Aims 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 (SMCs) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis are still uninvestigated. Methods Flt-1 expression and apoptosis in Wistar rat aortic intimal cells fifteen days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, Western blot and PCR. Anti-flt1 blocking antibody effects were compared to those of anti-PlGF and anti-VEGF antibodies. Results Rat aortic intimal cells fifteen days after injury exhibited increased flt-1 protein and mRNA and low smooth muscle markers compared to normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC50=16.5 ng/ml) and anti-PlGF (EC50=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-B), Inhibitor of Apoptosis Protein-1 (IAP-1) and reduced bax levels associated to anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-B activity inhibitor and IAP-1 interfering RNA. Blocking of NF-B activity reduced IAP-1 expression and prevented IAP-1 interfering RNA 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-B and 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, A., Ferlosio, A., Arcuri, G., Scioli, M., De Falco, S., Spagnoli, L.g. (2010). Flt-1 expression influences apoptotic susceptibility of vascular smooth muscle cells through the NF-kappaB/IAP-1 pathway. CARDIOVASCULAR RESEARCH, 85, 214-223 [10.1093/cvr/cvp288].
Flt-1 expression influences apoptotic susceptibility of vascular smooth muscle cells through the NF-kappaB/IAP-1 pathway.
ORLANDI, AUGUSTO;FERLOSIO, AMEDEO;SPAGNOLI, LUIGI GIUSTO
2010-01-01
Abstract
Aims 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 (SMCs) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis are still uninvestigated. Methods Flt-1 expression and apoptosis in Wistar rat aortic intimal cells fifteen days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, Western blot and PCR. Anti-flt1 blocking antibody effects were compared to those of anti-PlGF and anti-VEGF antibodies. Results Rat aortic intimal cells fifteen days after injury exhibited increased flt-1 protein and mRNA and low smooth muscle markers compared to normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC50=16.5 ng/ml) and anti-PlGF (EC50=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-B), Inhibitor of Apoptosis Protein-1 (IAP-1) and reduced bax levels associated to anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-B activity inhibitor and IAP-1 interfering RNA. Blocking of NF-B activity reduced IAP-1 expression and prevented IAP-1 interfering RNA 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-B and 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.