PDGFRb-dependent PI3K activation had been genetically disrupted. These findings show that the membrane ruffling and increased smooth muscle migration in smLRP2/2 mice is critically dependent upon PI3K activation, which is mediated by PDGFRb. Nevertheless, a caveat to this interpretation is that, although PI3K is the only known cellular signal transducer that interacts with pY739 and 750 of PDGFRb, 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 [21,32] or by loss of function mutations in TGFb receptor I or II [24,25]. TGFb signaling is abnormally elevated in fibrillin-1- deficient mice [22,23,33] and human aortas [34] as well as TGFb receptor I and II deficiency [24]. Previous data from our laboratory have shown nuclear accumulation of phosphorylated Smad2, an indicator of activation of TGFb signaling, in the LRP1(TbR-V)-deficient vascular wall [15]. 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 smLRP12/2; PDGFRb F2/F2 mice, however, the increased phosphorylation and nuclear translocation of Smad2 was not affected by the PDGFRb mutations. These findings indicate that TGFb activation through LRP1 precedes PDGFRb-dependent PI3K signaling, and that activation of TGFb signaling by itself is not sufficient to disrupt the vascular wall architecture. PDGFRb- dependent PI3K activation appears to be necessary for the expression of the Marfan-like phenotypes. Suppression of PI3K activation by PDGFRb prevents the Marfan-like phenotypic changes in the vascular wall in the presence of unabated TGFb signaling, suggesting a pivotal role of LRP1-controlled and PDGFRb-dependent PI3K activation in the pathogenesis of Marfan syndrome. Selective elimination of PDGFRb-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. Materials and Methods Generation of the mouse strains All experimental mice were maintained on a mixed C57BL/6/ 129 background. Transgenic mice expressing Cre recombinase specifically in smooth muscle cells (SM22 Cre) mated with LRP1 loxP/loxP mice to generate smLRP12/2 mice. Similarly, smLRP12/2; LDLR2/2 and smLRP12/2; LDLR2/2; PDGFRb F2/F2 mouse strains were established. Paired littermates were utilized throughout the study. Animal & aorta preparation Experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. All animals were maintained on standard rodent chow LRP1 Controls Vascular PI3K (Teklad 6% fat) with water ad libitum. Mice were sacrificed and blood samples were collected for lipid analysis following a six-hour fasting period. Aortas were removed intact from the root of the aortic arch to the iliac bifurcation and preserved in 4% paraformaldehyde (PFA) for conventional morphological study. Oil Red O staining Aortas were opened longitudinally under a dissecting micro- scope (Model Z30L, Cambridge Instruments). After fixation in 4% PFA, tissues were stained with 0.05% Oil Red O (s1848, Poly Scientific) at 60uC for 30 min. The aortas were then rinsed twice with 85% propylene glycol to develop the color. Hematoxylin-eosin (H&E), Masson’s Trichrome and Hart’s Elastin stainings were performed according to established textbook methods [35,36]. Aortic extract preparation and Western blotting (WB) The fat and connective tissue of the aorta were removed carefully. The aorta was homogenized in RIPA buffer with Proteinase Inhibitor Cocktail (P8430, Sigma) and Phosphatase Inhibitor Cocktail II (P5726, Sigma) to inhibit tyrosine protein phosphatases. After centrifugation at 20,000 xg for 30 minutes at 4uC, the supernatant was applied for Western blotting and the pellet was discarded. Briefly, aortic extract was resolved on SDS-PAGE gel and transferred to nitrocellulose membranes (HybondTM-C Extra, RPN303 E, Amersham Biosciences). Blots were blocked with 5% skim milk, probed with the appropriate primary antibodies (a- PDGFRb: #06-498, Upstate; a-p-Erk1/2: #9101, Cell Signaling; a-LRP1: 377, Herz Lab; a-TGFbRII: #06-227, Upstate; a-p- Smad2, #3108, Cell signaling) and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (NA934V, Amersham Biosciences). Immunoreactive bands were visualized using an enhanced chemiluminescence Western blotting detection kit (RPN 2132, Amersham Biosciences). Co-immunoprecipitation Aorta extracts were prepared as described above but using immunoprecipitation (IP) lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40) instead of RIPA buffer. Protein extract was pre-cleared with irrelevant non-immune serum. Nonspecific binding was precipitated with pre-swollen protein A-Agarose beads (P3476, Sigma). Supernatant was harvested by short spin and the appropriate specific primary antibodies were applied (a-PDGFRb: #06-498, Upstate; a-p- Tyrosine: #05-321, Upstate). The suspension was probed at 4uC for 2 hours and then precipitated again with protein A-Agarose beads. The antigen-antibody-protein A Agarose complex was collected by a brief centrifugation, washed three times with a solution containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% NP-40 and then three times with the same buffer without NP-40. The washed antigen-antibody-protein A Agarose complex was resolved on SDS-PAGE gel and immunoblotted with the relevant antibodies (a-p-Tyrosine: #05-321, Upstate; a-PI3K-p85: #06- 496/#05-217, Upstate; a-PDGFRb: #06-498, Upstate; a-LRP1: 377, Herz Lab; a-actin: A4700, Sigma). Primary SMC culture Mouse primary SMC culture was established using the explant technique as previously described [37,38]. All aortas were obtained from 8-week old male mice. Briefly, the aorta was dissected under sterile conditions, rinsed with PBS containing antibiotics, and the connective tissue and adventitia were removed PLoS ONE | www.plosone.org 9 September 2009 | Volume 4 | Issue 9 | e6922