Figure 1.
Effective deletion of the Lrp1 gene in macrophages from macLRP1-/- mice.
a) Bone marrow derived macrophages from LRP1+/+ and macLRP1-/- mice were analyzed for LRP1 expression by immunoblot analysis. b) Resident macrophages were isolated by peritoneal lavage and incubated with fluorescent labeled RAP for 1 h at 37 oC. RAP-positive, F4/80 positive cells were then determined by FACS analysis.
Figure 2.
LRP1 expressed in macrophages protects against vascular remodeling in the carotid-ligation model.
a) Photomicrographs showing the representative Elastic-Van Gieson staining of contralateral (left panels) and ligated vessels (right panels) in LRP1+/+ (top) and macLRP1-/- (bottom) mice. b) H&E staining of the serial sections of the same artery as in (a). c) Morphometric measurements of the total vessel area, adventitia area, media area and intima area were measured for LRP1+/+ (n = 20) and macLRP1-/- (n = 20) mice (*p = 0.03; **p = 0.009, Students t-test). d) Ratio of the Intima over the Media is represented for LRP1+/+ and macLRP1-/- mice (***p = 0.010, Students t-test).
Figure 3.
Immunohistochemical analysis of macrophage and α-SMC actin positive cells in the neointima.
Cross-sections of contralateral (a,d) and ligated (b,c,e,f) carotid arteries 2 weeks after ligation. Representative expression of Mac-2 (a,b,c) and α-SMA (d,e,f) antigen in contralateral and ligated vessels. Internal elastic lamina (IEL) is marked by a black arrow. Arrowhead indicates the cells positive for antibody staining. L shows lumen. g) Immunoblot analysis of LRP1 expression in contralateral and surgery vessels of LRP1+/+ (n = 2) and macLRP1-/- (n = 2) mice. Gels were analyzed using NIH imageJ h) Immunoblot analysis of Mac-2 expression in contralateral and ligated vessels of LRP1+/+ (n = 4) and macLRP1-/- (n = 4) mice. Gels were analyzed using NIH imageJ.
Figure 4.
qRT-PCR array analysis identifies increased expression of Tgf-β2, Pdgfa and Eln mRNA in macLRP1-/- vessels.
Total RNA was isolated from carotid arteries of control mice (no surgery) or mice undergoing carotid ligation. Carotid arteries from 3–4 mice were pooled to generate a single sample (n) for analysis. qRT-PCR array analysis using the atherosclerosis array from SABiosciences™ was performed. Only 3 of the 84 genes were significantly dependent upon genotype when ligated vessels were compared from LRP1+/+ mice with those from macLRP1-/- mice as assessed by 2-way ANOVA analysis. The relative levels of TGF-β2 (a), Pdgfa (b) and Eln (c) are shown for controls; LRP1+/+ (n = 3), macLRP1-/- (n = 3) and for surgery; LRP1+/+ (n = 3), macLRP1-/- (n = 3). Two way ANOVA revealed significant effects for genotype (*p = 0.009; **p = 0.023; ***p = 0.049) and treatment (*p = 0.002; **p = 0.002; ***p = 0.002) with no significant genotype X treatment interaction.
Table 1.
Genes whose expression levels change upon atreatment.
Figure 5.
Increased activation of the TGF-β signaling pathway during vessel remodeling in macLRP1-/- mice.
Representative images from contralateral (a,d) and ligated vessels (b,c,e,f) stained for TGF-β2 (a,b,c) or phospho-Smad 2/3 (d,e,f). Internal elastic lamina (IEL) is marked by a black arrow. Arrowheads indicate a cell positive for antibody staining while L marks the lumen. (g) five fields from each mouse were assessed for the number of phospho-Smad2/3 positive nuclei for LRP1+/+ (n = 2 mice) and macLRP1-/- (n = 2 mice). *p<0.0001, students t-test.
Figure 6.
Evaluation of matrix deposition in neointima.
Photomicrographs showing the representative Fibrin-Fraser-Lendrum stained sections of contralateral (a) and ligated vessels (b,c). Internal elastic lamina (IEL) is marked by black arrow. White asterisk show collagen matrix specific green color deposition. L identifies the lumen.
Figure 7.
Increased cell proliferation and PDGFR-βexpression during vascular remodeling.
Representative anti-PDGFR-β staining (a,b,c) and anti-PCNA staining (d,e,f) for proliferating cells of contralateral (a,d) and ligated carotid arteries (b,c,e,f). Internal elastic lamina (IEL) is marked by a black arrow. Arrowheads indicate the cells positive for antibody staining. L identifies the lumen.
Figure 8.
TNF-α attenuates Tgf-β2 expression in LRP1 expressing macrophages.
a) Bone marrow derived macrophages were treated with TNF-α overnight. Total mRNA from each sample was isolated and analyzed by qRT-PCR array analysis using the atherosclerosis array from SABiosciences™. The levels of Tgf-β2 (a) and SerpinB2 (b) are shown for LRP1+/+ and macLRP1-/- cells (n = 3 for each cell type; *p = 0.007, Students t-test).
Figure 9.
TGF-β2 induces SMAD and MAPK signaling in hAoSMCs and TGF-β-mediated ERK activation is inhibited in LRP1+/+ expressing macrophages.
a) hAoSMCs were serum starved for overnight and induced with 2 ng/ml TGF-β2 at indicated times. Cell extracts were analyzed for phospho-SMAD2 (S465/467), phospho-Erk1/2 (T202/Y204) or α/β tubulin for loading control. Results are representative of two independent experiments. b,c) Bone marrow derived macrophages from LRP1+/+ or macLRP1-/- mice were treated with TGF-β2 (b) or TGF-β1 (c). Cell extracts were then analyzed for phospho-Erk1/2 and/or phospho-SMAD2 by immunoblot analysis. α/β tubulin levels represents loading control measured by immunoblot analysis.
Figure 10.
LRP1 binds TGF-β and regulates its levels.
a) Surface Plasmon resonance confirms binding of TGF-β2 to LRP1 immobilized on SPR chip. The red line shows the best fit to a pseudo-first order process also containing a non-specific binding component. b) Rmax determined from the fits in (a) are replotted vs TGF-β2 concentrations. The line shows the best fit to a binding isotherm using non-linear regression analysis, revealing a KD of 222 nM. c) TGF-β2 co-immunoprecipitates with LRP1 in LRP1+/+ macrophages. Macrophages from LRP1+/+ or macLRP1-/- mice were incubated with 125I- TGF-β2 plus or minus RAP. Following crosslinking, cell extracts were subjected to immunoprecipitation and the immunoprecipitated proteins separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were then exposed to autoradiographic film. d) TGF-β2 accumulation in macrophage condition media analyzed by immunoblot analysis. Bone marrow derived macrophages were grown in serum-free media for 72 h. Concentrated culture media (0.5 ml) were subjected to western blot analysis for TGF-β2 expression. Gels were analyzed using NIH ImageJ, and integrated bands were normalized to total cell protein (n = 3 for each cell type, *p = 0.0168, Students t-test).