Flt-1 expression influences apoptotic susceptibility of vascular smooth muscle cells through the NF-kappaB/IAP-1 pathway.
OBJECTIVE
Flt-1 is an fms-like tyrosine kinase receptor which binds to vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Ligand activation and blocking of flt-1 influence several vascular smooth muscle cell (SMC) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis have still to be investigated.


RESULTS
Flt-1 expression and apoptosis in Wistar rat aortic intimal cells 15 days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, western blotting, and PCR. Anti-flt1 blocking antibody effects were compared with those of anti-PlGF and anti-VEGF antibodies. Rat aortic intimal cells 15 days after injury exhibited increased flt-1 protein and mRNA and lower smooth muscle markers compared with normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC(50) = 16.5 ng/mL) and anti-PlGF (EC(50) = 20.5 ng/mL) antibodies added to intimal cultures reduced serum-deprived apoptosis but not serum- and PDGF-BB-induced proliferation; the anti-VEGF antibody was ineffective. Sorted flt-1(+) cells were more clonogenic than flt-1(-) and whole intimal SMC populations. Increased nuclear factor-kappaB (NF-kappaB) and inhibitor of apoptosis protein-1 (IAP-1) and reduced bax levels associated with the anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-kappaB activity inhibitor and IAP-1 interfering RNA (RNAi). Blocking of NF-kappaB activity reduced IAP-1 expression and prevented IAP-1 RNAi effects. Increased flt-1 immunoreaction was also documented in human atheromatous lesions.


CONCLUSIONS
Our results show that anti-flt-1 blocking reduces apoptosis through NF-kappaB and the downstream IAP-1 pathway. The close link between flt-1, PlGF, and apoptotic susceptibility of intimal SMCs suggests new potential strategies aimed at influencing post-injury arterial remodelling.
Orlandi
Augusto
A
Department of Biopathology and Image Diagnostics, Anatomic Pathology, Tor Vergata University of Rome, Via Montpellier, Rome 00133, Italy. orlandi@uniroma2.it
Ferlosio
Amedeo
A
Arcuri
Gaetano
G
Scioli
Maria Giovanna
MG
De Falco
Sandro
S
Spagnoli
Luigi Giusto
LG
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000502:physiology*; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000473:pathology*; D016328:NF-kappa B / Q000502:physiology*; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000032:analysis / Q000235:genetics / Q000502:physiology*
D051034:Inhibitor of Apoptosis Proteins; D016328:NF-kappa B; C000608036:PGF protein, human; C000608038:Pgf protein, rat; D011257:Pregnancy Proteins; D000072483:Placenta Growth Factor; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; C494836:Birc3 protein, rat; C501164:Flt1 protein, rat; D040281:Vascular Endothelial Growth Factor Receptor-1