Endothelial TRPV4 channels prevent tumor growth and metastasis via modulation of tumor angiogenesis and vascular integrity

Abstract

Transient receptor potential vanilloid 4 (TRPV4) is a ubiquitously expressed polymodally activated ion channel. TRPV4 has been implicated in tumor progression; however, the cell-specific role of TRPV4 in tumor growth, angiogenesis, and metastasis is unknown. Here, we generated endothelial-specific TRPV4 knockout (TRPV4^ECKO) mice by crossing TRPV4^lox/lox mice with Tie2-Cre mice. Tumor growth and metastasis were significantly increased in a syngeneic Lewis lung carcinoma tumor model of TRPV4^ECKO mice compared to TRPV4^lox/lox mice. Multiphoton microscopy, dextran leakage, and immunohistochemical analysis revealed increased tumor angiogenesis and metastasis that were correlated with aberrant leaky vessels (increased width and reduced pericyte and VE-cadherin coverage). Mechanistically, increases in VEGFR2, p-ERK, and MMP-9 expression and DQ gelatinase activity were observed in the TRPV4^ECKO mouse tumors. Our results demonstrated that endothelial TRPV4 is a critical modulator of vascular integrity and tumor angiogenesis and that deletion of TRPV4 promotes tumor angiogenesis, growth, and metastasis.

Keywords: Endothelial cell; Metastasis; Transient receptor potential vanilloid 4; Tumor angiogenesis; Vascular endothelial growth factor receptor 2.

Figure 1.. Characterization of endothelial TRPV4 knockout (TRPV4^ECKO) deletion in mice.

A) Immunohistochemistry shows the colocalization (yellow; arrows) of the endothelial marker CD31 (green) with TRPV4 (red; arrowheads) in the TRPV4^lox/lox mouse aortae; this finding is absent in TRPV4^ECKO mouse aortae (only green). Scale bar: 10 μm. Note that TRPV4 (red) is present only in the smooth muscle layer of the TRPV4^ECKO mouse aortae. B) RT-PCR analysis of TRPV4, VEGFR2, and GAPDH expression in isolated mouse aortic endothelial cells from the TRPV4^lox/lox and TRPV4^ECKO mice. C) Representative average traces showing relative changes in cytosolic calcium in response to GSK1016790A (GSK101) in the Fluo-4/AM-loaded TRPV4^lox/lox and TRPV4^ECKO endothelial cells (n=64). The arrow represents the time when the cells were stimulated with GSK101 (F/F0 = ratio of normalized Fluo-4 fluorescence intensity relative to time). D) Quantitative analysis of cytosolic calcium influx induced by GSK101 in the TRPV4^lox/lox and TRPV4^ECKO endothelial cells. All data represented are the mean ± SEM from at least three independent experiments. Statistical significance is indicated by *** p≤0.001.

Figure 2.. Deletion of endothelial TRPV4 channels promotes tumor growth and abnormal vasculature.

A) Tumor growth curves of the TRPV4^lox/lox (n =9) and TRPV4^ECKO (n =11) mice bearing Lewis lung carcinoma (LLC) cells. B) Tumor volumes of the TRPV4^lox/lox and TRPV4^ECKO mice at day 21. C-D) Multiphoton microscopy images of tumor vasculature from the TRPV4^lox/lox and TRPV4^ECKO mice (n=3) showing the blood vessel diameter. Mice were infused with Alexa Fluor 594-conjugated isolectin B4 prior to tumor extraction. Scale bar: 10 μm. E) Representative immunofluorescence images of tumor sections of the TRPV4^lox/lox and TRPV4^ECKO mice stained with an endothelial marker (CD31, red) (n=3). Scale bar: 10 μm. F) Quantification of vessel density from sections of the TRPV4^lox/lox and TRPV4^ECKO mouse tumors. All data are presented as the mean ± SEM, and statistical significance is indicated by * p≤0.05, **p≤0.01, and *** p≤0.001.

Figure 3.. Endothelial TRPV4 channel deletion disrupts vascular integrity, leading to leaky vasculature.

A) Representative merged immunofluorescence images from tumor sections of the TRPV4^lox/lox and TRPV4^ECKO mice stained with an endothelial marker (CD31, red) and pericyte marker (NG2, green) (n=3). Scale bar: 10 μm. Quantification of pericyte coverage (NG2, pericytes in green/CD31, EC in red). B) Representative merged immunofluorescence images from the TRPV4^lox/lox and TRPV4^ECKO mouse tumor sections stained with an endothelial marker (CD31, red) and endothelial adherens junctional protein (VE-cadherin, green) (n=3). Scale bar: 10 μm. Quantification of VE-cadherin coverage (VE-cadherin, adherens junctional protein in green/CD31, ECs in red). C) Representative immunofluorescence images showing the leakage of Texas red dextran from the vasculature of the TRPV4^lox/lox and TRPV4^ECKO mouse tumors at day 21. Quantification of mean fluorescence intensity (MFI) (n=3). All data are presented as the mean ± SEM, and statistical significance is indicated by **p≤0.01 and *** p≤0.001.

Figure 4.. Loss of endothelial TRPV4 increases VEGFR2 expression in LLC tumors.

Western blot analysis (A) and quantification (B) of VEGFR2 expression in LLC tumors from the TRPV4^lox/lox and TRPV4^ECKO mice (n=3). C) Representative merged immunofluorescence images of tumor sections stained with endothelial markers (CD31, red) and VEGFR2 (green) from the TRPV4^lox/lox and TRPV4^ECKO mice. Scale bar: 10 μm. D) Quantification of VEGFR2-positive vessels in tumor sections from the TRPV4^lox/lox and TRPV4^ECKO mice (n=3). All data are presented as the mean ± SEM, and statistical significance is indicated by **p≤0.01 and ***p≤0.001.

Figure 5.. TRPV4 deletion in the endothelium promotes lung metastasis via increased MMP-9 expression in tumors.

Representative H&E-stained images of lung sections (A) and quantification of metastatic foci (B) from the LLC tumor-bearing TRPV4^lox/lox (n=9) and TRPV4^ECKO mice (n=11). Scale bar: 200 μm. qPCR analysis of relative MMP-9 (C) and MMP-2 (D) expression in the TRPV4^lox/lox and TRPV4^ECKO mouse tumors (n=3). All data are presented as the mean ± SEM, and statistical significance is indicated by *p≤0.05 and ** p≤0.01, NS: nonsignificant.