Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis

Abstract

PURPOSE. We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis. METHODS. Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca 2þ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording. RESULTS. TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca 2þ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model. CONCLUSIONS. TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

“…Although capsaicin receptor transcripts represent the most abundant vanilloid mRNAs in the human tissue, we find its expression in the mouse CE to be modest, with reporter expression in TRPV1 tdT corneas confined to a subset of CECs that may endow the CE with sensitivity to anandamide, protons, noxious heat, and capsaicin. 31,71,72 TdT always colocalized with TRPV4 but it remains to be seen whether the vanilloid subunits heteromerize, as recently reported for endothelial cells 73 but not retinal ganglion cells. 34 Consistent with the latter possibility, barrier permeability in CE monolayers is regulated by GSK101 but not the TRPV1 agonist capsaicin.…”

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

“…Finally, pharmacological (with capsazepine) and genetic (with a selective siRNA) inhibition of TRPV1 prevented 14,15-EET-induced Ca 2+ influx, NO release, in vitro tube formation and in vivo angiogenesis in HMECs (Su et al, 2014a). A recent investigation demonstrated that TRPV1 may assemble into a heteromeric complex with TRPV4 in mouse retinal microvascular endothelial cells (RMECs) (O'Leary et al, 2019). The TRPV1/TRPV4 heteromeric channel did not promote VEGF-induced extracellular Ca 2+ entry and sprouting angiogenesis, but it was per se able to mediate Ca 2+ influx and drive RMEC proliferation, motility and capillary-like tube formation (O'Leary et al, 2019).…”

“…A recent investigation demonstrated that TRPV1 may assemble into a heteromeric complex with TRPV4 in mouse retinal microvascular endothelial cells (RMECs) (O'Leary et al, 2019). The TRPV1/TRPV4 heteromeric channel did not promote VEGF-induced extracellular Ca 2+ entry and sprouting angiogenesis, but it was per se able to mediate Ca 2+ influx and drive RMEC proliferation, motility and capillary-like tube formation (O'Leary et al, 2019). While these studies hinted at extracellular Ca 2+ entry as the main mechanism whereby TRPV1 induces angiogenesis, an alternative signaling mode has been described in EA.hy926 cells (Hofmann et al, 2014).…”

Endothelial TRPV1 as an Emerging Molecular Target to Promote Therapeutic Angiogenesis

“…It has long been known that TRPV1 and TRPV4 subunits form heteromeric channel exclusively located on the plasma membrane [88]. Accordingly, TRPV4 was found to associate with TRPV1 into a functional heteromeric channel complex in mouse primary retinal microvascular endothelial cells [89]. Likewise, TRPV5 and TRPV6 may assemble into heteromeric complexes in the kidney and gastrointestinal tract, but they are yet to be reported in vascular endothelium [37,90].…”

“…TRPV1 expression has also been found in the ER in several cell types, including rat nociceptive neurons, [125] and breast [126] and prostate [127,128] cancer cell lines. Cytosolic localization of TRPV1 has been recently described in bovine retinal microvascular endothelial cells (RMECs) [89]. It is, however, still unclear whether intracellular TRPV1 contributes to endothelial Ca 2+ signaling.…”