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

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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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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1406 S. Coulter St., Amarillo, TX, 79106, USA

    Racheal Grace Akwii, Md. Sanaullah Sajib, Fatema Tuz Zahra & Constantinos M. Mikelis

  2. Department of Obstetrics and Gynecology, School of Medicine, Texas Tech University Health Sciences Center, Amarillo, TX, 79106, USA

    Paul Tullar

  3. Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, 79409, USA

    Masoud Zabet-Moghaddam

  4. Center and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, 45229, USA

    Yi Zheng

  5. Department of Pharmacology, UCSD, San Diego, CA, 92093, USA

    J. Silvio Gutkind

  6. Program in Exercise and Sport Science, College of Health Professions, Marian University Indianapolis, Indianapolis, IN, 46222, USA

    Colleen L. Doci

  7. Department of Pharmacy, University of Patras, Patra, 26504, Greece

    Constantinos M. Mikelis

Authors
  1. Racheal Grace Akwii
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  2. Md. Sanaullah Sajib
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  3. Fatema Tuz Zahra
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  4. Paul Tullar
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  5. Masoud Zabet-Moghaddam
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  6. Yi Zheng
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  7. J. Silvio Gutkind
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  8. Colleen L. Doci
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  9. Constantinos M. Mikelis
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Contributions

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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