July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) activate transient receptor potential vanilloid channel 1 (TRPV1) and 4 (TRPV4) in human corneal endothelial cells
Author Affiliations & Notes
  • Nina Ljubojevic
    Klinik für Augenheilkunde, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
  • Margarita Pushina
    Institute of Anatomy, University of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany
  • Jana Loeffler
    Institute of Anatomy, University of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany
  • Christoforos Dimitropoulos
    Klinik für Augenheilkunde, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
  • Christin Kupper
    Klinik für Augenheilkunde, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
  • Peter Sol Reinach
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China
  • Stefan Mergler
    Klinik für Augenheilkunde, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
  • Monika Valtink
    Institute of Anatomy, University of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany
  • Footnotes
    Commercial Relationships   Nina Ljubojevic, None; Margarita Pushina, None; Jana Loeffler, None; Christoforos Dimitropoulos, None; Christin Kupper, None; Peter Reinach, None; Stefan Mergler, None; Monika Valtink, None
  • Footnotes
    Support  ME 1706/18-1 and VA 179/4-1
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 1366. doi:
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      Nina Ljubojevic, Margarita Pushina, Jana Loeffler, Christoforos Dimitropoulos, Christin Kupper, Peter Sol Reinach, Stefan Mergler, Monika Valtink; Fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) activate transient receptor potential vanilloid channel 1 (TRPV1) and 4 (TRPV4) in human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1366.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The human corneal endothelium (HCE) sustains physiology and transparency of the cornea by Ca2+ dependent mechanisms and in response to various ambient stimuli. TRPV1 (heat or capsaicin receptor) and TRPV4 (moderate heat or osmosensor) are non-selective cation channels mainly regulated by Ca2+. They are functionally expressed in HCE cells. We hypothesized that these ion channels might play a role in apoptosis and interact with FGF receptors (FGFR-1 and -2) and EGF receptor (EGFR).

Methods : TRPV1 and TRPV4 were overexpressed in the human corneal endothelial cell line HCEC-12 by lentiviral transduction. TRPV1 and TRPV4 gene and protein expression after transduction were characterized by RT-PCR and Western blotting. Ca2+ imaging and planar patch-clamping in whole-cell configuration were used to functionally characterize these channels in overexpressing cells as well as in untransduced HCEC-12. Flow cytometric detection of sub-G1 DNA was used to quantify the number of apoptotic cells after stimulation with channel modulators.

Results : RT-PCR showed that overexpressing cells have higher levels of TRPV1 and TRPV4 mRNA and protein comparing to untransduced HCEC-12. Capsaicin (20 µmol/l) induced Ca2+ influx in TRPV1 overexpressing cells. This response was suppressed by TRPV1 channel blocker capsazepine (CPZ; 10 µmol/l). The specific TRPV4 agonist GSK1016790A (20 µmol/l) increased intracellular Ca2+ levels in overexpressing cells, which could be blocked by the specific TRPV4 antagonist RN 1734 (20 µmol/l). Whole-cell currents induced by FGF-2 and EGF (both 10 ng/ml) were at significantly higher level in TRPV1 and TRPV4 overexpressing cells compared to non-transduced cells. All whole-cell current increases could be suppressed by the unspecific TRP channel blocker lanthanum(III)-chloride (500 µmol/l). Inhibition of TRPV1 with CPZ (10 µmol/l) or activation of TRPV4 with GSK1016790A (20 µmol/l) increased the number of apoptotic cells more in overexpressing cells than in untransduced HCEC-12, while activating TRPV1 or inhibiting TRPV4 had nearly no effect.

Conclusions : There is evidence for a cross-talk between FGF-2 and EGF and TRPV1 and TRPV4 channels in HCE. These findings may have an impact on culture protocols for HCE cell cultures and donor cornea organ culture with respect to replacing serum with growth factors.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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