Interleukin 35 Delays Hindlimb Ischemia-Induced Angiogenesis Through Regulating ROS-Extracellular Matrix but Spares Later Regenerative Angiogenesis

Front Immunol. 2020 Oct 14;11:595813. doi: 10.3389/fimmu.2020.595813. eCollection 2020.


Interleukin (IL) 35 is a novel immunosuppressive heterodimeric cytokine in IL-12 family. Whether and how IL-35 regulates ischemia-induced angiogenesis in peripheral artery diseases are unrevealed. To fill this important knowledge gap, we used loss-of-function, gain-of-function, omics data analysis, RNA-Seq, in vivo and in vitro experiments, and we have made the following significant findings: i) IL-35 and its receptor subunit IL-12RB2, but not IL-6ST, are induced in the muscle after hindlimb ischemia (HLI); ii) HLI-induced angiogenesis is improved in Il12rb2-/- mice, in ApoE-/-/Il12rb2-/- mice compared to WT and ApoE-/- controls, respectively, where hyperlipidemia inhibits angiogenesis in vivo and in vitro; iii) IL-35 cytokine injection as a gain-of-function approach delays blood perfusion recovery at day 14 after HLI; iv) IL-35 spares regenerative angiogenesis at the late phase of HLI recovery after day 14 of HLI; v) Transcriptome analysis of endothelial cells (ECs) at 14 days post-HLI reveals a disturbed extracellular matrix re-organization in IL-35-injected mice; vi) IL-35 downregulates three reactive oxygen species (ROS) promoters and upregulates one ROS attenuator, which may functionally mediate IL-35 upregulation of anti-angiogenic extracellular matrix proteins in ECs; and vii) IL-35 inhibits human microvascular EC migration and tube formation in vitro mainly through upregulating anti-angiogenic extracellular matrix-remodeling proteins. These findings provide a novel insight on the future therapeutic potential of IL-35 in suppressing ischemia/inflammation-triggered inflammatory angiogenesis at early phase but sparing regenerative angiogenesis at late phase.

Keywords: IL-12Rβ2; IL-35; angiogenesis; endothelial cells; ischemia and hypoxia.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Apolipoproteins E / genetics
  • Cell Line
  • Cell Movement
  • Extracellular Matrix / immunology
  • Hindlimb / blood supply*
  • Humans
  • Interleukins / immunology*
  • Ischemia / immunology*
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Neovascularization, Pathologic
  • Neovascularization, Physiologic
  • Reactive Oxygen Species / immunology
  • Receptors, Interleukin-12 / genetics
  • Receptors, Interleukin-12 / immunology*


  • Apolipoproteins E
  • Il12rb2 protein, mouse
  • Interleukins
  • Reactive Oxygen Species
  • Receptors, Interleukin-12
  • interleukin-35, mouse