Vascular smooth muscle cell proliferation and regrowth after mechanical injury in vitro are Egr-1/NGFI-A-dependent.
Smooth muscle cell (SMC) proliferation is a key event in renarrowing of blood vessels after balloon angioplasty. Mechanical injury imparted to the arterial wall in experimental models induces the expression of the immediate-early gene, egr-1. Egr-1 binds to and activates expression from the proximal promoters of multiple genes whose products can, in turn, influence the vascular response to injury. Here, we used antisense strategies in vitro to inhibit rat vascular SMC proliferation by directly targeting Egr-1. A series of phosphorothioate antisense oligonucleotides of 15 base length and complementary to various theoretically accessible regions within Egr-1 mRNA were synthesized and assessed for their ability to selectively inhibit SMC proliferation in an Egr-1-dependent manner. Western blot analysis revealed that two oligonucleotides, AS2 and E11, inhibited Egr-1 synthesis in cells exposed to serum without affecting levels of the zinc finger protein Sp1. AS2 and E11 inhibited serum-inducible [(3)H]thymidine incorporation into DNA, as well as serum stimulation of total cell numbers. Size-matched phosphorothioate oligonucleotides with random, scrambled, sense or mismatch sequences failed to inhibit. Antisense Egr-1 inhibition was nontoxic and reversible. These oligonucleotides also inhibited SMC regrowth after mechanical injury in vitro. Egr-1 thus plays a key regulatory role in SMC proliferation and repair following injury.
Santiago
F S
FS
Centre for Thrombosis and Vascular Research, The University of New South Wales, Sydney, Australia Sydney, Australia.
Atkins
D G
DG
Khachigian
L M
LM
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051766:Early Growth Response Protein 1; D016650:Fluorescein-5-isothiocyanate; D015870:Gene Expression / Q000187:drug effects; D017874:Immediate-Early Proteins; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000293:injuries / Q000378:metabolism; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000493:pharmacokinetics; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis; D012333:RNA, Messenger / Q000187:drug effects; D051381:Rats; D013873:Thionucleotides / Q000493:pharmacokinetics / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D001798:Blood Proteins; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D016376:Oligonucleotides, Antisense; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D013873:Thionucleotides; D014157:Transcription Factors; C075278:platelet-derived growth factor A; D016650:Fluorescein-5-isothiocyanate