The capacity of vascular SMCs to modulate their phenotype in response to environmental stimuli plays a critical role in the homeostatic response of the cardiovascular system. ILK is ideally positioned in the vascular SMC to transduce extracellular signals received from the extracellular membrane through β-integrin cell surface receptors.44
It plays a role in the phosphorylation of vital substrates in the SMC which regulate cell morphology, migration, contraction, proliferation and survival.13, 45, 46
Therefore, it was of interest to examine the vascular phenotype of mice in which the Ilk
gene was selectively ablated in vascular SMCs. Surprisingly, Ilk
conditional mutant mice survived to birth, but succumb in the immediate postnatal period coincident with redirection of the fetal circulation. Ilk
conditional mutant mice exhibit profound abnormalities in structural organization of great arteries including the aorta and ductus arteriosus. Defects in morphogenetic development of the aorta were observed as early as E12.5. Examination of large elastic arteries in Ilk
mutant embryos and pups revealed profound disruption of the tunica media. SMC-specific deletion of ILK alters RhoA activation and cytoskeletal dynamics which is transduced, at least in part, to the nucleus via MRTF-A attenuating the transcription and expression of SMC genes associated with the contractile SMC phenotype. These data demonstrate that ILK plays a critical role in morphogenetic development of large elastic arteries and that ablation of the Ilk
gene in vascular SMCs severely disrupts the structural integrity of arterial structures required for postnatal survival.
Prior studies in genetically targeted Ilk
mice revealed a critical role for ILK in cardiovascular biology. Endothelial-specific deletion of the murine Ilk
gene inhibits vascularization and results in embryonic lethality.19
Targeted ablation of ILK in the murine heart ultimately results in a dilated cardiomyopathy and congestive heart failure.20
With regard to SMC biology, one prior study has demonstrated that angiotensin II increases ILK protein expression and kinase activity in vitro.24
Adenoviral gene transfer experiments using a dominant-negative construct suggested that ILK is necessary for angiotensin II-mediated SMC migration and proliferation in vitro. Other investigators have demonstrated that ILK may modulate calcium-independent myosin-mediated contraction of triton-skinned rat caudal artery preparations. More recently, it was also demonstrated that ILK protein levels were decreased following balloon injury of the rat carotid artery.25
ILK knockdown with a RNA silencing led to augmented cell movement and enhanced wound closure in an in vitro model, which contrasts with the prior published study which employed adenoviral over-expression constructs and found diminished SMC migration.24
The present study thus extends the prior work by specifically defining a role for ILK in the morphogenetic development of the aorta and in the structural integrity of large elastic arteries in vivo. In this regard it is noteworthy that alterations in development of the aorta may result from vascular SMC autonomous defects directly attributable to ILK signaling and/or indirectly via alterations in blood flow resulting from alterations in vascular SMC phenotype/contractility or vascular SMC-ECM interactions.
A recent study evaluated platelet derived growth factor receptor (PDGFR)–B-driven deletion of ILK in multiple vascular wall cells including dermal pericytes and vascular smooth muscle cells, resulting in embryonic demise associated with local hemorrhage and edema first evident around embryonic day E13.5.26
While extensive pathology in the embryonic dermal vasculature was noted, no pathology of the great vessels was described using the PDGFR-B-driven deletion of ILK in mice. Of note, the investigators also described a mechanism by which ILK knockdown in immortalized, murine SMCs activated Rho/ROCK signaling and induced the phosphorylation of myosin light chain, and abnormally enhanced VSMC contraction in vitro and in vivo. However, the observation of down-regulation of contractile SMC genes in the mutant ductus arteriosus (and aorta) of SM22Cre+/IlkFl/Fl
mice raises questions about the relevance of this finding in vivo. Undoubtedly, the use of different cell types and promoters contribute to these different findings, but together the data underscore a clear role for ILK in SMC homeostasis.
Our in vivo and in vitro results demonstrate that SMC-specific deletion of ILK represses the transcription and expression of SMC genes which are required for the maintenance of the contractile SMC phenotype. A particularly novel finding in ILK deletion in SMC in vitro relates to the observed effects on MRTF–A, which is believed to transduce Rho GTPase-dependent signaling from the cell membrane to the nucleus, and is critical to maintenance of the contractile SMC phenotype.1
Our present studies are consistent with prior work demonstrating that ILK deletion in leukocytes modulates the activationof small GTPases, which are also important for membraneruffling and lamellipodia formation, as well as chemokine-triggered cell movement. It has also been shown that pharmacological agents that inhibit actin polymerization and/or forced expression of nonpolymerizing actin mutant proteins stimulate the export of MRTF-A.1
However, disruption of MRTF-A signaling alone cannot explain the vascular derangements observed in ILK conditional mutant mice, as mice harboring a null mutation in MRTF-A appear phenotypically normal. This is not surprising as activity of SRF is modulated by multiple overlapping and redundant signaling pathways including signals transduced via the related transcriptional co-activators myocardin and MRTF-A.1
Additional abnormalities observed in the ILK-deficient mice may also be contributing in causal pathways contributing to the vascular pathology. Confocal imaging of the vessel wall demonstrated striking disruption of the extracellular matrix, consistent with prior studies demonstrating a role for ILK in FN matrix assembly.47
Matrix abnormalities could, which in turn, be influencing cell migration and proliferation in the ILK-deficient aortas. The observed changes in elastin staining are similarly notable, since elastin is known to affect actin polymerization through an integrin-independent but a RhoA-dependent pathway.48
It is tempting to speculate that these disruptions of vascular SMC-ECM signaling in SM22Cre+Ilk Fl/Fl
mutant embryos may have led to compensatory expression of fibrillin-1 highlighting the complex, but critical role that ECM signaling plays in regulating development of the cardiovascular system.
While our studies provide unequivocal evidence that ILK plays a critical role in the morphogenetic development and structural organization of the aorta required for survival beyond the perinatal period, the role of ILK in maintenance of cardiovascular homeostasis remains to be determined. Given these data and the critical role that ILK plays in transduction of external signals and the phenotypic abnormalities observed in ILK-deficient aortic SMCs, it is tempting to speculate that ILK may be involved directly or indirectly in the pathogenesis of vascular proliferative syndromes including atherosclerosis and/or the pathogenesis of congenital or acquired vascular aneurysms. In support of this hypothesis, we have recently observed that ILK expression is markedly diminished in a mouse model of abdominal aortic aneurysm disease (Online Fig. V
). In any case, further studies examining the function of ILK in the postnatal vasculature are warranted.