Characterization of kaposiform lymphangiomatosis tissue‐derived cells

Abstract

Background Kaposiform lymphangiomatosis (KLA) is a recently characterized systemic lymphatic anomaly. Activation of RAS/MAPK and PI3K/AKT/mTOR pathways may affect KLA pathogenesis, but the cellular basis of KLA is unclear. Abnormal‐spindle endothelial cells that express lymphatic endothelial cell (LEC) markers are characteristic of KLA histopathology. This study evaluated patient‐derived KLA cells to establish their morphological and biological characteristics. Procedure We established cell lines from primary KLA tissues of two patients with KLA and examined their morphological and functional characteristics, messenger RNA and protein expression profiles, gene mutations, and responses to inhibitors of the RAS/MAPK and PI3K/AKT/mTOR pathways. Results Both KLA cell lines showed spindle‐shaped morphology, stained positive for podoplanin (PDPN), and exhibited impaired tube‐formation properties. They expressed LEC marker PDPN and mesenchymal stem cell markers (CD90, CD105) in the absence of endothelial cell markers (CD34, CD31, VWF), per real‐time polymerase chain reaction. Both mTOR inhibitor rapamycin and MEK inhibitor trametinib inhibited growth of the two cell lines. A NRAS p.Q61R variant was found in one of two independent KLA tissue samples, but not in the KLA cells (per targeted next‐generation sequencing); and KLA cells with this variant had elevated AKT phosphorylation levels. ERK phosphorylation levels were undetectable in both KLA cell lines. Conclusions Inhibition of the RAS/MAPK and PI3K/AKT/mTOR pathways may represent potential therapeutic targets in KLA. These patient‐derived KLA cell lines will be useful research tools to elucidate KLA etiology, and could pave the way for basic, translational, and preclinical studies of this disease.

Treatment of severe Kaposiform lymphangiomatosis positive for NRAS mutation by MEK inhibition

“…The pathogenesis of the disorganized hyperproliferative phenotype of KLA lymphatic channels has been elucidated recently. Studies of KLA tissue derived cells demonstrated increased expression of proliferative mesenchyme stem-cell markers and the relative lack of endothelial cell markers, 17 , 18 while lymphatic endothelial cells from a GLA patient carrying NRAS p.Q61R demonstrated increased proliferation with decreased ability to form organized lymphatic vessels. A zebrafish model transduced to express the same mutation demonstrated abnormal development of the vascular and lymphatic systems caused by impaired migration of parachordal cells.…”

“…Current knowledge of molecular mechanisms driving KLA pathogenesis is derived mainly from the study of cell lines obtained from KLA patients as well as patients with different lymphatic anomalies carrying the typical NRAS mutation p.Q61R 5 , 6 , 17 , 18 . Transduction of HDLECs to express NRAS p.Q61R used here offers a reproducible method for the continued study of expression repertoire and the performance of drug screens .…”

Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells

“…A previous study examined the characteristics of cells isolated from two KLA patients, one of whom had the NRAS Q61R mutation [ 7 ]. A tube formation assay showed that KLA cells had significantly fewer tubes than normal HDLECs.…”

“…A recent study of human endothelial progenitor cells expressing the NRAS Q61R mutation reported the same effects on these pathways [ 19 ]. However, established cell lines from three KLA patients demonstrated various results, including the downregulation of p-ERK or upregulation of p-AKT [ 7 , 20 ]. Although it is challenging to predict whether these pathways are activated or inactivated in affected lesions, these established cell lines may contain mutated and non-mutated cells in pathological structures that have abnormal functions in the affected lesions.…”

“…Sirolimus is a first-line drug for refractory CLAs, and trametinib is an alternative drug candidate [ 21 , 22 ]. In previous studies, these drugs inhibited the proliferation of patient-derived cells [ 7 , 19 ]. The present study showed that the tube formation and cell proliferation of NRAS Q61R HDLECs improved after both sirolimus and trametinib treatment.…”