Selective Modulation of the Keratoconic Stromal Microenvironment by FSH and LH

The American Journal of Pathology · 7 authors, 4 centres

AI SUMMARY

FIDELITY 100%

POPULATIONPrimary human corneal stroma cells from healthy subjects (HCFs) and keratoconus patients (HKCs)

INTERVENTIONStimulation with FSH (2.5 or 10 mIU/mL) or LH (5 or 35 mIU/mL) in 2D and 3D cell cultures

COMPARISONUnstimulated controls (regular media only) and comparisons between HCFs and HKCs

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TL;DR

This study found that luteinizing hormone (LH) significantly increased proliferation and decreased fibrosis markers in keratoconic corneal cells, while follicle-stimulating hormone (FSH) had minimal effects. These findings suggest that gonadotropins, particularly LH, may play a role in keratoconus pathogenesis and progression. The results highlight the need for further research into hormonal influences on corneal health and potential therapeutic targets.

Full summary

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**Background:** Keratoconus (KC) is a progressive corneal ectatic disease that typically emerges during puberty and progresses into the mid-30s, with unpredictable severity. The etiology is multifactorial, and hormone imbalances have been increasingly recognized as significant modulators. Previous work identified alterations in gonadotropins (FSH and LH) and their receptors in KC. This study aimed to investigate the effects of exogenous FSH and LH on the keratoconic stromal microenvironment using an established three-dimensional in vitro model.

**Methods:** Primary corneal fibroblasts from healthy subjects (HCFs) and KC patients (HKCs) were isolated and cultured. For 2D studies, cells were stimulated with FSH (2.5 or 10 mIU/mL) or LH (5 or 35 mIU/mL) for 48 hours. Cell proliferation was assessed via BrdU assay, and morphology was monitored. For 3D studies, cells were cultured on Transwell plates with stable vitamin C to promote extracellular matrix assembly and stimulated with FSH 10 mIU/mL or LH 5 mIU/mL for 4 weeks. Protein expression was evaluated by Western blot for sex hormone receptors (AR, PR, ERα, ERβ, GnRHR), fibrotic markers (EDA-Fn, α-SMA), proteoglycans (keratocan, lumican), and gonadotropin signaling pathway components (CRH, GPER, cAMP, pan-AKT, PKA, JNK, ERK 1/2, CREB, CYP3A4). Statistical analysis used one-way ANOVA with significance at P < 0.05.

**Key Results:** In 2D cultures, GnRHR expression was significantly higher in HKCs stimulated with FSH 2.5 mIU/mL compared to LH 35 mIU/mL (P = 0.0242). BrdU assay showed significantly greater proliferation in HKCs stimulated with LH 5 mIU/mL compared to HCFs (P = 0.0127), while FSH had no effect. In 3D cultures, LH 5 mIU/mL significantly up-regulated PR (P = 0.0259) and ERβ (P = 0.0356) in HCFs, and ERβ in HKCs (P = 0.0043) compared to controls. AR expression was significantly higher in HKCs versus HCFs with FSH 10 mIU/mL (P = 0.0259), while PR and ERα were significantly lower in HKCs versus HCFs with LH 5 mIU/mL (P = 0.0338 and P = 0.0431, respectively). Fibrosis markers: In HKCs, α-SMA was significantly down-regulated with both FSH 10 mIU/mL (P = 0.0197) and LH 5 mIU/mL (P = 0.0084), and EDA-Fn was down-regulated with LH 5 mIU/mL (P = 0.0150). Both EDA-Fn and α-SMA were significantly higher in LH-stimulated HCFs compared to HKCs (P = 0.042 and P = 0.015, respectively). Proteoglycans: Keratocan was significantly up-regulated in HCFs with LH 5 mIU/mL (P < 0.0001) and was higher in HCFs versus HKCs with both FSH and LH (P = 0.0283 and P < 0.0001). Lumican was significantly down-regulated with both FSH and LH in HCFs and HKCs (all P ≤ 0.0073). Signaling pathways: In HKCs, GPER was significantly up-regulated with LH 5 mIU/mL versus control (P = 0.0030) and versus FSH (P = 0.0026), and was higher than in HCFs with LH (P = 0.0067). PKA was significantly down-regulated in HKCs with LH 5 mIU/mL (P = 0.0350). Pan-AKT was up-regulated in HCFs with FSH 10 mIU/mL versus control (P = 0.0175) and versus LH (P = 0.0169). JNK was significantly lower in HKCs versus HCFs with FSH 10 mIU/mL (P = 0.0129). CREB was significantly higher in HKCs versus HCFs with LH 5 mIU/mL (P = 0.0342).

**Clinical Implications:** This study demonstrates that exogenous gonadotropins, particularly LH, selectively modulate the keratoconic stromal microenvironment by increasing proliferation and reducing fibrosis markers in KC cells, while FSH has minimal effects. The findings suggest that LH may play a protective role in KC, potentially through GPER signaling. These results highlight the importance of hormonal regulation in corneal homeostasis and KC pathogenesis. Further in vivo and clinical studies are warranted to validate these findings and explore therapeutic strategies targeting gonadotropin pathways in KC patients.

PICO

PPOPULATION

Primary human corneal stroma cells from healthy subjects (HCFs) and keratoconus patients (HKCs)

IINTERVENTION

Stimulation with FSH (2.5 or 10 mIU/mL) or LH (5 or 35 mIU/mL) in 2D and 3D cell cultures

OOUTCOME

Cell proliferation (BrdU assay), protein expression of sex hormone receptors, fibrotic markers, proteoglycans, and gonadotropin signaling pathway components (Western blot)

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10726429

Selective Modulation of the Keratoconic Stromal Microenvironment by FSH and LH

The American Journal of Pathology

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ABSTRACT

Keratoconus (KC) affects the corneal structure, with thinning and bulging outward into a conelike shape. Irregular astigmatism and decreased visual acuity appear during puberty and progress into the mid-30s, with unpredictable disease severity. The cause of KC is recognized as multifactorial, but remains poorly understood. Hormone imbalances are a significant modulator of the onset of KC. This study sought to investigate the role of gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in KC, using a three-dimensional, self-assembled matrix in vitro model. Healthy corneal fibroblasts and human KC cells in the corneal stroma were isolated, cultured, and stimulated with stable vitamin C to promote extracellular matrix assembly. Cultures were further stimulated with 2.5 or 10 mIU/mL FSH and 5 or 35 mIU/mL LH. Samples were evaluated for cell proliferation and morphology via BrdU assay and imaging; protein expression was assessed via Western blot analysis. Proliferation was significantly greater in human KC cells compared to healthy corneal fibroblasts with LH stimulation, but no changes were found with FSH stimulation. Additionally, in sex hormone receptors, fibrotic markers, proteoglycans, and members of the gonadotropin signaling pathway were significantly changed, largely driven by exogenous LH. The impact of exogenous FSH/LH in the KC stromal microenvironment was demonstrated. These results highlight the need to further examine the role of FSH/LH in KC and in human corneal homeostasis.

CITATION EXPORT

Paulina Escandon, Sarah E. Nicholas, Brenda Vasini et al.. Selective Modulation of the Keratoconic Stromal Microenvironment by FSH and LH. The American Journal of Pathology. (2023). https://doi.org/10.1016/j.ajpath.2023.02.002

AUTHORS

7 · 4 CENTRES

Paulina Escandon, Sarah E. Nicholas, Brenda Vasini, Rebecca L. Cunningham, David A. Murphy, Kamran M. Riaz, Dimitrios Karamichos

∗North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas
†Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, Texas
‡Department of Ophthalmology, Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
§Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas