Previous studies have demonstrated that medial SMCs start to migrate toward the intima in the early phase (usually between 3 and 7 days) after vascular injury, then proliferate there to constitute the neointima [23
]. PN has been shown to serve as a ligand for select integrins including αν
β3 and αν
β5, both integrin receptors are upregulated at early time points after vascular injury [3
]. Consistent with the previous observations in rat carotid arteries after balloon injury [9
], we observed that PN expression is upregulated in mouse carotid arteries after wire injury, especially in the early phase (3d, 7d), and the upregulation is more prominent in the atherogenic apoE−/− mice than in normal C57Bl6 mice (Supplemental Fig. 1
). Therefore, we speculate that the enhanced PN in injured vessel wall may stimulate medial SMC migration via interaction with integrin receptors αν
β3 (and αν
β5), eventually contributing to neointima formation after vascular injury (). In this regard, the present study was conducted to explore the molecular mechanism by which the integrin–PN interaction mediates vascular SMC migration using well-defined cell culture and gene transfection system.
Fig. 6 Hypothetical mechanism whereby PN plays a role in the process of SMC migration and neointima induced by vascular injury. PN expression is induced in the injured arterial wall in the early phase after vascular injury. PN stimulates medial SMC migration (more ...)
Our study is the first to provide several lines of evidence demonstrating a significant role for PN in the regulation of SMC migration in vitro. We show that (1) aortic SMCs isolated from PN−/− mice exhibit a significantly reduced ability to migrate and proliferate in vitro, (2) adenovirus-mediated overexpression of PN resulted in a robust migration to a similar extent in PN−/− and wildtype mouse SMCs in the wound migration assay, and (3) recombinant human PN (rhPN) stimulated hASMC migration in a dose-dependent manner in the haptotaxis assay. It is well known that different ECM proteins stimulate SMC migration through distinct integrins. For example, vitronectin and osteopontin stimulate SMC migration predominantly through αν
β3 integrin, whereas fibronectin and collagen stimulate SMC migration through β1 integrins (e.g. α2β1 and α5β1) [26
]. PN is a unique ligand for integrins in that it does not contain an Arg-Gly-Asp (RGD) sequence motif, instead it binds to integrins through a fasciclin I (FAS1) domain [29
]. PN has been shown to be a ligand for several integrins in human tumors, endothelial cells, and human autosomal dominant polycystic kidney cells [9
]. Lindner et al. reported that overexpression of periostin enhanced cell migration in C3H10T1/2 cells (mouse fibroblast cell lines) [10
]. However, little is known about which integrin receptor(s) mediates migration of vascular SMCs induced by PN. The present study extends previous studies by demonstrating that interactions of PN-αν
β3, and to a lesser extent PN-αν
β5, but not PN-β1, are required for migration of human SMCs in vitro. It is worth to note that while our data support the view that such integrin subsets are needed for PN-induced migration (i.e. the role of integrins), more work still needs to understand whether PN–integrin interaction is required for PN-dependent migration and the relative effect of the same anti-integrin antibodies on cell migration under more complex conditions in the presence of serum or PDGF, or other integrin ECM ligands. Although it is well established that integrin αν
β3 in the vessel wall is upregulated in response to vascular injury, the mechanism remains poorly understood. In cultured vascular SMCs, αv
expression appears to be stimulated by PDGF-BB, thrombin, and osteopontin [28
]. In this study, we show that the adenoviral overexpression of PN resulted in an increase in expression of β3-integrin in cultured mouse SMCs. Therefore, we speculate that the enhanced PN in the injured arterial wall might contribute to the upregulation of αν
β3 integrin in the response to vascular injury.
FAK is a non-receptor cytoplasmic protein tyrosine kinase that localizes to focal adhesion plaques, it becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix proteins [32
]. A critical event in integrin-mediated FAK signaling is phosphorylation of Tyr397 [33
]. When phosphorylated, Y397 becomes a binding site for the tyrosine kinase Src, which phosphorylates FAK at Y576 and Y577 to further activate FAK kinase activity [32
]. Several recent studies have demonstrated a role for PN in integrin-mediated FAK signaling. Shimazaki et al. [34
] showed that PN is essential for cardiac healing after acute myocardial infarction through FAK-alphaV-integrin signaling. Cardiac healing was impaired in PN−/− mice, caused by a reduced number of alpha smooth muscle actin-positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes can be rescued by gene transfer of PN. The inhibition of FAK or alphaV-integrin blocked the PN-promoted cardiac fibroblast migration. Shao et al. [35
] showed that PN-stimulated FAK tyrosine phosphorylation in cultured human microvascular endothelial cells via αν
β3 integrin. Altogether these observations reveals that alphaV-integrin and FAK are involved in PN signaling. In the present study, we extend previous findings by demonstrating that activation of FAK pathway is required for PN-mediated SMC migration in vitro. Our results show that (1) the phosphorylation levels of FAK at Tyr397 was reduced in PN−/− SMCs after transient stimulation with PDGF-BB, and (2) the adenovirus-mediated overexpression of PN enhanced the phosphorylation levels of FAK at Tyr397 in Ad-PN infected wildtype SMCs. One limitation of our study was the lack of data about the effect of PN overexpression on FAK phosphorylation in PN−/− SMCs. Furthermore, we investigated the involvement of FAK/FRNK in the PN-induced cell migration in cultured human hASMCs. FAK activity is known to be required for integrin- and growth factor-induced SMC migration. It is well characterized that FRNK, an endogenous inhibitor of FAK, inhibits integrin-mediated cell migration through blocking FAK activation and FAK-mediated signaling [16
]. Our data show that the rh-PN can induce FAK (Tyr397) phosphorylation and cell migration in cultured hASMCs, and these effects can be inhibited by means of overexpressing FRNK using Ad-FRNK gene transfer. Taken together, these findings reveal a mechanism whereby enhanced PN expression mediates vascular SMC migration through interaction with alphaV-integrins (mainly αν
β3) and subsequent activation of FAK.
In conclusion, the present study demonstrates for the fist time that enhanced PN expression plays a significant role in vascular SMC migration induced either by mechanic injury or PDGF stimulation, through interaction with alphaV-integrins (mainly ανβ3) and subsequent activation of FAK pathway. Whether the enhanced PN in the injured vessel wall substantially contributes to the development of neointima formation in vivo is now under investigation.