title: Knockdown of mouse VCAM-1 by vector-based siRNA.
abstract: Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.
authors.affiliation: Department of Immunology, Division of Medicine, Imperial College London, Hammersmith Campus, London W12 0NN, UK.
authors.forename: A K M Shamsul
authors.initials: AK
authors.lastname: Alam
authors.forename: Oliver
authors.initials: O
authors.lastname: Florey
authors.forename: Michele
authors.initials: M
authors.lastname: Weber
authors.forename: Radhakrishna G
authors.initials: RG
authors.lastname: Pillai
authors.forename: Cliburn
authors.initials: C
authors.lastname: Chan
authors.forename: Peng H
authors.initials: PH
authors.lastname: Tan
authors.forename: Robert I
authors.initials: RI
authors.lastname: Lechler
authors.forename: Myra O
authors.initials: MO
authors.lastname: McClure
authors.forename: Dorian O
authors.initials: DO
authors.lastname: Haskard
authors.forename: Andrew J T
authors.initials: AJ
authors.lastname: George
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D003315:Cornea / Q000378:metabolism; D017931:DNA Primers; D042783:Endothelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005821:Genetic Techniques / Q000009:adverse effects*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D018384:Oxidative Stress / Q000502:physiology; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D017931:DNA Primers; D034741:RNA, Small Interfering; D019010:Vascular Cell Adhesion Molecule-1