Vascular SMC migration contributes to the intimal thickening in occlusive vascular diseases. Following vascular injury, SMC migratory response to various stimuli begins with activation of cell surface receptors followed by remodeling events that involve focal adhesion complex and filament actin remodeling. Various cytokines, peptide growth factors, and components of the extracellular matrix (ECM) have been characterized as pro-migratory molecules.
PDGF is a very potent stimulant for SMC migration through activation of several signaling pathways [26–28]. In vascular SMC, PDGF activates multiple signaling cascades including the PI3K/Akt, protein kinase A (PKA), Src, Ras, and MAPKs signaling. The family of MAPKs comprises three distinct protein kinases, p38 MAPK, JNK, and Erk1/2, all of which have been shown to be involved in vascular diseases and remodeling .
On another note, integrin clustering and its downstream signals are necessary for SMC adhesion to ECM, an event that also initiates pro-migratory responses within the vascular wall. Mawatari and colleagues have shown that in human saphenous vein SMCs beta-3 integrin is required for growth factor and cytokine-induced proliferation . It is believed that a coordinated cross-talk between integrins and growth factor tyrosine kinase receptors is necessary for the effective modulation of cell survival, growth, proliferation, and migration .
ILK is a key protein of focal adhesion complex connecting β1 and β3 integrins in plasma membrane to the actin cytoskeletal machinery. ILK also interacts with growth factor receptors through the NH2-terminal ankyrin repeat domain and small GTPase signaling molecules such as Nck-2 [14, 31]. Previous reports on the effect of PDGF on ILK expression and activity in non-muscle cell cultures were controversial. While Campana et al reported on a PDGF-induced increase in ILK activity in primary rat Schwann cells , Janji and colleagues proposed that PDGF had no effect on ILK expression in melanoma HT-144 cells , indicating that the outcome of PDGF treatment on ILK signaling is cell specific.
In the present study, we have shown that PDGF-BB treatment results in an increase in ILK kinase activity in mouse aortic SMC culture. It is of importance that the increase in ILK kinase activity in SMCs is associated with a considerable increase in expression of both β1 and β3 integrins (Esfandiarei & van Breemen, unpublished observation). It is well known that the adhesive function of integrin receptors affects cytoskeleton rearrangement and cell motility. Therefore, it is presumable that increase in β1 and β3 integrins expression contributes to the pro-migratory effect of PDGF-BB in SMC culture. However, further investigation is required to evaluate the potential cross-talk between β1 and β3 integrins and PDGF receptors and the significance of such events in vascular SMC migration.
Numerous studies have provided a wealth of information on the crucial regulatory role for ILK as a protein kinase and also as a molecular scaffold and adaptor protein during cell spreading and migration in a variety of cancer cells [13, 34]. Moreover, A recent study has shown that an increase in ILK expression coincides with higher rate of SMC migration within the atherosclerotic plaque in ApoE -/- mice, a reliable model that mimics the progression of atherosclerosis in humans . On the contrary, Ho et al has shown that following balloon catheter injury of the rat carotid artery, a significant decrease in ILK expression is associated with an elevation in vascular SMC proliferation and migration . Nevertheless, there is no clear understanding as to how ILK contributes to the intimal thickening in response to growth factors and cytokines released during the development of the atherosclerotic plaque, and the mechanisms by which ILK may modulate vascular SMC migration to the sub-intimal layer of the vascular wall.
In this study, we examined the role of ILK in cell migration, by inhibiting (using siRNA) or increasing (using an adenoviral vector expressing wild type ILK) ILK expression. Inhibition of ILK by different means significantly decreased cell migration in response to PDGF-BB, demonstrating that ILK plays an important role in regulating PDGF-induced vascular SMC migration. Expression of a wild type form of ILK increased SMC migration even in the absence of growth factor. It is noteworthy that the effect of wild type ILK in increasing SMC migration is more evident when SMCs were treated with PDGF-BB (figure 6A). These observations are in agreement with previous report by Dwivedi et al  that over-expression of a dominant negative form of ILK markedly reduced intimal thickening in human saphenous vein organ culture.
ILK mutation within the catalytic domain (E359K) disrupts the interaction and binding of ILK with two important components of focal adhesion complex, β-parvin and paxillin. This interaction is required for the assembly of a functional focal adhesion complex and subsequent actin polymerization and reorganization [38, 39]. Over-expression of the E395K mutant of ILK resulted in significant reduction in PDGF-induced SMC migration. The effect was fully reversed by a wild type form of ILK. These findings further underscore the crucial function of ILK as molecular scaffold during SMC migration.
In an effort to understand how ILK mediates the pro-migratory effect of PDGF-BB in primary mouse SMC using RNA silencing technique, we were able to show that ILK inhibition could lead to a considerable decrease in p38 MAPK phosphorylation, while having no visible effect on Erk1/2 and JNK phosphorylation in response to PDGF-BB treatment. We also demonstrated that over-expression of an active form of MKK3/6 (specific upstream activator of p38 MAPK) dramatically reversed the inhibitory effect of ILK inhibition on SMC migration. Expression of a wild type form of p38 MAPK markedly increased SMC migration in the presence of PDGF-BB; however, it could not restore migratory response in cells over-expressing a kinase deficient form of ILK. It is of importance that the latter observation accentuates the view that the presence of a functional ILK is required for the initiation of a p38 MAPK-dependent migratory response to PDGF-BB in mouse aortic SMCs. Further studies are required to characterize the precise mechanisms underlying the ILK-mediated regulation of p38 MAPK in SMCs.
Actin polymerization and reorganization is a crucial step in cell migration. We have shown that PDGF increases actin polymerization and filament reorganization in SMCs. This event was significantly blocked by both ILK siRNA and p38 MAPK inhibitor, confirming the importance of this pathway in regulating SMC migration. The actual mechanism by which p38 MAPK may regulate cell migration is not well understood. Actin polymerization and reorganization is under rigorous regulation by a group of protein kinases and/or phosphatases that coordinate the constant turnover of actin filaments in a stimulated cell. Among these, cofilin is a conserved actin-binding protein that enhances actin filament reorganization through de-polymerization and severing of preexisting actin filaments . It is well known that phosphorylation of cofilin (serine 3) by LIM kinase (LIMK) and testicular protein kinase 2 (TESK-2) results in deactivation of cofilin and subsequent inhibition of actin reorganization [41, 42]. In mouse aortic SMCs, PDGF treatment resulted in rapid de-phosphorylation and activation of cofilin around 5-15 minutes post-treatment (additional file 3, figure S3). PDGF also increased LIMK phosphorylation leading to phosphorylation and deactivation of cofilin (additional file 3, figure S3). The feedback mechanism provides a strict control system for cell responses to migratory stimuli such as PDGF. Our preliminary observation suggests that p38 MAPK may control actin reorganization partly via regulating the de-phosphorylation and consequent activation of cofilin (Esfandiarei & van Breemen, unpublished observation). Further detailed studies are ongoing in our laboratory to characterize the signaling network and downstream pathways mediating such regulatory events.