Abstract
The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.
Highlights
In order to shed light on the role of transient receptor potential vanilloid 4 (TRPV4) expression levels in mediating responses underlying control of human corneal endothelium (HCE) homeostasis, we evaluated the effects of its overexpression on some responses that underlie its normal function in vitro
human corneal endothelial cells (HCEC)-12 and the control cell line HT1080 were transduced with Lentiviral vector particles (LVPs) encoding for human TRPV4 (Figure 1a), resulting in 51% and 60% transduced cells, respectively, as indicated by DsRedEx2 expression at 3 dpi (Figure 1b,c)
Quantitative real-time PCR analysis with exon-spanning primers for the detection of all TRPV4 transcript variants showed that TRPV4 was expressed at a level that was 1000-fold higher (p = 0.001) in 12V4 cells compared to the parent HCEC-12 counterpart (Figure 2a)
Summary
Corneal transparency and visual acuity are both dependent on the maintenance of the structural and functional integrity of the endothelial monolayer array, which lines the posterior cornea facing the anterior chamber of the eye. Another feature that contributes to the ability of the endothelial layer to mediate sufficient fluid efflux is its low paracellular junctional resistance [2] The maintenance of this resistance is dependent on the extracellular presence of calcium, since exposure of the HCE to either a calcium-free solution or a medium containing a calcium channel antagonist disrupts the junctional structural integrity and its permeability and selectivity [3,4]. The maintenance of tissue transparency is dependent on the ability of the endothelium to express a wide range of ion transporter ATPases, co-transporters and ion channels Their coordinated activity establishes the ionic gradients across the paracellular and cellular pathways, which underlie osmotically coupled outward fluid egress from the stroma into the anterior chamber. In order to shed light on the role of TRPV4 expression levels in mediating responses underlying control of HCE homeostasis, we evaluated the effects of its overexpression on some responses that underlie its normal function in vitro
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