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Updated: Aug 9, 2026
Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Published on: February 15, 2022
Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation
Henrik ten Freyhaus1, Michael Huntgeburth, Kirstin Wingler
1Klinik III für Innere Medizin der Universität zu Köln, Germany.
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).
Methods And 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.
Insights
The novel Nox inhibitor VAS2870 effectively blocks platelet-derived growth factor (PDGF)-induced reactive oxygen species (ROS) production in vascular smooth muscle cells (VSMC). While VAS2870 inhibits PDGF-driven VSMC migration, it does not impact DNA synthesis, highlighting a role for Src signaling.
Area of Science:
- Cardiovascular Research
- Cell Signaling
- Oxidative Stress Biology
Background:
- NAD(P)H oxidases (Nox) generate reactive oxygen species (ROS), critical in cardiovascular disease pathophysiology.
- Growth factors like platelet-derived growth factor (PDGF) regulate NAD(P)H oxidase expression and activity.
Purpose of the Study:
- To investigate the effects of the novel Nox inhibitor VAS2870 on PDGF-induced ROS production and cellular responses in vascular smooth muscle cells (VSMC).
Main Methods:
- Assessed NAD(P)H oxidase activity and intracellular ROS using lucigenin-enhanced chemiluminescence and 2,7-DCF.
- Quantified VSMC migration via modified Boyden chamber assays and DNA synthesis using BrdU incorporation.
- Analyzed PDGF-dependent signaling pathways (Akt, Erk, Src) using Western blot with phospho-specific antibodies.
Main Results:
- PDGF-BB significantly increased NAD(P)H oxidase activity and ROS levels in VSMC.
- VAS2870 completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production.
- PDGF-induced VSMC migration was completely inhibited by VAS2870, while DNA synthesis remained unaffected.
- VAS2870 blocked PDGF-dependent Src activation but not Akt or Erk activation.
Conclusions:
- VAS2870 is an effective inhibitor of growth factor-mediated ROS liberation in VSMC.
- The inhibitor selectively abrogates PDGF-dependent VSMC migration by targeting redox-sensitive Src activity, distinct from its effects on cell cycle progression.
- These findings underscore the critical role of Src signaling in PDGF-induced chemotaxis and its sensitivity to ROS modulation.
