Mechanosensitive TRPV4 channels stabilize VE-cadherin junctions to regulate tumor vascular integrity and metastasis

Abstract

The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900 Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis.

Keywords: Angiogenesis; Mechanotransduction; Stiffness; TRPV4; VE-cadherin; Vascular integrity.

Figure 1.. TRPV4 modulates VE-cadherin localization at cell-cell junctions

Representative immunofluorescence images of NEC, TEC, and TRPV4KO EC showing localization of VE-cadherin (green). Nuclei were stained with DAPI (blue). B) The graph represents the quantification of VE-cadherin coverage at the cell-cell junctions of NEC, TEC and TRPV4KO EC. Data represented are mean ± SEM of 3 independent experiments. The significance was set at p≤0.05. Scale bar = 10 µm.

Figure 2.. TRPV4 regulates angiogenesis in vivo in response to matrix stiffness

A) Representative bright field images of H&E stained Matrigel plugs of varying stiffness (700–900 Pa) excised from WT and TRPV4KO mice. B) Quantitative analysis of the average number of nuclei per field in Matrigel plugs showing a significant (p≤0.05) stiffness-dependent (700–900 Pa) increase of nuclei in TRPV4KO plugs in comparison to WT. Data represented are mean ± SEM of 3 independent experiments. The significance was set at p≤0.05. Scale bar = 100 µm.

Figure 3.. Deletion of TRPV4 results in poor vessel integrity in response to varying matrix stiffness in vivo

A) Representative immunofluorescence images (20X) showing CD31 (red) to visualize physiological angiogenesis in response to changing stiffness (700–900 Pa) in WT and TRPV4KO mice. B) Quantitative analysis of the area of endothelium per vessel (µm²) showing a significant (p≤0.05) stiffness-dependent (700–900 Pa) increase in vessel area in TRPV4KO Matrigel plugs. Data represented are mean ± SEM of 3 independent experiments. The significance was set at p≤0.05. Scale bar = 10 µm.

Figure 4.. TRPV4KO mice exhibit decreased VE-cadherin coverage in Matrigel plugs of varying stiffness

A) Representative immunofluorescence merged (yellow) images (20X) of CD31 (red) co-localized with adherens junction protein, VE-cadherin (green), in Matrigel plugs of different stiffness (700–900 Pa) among WT and TRPV4KO mice. B) Quantitative analysis of VE-cadherin covered vessels showing a significant (p≤0.05) reduction in endothelial junctions in TRPV4KO Matrigel plugs in comparison to WT Matrigel plugs. Data represented are mean ± SEM of 3 independent experiments. The significance was set at p≤0.05. Scale bar = 10 µm.

Figure 5.. TRPV4 deletion reduces VE-cadherin expression and destabilizes tumor vessel integrity

A) Vessel integrity was assessed with immunofluorescence in tumor sections from WT and TRPV4KO mice. Vessels were visualized by co-staining with CD31 (red) and cell-cell junctions were stained with VE-cadherin (green) [n≥3]. B) Quantitative analysis of VE-cadherin covered vessels showing a significant (p≤0.05) reduction in tumor vasculature in TRPV4KO mice in comparison to WT. Scale bar = 10 µm.

Fig.6.. Absence of TRPV4 promotes lung metastasis of subcutaneous LLC tumor

A) Brightfield images (2X and 20X) of H&E stained lung tissue collected from tumor bearing WT and TRPV4KO mice to visualize metastases (secondary tumor growth) in the lung. B) Quantitative analysis of average number of lung metastasis from WT and TRPV4KO mice with LLC tumors [n≥4]. Data represented are mean ± SEM of 3 independent experiments. The significance was set at p≤0.05. Scale bar = 1 mm.