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Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
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Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth

Won Gil Cho1, Romulo J C Albuquerque, Mark E Kleinman

  • 1Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40536, USA.

Proceedings of the National Academy of Sciences of the United States of America
|April 11, 2009
PubMed

Insights

Short small interfering RNAs (siRNAs) activate Toll-like receptor 3 (TLR3), suppressing both blood and lymphatic vessel growth. This discovery offers a new therapeutic approach but also highlights potential risks of siRNA use.

Area of Science:

  • Endothelial biology
  • Immunology
  • RNA interference therapeutics

Background:

  • Neovascularization, involving blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessel growth, is crucial for tissue repair but also implicated in disease.
  • Small interfering RNAs (siRNAs) of 21 nucleotides or longer were previously shown to suppress hemangiogenesis via Toll-like receptor 3 (TLR3) activation, independent of RNA interference.
  • Toll-like receptor 3 (TLR3) is an immune receptor recognizing double-stranded RNA, expressed on the surface of endothelial cells.

Purpose of the Study:

  • To investigate the efficacy of 21-nucleotide (nt) nontargeted small interfering RNAs (siRNAs) in suppressing both hemangiogenesis and lymphangiogenesis.
  • To determine if TLR3 activation by siRNAs affects lymphatic endothelial cells and induces apoptosis.
  • To evaluate the potential of TLR3 activation as a unified strategy for inhibiting neovascularization and to assess associated risks.

Main Methods:

  • Utilized mouse models of neovascularization, including corneal sutures and hindlimb ischemia.
  • Administered 21-nt and 7-nt nontargeted siRNAs, as well as a 21-nt siRNA targeting vascular endothelial growth factor-A (VEGF-A).
  • Assessed neovascularization suppression and investigated siRNA-induced TLR3 activation, phosphorylation, and apoptosis in lymphatic endothelial cells.

Main Results:

  • A 21-nt nontargeted siRNA suppressed both hemangiogenesis and lymphangiogenesis as effectively as a VEGF-A targeting siRNA.
  • A 7-nt nontargeted siRNA, too short to activate TLR3, did not inhibit neovascularization.
  • Exposure to 21-nt siRNA triggered TLR3 phosphorylation on lymphatic endothelial cells and induced apoptosis, independent of cellular internalization.

Conclusions:

  • Activation of Toll-like receptor 3 (TLR3) by 21-nt siRNAs provides a method for jointly suppressing blood and lymphatic neovascularization.
  • These findings suggest a novel therapeutic strategy targeting TLR3 for controlling aberrant vessel growth.
  • The study raises concerns regarding potential adverse effects of siRNAs on both blood and lymphatic circulatory systems due to TLR3 activation.

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