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.