Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4

Abstract

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel non-canonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

Extracellular Vesicles From Pathological Microenvironment Induce Endothelial Cell Transformation and Abnormal Angiogenesis via Modulation of TRPV4 Channels

“…Further, TRPV4-mediated calcium influx was significantly attenuated in these cells. Downregulation of TRPV4 has been shown to activate VEGF/VEGFR2 pathway via Rho kinase/YAP (Kanugula et al, 2019), and indeed, we found that TCM treatment significantly reduced perinuclear VEGFR2 suggesting activation of VEGFR2 in hTEC (Manickam et al, 2011;Wang et al, 2017). Further, pharmacological inhibition of Rho kinase was able to normalize abnormal angiogenesis exhibited by hTEC.…”

“…Further, calcium imaging revealed that TRPV4-mediated calcium influx in response to the specific agonist GSK1016790A, was significantly lower in hTEC (p ≤ 0.0001) compared to hNEC (Figures 2C,D). We next asked if the reduction in TRPV4 altered vascular endothelial growth factor receptor 2 (VEGFR2) or Rho/Rho kinase pathways in hTEC, which we have previously shown to be activated in TRPV4 null (TRPV4KO) or TRPV4 siRNA knocked down EC (Kanugula et al, 2019). Indeed, immunostaining revealed a significant reduction of perinuclear VEGFR2 levels in hTEC compared to hNEC (p ≤ 0.0001) (Figures 3A,B).…”

“…Human microvascular endothelial cells (HMEC-1) were purchased from ATCC (Manassas, VA, United States), cultured in MCDB-131 media supplemented with 10% (FBS), 1% penicillin streptomycin, 1% L-glutamine, 1 µg/mL hydrocortisone, and 10 ng/mL human vascular endothelial growth factor (Kanugula et al, 2019). HMEC-1 were denoted as human normal endothelial cells (hNEC).…”

Endothelial Transient Receptor Potential Channels and Vascular Remodeling: Extracellular Ca2 + Entry for Angiogenesis, Arteriogenesis and Vasculogenesis

“…Early work demonstrated that mechanical stimulation of TRPV4 by cyclic strain induced bovine capillary endothelial cells to realign perpendicular to the direction of the strain upon PI3K-dependent β1 integrin recruitment, stress fiber and focal adhesion rearrangement (Thodeti et al, 2009). Interestingly, the same group recently demonstrated that genetic silencing of TRPV4 increased the cell surface expression and phosphorylation at Y1175 of VEGFR2 in HMECs, thereby stimulating VEGF-induced migration (Kanugula et al, 2019). The emerging role played by TRPV4 in angiogenesis was further corroborated by Hatano et al (2013), who found that TRPV4 activation by 4α-PDD induced proliferation in human brain capillary endothelial cells.…”

The Role of TRPV4 in Regulating Innate Immune Cell Function in Lung Inflammation

“…Specifically, the TRP family member, TRPV4, is a ubiquitously-expressed, plasma membrane-based, calcium-permeable channel that is sensitized and activated by both chemical [5,6-Epoxyeicosatrienoic acid (EET), 4 alphaphorbol 12,13-didecanoate (4-αPDD)] and physical stimuli (temperature, stretch, and hypotonicity) (40)(41)(42)(43). TRPV4 can initiate intracellular, celltype and context-specific signals that depend on local increases in intracellular calcium which could act as a second messenger, and/or induce heterodimerization with other channels, activate kinases, and/or directly interact with cytoskeletal proteins via intracellular amino-(NH 2 ) and carboxy-(COOH) terminal tails (44)(45)(46)(47).…”