Lymphatic Malformations With Activating KRAS Mutations Impair Lymphatic Valve Development Through Matrix Metalloproteinases

BACKGROUND: Lymphatic malformations are lesions that can be due to inherited or somatic mutations, and they lead to a defective lymphatic vasculature. Activating KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations have been identified recently in patients with lymphatic malformation with lymphedema, chylous ascites, or life-threatening chylothorax. In a lymphatic malformation mouse model, KRAS mutations are associated with a loss of lymphatic valves, which has been proposed to cause chylothorax via retrograde lymph flow into the pleural space. However, the mechanisms underlying the loss of lymphatic valves are unknown. METHODS: To investigate the mechanisms leading to valve loss, we combined the lymphatic-specific and tamoxifen-inducible Flt4CreER T2 with Kras-loxP-stop-loxP-G12D ( Kras +/G12D ) mice and Prox1GFP reporter mice to induce the restricted expression of KRAS-G12D and enable valve quantification in postnatal pups. Human dermal lymphatic endothelial cells expressing KRAS-G12D were probed for changes in mRNA and protein expression with quantitative real-time polymerase chain reaction, Western blot, and gel zymography, and mechanistic studies were performed using 3-dimensional cell culture in collagen matrices. RESULTS: Our data showed that lymphatic-specific expression of KRAS-G12D significantly attenuated valve development in the mesentery, diaphragm, and ear skin. Quantitative real-time polymerase chain reaction, Western blot, and gel zymography using human dermal lymphatic endothelial cells expressing KRAS-G12D revealed the upregulation of the PA (plasminogen activator) pathway and MMPs (matrix metalloproteinases). The MMPs were sufficiently activated by plasmin, the product of the PA pathway, in human dermal lymphatic endothelial cells grown in a 3-dimensional collagen matrix, indicating a role for MMPs in the degradation of valve ECM (extracellular matrix) core. Furthermore, a broad-spectrum MMP inhibitor given to Flt4CreER T2 ;Kras +/G12D mice rescued lymphatic valve development. CONCLUSIONS: We conclude that hyperactive KRAS signaling upregulates MMPs that become excessively activated by the upregulation of the PA pathway. MMPs then degrade the lymphatic valve ECM core, preventing valve formation.