Human Lymphatic Endothelial Cells Expressing NRAS^Q61R Model Characteristics of Kaposiform Lymphangiomatosis

Abstract

Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly characterized by lymphatic overgrowth and dysfunction. The goal of this study was to determine how the NRAS^Q61R variant found in the tissue of KLA patients impacts key characteristics of lymphatic endothelial cells (LEC). LEC expressing NRAS^Q61R had a spindled morphology, increased migration, increased proliferation, increased MAPK signaling, and elevated angiopoietin-2 production. In an in vitro lymphangiogenesis model, LEC expressing NRAS^Q61R showed increased sprouting and formed enlarged and disorganized vascular structures. This model of LEC expressing NRAS^Q61R can be used to give insights into KLA pathogenesis and test new treatments.

Introduction

Activating mutations in RAS genes can signal through mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) to drive dysregulated cell proliferation that can result in cancers and vascular anomalies (1). A somatic activating pathogenic variant NRAS p.Q61R (NRAS^Q61R) has been associated with kaposiform lymphangiomatosis (KLA), a complex lymphatic anomaly with high morbidity and mortality (2, 3). KLA pathology shows spindled lymphatic endothelial cells (LEC) accompanying overgrowths of malformed lymphatic vessels that can cause pleural and/or pericardial effusions and coagulopathy (4). Endothelial progenitor cells (EPCs) expressing NRAS^Q61R support its role as a driver of KLA pathology (5, 6). However, since KLA is a lymphatic anomaly, it is also important to determine whether LEC expressing NRAS^Q61R recapitulate characteristics of KLA. Therefore, the objective of this study was to generate LEC with inducible NRAS^Q61R expression (NRAS^Q61R LEC) to be used in a novel lymphangiogenesis model to study the pathogenesis of KLA and test targeted drugs in the future.

Methods

Cell Culture and characterization:

Human LEC were isolated from neonatal foreskin by sorting for podoplanin (7). LEC (passage 11–14) were plated on fibronectin-coated plates and cultured in microvascular endothelial growth medium-2 (EGM-2MV) media with 20% fetal calf serum (Lonza). LEC were transduced with doxycycline (Dox)-regulated pCW-NRAS^Q61R or pCW-NRAS^WT (control) lentiviral vectors, puromycin selected, and expanded (5). LEC were seeded to 6-well plates, Dox was added to the culture media, images were captured at 48 and 72 hrs, and cell circularity was measured (5). Culture media and LEC lysates were harvested at 48 hours, 1 week, and 2 weeks after Dox was added, with media changes every 3 days. For immunofluorescence, LEC were grown in a 24-well plate on glass coverslips and fixed at 48 hours, 1 week, and 2 weeks after Dox was added to the media. Primary antibodies were against PROX1 (1:250; R&D Systems AF2727) and PDPN (1:250; Sigma-Aldrich P5374), and secondary antibodies were donkey anti-rabbit 488 (1:200; Invitrogen A21206) and donkey anti-goat 594 (1:200; Invitrogen A11058).

Western blot analysis:

Western blots of LEC lysates were performed (5) and probed for NRAS^Q61R protein (1:400; Abcam 227658), phospho-ERK (1:4000; Cell Signaling 4370), ERK (1:2000; Cell Signaling 9102), phospho-S6 Kinase Ser 235/236 (1:2000; Cell Signaling 4858), S6 kinase (1:1000; Cell Signaling 2217), and actin (1:10,000; Seven Hills Bioreagents C4). Anti-rabbit, anti-goat, and anti-mouse (Calbiochem; 1:10,000) secondary antibodies were used (Supplemental Fig 4). Angiopoietin-2 (Ang-2) levels were measured in the cell culture media by ELISA (DANG20; R&D Systems) (5).

LEC migration and proliferation:

For migration studies, LEC were seeded on both sides of a silicone insert in a 24-well plate and proliferation was blocked with hydroxyurea (5). Dox was added to the media. The insert was removed, images of the gap were taken every 2 hours for 16 hours, and the gap area was measured at each time point (5). For proliferation studies, LEC were plated (4,000 cells/well) in a 96-well plate with Dox in the media and stained with NucBlue (2 drops/40 mL, R37605; Invitrogen). Using an Agilent Cytation 5 microscope, images were captured in the DAPI channel every hour for 72 hours. Agilent Gen5 software was used to count cells. Each time point was compared to time point t = 0.

LEC sprouting and vessel formation:

A 3D fibrin bead lymphangiogenesis model was performed (8). Dox was added to the media immediately after embedding LEC coated beads (Cytiva 17048501) in the fibrin gels. Images were acquired at 24 hours, 48 hours and 14 days using an EVOS M7000 system. Sprouts were measured and counted per bead at 24 and 48 hours (9). Vascular area was measured at 14 days (5).

Statistical Analysis:

Data are expressed as mean ± standard deviation (SD). One-way ANOVA with Tukey’s post hoc test was used for multiple comparisons. A repeated measures two-way ANOVA followed by a post hoc test was used for correlated observations. All calculations were performed using GraphPad Prism. Differences were considered significant for p-value < 0.05.

Results

NRAS^Q61R expression alters cell morphology, migration and proliferation of LECs:

NRAS^Q61R LEC adopted a spindled morphology with decreased circularity index compared to NRAS^WT LEC (Fig. 1 A–B). NRAS^Q61R LEC migrated and proliferated at a faster rate compared to NRAS^WT LEC (Fig. 1C–E). Ang-2 production by NRAS^Q61R LEC was elevated compared to NRAS^WT LEC (Fig 1F).

Figure 1:

Panel A: Morphology of human lymphatic endothelial cells (LECs) expressing NRAS^WT and NRAS^Q61R. Representative images of LEC monolayer without doxycycline (No Dox) and 48 and 72 hours after addition of doxycycline to the cell culture media. Scale bar = 200 μm.

Panel B: Circularity index measurements for NRAS^WT and NRAS^Q61R LEC in media with and without Dox at 72 hours. Measurements for each cell is shown. One-way Anova followed by Tukey’s test was performed for multiple comparisons. *p < .0001.

Panel C: Representative images LEC migration. Images at t = 0 and t = 16 hours are shown. Scale bar: 200 μm.

Panel D: Quantification of gap closure area of NRAS^WT and NRAS^Q61R LEC. The gap closure was measured every 2 hours from 0 to 16 hours after removing the insert. Data points are mean ± SD. Two-way Anova followed by Bonferroni test for multiple comparisons was performed. *p < .0001 for times ≥ 8 hours.

Panel E: Quantification of the proliferation of NRAS^WT and NRAS^Q61R LEC. Data points are mean ± SD; n=8 wells. Two-way Anova followed by Bonferroni test for multiple comparisons was performed. *p < 0.0001 for times ≥ 21 hours.

Panel F: Angiopoietin-2 levels (pg/mL) in cell culture media of NRAS^WT and NRAS^Q61R LEC at 48 hours, 1 week, and 2 weeks on Dox and no Dox control (–) at 48 hours. Data points are mean ± SD. n = 6 wells per group. One-way analysis of variance (ANOVA) was performed with Tukey’s multiple comparison test. *p < .0001.

NRAS^Q61R LECs show increased MAPK and PI3K signaling, lymphatic marker expression, increased sprouting, and enlarged vascular structures:

Immunoblotting showed induction of NRAS^Q61R protein expression in the Dox-treated NRAS^Q61R LEC at all time points (Fig. 2A). In these NRAS^Q61R expressing LEC there were sustained increases in p-ERK and p-S6 indicating elevated MAPK and PI3K signaling. Immunofluorescence verified that the NRAS^Q61R LEC retained strong expression of lymphatic markers PDPN and PROX-1 (Fig. 2B). In the in vitro lymphangiogenesis model NRAS^Q61R LEC had increased number of sprouts, total sprout length (Fig. 2 C–D), and formed enlarged vascular structures (Fig. 2 E–F).

Figure 2:

Panel A: Western blot analysis of NRAS^Q61R, p-ERK, ERK (total), p-S6, S-6 (total) and Actin in induced NRAS^WT or NRAS^Q61R LEC 48 hours, 1 and 2 weeks on doxycycline (Dox).

Panel B: Representative confocal images of NRAS^WT and NRAS^Q61R LEC stained for PROX1 and PDPN. Scale Bar = 50 um.

Panel C: Representative images of NRAS^WT and NRAS^Q61R LEC sprouting. Scale bar = 250 μm.

Panel D: Quantification of LEC sprouting at 48 hours on Dox. Data points are mean ± SD. One-way analysis of variance (ANOVA) was performed with Tukey’s multiple comparison test. *p < .0001.

Panel E: Representative images of vascular structures formed by NRAS^WT and NRAS^Q61R LEC at 14 days on Dox. Scale bar = 250μm.

Panel F: Quantification of vascular area of NRAS^WT and NRAS^Q61R LEC at 14 days on Dox. Data points are mean ± SD. One-way analysis of variance (ANOVA) was performed with Tukey’s multiple comparison test. *p < .0001.

Discussion

The discovery of NRAS^Q61R a pathogenic variant associated with KLA has led to the development of models that can be used to study the disease (2, 5, 6). Here, human LEC were transduced with lentiviral vectors for Dox-regulated NRAS^Q61R and NRAS^WT expression. NRAS^Q61R LEC exhibited abnormal characteristics including spindled morphology, increased migration and proliferation, increased Ang-2 production, increased MAPK signaling, and formed enlarged vascular structures in vitro. These features recapitulate key features of KLA (4, 10, 11).

While previous studies have studied NRAS^Q61R expression in EPC, since KLA is a lymphatic disease, it is more relevant to determine the impact of NRAS^Q61R expression on LEC (5, 6). NRAS^Q61R LEC showed many of the same features as NRAS^Q61R EPC. In this study we also assessed LEC sprouting in an in vitro lymphangiogenesis model, which was not studied previously. We observed more and longer cell sprouts in NRAS^Q61R LEC, which correlated with formation of larger vascular structures in the angiogenesis model. This finding provides novel insights into how NRAS^Q61R expression could be impacting lymphatic development, which could be contributing to KLA pathobiology.

In a study by another group LEC were transduced with a lentiviral vector driving constitutive NRAS^Q61R expression. They reported impaired tube formation, increased migration, but that cell proliferation was reduced (12). While some of these findings align with our study, the finding of decreased proliferation rates of NRAS^Q61R LEC is inconsistent with our study (12). This could be due to the differences in experimental setup. Yasue et al. used a higher seeding density (10,000 cells/well) than the present study (4,000 cells/well). LEC are known to exhibit contact inhibition, so higher seeding density may reduce proliferative capabilities (13). Given that MAPK and PI3K signaling pathways are known to drive cell proliferation in other cells (14), the increased MAPK and PI3K signaling that we observed by Western blot in the NRAS^Q61R LEC is consistent with increased, not decreased proliferation, and a possible role in lesion growth in patients. Sirolimus (rapamycin) which inhibits mTOR and is downstream of PI3K signaling has been commonly used for treatment of patients with KLA, although the response may be partial (15). Treatment with the MEK inhibitor trametinib has yielded good clinical responses in case reports and suggests that MAPK signaling may also be contributing to the disease process and lesion growth in patients with KLA (16, 17).

Other genetic variants besides NRAS^Q61R have been identified in some patients with KLA. There is one report of a KLA patient with a CBL gene variant (17), and HRAS and KRAS gene variants have also been associated with KLA (18, 19). The LEC model that we present here could be adapted in the future to test these other genetic variants and give insights into their role in KLA.

In summary, NRAS^Q61R LEC in culture mimic some features of KLA including spindled cell morphology, increased Ang-2 production, and disorganized LEC overgrowth (4). This novel in vitro model which allows precise regulation of NRAS^Q61R expression in LEC can used to provide further insights into the disease process and test new treatments for KLA.

Supplementary Material

Supplemental Figure 1

Supplemental figure S1. Circularity index measurements for NRAS^WT and NRAS^Q61R LEC in media with and without Dox at 48 hours. Measurements for each cell is shown. One-way Anova followed by Tukey’s test was performed for multiple comparisons. *p < .0001.

Supplemental Figure 4

Supplemental figure S4. Quantification of LEC sprouting at 24 hours on Dox. Data points are mean ± SD. One-way analysis of variance (ANOVA) was performed with Tukey’s multiple comparison test. *p < .0001.

Supplemental Figure 2

Supplemental figure S2. Representative images LEC proliferation. Images at t = 0 and t = 72 hours are shown. Scale bar = 200 μm.

Supplemental Figure 6

Supplemental figure S6. Figure 2 panel A: NRAS^Q61R protein western blot.

Supplemental Figure 5

Supplemental figure S5. Quantification of Vascular Area at 2 weeks on Dox. Data points are mean ± SD. One-way analysis of variance (ANOVA) was performed with Tukey’s multiple comparison test. *p < .0001.

Supplemental Figure 7

Supplemental figure S7. Figure 2 panel A: phospho-ERK and phospho-S6 kinase western blot.

Supplemental Figure 8

Supplemental figure S8. Figure 2 panel A: total ERK western blot.

Supplemental Figure 9

Supplemental figure S9. Figure 2 panel A: actin and total S6 kinase western blot.

Supplemental Figure 3

Supplemental figure S3. Representative images of cells stained for lymphatic markers podoplanin (PDPN) and PROX-1. Images taken at 48 hours, 1 week, and 2 weeks. Both individual channels and merged images shown. Scale bar = 50 μm.

Abbreviation

Ang-2

Angiopoietin-2

KLA

Kaposiform lymphangiomatosis

LEC

Lymphatic endothelial cells

EPC

Endothelial progenitor cells

ELISA

Enzyme Linked Immunosorbent Assay

NRAS

Neuroblastoma RAS viral oncogene homolog

MEK

Mitogen-activated protein kinase kinase

MAPK

Mitogen-activated protein kinase

ERK

Extracellular signal-regulated kinases

CCHMC

Cincinnati Children’s Hospital Medical Center

ANOVA

Analysis of variance

p-S6

Phosphorylated S6 kinase

PI3K

Phosphoinositide 3-kinase

KRAS

Kirsten RAS viral oncogene homolog

PROX-1

Prospero homeobox protein 1

PDPN

Podoplanin

EGM-2MV

Microvascular endothelial growth medium-2