P712Imatinib counteracts hypoxia-induced human vascular smooth muscle cell migration through LRP1-pPyK2-MMP-9 axis impairment

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Abstract

Purpose. Hypoxia-related vascular diseases are associated with an imbalance in vascular smooth muscle cell migration. Low-density lipoprotein receptor-related protein 1 (LRP1) is essential to promoting human VSMCs (hVSMCs) migration under hypoxic conditions since this receptor is a crucial player in hypoxia-induced proline-rich tyrosine kinase 2 (Pyk2) phosphorylation and metalloproteinase-9 (MMP-9) activation. Imatinib is a small molecule that blocks BCR/ABL oncogene. Recent studies showed a protective effect of Imatinib on atherosclerosis.The aim of this work was to study the effect of Imatinib in LRP1-pPyk2-MMP-9 axis activation and hVSMC migration induced by hypoxia in human VSMC.

Methods. The hVSMCs were maintained in medium 199 supplemented with 20 % fetal bovine serum, exposed to normoxia (21% O2) or hypoxia (1% O2) and treated with several Imatinib dose for different times. LRP1, pLRP1, pPyk2, total Pyk2, and β-tubulin protein levels were measured by Western blot. The formation of protein-protein complexes was analyzed by immunoprecipitation followed by western blot experiments. MMP-9 and MMP-2 mRNA expression and activity were tested by Real time PCR and zimography, respectively. Migration studies were performed using the technique of wound repair assays after cell monolayer scratching.

Results. Imatinib (20 μM) partially prevented hypoxia-induced LRP1 mRNA and LRP1 protein levels. In contrast, Imatinib did not exert any significant effect in other lipoprotein receptors such as very low density lipoprotein receptor (VLDLR) or low density lipoprotein receptor (LDLR). The inhibitor completely impaired hypoxia-induced LRP1 and Pyk2 phosphorylation in hVSMC. In line with this results, the high amounts of pLRP1/pPyk2 complexes induced by hypoxia were not present in Imatinib-treated hVSMC. MMP-9 expression and activation induced by hypoxia were abolished in Imatinib-treated hVSMC. In contrast, MMP-2 mRNA expression or MMP-2 activity were not altered by hypoxia or Imatinib treatment in hVSMC. Wound repair assays showed that Imatinib prevented the increase on hVSMCs migration induced by hypoxia.

Conclusions. Imatinib effectively prevented hypoxia-induced hVSMC migration. Therefore, Imatinib could be particularly useful to counteract the deleterious effects of hypoxia on vascular remodeling. These results highlight a new mechanism underlying the potential beneficial effects of Imatinib in atherosclerosis.

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