Mammals transport blood through a high-pressure, closed vascular network and lymph through a lowpressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life.

SUMMARY The cerebral cavernous malformation (CCM) pathway is required in endothelial cells for normal cardiovascular development and to prevent postnatal vascular malformations, but its molecular effectors are not well defined. Here we show that loss of CCM signaling in endocardial cells results in mid-gestation heart failure associated with premature degradation of cardiac jelly. CCM deficiency dramatically alters endocardial and endothelial gene expression, including increased expression of the Klf2 and Klf4 transcription factors and the Adamts4 and Adamts5 proteases that degrade cardiac jelly. These changes in gene expression result from increased activity of MEKK3, a mitogen-activated protein kinase that binds CCM2 in endothelial cells. MEKK3 is both necessary and sufficient for expression of these genes, and partial loss of MEKK3 rescues cardiac defects in CCM-deficient embryos. These findings reveal a molecular mechanism by which CCM signaling controls endothelial gene expression during cardiovascular development that may also underlie CCM formation.

Neonatal mice lacking lymphatic vessels due to loss of lymphangiogenic factor CCBE1 or VEGFR3 function fail to inflate their lungs, suggestive of respiratory failure in infants with congenital pulmonary lymphangiectasia.

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