Rescue of Angiopoietin-2 Inhibits Proliferation of Lymphatic Malformation Endothelial Cells

Cystic lymphatic malformations (LMs) are congenital anomalies characterized by the formation of dilated lymphatic channels that can infiltrate adjacent structures and cause significant morbidity. Somatic activating single nucleotide variants in PIK3CA have been identified in the majority of these lesions, yet the precise mechanisms underlying the role of these variants in LM progression remain unclear. Here, we investigated the role of Angiopoietin-2 (Ang2) downregulation downstream of pathogenic PIK3CA variants in the pathogenesis of LMs and delineated a novel inhibitory mechanism for autocrine Ang2 in the lymphatic vasculature. Transcriptomic profiling of patient-derived LM endothelial cells (LMECs) with PIK3CA variants revealed Ang2 as one of the most significantly downregulated genes. Overexpression of ANG2 in LMECs and normal human dermal lymphatic endothelial cells significantly suppressed their proliferation and inhibited VEGFR3 expression. In contrast, non-cell autonomous derived or exogenous ANG2 significantly promoted LMEC viability and increased AKT activity. Treatment of LMECs with the PI3K inhibitor alpelisib, but not the mTOR inhibitor sirolimus (rapamycin), rescued autocrine ANG2 expression and significantly downregulated VEGFR3. Alpelisib or sirolimus in combination with ANG2 overexpression significantly reduced LMEC viability compared to ANG2 overexpression or drug treatment alone. Finally, ANG2 overexpression in LMECs suppressed the LM phenotype in a murine xenograft model. Together, our findings suggest that targeting autocrine Ang2 signaling may be a viable option for suppressing pathological lymphangiogenesis.