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Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the β1 integrin-RhoA-formin axis

Abstract

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

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Acknowledgements

The authors thank Dr. Guillermo Oliver (Northwestern University) for providing the Prox1-CreERT2 mice and the members of the TTUHSC animal facility in Amarillo for their support. This work was supported in part by the National Institutes of Health Grant (NCI) R15CA231339 and Texas Tech University Health Sciences Center (TTUHSC) School of Pharmacy Office of the Sciences grant. The common equipment used was obtained through the Cancer Prevention Research Institute of Texas (CPRIT) Grants RP110786, RP190524 and RP200572. The funders had no role in study design, decision to write, or preparation of the manuscript.

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Conceptualization, RGA and CMM; Methodology, RGA, MSS; Validation, FTZ, CMM; Investigation, RGA, MSS, FTZ, MZM and CMM; Writing—Original Draft, RGA and CMM; Writing—Review & Editing, FTZ, MSS, PT, MZM, YZ, JSG and CLD; Resources, PT, YZ, JSG and CLD; Supervision, CMM; Funding Acquisition: CMM.

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Correspondence to Constantinos M. Mikelis.

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Supplementary Information

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10456_2022_9831_MOESM1_ESM.pdf

Supplementary Figure 1: Ang2 does not affect Human Umbilical Vein Endothelial Cell (HUVEC) angiogenic functions. (A) Quantification of Ang2-induced HUVEC proliferation (n = 3). (B) Quantification of Ang2-induced HUVEC migration (n = 3). (C-F) Quantification of Ang2-induced sprout formation on HUVEC, assessed by the number of nodes (C), junctions (D) and total sprout length (E), and representative images (F) (n = 3), ns: not significant. Supplementary Figure 2: Ang2 affects Human Dermal Lymphatic Endothelial Cell (HDLEC) proliferation, but not sprout formation. (A) Quantification of Ang2-induced HDLEC proliferation (n = 3). (B-E) Quantification of Ang2-induced (20 ng/ml) sprout formation on HDLEC, assessed by number of nodes (B), junctions (C) and total sprout length (D), and representative images (E) (n = 4). *P < 0.05; **P < 0.01; ns: not significant. Supplementary Figure 3. Ang2 induces Human Umbilical Vein Endothelial Cell (HUVEC) RhoA activation. (A, B) Representative images (A) and quantification (B) upon dose-response of Ang2 treatment (n = 3); ns: not significant. Supplementary Figure 4: Ang2 treatment induces Tie1 and inhibits Tie2 phosphorylation in HDLEC. (A) Representative images and (B) Quantification of Tie1 and Tie2 phosphorylation in HDLEC upon Ang2 (20 ng/ml) treatment (n = 2 for Tie1 and n = 3 for Tie2). ***P < 0.001. Supplementary Figure 5: Toxicity and efficiency evaluation of Src and FAK inhibitors. (A) Quantification of HDLEC cell number upon treatment (5 μM and 10 μΜ) with SU-6656, PP2 or PF-573 (n = 2). (B, C) Representative images of Src (B) and FAK (C) phosphorylation in the presence of PP2 (B) and PF-573 (C) inhibitors respectively (n = 3). (D, E) Quantification of Ang2-induced (20 ng/ml) cell migration upon treatment with Fasudil (10 μM) or Y-27632 (10 μΜ) at different time points (1, 6, 16 h) (F) Representative images of HDLEC with phalloidin staining (red) after treatment of Ang2 (20 ng/ml) upon Fasudil (10 μΜ) (D) or Y-27632 (10 μΜ) (E) treatment (n = 2) (blue: nuclei). *P < 0.05; ***P < 0.001; ns: not significant. Supplementary Figure 6: Toxicity evaluation of formin activator and inhibitor. (A) Quantification of HDLEC cell number upon treatment with IMM01 (10 μM and 100 μΜ) and with SMIFH2 (5 μΜ and 10 μΜ) (n = 2). *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Supplementary Figure 7: Evaluation of Ang2-induced lymphangiogenesis in the ear sponge assay after 21 days of sponge implantation. (A) Schematic diagram of the ear sponge assay model. (B, C) Representative images (B) and quantification (assessed as lymphatic vessel area and density) (C) of Ang2-induced lymphangiogenesis after 21 days of sponge incubation (n = 2). (D) Representative images of ear sponge stained for LYVE1 and Tie2 (n = 2). White arrows denote representative areas with colocalized signal. (E) Representative images of RhoA expression in isolated dermal lymphatic endothelial cells from tamoxifen-treated RhoAiΔLEC mice and littermate controls. Scale bars, 500 μm, 100 μm. *P < 0.05; ns: not significant. Supplementary Table 1: List of proteins interacting with Angiopoietin-2. Text in Bold shows the interaction between Ang2 and integrin beta-1 upon Ang2 treatment. Supplementary Table 2: List of proteins interacting with Tie2. Text in Bold shows the interaction between Tie2 and integrin beta-1 upon Ang2 treatment. Supplementary file1 (PDF 1505 kb)

Appendix

Appendix

Key Resources Table

Reagents or resources Source Identifier
Cells and media
 HDLEC PromoCell Cat# C-12216
 Endothelial cell growth supplement R&D systems Cat# CCM027
 Endothelial cell base growth media R&D systems Cat# CCM028
 M199 medium Corning Cat# MT10060CV
 Fetal Bovine Serum GIBCO™ Cat# 10438026
 DMEM Life Technologies Corporation Cat# 11995073
Antibodies   
 RhoA (67B9) Cell Signaling Technology Cat# 2117; RRID: AB_1069392
 Tie1 (D2K2T) Cell Signaling Technology Cat# 23111; RRID: AB_2798856
 Tie2 R&D Systems Cat# AF313; RRID: AB_355295
 Integrin beta-1 (D6S1W) Cell Signaling Technology Cat# 34971; RRID: AB_2799067
 mDia1 (Diap1) Cell Signaling Technology Cat# 5486; RRID: AB_10828440
 FHOD1 ECM Biosciences Cat# FM3521; RRID: AB_2104508
 Angiopoietin-2 Cell Signaling Technology Cat# 2948; RRID: AB_2289507
 Angiopoietin-2 Santa Cruz Biotechnology Cat# sc-74403, RRID: AB_1118956
 Tubulin Cell Signaling Technology Cat# 2146; RRID: AB_2210545
 Actin Cell Signaling Technology Cat# 3700; RRID: AB_2242334
 GEF-H1 Cell Signaling Technology Cat# 4076; RRID: AB_2060032
 PDZ-Rho GEF Abcam Cat# ab110059; RRID: AB_10863676
 LARG Abcam Cat# ab136072; RRID: AB_2828035
 FAK Cell Signaling Technology Cat# 3285; RRID: AB_2269034
 Phospho-FAK (Tyr397) (D20B1) Cell Signaling Technology Cat# 8556; RRID: AB_10891442
 Src Cell Signaling Technology Cat# 2108; RRID: AB_331137
 Phospho-Src Cell Signaling Technology Cat# 2101; RRID: AB_331697
 Myosin light chain 2 Cell Signaling Technology Cat# 3672; RRID: AB_10692513
 Phospho-myosin light chain 2 Cell Signaling Technology Cat# 3674; RRID: AB_2147464
 Akt Cell Signaling Technology Cat# 9272; RRID: AB_329827
 Phospho-Akt (Ser473) Cell Signaling Technology Cat# 4060; RRID: AB_2315049
 PY—4G10 Millipore Cat# 05-1050X; RRID: AB_916370
 Anti-rabbit Southern Biotech Cat# 4010-05; RRID: AB_2632593
 Anti-goat Southern Biotech Cat# 6420-05; RRID: AB_2796335
 Anti-mouse Southern Biotech Cat# 1010-05; RRID: AB_2728714
 LYVE1 R&D systems Cat# AF2125; RRID: AB_2297188
 IgG mouse isotype control Santa Cruz Biotechnology Cat# sc-2025; RRID: AB_737182
 LYVE-1 ReliaTech Cat# 103-PA50; RRID: AB_2783787
 VE-cadherin Cell Signaling Technology Cat# 2500; RRID: AB_10839118
 ZO-1 Invitrogen Cat# 33-9100; RRID: AB_87181
siRNAs   
 RhoA 1, s758 Ambion Cat# 4390826
 RhoA 2, s759 Ambion Cat# 4390826
 Tie1 a, s14142 Ambion Cat# 4392420
 Tie1 b, s14141 Ambion Cat# 4392420
 Tie 2 a, s13984 Ambion Cat# 4457298
 Tie 2 b, s13984 Ambion Cat# 4390824
 Integrin beta 3, s7575 Ambion Cat# 4390824
 Integrin beta 1, s112581 Ambion Cat# AM51331
 mDia1 Dharmacon Cat# M-010347-02-0005
 FHOD1 Dharmacon Cat# M-013709-01-0005
 Silencer® Select Negative Control siRNA Thermo Fisher Scientific Cat# 4390844
Other reagents & materials   
 DharmaFECT 1 Dharmacon Cat# T-2001
 Tamoxifen Alfa-Aesar Cat# 10540-29-1
 JumpStart REDTaq Ready-Mix Reaction Mix Millipore Sigma Cat# P0982-100-RXN
 Antibiotic–antimycotic solution GIBCOTM Cat# 15240-062
 Glutathione Sepharose 4B beads GE-Healthcare Cat# 45-000-139
 Protease and phosphatase inhibitor cocktail Thermo Scientific Cat# 1861281
 Immobilon Western Chemiluminescent HRP substrate Millipore Cat# WBKLS0500
 Immobilon P Millipore Cat# IPVH304F0
 Polycarbonate membranes NeuroProbe Cat# PFB8
 Trypsin–EDTA Life Technologies Cat# 25300-054
 RGF-basement membrane extract Trevigen Cat# 3433
 Qiagen’s RNeasy mini kit Qiagen Cat# 74101
 Verso cDNA synthesis kit Thermo Scientific Cat# AB-14531/A
 SYBR Green PCR MasterMix Thermo Fisher Scientific Cat# 4309155
 Dyna beads A Invitrogen Cat# 10004D
 Dyna beads G Invitrogen Cat# 10002D
 Human Ang2 Peprotech Cat# 130-07
 Mouse Ang2 Fisher Scientific Cat# 7186-AN
 C3 toxin Fisher Scientific Cat# NC9317720
 RGD Fisher Scientific Cat# NC0210557
 PP2 Fisher Scientific Cat# 52-957-31MG
 PF-573228 Fisher Scientific Cat# 50-101-3643
 SU-6656 Fisher Scientific Cat# 57-263-51MG
 Y-27632 Fisher Scientific Cat# 12-541-0
 Fasudil LC Laboratories Cat# F4660
 IMM01 Sigma Aldrich Cat# SML1064
 SMIFH2 Sigma Aldrich Cat# S4826
 Dispase Fisher Scientific Cat# 17105-041
 Collagenase Type II Fisher Scientific Cat# 17101-015
qPCR oligonucleotides (Primers)   
 Human RhoA Forward: 5′-AGCCAAGATGAAGCAGGAGC-3′ Integrated DNA Technologies  
 Human RhoA Reverse: 5′-TTCCCACGTCTAGCTTGCAG-3′ Integrated DNA Technologies
 Human Ang2 Forward: 5′-AAGAGAAAGATCAGCTACAGG-3′ Integrated DNA Technologies
 Human Ang2 Reverse: 5′- CCTTAGAGTTTGATGTGGAC-3′ Integrated DNA Technologies
 Human Actin Forward: 5′-CTCTTCCAGCCTTCCTTCCTG-3′ Integrated DNA Technologies
 Human Actin Reverse: 5′- CAGCACTGTGTTGGCGTACAG-3′ Integrated DNA Technologies
Genotyping oligonucleotides (Primers)  
 RhoAf/f Forward: 5′-TCTCTGCACTGAGGGAGTTAGG-3′ Integrated DNA Technologies
 RhoAf/f Reverse: 5′-GTACATACAGGGAATGGAAACAAGG-3′ Integrated DNA Technologies
 Tom-GFP Wt Forward: 5′-CTCTGCTGCCTCCTGGCTTCT-3′ Integrated DNA Technologies
 Tom-GFP Wt Reverse: 5′-CGAGGCGGATCACAAGCAATA-3′ Integrated DNA Technologies
 Tom-GFP Mut Reverse: 5′-TCAATGGGCGGGGGTCGTT-3′ Integrated DNA Technologies
 Prox1-CreERT2 Forward: 5′-AACTCGAGCTCTTTCTCTCTACAGTTCAACAGATGCATTACC-3′ Integrated DNA Technologies
 Prox1-CreERT2 Reverse: 5′-GGGGGAGGGAGAGGGGCGGAATTGCTACTCGTGAAGGAGTTC-3′ Integrated DNA Technologies

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Akwii, R.G., Sajib, M.S., Zahra, F.T. et al. Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the β1 integrin-RhoA-formin axis. Angiogenesis 25, 373–396 (2022). https://doi.org/10.1007/s10456-022-09831-y

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Keywords

  • Angiopoietin-2
  • Ang2
  • RhoA
  • Integrins
  • Formins
  • Lymphangiogenesis
  • HDLEC
  • Lymphatic endothelial cells
  • Small GTPase
  • Cell migration