TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

Christopher N. Rudzitis; Mónika Lakk; Ayushi Singh; Sarah N. Redmon; Denisa Kirdajová; Yun‐Ting Tseng; Michael Lucio De Ieso; William Daniel Stamer; Samuel Herberg; David Križaj

doi:10.7554/elife.104894.3

bioRxivNov 5, 2024Physiology

TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

Christopher N. Rudzitis iD, Mónika Lakk iD, Ayushi Singh iD, Sarah N. Redmon iD, Denisa Kirdajová iD, Yun‐Ting Tseng iD, Michael Lucio De Ieso iD, William Daniel Stamer iD, Samuel Herberg iD, David Križaj iD

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Abstract

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-{beta}2 (TGF{beta}2) in the aqueous humor. Effective treatment of POAG requires detailed understanding of interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGF{beta}2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure (IOP) regulation in a mouse model of TGF{beta}2 -induced OHT. TGF{beta}2 upregulated expression of TRPV4 and PIEZO1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 agonists induced contractility of TM-seeded hydrogels whereas pharmacological inhibition suppressed TGF{beta}2-induced hypercontractility and abrogated OHT in eyes overexpressing TGF{beta}2. Trpv4-deficient mice resisted TGF{beta}2-driven increases in IOP, but nocturnal OHT was not additive to TGF{beta}-evoked OHT. Our study establishes the fundamental role of TGF{beta} as a modulator of mechanosensing in nonexcitable cells, identifies the TRPV4 channel as the final common mechanism for TM contractility and circadian and pathological OHT, and offers insights for future treatments that can lower IOP in the sizeable cohort of hypertensive glaucoma patients that resist current treatments.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (19 May 2025) · CC BY 4.0

TGFβ2 raises TRPV4 expression and activity in human trabecular meshwork cells; TRPV4 blockade or deletion lowers TGFβ2-driven ocular hypertension and contractility in mice and hydrogels.

How trabecular meshwork pressure sensing interacts with TGFβ2, a glaucoma risk factor, was unclear, and earlier reports disagreed on whether TRPV4 raises or lowers intraocular pressure (IOP). This work tests TRPV4 as a shared effector of TGFβ2-driven and nocturnal ocular hypertension.

Key results

ShownChronic TGFβ2 exposure raised transcripts of the mechanosensitive channels TRPV4 and PIEZO1 in primary human trabecular meshwork (pTM) cells, but not TRPC1 or KCNK2, alongside fibrotic marker changes.
1 ng/mL TGFβ2 for five days: TRPV4 +102.5% (P = 0.0193), PIEZO1 +78.9% (P = 0.0114) across 8 replicates including 7 pTM strains; TRPC1 P = 0.261; KCNK2 P = 0.293. HC-06 did not change this upregulation. · TGFβ2 drives overexpression of genes that encode…; Figure legends
ShownFive-day TGFβ2 treatment strongly potentiated TRPV4 agonist-evoked calcium responses, whereas 24-hour treatment did not; the effect is time-dependent.
GSK101 (10 nM) response was 258.4% ± 61.7% of control (P = 0.0046; 5/5 strains) after five days; 24 h at 1 ng/mL gave 117.0% ± 23.6% and at 5 ng/mL 133.6% ± 34.5% of control, not significant. · TGFβ2 exposure time-dependently augments…; Figure legends
ShownTGFβ2 increased contraction of pTM-seeded collagen hydrogels; the TRPV4 antagonist reduced this, and the agonist given after antagonist washout caused transient contraction, so TRPV4 is needed for TGFβ2 hypercontractility.
TGFβ2 (5 ng/mL) raised contraction (P < 0.0003); HC-06 (5 µM) reduced it (P < 0.0001); GSK101 (25 nM) after HC-06 washout gave contraction at 15 min (P < 0.01); consistent in 3 pTM strains, n = 6 hydrogels/group. · TGFβ2-Induced TM contractility requires TRPV4…; Figure legends
ShownIn mice with lentiviral TGFβ2 overexpression, intracameral TRPV4 antagonist reversibly normalized IOP in TGFβ2 eyes without changing control eyes, while Trpv4-null mice showed only mild OHT.
At 2 weeks LV-TGFβ2 19.9 ± 4.7 vs LV-Ctrl 14.0 ± 1.2 mm Hg (P = 0.0002; N = 5). After HC-06 (100 µM): 12.2 ± 1.7 vs 12.6 ± 1.9 mm Hg; OHT returned by 1 week. Trpv4-/- (N = 6) vs WT: ΔTGFKO-TGFWT = -3.1 mm Hg (P = 0.0009). · TRPV4 activation is required to maintain…; Discussion
ShownTGFβ2-induced OHT was present in the daytime but not additive to nocturnal OHT, and TRPV4 inhibition lowered nocturnal IOP in both control and TGFβ2 eyes, pointing to a shared TRPV4-dependent pathway.
Diurnal ΔTGFβ-Ctrl = 7.9 mm Hg (P < 0.0001) vs nocturnal 0.2 mm Hg (N = 4 per group). HC-06 lowered nocturnal IOP by about 5 mm Hg (P < 0.01) and blocked daytime TGFβ2 OHT (P < 0.001); N = 4 eyes/group. · TGFβ2-induced and nocturnal OHT are non-additive…; Figure legends

Methods, data and reproducibility

Primary human TM cells were treated with TGFβ2 for qPCR, western blot, Fura-2 calcium imaging and patch clamp, and tested in collagen hydrogel contraction assays with a TRPV4 agonist and antagonist. IOP was measured by rebound tonometry in mice with lentiviral TGFβ2 overexpression, intracameral HC-067047 and Trpv4 deletion.

Primary TM cell strains from postmortem donors without glaucoma history, used for qPCR after TGFβ2 treatment · human · n = 3-7 pTM strains; 4-8 samples per gene
pTM strains for Fura-2 GSK101 calcium imaging after five-day TGFβ2 treatment · human · n = N = 5 pTM strains; 3-5 slides/condition/day
pTM-seeded collagen type I hydrogels for contraction assays (TGFβ2, HC-06, GSK101) · human cells in rat tail collagen · n = 3 pTM strains; n = 6 hydrogels/group
Membrane fraction western blot for TRPV4 from pooled pTM samples · human · n = N = 2 pooled samples (3 strains each)
C57BL/6J wild-type mice with LV-TGFβ2 vs LV-Ctrl contralateral eyes, with HC-06 or PBS intracameral injection · mouse · n = N = 5 eyes/group
Trpv4-/- mice injected with LV-TGFβ2 and LV-Ctrl, compared with pooled WT cohorts · mouse · n = N = 6 Trpv4-/- eyes/group; WT N = 8-15
Day vs nocturnal IOP in WT mice with LV-TGFβ2 or LV-Ctrl, with PBS or HC-06 crossover · mouse · n = N = 4 eyes/group (4-6 in awake cohort)

Methods & tools: SYBR Green qPCR (comparative CT, GAPDH normalization), Fura-2 AM ratiometric calcium imaging, Patch clamp (Multiclamp 700B, pClamp 10.6, Clampfit 10.7), GSK1016790A (TRPV4 agonist), HC-067047 (TRPV4 antagonist), Collagen type I hydrogel contraction assay, FIJI, TonoLab rebound tonometer, Lentiviral hTGFB2 (C226,228S) overexpression, GraphPad Prism 9
Reagents & environment: Analysis in GraphPad Prism 9; electrophysiology in pClamp 10.6 and Clampfit 10.7; hydrogel size quantified in FIJI; lentiviral construct VB170816-1094fnw (VectorBuilder).
Reproducibility gaps: Further data only on reasonable request to corresponding authors; individual in vivo datapoints and uncropped western blots are in supplementary files; no code reported.

Limitations (4)

Primary TM cell strains were selected according to what was available when experiments were run, and only 3 to 7 donor strains were used per gene. · stated by the authors
The mechanisms that make circadian TRPV4 activation reversible are unresolved and are only conjectured (suprachiasmatic nucleus, hormonal and cytoskeletal modulation). · stated by the authors
The TRPV4 protein upregulation rests on a western blot of two pooled samples (3 strains each), and the 24-hour current data came from few cells (n = 10 and 11). · noted in this summary
Pharmacology relies on one antagonist (HC-06) and a global Trpv4 knockout, so TM-specific causality is not isolated; in vivo cohorts are small (4 to 6 eyes) and mixed in sex. · noted in this summary

Read this if you study trabecular meshwork mechanotransduction or are weighing TRPV4 antagonists as an intraocular pressure target in glaucoma.