1. Cell Biology
  2. Medicine

ErbB signaling is a potential therapeutic target for vascular lesions with fibrous component

  1. Suvi Jauhiainen
  2. Henna Ilmonen
  3. Pia Vuola
  4. Heta Rasinkangas
  5. Heidi H Pulkkinen
  6. Sara Keränen
  7. Miika Kiema
  8. Jade J Liikkanen
  9. Nihay Laham-Karam
  10. Svetlana Laidinen
  11. Mustafa Beter
  12. Einari Aavik
  13. Kimmo Lappalainen
  14. Jouko Lohi
  15. Johanna Aronniemi
  16. Tiit Örd
  17. Minna U Kaikkonen
  18. Päivi Salminen
  19. Erkki Tukiainen
  20. Seppo Ylä-Herttuala
  21. Johanna P Laakkonen  Is a corresponding author
  1. A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Finland
  2. Department of Plastic Surgery, Helsinki University Hospital and University of Helsinki, Finland
  3. VASCERN VASCA European Reference Centre, Helsinki University Hospital, Finland
  4. Department of Radiology, HUS Diagnostic Center and Helsinki University Hospital and University of Helsinki, Finland
  5. Department of Pathology, HUSLAB, Helsinki University Hospital and University of Helsinki, Finland
  6. Department of Pediatric Surgery, New Children's Hospital, Helsinki University Hospital and University of Helsinki, Finland
  7. Science Service Center, Kuopio University Hospital, Finland
  8. Gene Therapy Unit, Kuopio University Hospital, Finland
https://doi.org/10.7554/eLife.82543
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Cite this article
  1. Suvi Jauhiainen
  2. Henna Ilmonen
  3. Pia Vuola
  4. Heta Rasinkangas
  5. Heidi H Pulkkinen
  6. Sara Keränen
  7. Miika Kiema
  8. Jade J Liikkanen
  9. Nihay Laham-Karam
  10. Svetlana Laidinen
  11. Mustafa Beter
  12. Einari Aavik
  13. Kimmo Lappalainen
  14. Jouko Lohi
  15. Johanna Aronniemi
  16. Tiit Örd
  17. Minna U Kaikkonen
  18. Päivi Salminen
  19. Erkki Tukiainen
  20. Seppo Ylä-Herttuala
  21. Johanna P Laakkonen
(2023)
ErbB signaling is a potential therapeutic target for vascular lesions with fibrous component
eLife 12:e82543.
https://doi.org/10.7554/eLife.82543
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8 figures, 5 tables and 3 additional files

Figures

Figure 1 with 2 supplements
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Genes involved in ErbB signaling pathway are upregulated in patient-derived endothelial cells (ECs) in vascular lesions with the venous components.

(A) Magnetic resonance images of an angiomatosis of soft tissue (AST) lesion (arrows) in the soleus muscle show the replacement of the normal muscle by dilated venous channels, diffusely enhancing …

Figure 1—figure supplement 1
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Highest expression of TGFA and EPGN is shown in patient ECs positive for oncogenic PIK3CA variants.

RNA-seq data revealed that the highest levels of transforming growth factor A (TGFA) and epigen (EPGN) mRNA were detected in patient-derived endothelial cells (ECs) having an oncogenic PIK3CA …

Figure 1—figure supplement 2
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TGFA and genes related to cell migration, proliferation, and ECM organization, are regulated in patient ECs with oncogenic PIK3CA variant.

(A) Further analysis demonstrated 818 differentially expressed genes (DEGs) between PIK3CAmut+ Patient endothelial cells (ECs) and HUVEC, and 327 DEGs between PIK3CAmut+ Patient ECs and human …

Figure 2 with 3 supplements
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Epidermal growth factor receptor (EGFR)/ErbB1 ligand transforming growth factor A (TGFA) is upregulated in venous malformation (VM) and angiomatosis of soft tissue (AST) patient tissue samples.

(A) RT-qPCR analysis showed significantly higher expression of TGFA mRNA (with two different assays) in VM and AST tissue than in the control group. Mean and SEM are presented (lesions n=11; control …

Figure 2—figure supplement 1
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TGFA expression in patient lesions normalized to vascular EC marker.

RT-qPCR analysis showed a trend (p = 0.0782) toward higher expression of transforming growth factor A (TGFA) mRNA in venous malformation (VM) and angiomatosis of soft tissue (AST) patient tissues …

Figure 2—figure supplement 1—source data 1

Raw data for western blot images.

https://cdn.elifesciences.org/articles/82543/elife-82543-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
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TGFA and EGFR are expressed in patient cells.

A) RT-qPCR analysis of ErbB1 ligand amphiregulin (AREG) mRNA in venous malformation (VM) and angiomatosis of soft tissue (AST) lesions. Mean and SEM are presented (lesions n=10; control group, n=4; …

Figure 2—figure supplement 3
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scRNAseq data from mouse lower limb skeletal muscle presenting clusters positive for EGFR and TGFA.

scRNAseq data from mouse lower limb skeletal muscle (Tabula Muris, czbiohub.org) of epidermal growth factor receptor (EGFR) (A–B) and transforming growth factor A (TGFA) (C–D) expression. EGFR was …

Figure 3 with 3 supplements
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Oncogenic PIK3CAH1047R induces enrichment of hallmark hypoxia.

(A) Several shared MSigDB Hallmarks were found in bulk RNA-seq data from (i) patient-derived endothelial cells (ECs) vs control ECs (left panel), and (ii) ECs expressing PIK3CAwt or most common …

Figure 3—figure supplement 1
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Oncogenic PIK3CAH1047R induces morphological and transcriptional changes in transduced ECs.

(A) In-line with previous publications (Limaye et al., 2015), expression of oncogenic PIK3CAH1047R induced morphological changes in HUVECs. Representative images of HUVECs transfected with a …

Figure 3—figure supplement 2
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Comparison of bulk RNA-sequencing data of patient-derived endothelial cells and PIK3CAH1047R transduced primary endothelial cells.

Gene set enrichment analysis (GSEA) for RNAseq data from done for upregulated (A–B) or down-regulated (C–D) differentially expressed genes (DEGs) confirms that the RNA-seq data from HUVECs …

Figure 3—figure supplement 3
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VEGF-A is upregulated and secreted from patient-derived stromal cells.

(A) RT-qPCR analysis of VEGFA mRNA expression in patient stromal cells (SCs) and control fibroblasts (SCs, n=6; HPF-c, n=3). Two-tailed Mann-Whitney U test (data not normally distributed). *, p<0.05.…

Figure 4 with 2 supplements
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Patient stromal cells (SCs) and transforming growth factor A (TGFA) induce an angiogenic endothelial cell (EC) phenotype together with VEGF-A.

(A–B) Venous malformation (VM) patient SCs induce sprouting of genotypically normal ECs. HUVECs on collagen-coated beads were embedded into a fibrin gel and patient SCs or control HPF-c cells were …

Figure 4—figure supplement 1
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rhTGFA stimulation induces VEGFA in normal fibroblasts.

rhTGFA stimulation of HPF-c cells increased VEGFA mRNA expression (A, 6 hr) and VEGF-A protein secretion (B, 24 hr). The data is from three independent experiments done in triplicates. Two-tailed …

Figure 4—figure supplement 2
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Quantification for the proliferation of cells in co-culture experiments combining endothelial cells (ECs) expressing PI3KCAH1047R or PI3KCAwt wt+/−TGFA and genetically normal HPF-c.

Knock-down of transforming growth factor A (TGFA) was performed using siRNA targeting to TGFA and non-targeting control siRNA as a control. Just prior to imaging, HUVECs and HPF-c were mixed at a …

Figure 5 with 1 supplement
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Fibroblasts induce vascularization in a mouse xenograft model for vascular lesions.

(A) Subcutaneous injection of matrigel with HUVECs transduced with PIK3CAwt or PIK3CAH1047R encoding lentivirus vectors, with or without primary fibroblasts, was performed in athymic Nude-Foxn1nu …

Figure 5—figure supplement 1
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EGFR expression in mouse xenograft sections.

Subcutaneous injection of matrigel with HUVECs transfected with wt or PIK3CAH1047R expressing lentivirus vectors with/without primary fibroblasts was performed in athymic Nude-Foxn1nu mice. (A) …

Figure 6
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Afatinib reduces VEGF-A secretion, angiogenesis, and lesion size.

(A) Afatinib decreased EGFR/ErbB1-phosphorylation in rhTGFA-stimulated HPF-c cells. Total epidermal growth factor receptor (EGFR) was used to control equal loading of the samples. (B) VEGF secretion …

Figure 6—source data 1

Raw data for western blot images.

https://cdn.elifesciences.org/articles/82543/elife-82543-fig6-data1-v2.zip
Figure 7
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Afatinib reduces lesion size in matrigel plug-in assay.

Subcutaneous injection of matrigel with HUVECs transfected with PIK3CAH1047R expressing lentivirus vectors with primary fibroblasts was performed in athymic Nude-Foxn1nu mice. After lesions reached …

Figure 8
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Proposed model for the paracrine signaling of TGFA/VEGF-A in the vascular lesion.

Schematic illustration showing the general structure of venous malformation or angiomatosis of soft tissue. Pathological vasculature in the lesion (dark blue) is surrounded by a disorganized …

Tables

Table 1
Demographics of patients with AST patients.
PatientGenderAgePathological diagnosis# of lesionsTissue*LocationSomatic mutation(Fractional abundance §)
1 †, ‡F11AST1imcalfND
2 †,‡M34AST1imshoulderPI3KCA p.E542K (WTL: 10.25; EC: 50.65; SC: none)
3 †,†M16AST1imcalfPI3KCA p.H1047R (WTL: 18.80; EC: 44.00; SC: none)
4 †, ‡F16AST1scshinPI3KCA p.H1047L (WTL: 8.30; EC: 48.95; SC: none)
5†F17AST1imbackND
6M34AST1imthighND
7M17AST1imthighPI3KCA p.E542K (WTL: 7.32)
8F31AST1imthighPI3KCA p.H1047R (WTL: 5.07)
9F22AST1imthighPI3KCA p.H1047R (WTL: 13.10)
10F13AST1im, scfootPI3KCA p.E545K (WTL: 11.65)
11M19AST1imthighPI3KCA p.H1047R (WTL: 8.89)
12M13AST1im, scanklePI3KCA p.H1047R (WTL: 12.60)
13F23AST1imcalfPIK3CA p.Y644H
14F41AST1scshinND
15F25AST1imfootPI3KCA p.E545K (WTL: 8.93)
16F16AST1im, scanklePI3KCA p.H1047R (WTL: 5.98)
17F46AST1im, scbackPI3KCA p.E542K (WTL: 9.83)
18F18AST1im, scanklePI3KCA p.E545K (WTL: 11.35)
19F13AST1imcalfND
20F24AST1im, smthighPI3KCA p.E542K (WTL: 5.64)
  1. *

    Tissue: im, intramuscular; sc, subcutaneous; sm, synovial membrane.

  2. †

    ECs and SCs isolated for cell experiments.

  3. ‡

    used in RNA-seq experiment.

  4. §

    Fractional abundance of the mutation in WTL, whole tissue lysate; EC, endothelial cells; SC, intervascular stromal cells; ND, not detected.

  5. mutation detected by whole-exome sequencing.

Table 2
Demographics of patients with VM.
PatientGenderAgePathological diagnosis# of lesionsTissue*LocationSomatic mutation(Fractional abundance in whole tissue lysate) §
21M34VM1imthighTEK p.Y1108X
22 ,†, ‡F77VM6scneck, fossa cubitalis, chest, hip, big toeTEK p.L914F (5.63)
23M40VM2imchest, backND
24F69VM3im, scforearm **, handTEK p.L914F (10.01)
25M24VM2scankle, solePI3KCA p.H1047L (3.83)
26M14VM1sclipTEK p.L914F (16.31)
27F39VM1imchestND
28M28VM1scclavicleTEK p.L914F (5.95)
29M46VM1scshinND
30M31VM1sckneeTEK p.L914F (10.60)
31F21VM1im, sc, smkneeND
32M16VM1im, sc, smthigh, kneeND
33M9VM1sc, smkneeND
34F35VM1im, scbladeTEK p.L914F (12.10)
35F16VM1scanklePI3KCA p.E545K (4.34)
36M21VM/AST1imupper armPI3KCA p.H1047R (3.91)
37F41VM/AST1scankleKRAS p.Q61R
38F25VM/AST1sccalf, legPI3KCA p.H1047L (5.23)
  1. *

    Tissue: im, intramuscular; sc, subcutaneous; sm, synovial membrane.

  2. †

    ECs and SCs isolated for cell experiments.

  3. ‡

    used in RNA-seq experiment.

  4. §

    ND, not detected.

  5. mutation detected by whole-exome sequencing.

  6. **

    The patient had multiple lesions but only a lesion located in the forearm was operated.

Table 3
Selected cell signaling pathways regulated in patient-derived ECs.
GOTermGenes*
GO:0038127ERBB signaling pathwayPRKCE,TNRC6C,PDE1A,AREG,
RPS27A,TGFA,KITLG,ERBB2,
PTK2B,DGKD,PRKAR2B,NRG1,
RPS6KA5,PRKACB
GO:0043122regulation of I-kappaB kinase/
NF-kappaB signaling		GREM1,LURAP1,BIRC3,TNFAIP3,
S100A4,MALT1,LITAF,PRKCE,
LPAR1,C18orf32,ZFAND6,RPS27A,
PLK2,TLR4,F2RL1,CASP1,S100A13
GO:0008277regulation of G-protein coupled receptor protein signaling pathwayRGS4,RGS9,DYNLT1,RGS11,CXCL8,RGS10,RGS20,RGS5,RAMP2,RGS7,
PLCB1,ADRBK2
GO:0043410positive regulation of MAPK cascadePAK1,MAP4K2,SEMA4C,TGFA,
GADD45G,KSR2,ERBB2,NENF,
PLCB1,TPD52L1,GLIPR2,ICAM1,
CD74,PRKCE,LPAR1,PDCD10,INSR,
IGF1R,GADD45A,RPS27A,PTK2B,
KITLG,HGF,F2RL1,TLR4,PIK3CG,
ZEB2,EPGN
GO:0046328regulation of JNK cascadePAK1,MAP4K2,GADD45A,IGF1R,
GADD45G,TNXB,PTK2B,CBS,SFRP1,PLCB1,F2RL1,TLR4,TPD52L1,ZEB2
GO:0007219Notch signaling pathwayHEY2,NOTCH1,TNRC6C,RBX1,
TMEM100,LFNG,RPS27A,E2F1,
DTX3,NOTCH2,MESP1,DNER,
FOXC1,SNAI2,HDAC9
  1. *

    ErbB pathway receptors and ligands are marked in bold.

Table 4
Taqman assays used in qRT-PCR analysis.
GeneDescriptionAssay ID
GAPDHGlyceraldehyde-3-phosphate dehydrogenase4352934E
B2MBeta-2-microglobulinHs00187842_m1
TGFATransforming growth factor A [assay 1]Hs00608187_m1
TGFATransforming growth factor A [assay 2]Hs00177401_m1
TGFATransforming growth factor A [assay 3]HsaCEP0053322
ERBB1Protein tyrosine kinase ERBB1, epidermal growth factor receptorHs01076090_m1
AREGAmphiregulinHs00950669_m1
NRG1Neuregulin 1Hs00247620_m1
EPGNEpithelial mitogen, epigenHs02385424_m1
VEGF-AVascular endothelial growth factor AHs00900055_m1
PIK3CAPhosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit-αHsaCEP0050716
Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, female)Hsd: Athymic Nude-Foxn1nuEnvigoAutosomal recessive mutation on nu locus on chromosome 11; T-cell deficient; accepts xenograft transplantation.
Cell line (Homo sapiens)HUVECThis paperUmbilical cord endothelial cells isolated in house; see materials and methods section
Cell line (Homo sapiens)HSaVECPromoCell# C-12231Saphenous vein endothelial cells
Cell line (Homo sapiens)HPF-cPromoCell# C-12360Pulmonary fibroblast
Transfected construct (Homo sapiens)LV-PIK3CAThis paperPIK3CAwtLentiviral construct to transfect and express PIK3CA wild-type; produced using Addgene plasmid #116771
Transfected construct (Homo sapiens)LV-PIK3CA-H1047RThis paperPIK3CAH1047RLentiviral construct to transfect and express PIK3CA with oncogenic point mutation on p.H1047R; produced using Addgene plasmid #116500
Biological sample (Homo sapiens)Patients’ AST/VM tissueThis paperPatients’ lesionTissue material collected during surgery; see materials and methods section
Biological sample (Homo sapiens)Endothelial cells from patients’ AST/VMThis paperPatient EC (CD31+)Freshly isolated from AST/VM tissue; see materials and methods section
Biological sample (Homo sapiens)Stromal cells from patients’ AST/VMThis paperPatient SC (CD31-/ Vimentin+)Freshly isolated from AST/VM tissue; see materials and methods section
Recombinant DNA reagentpHAGE-PIK3CAAddgene# 116771Plasmid encoding PIK3CA wild-type
Recombinant DNA reagentpHAGE-PIK3CA-H1047RAddgene# 116500Plasmid encoding PIK3CA with oncogenic point mutation on p.H1047R
Sequence-based reagentsiRNA: nontargeting control #1Thermo Fisher Scientific# 4390844Silencer Select siRNA
Sequence-based reagentsiRNA: nontargeting control #2Thermo Fisher Scientific# 4390847Silencer Select siRNA
Sequence-based reagentsiRNA: targeting to human TGFAThermo Fisher ScientificAssay ID: s14053Silencer select siRNA
Sequence-based reagentPrimePCR ddPCR mutation detection assays for PIK3CA c.3140A>G (p.H1047R)Bio-RadAssay IDs: dHsaMDM5225715851 (mut) and dHsaMDW5225715853 (wt)
Sequence-based reagentPrimePCR ddPCR mutation detection assays for PIK3CA c.3140A>T (p.H1047L)Bio-RadAssay IDs: dHsaMDM2916088171 (mut) and dHsaMDW2916088173 (wt)
sequence-based reagentPrimePCR ddPCR mutation detection assays for PIK3CA c.1633G>A (p.E545K)Bio-RadAssay IDs: dHsaMDM9869636521 (mut) and dHsaMDW9869636523 (wt)
sequence-based reagentPrimePCR ddPCR mutation detection assays for PIK3CA c.1624G>A (p.E542K)Bio-RadAssay IDs: dHsaMDM3010833491 (mut) and dHsaMDW3010833493 (wt)
sequence-based reagentCustom-design Taqman SNP Genotyping assays for TEK c.2740C>T (p.L914F)Thermo Fisher Scientific# 4331349fwd 5’-CTTCCCTCCAGGCTACTT-3’, rev 5’-AATGCTGGGTCCGTCT-3’, reporter 1 (HEX) 5’-CTTGCGAAGGAAGTCCAGAAGGTTTC-3’, and reporter 2 (FAM) 5’- CTTGCGAAAGAAGTCCAGAAGGTTTC-3’
peptide, recombinant proteinrhTGFASigma-Aldrich# GF313
commercial assay or kitHuman CD31 microbead kitMiltenyi Biotec# 130-091-935
commercial assay or kitRNeasy Mini KitQiagen# 74106
commercial assay or kitHuman Quantikine TGFA ELISAR&D Systems# DTGA00
commercial assay or kitHuman Quantikine VEGF ELISAR&D Systems# DVE00
chemical compound, drugAfatinibMedChem Express# HY-10261
software, algorithmSproutAngiohttps://github.com/mbbio/SproutAngio
(Beter et al., 2023)
Open access tool for quantitation

Additional files

Supplementary file 1

NGS experiments.

https://cdn.elifesciences.org/articles/82543/elife-82543-supp1-v2.xlsx
Supplementary file 2

gBlock gene fragments.

https://cdn.elifesciences.org/articles/82543/elife-82543-supp2-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/82543/elife-82543-mdarchecklist1-v2.docx

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