A 3D lymphatics-on-a-chip reveals matrix stiffness dependent VEGFR-2 and VEGFR-3 contributions to lymphangiogenesis

The lymphatic system regulates fluid homeostasis and host immunity by draining interstitial fluid and immune cells and transporting them to the lymph nodes. Lymphatic capillaries, essential for capturing interstitial material, undergo lymphangiogenesis to form new lymphatic vessels from existing ones. However, the roles of the vascular endothelial growth factor (VEGF) family and their receptors in lymphangiogenesis under varying matrix stiffness conditions remain unclear. We examine lymphangiogenesis in response to VEGF-A, VEGF-C, and VEGF-C156S across different matrix stiffnesses and evaluate the effects of VEGFR-3 and VEGFR-2 inhibition in a three-dimensional (3D) lymphatics-on-a-chip model. In the model, VEGF-C induced the greatest lymphangiogenesis across all tissue stiffnesses. VEGF-A driven lymphangiogenesis was also notable in compliant and stiff matrices. VEGF-C156S-induced lymphangiogenesis was weaker than that in VEGF-A and VEGF-C conditions. Given that VEGF-C promoted the greatest sprouting across all matrix conditions and could interact with both VEGFR-3 and VEGFR-2, we examined VEGF-C driven lymphangiogenesis under VEGFR-3 and VEGFR-2 inhibition. In a soft matrix, VEGF-C driven lymphangiogenesis was significantly blocked by the VEGFR-3 inhibitor but less extensively blocked by the VEGFR-2 inhibitor; in a stiff matrix, either the VEGFR-3 or VEGFR-2 inhibitors significantly blocked lymphangiogenesis, indicating that lymphangiogenic VEGFR-3 pathways mediate VEGF-C induced lymphangiogenesis the most in a soft matrix. VEGFR-2 can similarly contribute to lymphangiogenesis in a stiff matrix, showing VEGF-C mediated sprouting loses stiffness resilience under VEGFR-2 inhibition. Ultimately, VEGF-C/VEGFR-3 signaling has a greater impact on lymphangiogenesis in a compliant matrix, but VEGF-C/VEGFR-2 signaling mediates stiffness-resistant lymphangiogenesis in a stiff matrix. STATEMENT OF SIGNIFICANCE: The lymphatic system preserves fluid homeostasis by draining interstitial fluid. Lymphatic capillaries form new vessels through lymphangiogenesis across tissues spanning soft to stiff environments, yet how VEGF signaling functions under varying matrix stiffness remains unclear. Using a 3D lymphatics-on-a-chip model, we investigated lymphangiogenesis induced by VEGF-A, VEGF-C, and VEGF-C156S and assessed VEGFR-3 and VEGFR-2 inhibition. VEGF-C produced the strongest sprouting across all stiffness conditions; VEGF-A elicited weaker responses, and VEGF-C156S was effective only in soft matrices. In compliant matrices, VEGF-C-driven sprouting depended primarily on VEGFR-3. In stiff matrices, inhibition of either VEGFR-3 or VEGFR-2 significantly reduced lymphangiogenesis, revealing stiffness-dependent receptor contributions to VEGF-C-mediated sprouting, suggesting distinct therapeutic strategies for varying ECM stiffness in different organs and diseases.