TRPV4 channels regulate tumor angiogenesis via modulation of Rho/Rho kinase pathway

Abstract

Targeting angiogenesis is considered a promising therapy for cancer. Besides curtailing soluble factor mediated tumor angiogenesis, understanding the unexplored regulation of angiogenesis by mechanical cues may lead to the identification of novel therapeutic targets. We have recently shown that expression and activity of mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) is suppressed in tumor endothelial cells and restoring TRPV4 expression or activation induces vascular normalization and improves cancer therapy. However, the molecular mechanism(s) by which TRPV4 modulates angiogenesis are still in their infancy. To explore how TRPV4 regulates angiogenesis, we have employed TRPV4 null endothelial cells (TRPV4KO EC) and TRPV4KO mice. We found that absence of TRPV4 (TRPV4KO EC) resulted in a significant increase in proliferation, migration, and abnormal tube formation in vitro when compared to WT EC. Concomitantly, sprouting angiogenesis ex vivo and vascular growth in vivo was enhanced in TRPV4KO mice. Mechanistically, we observed that loss of TRPV4 leads to a significant increase in basal Rho activity in TRPV4KO EC that corresponded to their aberrant mechanosensitivity on varying stiffness ECM gels. Importantly, pharmacological inhibition of the Rho/Rho kinase pathway by Y-27632 normalized abnormal mechanosensitivity and angiogenesis exhibited by TRPV4KO EC in vitro. Finally, Y-27632 treatment increased pericyte coverage and in conjunction with Cisplatin, significantly reduced tumor growth in TRPV4KO mice. Taken together, these data suggest that TRPV4 regulates angiogenesis endogenously via modulation of EC mechanosensitivity through the Rho/Rho kinase pathway and can serve as a potential therapeutic target for cancer therapy.

Keywords: Rho/Rho kinase; TRPV4; endothelial cell; mechanotransduction; tumor angiogenesis.

Figure 1. TRPV4 deletion induces abnormal EC proliferation, ERK1/2 phosphorylation, migration, and Rho activity

(A) Quantitative analysis showing increased percentage of BrdU incorporation in TRPV4KO EC compared to WT EC. (B) Above: Western blot showing increased basal phosphorylation of ERK1/2 in TRPV4KO EC. Below: Densitometric analysis of ERK1/2 activity (normalized to total ERK1/2) showed a significant increase in TRPV4KO EC. (C) Above: Representative Brightfield images (4×) of scratch wounds taken at 0 and 12 hr. Scale bar = 100 μm. Below: Quantitative analysis showing increased migration of TRPV4KO EC compared to normal EC (NEC). (D) Above: Western blot showing increased basal levels of active RhoA in TRPV4KO EC compared to WT EC. Below: Densitometric analysis of RhoA activity (normalized to total RhoA) showed a significant increase in TRPV4KO EC.

Figure 2. Absence of TRPV4 induces abnormal angiogenesis in vitro, ex vivo, and in vivo

(A) Phase contrast images (4×) showing the angiogenic behavior of WT EC and TRPV4KO EC when plated on 2D Matrigel at high density (8 × 10⁴/well) at 4, 6, and 8 hours. (B) Above: Representative images (4×) showing sprouting angiogenesis from aortic explants isolated from WT and TRPV4KO mice. Below: Quantitative analysis showing significantly (p ≤ 0.05) increased vascular sprouting in aortic explants from TRPV4KO mice on Day 5. (C) Images of Matrigel plugs extracted from WT and TRPV4KO mice. Immunofluorescence images from Matrigel plugs sections (WT and TRPV4KO) showing vessel formation, as visualized by CD31 staining. Green = CD31; Blue = DAPI staining for nuclei. Scale bar = 100 μm for all images.

Figure 3. Rho kinase (ROCK) inhibition normalizes abnormal mechanosensitivity and angiogenesis exhibited by TRPV4KO EC

(A) Above: Phase contrast micrographs (20X) showing the normalizing effects of Rho kinase inhibitor, Y-27632 (10 μM) on TRPV4KO EC spreading. Cells were equally plated on ECM gels of varying stiffness (98, 370, and 2288 Pa) and representative images were taken 6 h after plating. Scale bar = 100 μm. Below: Quantification of cell area revealed that while treatment with Y-27632 (10 μM) had no effect on NEC spreading, Y-27632 significantly attenuated abnormal TRPV4KO EC spreading at intermediate and high stiffness gels. (B) Above: Phase contrast micrographs (4×) showing the normalizing effects of Rho kinase inhibitor, Y-27632 (10 μM) on TRPV4KO EC angiogenic behavior, when plated on Matrigel at high density (8 × 10⁴ well). WT EC were used as a control. Representative images were taken 8 h post plating, showing the formation of stable tubes upon inhibition of the Rho kinase pathway. Scale bar = 100 μm. Below: Quantification of tube length revealed a significant increase (p ≤ 0.05) in tube formation in TRPV4KO EC+Y-27632 compared to untreated TRPV4KO EC controls. Further, the measured tube lengths of TRPV4KO EC treated with Y-27632 were comparable to WT EC.

Figure 4. ROCK inhibitor, Y-27632, in conjunction with Cisplatin, reduces tumor growth in vivo in TRPV4KO mice

(A) Time-dependent growth of tumors in mice injected with saline (CON), Y-27632, Cisplatin, or Y-27632 + Cisplatin. Syngeneic tumors (LLC) were injected in the back of TRPV4KO mice and tumor growth was monitored for 21 days. Rho kinase inhibitor, Y-27632, was injected (i.p.) (10 mg/kg) every day starting from Day 7 until Day 21. Cisplatin was injected (i.p.) (3 mg/kg) once per week starting 2–4 days after the injection of Y-27632. Note that tumor growth was significantly reduced in Y-27632+Cisplatin treated mice, but not Y-27632 or Cisplatin alone. (B) Frozen sections of tumors from Control, Y-27632, Cisplatin, and Y-27632+Cisplatin treated mice (10 μm thickness; from Day 21) were stained with CD31 (red) and α-SMA (green) to measure pericyte coverage (matured vessels). Scale bar = 100 μm. (C) Quantitative analysis of pericyte covered micro-vessels showing increased pericyte coverage in the tumor vessels treated with Y-27632 or Y-27632 + Cisplatin, but not in Cisplatin alone treated mice.

Figure 5. TRPV4-dependent mechanotransduction during angiogenesis

A schematic representation of TRPV4-mediated mechanical signaling in normal and TRPV4KO/TRPV4-deficient (tumor) endothelial cells. In normal endothelial cells, TRPV4 senses mechanical force (ECM stiffness) and induces optimal Rho/Rho kinase activation necessary for endothelial migration and contraction which is required for partial cell rounding and angiogenesis. However, absence (TRPV4KO EC) or reduction (Tumor EC) in TRPV4 expression and function results in high basal Rho/Rho kinase activation, leading to abnormal (tumor) angiogenesis. This abnormal tumor vasculature can be normalized by restoring mechanosensitivity through pharmacological activation of TRPV4 in tumor endothelial cells (TRPV4-deficient) with GSK1016790A or inhibition of Rho kinase in TRPV4KO EC with Y-27632. Overall, these findings suggest that targeting TRPV4/Rho kinase-mediated mechanotransduction may be a novel therapy for tumor vascular normalization and improving anti-cancer drug delivery.