Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3-CA H1047R mutation, resulting in constitutive activation of the p110α PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA H1047R-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110α activation determining the LM subtype. In the postnatal vasculature, PIK3CA H1047R promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by coinhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways.

Paladin (Pald1, mKIAA1274 or x99384) was identified in screens for vascular-specific genes and is a putative phosphatase. Paladin has also been proposed to be involved in various biological processes such as insulin signaling, innate immunity and neural crest migration. To determine the role of paladin we have now characterized the Pald1 knock-out mouse in a broad array of behavioral, physiological and biochemical tests. Here, we show that female, but not male, Pald1 heterozygous and homozygous knock-out mice display an emphysema-like histology with increased alveolar air spaces and impaired lung function with an obstructive phenotype. In contrast to many other tissues where Pald1 is restricted to the vascular compartment, Pald1 is expressed in both the epithelial and mesenchymal compartments of the postnatal lung. However, in Pald1 knock-out females, there is a specific increase in apoptosis and proliferation of endothelial cells, but not in non-endothelial cells. This results in a transient reduction of endothelial cells in the maturing lung. Our data suggests that Pald1 is required during lung vascular development and for normal function of the developing and adult lung in a sex-specific manner. To our knowledge, this is the first report of a sex-specific effect on endothelial cell apoptosis.

Oncogenic mutations in PIK3CA, encoding p110α-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 involving 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.

Our studies revealed decreased cytotoxicity of the nanoparticle-formulated epirubicin compared to the free drug as well as a noticeable change in the drug's intracellular localization. Epirubicin-loaded nanoparticles were internalized via endocytosis, accumulated inside endosomal vesicles and induced a two-fold stronger pro-apoptotic signal when compared to the free drug. The level of the tumor suppressor protein p53 in HeLa cells increased significantly upon treatment with free epirubicin, but remained relatively unchanged when cells were treated with equivalent dose of nanoparticle-loaded drug, suggesting a possible shift from p53-dependent DNA/RNA intercalation-based induction of cytotoxicity by free epirubicin to a caspase 3-induced cell death by the epirubicin-loaded PBCA formulation.