

“…To our knowledge, this is the first study to demonstrate that topical cornea treatment with FGF-1 has an anti-inflammatory role that reduces corneal keratopathy in a mouse model of primary and recurrent ocular herpes. The decreased frequency and function of pro-inflammatory macrophages M1 infiltrating the cornea was associated with reduced HSV-1-induced corneal immunopathology observed in both primary and recurrent ocular herpes.Fibroblast growth factor-1 (FGF-1), a naturally occurring protein, promotes tissue repair and regenerates corneal tissue(23). The FGF family consists of a group of homologous growth-promoting polypeptides that increase proliferation, angiogenesis, and wound healing(24)(25)(26).…”