In this study we have investigated the role of LRP1 for PI3K activation by PDGFRβ in SMCs, and the impact this LRP1 ‘checkpoint’ has for preventing atherosclerotic lesion formation and progression, as well as for the maintenance of vascular wall integrity. We found that the selective genetic blockade of PI3K activation by PDGFRβ substantially suppressed spontaneous atherosclerotic lesion development, which is prominent in smLRP1−/−; LDLR−/− mice. Furthermore, vascular wall elongation and medial thickening, due to SMC hyperproliferation, increased SMC migration and disruption of elastic layers are normalized throughout the entire aorta. Our findings suggest that PI3K is the main driving force that promotes SMC proliferation and migration, elastolysis, spontaneous atherosclerosis and lesion progression in the absence of LRP1.
Prominent atherosclerotic lesions preexisted in smLRP1−/−; LDLR−/−
mice maintained on standard, low-fat and cholesterol-free rodent chow, but not in smLRP1−/−
animals of comparable age. These data suggest that in the presence of an intact endothelium and low plasma cholesterol levels, proliferative signals alone are not sufficient to initiate the pathogenic mechanisms that culminate in classic atherosclerotic plaques. By contrast, the aorta of LRP1+/+; LDLR−/−
mice appears histoanatomically normal despite increased plasma cholesterol levels on the same chow, and extensive atherosclerotic lesions develop only after feeding of a high-cholesterol diet for several months 
. Thus, LRP1 in SMCs functions cell autonomously in the maintenance of vascular wall integrity and protection from cholesterol-induced atherosclerosis.
In the absence of smLRP1, the mouse aorta undergoes hyperplastic and hypertrophic changes that were apparent in young (7 weeks) as well as older (11 months old) mice indicating that they are not the result of aging, but the manifestation of an intrinsic change of smooth muscle phenotype. This is most likely caused by the increased expression and activation of PDGFRβ in smLRP1−/− mice and an accompanying increase in PI3K association with PDGFRβ. Disruption of an obligatory proatherogenic proliferative pathway, involving PI3K and PDGFRβ, prevents or greatly reduces lesion development at sites of high shear stress, such as the aortic arch and the abdominal aorta, where endothelial integrity is easily compromised. Thus, by selectively controlling SMC proliferation and migration independent of endothelial integrity and plasma cholesterol levels in a novel genetically complex animal model, we have been able to isolate and demonstrate the pivotal and interdependent roles of two central mechanisms of atherosclerotic lesion development.
Activation of the PDGFRβ results in actin reorganization in the forms of membrane ruffling and chemotaxis 
and thus provides an excellent functional assay for the physiological activation of PDGFRβ through other genetic manipulations, such as the disruption of LRP1. PI3K binding to the cytoplasmic domain of activated PDGFRβ receptors requires phosphorylation at residues 739 and 750 and this interaction in turn activates the kinase 
. Replacement of these tyrosines by non-phosphorylatable phenylalanines prevents binding of PI3K and fails to mediate membrane ruffling and cell migration 
. As a result, the pronounced edge ruffling and circular membrane ruffling as well as greatly enhanced SMC migration that were observed in the absence of LRP1 were virtually normalized in mice in which PDGFRβ-dependent PI3K activation had been genetically disrupted. These findings show that the membrane ruffling and increased smooth muscle migration in smLRP−/−
mice is critically dependent upon PI3K activation, which is mediated by PDGFRβ. Nevertheless, a caveat to this interpretation is that, although PI3K is the only known cellular signal transducer that interacts with pY739 and 750 of PDGFRβ, this do not exclude the possibility that another unknown signal modulator also interacts with this site and contributes to the pathogenic mechanism.
Marfan syndrome, a disorder of connective tissue architecture with prominent manifestations in the skeletal, ocular and cardiovascular systems, is caused by mutations in the fibrillin-1 gene 
or by loss of function mutations in TGFβ receptor I or II 
. TGFβ signaling is abnormally elevated in fibrillin-1-deficient mice 
and human aortas 
as well as TGFβ receptor I and II deficiency 
. Previous data from our laboratory have shown nuclear accumulation of phosphorylated Smad2, an indicator of activation of TGFβ signaling, in the LRP1(TβR-V)-deficient vascular wall 
. In the present study, we have reconfirmed these Marfan syndrome-like phenotypes, including elastic layer disruption, aorta elongation, and aneurysm formation in the presence of increased Smad2 phosphorylation when LRP1 is deficient in the SMCs. These phenotypic manifestations in the vascular wall were essentially abolished in smLRP1−/−; PDGFRβ F2/F2
mice, however, the increased phosphorylation and nuclear translocation of Smad2 was not affected by the PDGFRβ mutations. These findings indicate that TGFβ activation through LRP1 precedes PDGFRβ-dependent PI3K signaling, and that activation of TGFβ signaling by itself is not sufficient to disrupt the vascular wall architecture. PDGFRβ-dependent PI3K activation appears to be necessary for the expression of the Marfan-like phenotypes. Suppression of PI3K activation by PDGFRβ prevents the Marfan-like phenotypic changes in the vascular wall in the presence of unabated TGFβ signaling, suggesting a pivotal role of LRP1-controlled and PDGFRβ-dependent PI3K activation in the pathogenesis of Marfan syndrome. Selective elimination of PDGFRβ-dependent PI3K activation thus could be a potential therapeutic target for both atherosclerosis and Marfan syndrome.
In conclusion, the current study reveals a novel PI3K-dependent mechanism by which LRP1 is essential for controlling the integrity of the vascular wall, and by which this multifunctional receptor potently protects against atherosclerosis and Marfan syndrome. The findings we have presented here shed new light on the molecular mechanisms that control cellular growth and migration, and which are thereby essential to the remodeling and repair of the vascular wall and for slowing or preventing degenerative disorders of the vascular wall.