PROJECT SUMMARY The lymphatic system is a unidirectional vasculature that absorbs interstitial fluid in the lymphatic capillaries to transfer the lymph fluid through the collecting lymphatic vessels, the lymph nodes, and then back into the blood circulation. To prevent reflux, the collecting vessels form intraluminal bicuspid valves with an extracellular matrix (ECM) core in response to oscillatory shear stress generated by lymph flow. Lymphatic malformations (LMs) are lesions due to inherited or somatic mutations that lead to a defective lymphatic vasculature with the overall incidence as high as 1:2000 live births. Multiple somatic activating mutations in KRAS have been recently identified in LM patients who commonly present with lymphedema, chylous ascites, or life-threatening chylothorax. KRAS mutations are associated with a loss of lymphatic valves in a mouse model of LMs and valve regression has been proposed to cause chylothorax via retrograde lymph flow into the chest. To further investigate, we combined the tamoxifen-inducible, lymphatic-specific Flt4CreERT2 with Kras-LSL-G12D mice to induce restricted expression of KRAS-G12D in postnatal pups. Our preliminary data show that lymphatic vessels expressing KRAS-G12D are almost devoid of valves compared to controls in multiple tissues. RNA-sequencing of human dermal lymphatic endothelial cells (hdLECs) overexpressing KRAS-G12D or mCherry as a vector control reveal the upregulation of several components of the plasminogen activator (PA) pathway and matrix metalloproteinases (MMPs) that was confirmed by qRT-PCR. Plasmin, the product of the PA pathway, can activate the same MMPs, which can then cleave the ECM proteins found in the valve leaflet core (e.g. laminin- a5, collagen, and fibronectin-EIIIA). We hypothesize that hyperactive KRAS signaling increases the expression of key PA enzymes that then activate several upregulated MMPs that degrade the ECM core of lymphatic valves. In Aim 1, we will investigate the PA pathway by assessing the expression of key PA enzymes, and by utilizing in vitro zymography approaches to assess MMP activation. In Aim 2, we will attempt to rescue lymphatic valve defects by genetically targeting the PA pathway or by pharmacological inhibition of MMPs.