Abstract Body (Do not enter title and authors here):
Background: VEGF-C is a central regulator of lymphangiogenesis, the formation of new lymphatic vessels, by binding to VEGFR-3 on lymphatic endothelial cels (LECs), triggering signaling pathways promoting their migration, and forming new lymphatic vessels. While we reported that reactive oxygen species (ROS) from NADPH oxidases (NOXs) and mitochondria, as well as physiological copper (Cu) levels regulated by the Cu uptake transporter CTR1, are important in angiogenesis, their roles in VEGF-C-induced VEGFR3 signaling and lymphangiogenesis remain unknown.
Results: Here we show that VEGF-C stimulation induced a biphasic ROS response in human LECs, an early transient increase within 30 min and a sustained late-phase elevation up to 4 hrs. Overexpression of mitochondria-targeted catalase (mito-catalase), but not NOX4 knockdown, suppressed both VEGF-C-induced ROS production and key lymphangiogenic responses including EC proliferation (1.9 fold), migration (2.5 fold), and tube formation (1.2 fold), indicating a predominant role for mitochondrial ROS (mitoROS). Mito-catalase also attenuated VEGFR3 phosphorylation and downstream ERK1/2 and Akt activation. We then examined the role of Cu and CTR1 and found that CTR1 knockdown significantly impaired VEGF-C-induced VEGFR3, ERK1/2, and Akt phosphorylation, late-phase ROS production, and lymphangiogenic responses without affecting early ROS generation. In contrast, Cu chelation with BCS inhibited late-phase ROS and lymphangiogenesis without affecting VEGFR3 activation, suggesting a Cu-independent role of CTR1 in initiating VEGFR3 signaling. Mechanistically, VEGF-C promoted rapid CTR1-VEGFR3 association and co-internalization within 30 min, supporting receptor activation and sustained ROS generation. In vivo, VEGF-C-induced lymphangiogenesis and inflammatory cell recruitment (Lyve1+, VEGFR3+, and CD45+ cells) were significantly diminished in Ctr1+/- mice using a Matrigel plug model.
Conclusion: These findings uncover a novel biphasic mechanism whereby VEGF-C promotes lymphangiogenesis through mitoROS-CTR1-VEGFR3 axis which initiates VEGFR3 signaling independent of Cu, followed by Cu-dependent ROS amplification to sustain downstream migration and tube formation. This study reveals CTR1 as a critical regulator of VEGFR3 signaling and lymphatic vessel formation.