Figures and data
TGFβ2 induces a fibrotic phenotype in pTM cells and increases expression and membrane insertion of the TRPV4 channel.
(A-B) Five-day TGFβ2 treatment (1ng/mL) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. (C) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4 - 8 experiments, each gene tested in 3-7 different pTM strains (See Table 1). Two-tailed one sample t-test of TGFβ2-induced gene expression levels as a percent of control samples. (D) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. * P < 0.05, ** P < 0.01.
Donor information for primary human trabecular meshwork (pTM) strains used in this study.
TRPV4-mediated Ca2+ influx is potentiated by five-day TGFβ2 treatment.
(A) Five-day TGFβ2 treatment (1 ng/mL) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca2+ influx in pTM cells compared to serum-free media alone treated cells tested on the same day (N = 5 pTM strains, n = 3 - 5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one sample t- test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. (B) Violin plots showing the distribution of GSK101-induced Ca2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. (C) Representative traces showing TRPV4 agonist-induced Ca2+ influx (seen as an increase in F340/F380) in pTM (mean ± SEM of 4 representative cells/ group), alongside example Fura-2-loaded pTM cells before (i), during (ii), and after (iii) GSK101 application. Scale bar = 50 µm. ** P < 0.01
TGFΒ2-induced TRPV4 potentiation is not seen at a shorter time period, regardless of treatment strength.
(A) TGFβ2 treatments for 24 hours at 1 ng/mL (N = 6 pTM strains, n = 3 - 5 slides/condition/day) or 5 ng/mL (N = 5 pTM strains, n = 3 - 5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca2+ influx (SI Appendix, Figure S1) and were significantly lower than cells treated with TGFβ2 for 5d at 1ng/mL (5d TGFβ2 results from Figure 2A). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1d treatment groups compared to control groups shown in Figure S1. (B) Representative traces for GSK101 response following 24-hour TGFβ2 treatment, traces show mean ± SEM of 3-4 cells. (C) Average current density in response to GSK101 (24-hour control: n = 11 cells, 24-hour TGFβ2: n=10 cells) shows generally increased current in TGFβ2-treated cells. Data shows mean ± SEM (D - E) Violin plots of individual cell strains shown in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ** P < 0.01, *** P < 0.001
Effects of TRPV4 inhibition/activation on TGFβ2-induced contraction of TM cells.
(A) Representative longitudinal 24-well plate scans of collagen type I hydrogels seeded with pTM subjected to the different treatments (dashed lines outline size of contracted constructs). (B) Longitudinal quantification of hydrogel construct size compared to the control group at the 0 minute time point. (C) Detailed comparisons between groups at each experimental time point. n = 6 hydrogels/group. One-way ANOVA with Tukey’s multiple comparisons test, data in (B,D) shows individual data points over mean ± S.D. One pTM strain shown: TGFβ2-induced contractility induction, HC-06-mediated rescue of hypercontractility, and GSK101-induced transient (15 min) contraction were consistent across (3/3) pTM strains tested (Figure S2). ** P < 0.01, *** P < 0.001, **** P < 0.0001.
TRPV4 activation is necessary to maintain LV-TGFβ2-induced ocular hypertension.
(A) Intravitreal injection of LV-TGFβ2 (week 1), but not LV-Control, elevates IOP in WT mice (N = 5 eyes/group) as early as one-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2 treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post-hoc analysis (B) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A. Two-way ANOVA with Bonferroni post-hoc analysis (C) Intravitreal injection of LV-TGFβ2 in Trpv4-/- mice (N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A-B, N = 8-15 eyes/group). (D) Statistical comparison of the IOP values shown in C. The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4-/- eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4-/- eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post-hoc analysis. (A, C) shows mean ± SEM. Data in (B, D) shows individual data points over mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
TRPV4 inhibition inhibits nocturnal IOP elevation in control and TGFβ2 overexpressing eyes.
(A) ∼2 months post-LV injection daytime (12-2:00 P.M) and nocturnal (9-10:00 P.M.) IOP compared in WT mice (N = 4 eyes/group) before drug treatment. LV-TGFβ2 eyes were elevated at daytime, but nocturnal OHT was not significantly different between LV-Ctrl and LV-TGFβ2 eyes. One- way ANOVA with Tukey’s multiple comparisons test. (B-C) PBS-injected eyes did not exhibit changes in daytime or nighttime intraocular pressure; however, HC-06 injection reduced TGFβ2-induced IOP elevations during the day and LV-Ctrl and LV-TGFβ2 nocturnal IOPs (N = 4 Eyes/Group); Two-way ANOVA with Bonferroni post-hoc analysis. Figures show datapoints over mean ± SEM, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
TGFβ2-TRPV4 interactions in TM remodeling and ocular hypertension.
Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate Rho/ROCK signaling, augment cytoskeletal contractility, and stimulate ECM release to increase the flow resistance of the conventional pathway. Increased contractility drives OHT, resulting in a feedforward vicious TRPV4-dependent circle loop that maintains ocular hypertension. Schematic made using Biorender.com.
