Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation

Oncogenic mutations in PIK3CA, encoding p110a-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs, but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk between iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes. Overall design: Dermal lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) were FACS-sorted from the ear skin of 4-OHT-treated 5-week-old Pik3caH1047R;Cdh5-CreERT2 (n=5) and Cre- littermate control mice (n=2). One wild type C57BL/6J mouse, not treated with 4-OHT (n=1), was included.