Abstract
Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.
Highlights
Angiogenesis is a process of fundamental biological importance, being essential for several normal functions, including embryonic development and tissue modeling and repair (1)
We report that TTX-R Voltage-gated sodium channels (VGSCs) activity modulates the membrane potential of human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor (VEGF) and subsequent Ca2ϩ signaling events, namely protein kinase C (PKC)␣ and ERK1/2 activation
Real-time PCR (RT-PCR) revealed that the VGSC␣ subtypes Nav1.2, Nav1.4, Nav1.8, Nav1.9, and NaX were present, but these constituted less than 0.2% of the total VGSC␣ mRNA
Summary
Angiogenesis (the development of new blood vessels) is a process of fundamental biological importance, being essential for several normal functions, including embryonic development and tissue modeling and repair (1). There is a need to further elucidate the complex molecular mechanisms controlling angiogenic activities of endothelial cells (ECs).. Based on the parallels between the processes involved in angiogenesis and tumor cell invasion (19) and the similarities between endothelial and neuronal guidance (20), we hypothesized that VGSC activity may be involved in the angiogenic properties of ECs. VGSC expression has been reported in human umbilical vein endothelial cells (HUVECs) (11, 14). It has been speculated that ion channel activity may be involved in angiogenesis (22), the specific functional roles of the VGSCs expressed in ECs (and non-excitable cells generally) and the associated molecular signaling mechanism(s) are unclear. We show, for the first time, that functional VGSCs expressed in HUVECs exert significant control upon the cells’ angiogenic activities and provide insights into underlying mechanisms. We report that TTX-R VGSC activity modulates the membrane potential of HUVECs in response to VEGF and subsequent Ca2ϩ signaling events, namely PKC␣ and ERK1/2 activation
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