Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling

Sheng Xia, Heather L. Menden, Thomas R. Korfhagen, Tsutomu Kume, Venkatesh Sampath*

*Corresponding author for this work

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Key points: The mechanisms by which bacteria alter endothelial cell phenotypes and programme inflammatory angiogenesis remain unclear. In lung endothelial cells, we demonstrate that toll-like receptor 4 (TLR4) signalling induces activation of forkhead box protein C2 (FOXC2), a transcriptional factor implicated in lymphangiogenesis and endothelial specification, in an extracellular signal-regulated kinase (ERK)-dependent manner. TLR4-ERK-FOXC2 signalling regulates expression of the Notch ligand DLL4 and signals inflammatory angiogenesis in vivo and in vitro. Our work reveals a novel link between endothelial immune signalling (TLR pathway) and a vascular transcription factor, FOXC2, that regulates embryonic vascular development. This mechanism is likely to be relevant to pathological angiogenesis complicating inflammatory diseases in humans. Abstract: Endothelial cells (ECs) mediate a specific and robust immune response to bacteria in sepsis through the activation of toll-like receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis programme EC specification and altered angiogenesis remain unclear. We postulated that the forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung ECs, lipopolysaccharide (LPS) induced ERK phosphorylation, FOXC2, and delta-like 4 (DLL4, the master regulator of sprouting angiogenesis expression) in a TLR4-dependent manner. LPS-mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung ECs. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK inhibition. FOXC2 +/− mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2 +/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programmes EC specification and altered angiogenesis.

Original languageEnglish (US)
Pages (from-to)1397-1417
Number of pages21
JournalJournal of Physiology
Volume596
Issue number8
DOIs
StatePublished - Apr 15 2018

Fingerprint

Toll-Like Receptor 4
Extracellular Signal-Regulated MAP Kinases
Lipopolysaccharides
Endothelial Cells
Toll-Like Receptors
Phosphorylation
Forkhead Transcription Factors
Sepsis
Lung
Pathologic Neovascularization
Lymphangiogenesis
Ligands
Bacteria
Mitogen-Activated Protein Kinase 1
Threonine
Serine
Small Interfering RNA
Transcriptional Activation
Embryonic Development
Transfection

Keywords

  • FOXC2 and inflammatory angiogenesis
  • TLR and EC specification
  • TLR4 regulates FOXC2
  • TLR4-ERK-FOXC2-DLL4 axis

ASJC Scopus subject areas

  • Physiology

Cite this

@article{08cc20dd02f2401bb5af48179aa835de,
title = "Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling",
abstract = "Key points: The mechanisms by which bacteria alter endothelial cell phenotypes and programme inflammatory angiogenesis remain unclear. In lung endothelial cells, we demonstrate that toll-like receptor 4 (TLR4) signalling induces activation of forkhead box protein C2 (FOXC2), a transcriptional factor implicated in lymphangiogenesis and endothelial specification, in an extracellular signal-regulated kinase (ERK)-dependent manner. TLR4-ERK-FOXC2 signalling regulates expression of the Notch ligand DLL4 and signals inflammatory angiogenesis in vivo and in vitro. Our work reveals a novel link between endothelial immune signalling (TLR pathway) and a vascular transcription factor, FOXC2, that regulates embryonic vascular development. This mechanism is likely to be relevant to pathological angiogenesis complicating inflammatory diseases in humans. Abstract: Endothelial cells (ECs) mediate a specific and robust immune response to bacteria in sepsis through the activation of toll-like receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis programme EC specification and altered angiogenesis remain unclear. We postulated that the forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung ECs, lipopolysaccharide (LPS) induced ERK phosphorylation, FOXC2, and delta-like 4 (DLL4, the master regulator of sprouting angiogenesis expression) in a TLR4-dependent manner. LPS-mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung ECs. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK inhibition. FOXC2 +/− mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2 +/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programmes EC specification and altered angiogenesis.",
keywords = "FOXC2 and inflammatory angiogenesis, TLR and EC specification, TLR4 regulates FOXC2, TLR4-ERK-FOXC2-DLL4 axis",
author = "Sheng Xia and Menden, {Heather L.} and Korfhagen, {Thomas R.} and Tsutomu Kume and Venkatesh Sampath",
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Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling. / Xia, Sheng; Menden, Heather L.; Korfhagen, Thomas R.; Kume, Tsutomu; Sampath, Venkatesh.

In: Journal of Physiology, Vol. 596, No. 8, 15.04.2018, p. 1397-1417.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling

AU - Xia, Sheng

AU - Menden, Heather L.

AU - Korfhagen, Thomas R.

AU - Kume, Tsutomu

AU - Sampath, Venkatesh

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AB - Key points: The mechanisms by which bacteria alter endothelial cell phenotypes and programme inflammatory angiogenesis remain unclear. In lung endothelial cells, we demonstrate that toll-like receptor 4 (TLR4) signalling induces activation of forkhead box protein C2 (FOXC2), a transcriptional factor implicated in lymphangiogenesis and endothelial specification, in an extracellular signal-regulated kinase (ERK)-dependent manner. TLR4-ERK-FOXC2 signalling regulates expression of the Notch ligand DLL4 and signals inflammatory angiogenesis in vivo and in vitro. Our work reveals a novel link between endothelial immune signalling (TLR pathway) and a vascular transcription factor, FOXC2, that regulates embryonic vascular development. This mechanism is likely to be relevant to pathological angiogenesis complicating inflammatory diseases in humans. Abstract: Endothelial cells (ECs) mediate a specific and robust immune response to bacteria in sepsis through the activation of toll-like receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis programme EC specification and altered angiogenesis remain unclear. We postulated that the forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung ECs, lipopolysaccharide (LPS) induced ERK phosphorylation, FOXC2, and delta-like 4 (DLL4, the master regulator of sprouting angiogenesis expression) in a TLR4-dependent manner. LPS-mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung ECs. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK inhibition. FOXC2 +/− mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2 +/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programmes EC specification and altered angiogenesis.

KW - FOXC2 and inflammatory angiogenesis

KW - TLR and EC specification

KW - TLR4 regulates FOXC2

KW - TLR4-ERK-FOXC2-DLL4 axis

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