VEGF-A/C co-stimulation, without shear stress, triggers the polarization of lymphatic microvessels

The lymphatic system maintains interstitial fluid homeostasis and supports immune function through dynamic regulation of its architecture mediated by molecular signals—such as vascular endothelial growth factors (VEGFs)—and physical cues. While VEGFs are known to promote endothelial proliferation, their broader roles in tissue organization remain under investigation. Using a lymphatic vessel-on-a-chip platform, we examine how lymphatic endothelial cells (LECs) respond to VEGF-A, VEGF-C, or their combination. We find that co-stimulation synergistically enhances lymphangiogenic sprouting while preserving barrier integrity. Co-stimulation also induces axial polarization of the tissue along the vessel axis, even in the absence of external mechanical stimuli. This polarization requires activation of the VEGFR2/VEGFR3 heterodimer and is disrupted by inhibition of the Src-dependent mechanotransduction pathway. Further co-stimulation enhances LEC motility and triggers vessel contraction. Modeling suggests that the tubular geometry of the lymphatic monolayer imposes intrinsic mechanical anisotropy—softer in the circumferential than axial direction. This geometrically-encoded stiffness landscape directs cell migration along the stiffer axis, uncovering a form of durotaxis driven by curvature-induced anisotropy. These results highlight a previously unrecognized mechanism by which biochemical and biophysical cues direct lymphatic tissue polarization, offering new insight into how geometry and mechanosensing shape lymphatic function.