carefully. The aorta was opened longitudinally and the intima was scraped on luminal surface. Then the aorta was minced into small pieces and placed into a T25 flask with high glucose (4.5 g/L) DMEM containing 15% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 20 mM L-glutamine. Both explants and cells were cultured at 37uC in 5% carbon dioxide (CO2). Cells were detached by incubation with 0.25% trypsin-EDTA solution. Passages 5–15 were used in this study. Immunocytochemistry Subcultured SMCs from the mouse aortic explants were allowed to grow on glass coverslips for 24 hours after trypsiniza- tion. The cells were fixed in situ with 95% ethanol, blocked with 5% non-immune goat serum, and probed with anti-a-smooth muscle actin (A2547, Sigma) and LRP1 (377, Herz Lab) antibodies. After three washes in PBS, the cells were incubated with Alexa Fluor 594 goat anti-mouse (A11032, Molecular Probes) and Alexa Fluor 488 goat anti-rabbit IgG antibodies (A11034, Molecular Probes). After three more washes in PBS, coverslips were mounted on glass slides using a DAPI-containing mounting medium (VectashieldR Hard SetTM, H-1500, Vector) and analyzed using a fluorescence microscope (Axioplan 2 Imaging, Carl Zeiss MicroImaging Inc.). Boyden chamber transmigration assay SMC migration was measured using a 12-well modified Boyden chamber (AA12, Neuro Probe) hosting a polycarbonate filter with 8-mm pores (PFB8, Neuro Probe) as described [39]. 36104 cells in 100 ml were loaded into the top chamber of each well while the lower chambers were filled with SMC medium. After incubating at 37uC in 5% CO2 for 6 hours, non-migrated cells were scraped from the upper surface of the filter. Cells on the lower surface were fixed with 95% ethanol and stained with Harris Modified Hematoxylin (HHS-16, Sigma). The number of SMCs on the lower surface of the filter was determined by counting five continuous high-power (2006) fields of constant area per well. Experiments were performed three times in duplicate wells. Two-dimensional migration (scratch) assay Subcultured 36105 SMCs were seeded into 60 mm Petri dishes. 10 mg/ml mitomycin C was applied to inhibit cell proliferation. Cells were incubated at 37uC in 5% CO2 overnight. The next day, part of the dish was denuded by scratching along a straight line. The dishes were put back to the incubator for 24 hours. The cells which appeared on the denuded area were treated as migrated cells. To quantify the migrated cells, the cells in five continuous high-power (2006) fields were counted and statistical analysis was performed. References 1. Herz J, Strickland DK (2001) LRP: a multifunctional scavenger and signaling receptor. J Clin Invest 108: 779–784. 2. Lillis APVDL, Murphy-Ullrich JE, Strickland DK (2008) LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. Physiol Rev 88: 887–918. 3. Beisiegel U, Weber W, Ihrke G, Herz J, Stanley KK (1989) The LDL-receptor- related protein, LRP, is an apolipoprotein E-binding protein. Nature 341: 162–164. 4. Kowal RC, Herz J, Goldstein JL, Esser V, Brown MS (1989) Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins. Proc Natl Acad Sci U S A 86: 5810–5814. 5. Hussain MM, Maxfield FR, Mas-Oliva J, Tabas I, Ji ZS, et al. (1991) Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. J Biol Chem 266: 13936–13940. 6. Swertfeger DK, Bu G, Hui DY (2002) Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration. J Biol Chem 277: 4141–4146. LRP1 Controls Vascular PI3K PDGF-BB chemotaxis assay This experiment was performed as described above in the Boyden chamber transmigration assay with the exception that only 16104 cells were seeded. In addition, 10 ng/ml PDGF-BB was administrated to the lower chambers as an attractant. Immunohistochemistry Aortas were isolated and fixed in 4% PFA for 1 hour. The tissues were sliced into 8 mm cross sections after embedded in OCT. The mounted sections were treated with 0.1% Triton X-100 for 5 minutes, blocked with 5% non-immune goat serum, and probed with anti-p-Smad2 (#3108, Cell signaling) rabbit antibody. After three washes in PBS, the sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibodies (A11034, Molecular Probes). After three more washes in PBS, coverslips were mounted on glass slides with a DAPI-containing mounting medium (ProLong Gold antifade reagent with DAPI, P36935, Invitrogen). Statistical analysis Statistical analyses were performed using two-tail Students’ t-test. Results are given as mean6SD. A p,0.05 was considered significant. Supporting Information Figure S1 Plasma Lipoprotein Profile. A significant increase of LDL-cholesterol was observed in LDLR2/2 and smLRP12/2; LDLR2/2 mice. LRP1-deficiency in smooth muscle cells had no effect on the plasma lipoprotein profile. Found at: doi:10.1371/journal.pone.0006922.s001 (1.21 MB TIF) Figure S2 Light microscopy of cryostat sections. Aortas of 7-week old mice were dissected out. 8 mm-thick cryostat sections were stained with DAPI. Aortic wall thickening was observed in smLRP12/2 mice. Found at: doi:10.1371/journal.pone.0006922.s002 (4.56 MB TIF) Acknowledgments We thank John Shelton for excellent assistance in photography. We also thank Wenling Niu, Isaac Rocha and Elida Priscilla Rodriguez for their helpful technical support. Author Contributions Conceived and designed the experiments: LZ YT JH. Performed the experiments: LZ YT. Analyzed the data: LZ PB MDT JH. Contributed reagents/materials/analysis tools: LZ YT PB MDT JH. Wrote the paper: LZ JH. 7. Boucher P, Gotthardt M, Li WP, Anderson RG, Herz J (2003) LRP: role in vascular wall integrity and protection from atherosclerosis. Science 300: 329–332. 8. Tallquist M, Kazlauskas A (2004) PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15: 205–213. 9. Okkenhaug K, Vanhaesebroeck B (2001) New responsibilities for the PI3K regulatory subunit p85 alpha. Sci STKE 2001: PE1. 10. Arrieumerlou C, Donnadieu E, Brennan P, Keryer G, Bismuth G, et al. (1998) Involvement of phosphoinositide 3-kinase and Rac in membrane ruffling induced by IL-2 in T cells. Eur J Immunol 28: 1877–1885. 11. Brachmann SM, Yballe CM, Innocenti M, Deane JA, Fruman DA, et al. (2005) Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement. Mol Cell Biol 25: 2593–2606. 12. Cantley LC (2002) The phosphoinositide 3-kinase pathway. Science 296: 1655–1657. 13. Huang SS, Ling TY, Tseng WF, Huang YH, Tang FM, et al. (2003) Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1. Faseb J 17: 2068–2081. PLoS ONE | www.plosone.org 10 September 2009 | Volume 4 | Issue 9 | e6922