Rescue of Angiopoietin-2 inhibits lymphatic malformation endothelial cell proliferation through antagonism of VEGFR3 signaling

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 their role in LM progression remain unclear. Here, we investigated the role of Angiopoietin-2 (Ang-2) downregulation downstream of hotspot PIK3CA variants in the pathogenesis of LMs and delineated a novel inhibitory mechanism for Ang-2 in the lymphatic vasculature. Transcriptomic profiling of patient-derived lymphatic malformation endothelial cells (LM-ECs) revealed Ang-2 as one of the most significantly downregulated genes. Lentiviral overexpression of Ang-2 in LM-ECs and normal human dermal lymphatic endothelial cells significantly attenuated their proliferation by inhibiting VEGFR3 signaling via the Akt pathway. Treatment of LM-ECs with the PI3K inhibitor alpelisib, but not the mTOR inhibitor sirolimus (rapamycin), rescued Ang-2 expression and downregulated VEGFR3. Alpelisib in combination with Ang-2 overexpression produced a synergistic reduction in LM-EC proliferation compared to Ang-2 overexpression or alpelisib treatment alone. Finally, Ang-2 overexpression in LM-ECs antagonized lymphangiogenesis in a murine xenograft model of LM. Together, our findings suggest that targeting the Ang-2/VEGFR3 axis may be a viable option for the suppression of pathological lymphangiogenesis. Overall design: LM tissue and cystic fluid were collected from patients (n=6) undergoing surgical resections. LM-ECs (n=9) were isolated from LM tissue and cystic fluid as previously described. HDLEC lines (n = 3, isolated from different patients) were purchased from PromoCell (PromoCell, Germany) and used as controls. All cells were cultured in EBM-2 medium (Lonza Group Ltd, Switzerland) supplemented with growth factors, 20% fetal bovine serum (R&D Systems, Inc. Minneapolis, MN), and 1% antibiotic-antimycotic (ThermoFisher Scientific, Waltham, MA) on fibronectin-coated plates and maintained at 37°C in a 5% CO2 humidified chamber. Cells were cultured to 80% confluence, trypsinized, and collected as pellets for RNA isolation.