Overexpression of mutated IkappaBalpha inhibits vascular smooth muscle cell proliferation and intimal hyperplasia formation.
OBJECTIVE
Vascular injury and inflammation are associated with elaboration of a number of cytokines that signal through multiple pathways to act as smooth muscle cell (SMC) mitogens. Activation of the nuclear factor-kappa B (NF-kappaB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-kappaB is controlled by several upstream regulators, including the inhibitors of kappa B (IkappaB). These proteins bind to and keep NF-kappaB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IkappaBalpha super-repressor (AdIkappaBalphaSR) could inhibit development of intimal hyperplasia in vivo and to investigate how over-expression of this construct influences in vitro SMC proliferation and cell cycle regulatory proteins.


METHODS
A rat carotid injury model was used to study prevention of intimal hyperplasia. Arteries were assayed 14 days after injury and infection with AdIkappaBalphaSR or adenoviral beta-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIkappaBalphaSR were stimulated with 10% fetal bovine serum, interleukin-1beta, or tumor necrosis factor-alpha. Electrophoretic mobility shift assays were used to assay for NF-kappaB activation. Protein levels of IkappaBalpha and cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1) were determined with Western blot analysis. Proliferation was measured with (3)H-thymidine incorporation assays.


RESULTS
AdIkappaBalphaSR inhibited the development of intimal hyperplasia by 49% (P <.05). Infection with AdIkappaBalphaSR significantly suppressed in vitro SMC proliferation when stimulated with serum, interleukin 1, or tumor necrosis factor alpha, and did not result in cell death. Inhibition of proliferation was associated with increased p21(Cip1/Waf1) and p27(Kip1) protein levels.


CONCLUSIONS
Gene transfer of IkappaBalpha super-repressor inhibited development of intimal hyperplasia in vivo and SMC proliferation in vitro. The antiproliferative activity may be related to cell cycle arrest through upregulation of the cyclin-dependent kinase inhibitors p21 and p27. Overexpression of IkappaBalpha may be a future therapeutic option in treatment of vascular diseases.
Zuckerbraun
Brian S
BS
Department of Surgery, University of Pittsburgh School of Medicine, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA. zuckerbraun@msx.upmc.edu
McCloskey
Carol A
CA
Mahidhara
Raja S
RS
Kim
Peter K M
PK
Taylor
Bradley S
BS
Tzeng
Edith
E
D000256:Adenoviridae; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D002339:Carotid Arteries / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005312:Fetal Blood / Q000502:physiology; D005822:Genetic Vectors; D006965:Hyperplasia; D020672:I-kappa B Proteins / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D017539:Tunica Intima / Q000166:cytology*; D001616:beta-Galactosidase / Q000235:genetics / Q000502:physiology
C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007375:Interleukin-1; D015850:Interleukin-6; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D018809:Proliferating Cell Nuclear Antigen; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D001616:beta-Galactosidase