MEK / ERK inhibitor effectively impact generalized lymphatic anomaly (GLA) cells growth through EGFR / MEK /ERK signaling pathway CURRENT STATUS: POSTED

Background Generalized lymphatic anomaly is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation. Studies on GLA are frequently hampered by a lack of appropriate models to test the effects of potential treatments or decipher the mechanism of pathology. Moreover, diverse phenotypes observed with GLA require a large number of samples to be analyzed to obtain statistically informative results. Due to the very limited experimental material, most of the research is restricted to single case report. Methods We first time used two-step endothelial cell isolation technique (step 1: single cells were first sorted with a-human CD31 magnetic beads; step 2: collected CD31 Pos cells from step1 were sorted with a-human PDPN magnetic beads) to generate two GLA-LEC cell lines, and purified normal-LEC from normal liver tissue in the same case. To characterize the aberrant phenotype of generalized lymphatic anomaly lymphatic endothelial cells (GLA-LEC#1, and GLA-LEC#2). We investigated GLA-LECs growth curve, cell cycle, apoptosis, and sprouting angiogenesis in vitro . Matrigel plug assay was applied in immunodeficient mice to monitor the GLA-LECs formed vasculature in vivo . Rapamycin and dual MEK / ERK inhibitor were tested to investigate the efficacy on inhibiting GLA-LEC proliferation and downstream signaling pathway.

Generalized lymphatic anomaly is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation. Studies on GLA are frequently hampered by a lack of appropriate models to test the effects of potential treatments or decipher the mechanism of pathology. Moreover, diverse phenotypes observed with GLA require a large number of samples to be analyzed to obtain statistically informative results. Due to the very limited experimental material, most of the research is restricted to single case report.

Methods
We first time used two-step endothelial cell isolation technique (step 1: single cells were first sorted with a-human CD31 magnetic beads; step 2: collected CD31 Pos cells from step1 were sorted with a-human PDPN magnetic beads) to generate two GLA-LEC cell lines, and purified normal-LEC from normal liver tissue in the same case. To characterize the aberrant phenotype of generalized lymphatic anomaly lymphatic endothelial cells (GLA-LEC#1, and GLA-LEC#2). We investigated GLA-LECs growth curve, cell cycle, apoptosis, and sprouting angiogenesis in vitro . Matrigel plug assay was applied in immunodeficient mice to monitor the GLA-LECs formed vasculature in vivo . Rapamycin and dual MEK / ERK inhibitor were tested to investigate the efficacy on inhibiting GLA-LEC proliferation and downstream signaling pathway.

Results
We have successfully purified GLA-LECs from GLA tissues with > 99% purity. These cells also expressed the lymphatic markers lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and podoplanin (PDPN). GLA-LECs showed significantly higher proliferation rate compared to normal-LECs in both cases. Cell cycle analysis of cell distribution suggested that compared with normal-LECs, GLA-LECs showed increased proportion of cells in S phase and less G0/G1 phase. When GLA-LECs and normal-LECs apoptosis induced by serum deprivation, more Annexin V positive population of endothelial cells were observed in normal-LECs but not GLA-LECs. Hyper-activated epidermal growthfactor receptor (EGFR) signaling was observed in both cases of GLA-LECs, endogenously highly expression of EGF receptor and EGF induced phosphorylation of EGFR (phosphor Y1068) were found in both GLA cell lines. GLA-LECs are sensitive to both rapamycin and MEK / ERK dual inhibitor treatment.
In vivo, by using Matrigel plug assay, we found both GLA-LECs and immortalized GLA-LEC (SV40) grew 3 robust vessel-like structure.

Conclusions
In vitro , both GLA-LECs cell lines are highly proliferative as compared with normal-LECs. Rapamycin and dual MEK / ERK inhibitor dose-dependently inhibited GLA-LECs proliferation. In vivo , GLA-LECs showed angiogenic phenotype, and grew robust vessel-like structure in immunodeficient mice.

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However, the manuscript can be downloaded and accessed as a PDF.  Western blot for phosphorylated and total ERK in GLA-LEC#2 serum-starved overnight and exposed to 100 ng/ml EGF for 10 minutes.  GLALEC# 1 and GLA-LEC#2 were treated by different dose of rapamycin (10ng/ml, 25ng/ml, 50ng/ml, 100 ng/ml). CyQUANT assay was used to determine the cell proliferation. ***p < 0.001, N=3. (C, G) Western blotting was 13 applied to study downstream signaling pathway involved in rapamycin treatment. (B, F) GLA-LEC#1 and #2 were treated by different dose of dual MEK / ERK inhibitor RO5126766 (10nM, 50nM, 250nM, 1000nM). CyQUANT assay was used to determine the cell proliferation. ***p < 0.001, N=3. Western blotting was applied to investigate the downstream signaling pathway involved in MEK / ERK inhibitor treatment.

Supplementary Files
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