Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation.
OBJECTIVE
Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC).


RESULTS
PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229+/-9% and 362+/-54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 microM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2+/-0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2+/-0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 microM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 microM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner.


CONCLUSIONS
VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which-in contrast to Akt and Erk-appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.
Klinik III für Innere Medizin der Universität zu Köln, Germany.
Henrik
H
ten Freyhaus
Michael
M
Huntgeburth
Kirstin
K
Wingler
Jessika
J
Schnitker
Anselm T
AT
Bäumer
Marius
M
Vantler
Mohamed M
MM
Bekhite
Maria
M
Wartenberg
Heinrich
H
Sauer
Stephan
S
Rosenkranz
D000818:Animals; D001013:Aorta, Thoracic; D000077214:Becaplermin; D001583:Benzoxazoles / Q000494:pharmacology*; D015153:Blotting, Western / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000502:physiology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology*; D019061:src-Family Kinases / Q000378:metabolism
C511179:3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine; D001583:Benzoxazoles; D004791:Enzyme Inhibitors; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species; D014230:Triazoles; D000077214:Becaplermin; D004247:DNA; D019255:NADPH Oxidases; D019061:src-Family Kinases