Fibroblast growth factor-2/platelet-derived growth factor enhances atherosclerotic plaque stability

Abstract

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor-A (VEGF)-A, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB and FGF-2 + PDGF-BB. Lentivirus was percutaneously injected into the media-adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque-rupture rate, plaque-vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF-2/PDGF-BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF-A- and FGF-2-overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF-2/PDGF-BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF-2/PDGF-BB induced epsin-2 expression and enhanced the VEGF receptor-2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF-2 and PDGF-BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.

Keywords: atherosclerosis; fibroblast growth factor (FGF-2); pericytes; platelet-derived growth factor-BB (PDGF-BB); vascular normalization.

Figure 1

General condition of rabbits. A, Animal grouping and time course for the in vivo studies. B, IVUS images and measurement of external elastic membrane area (EEMA), lumen area (LA), plaque area (PA) and plaque burden (PB) (mean ± SEM; n = 3). Red line indicated the area of EEMA. Yellow line indicated the area of LA. C, Bars indicate the number of animals with IPH and plaque rupture in the sham (5/15), vector (6/13), VEGF‐A (12/15), FGF‐2 (9/13), PDG‐BB (3/15) and FGF‐2 + PDGF‐BB (1/15) groups. D, Representative images of the necrotic core and quantification of necrotic core area and necrotic ratio in rabbit atherosclerotic plaques (mean ± SEM; n = 5). Bar, 20 μm. Arrow indicated lumen surface. ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB

Figure 2

The effects of growth factors on atherosclerotic plaque vulnerability. A, Immunostaining of sections with the anti‐α‐SMC actin and anti‐RAM‐11 antibodies, Oil Red O staining and Sirius Red staining. Bar, 20 μm. B‐F, Quantification of α‐SMC+ and RAM‐11+ (macrophages) cells and quantification of Oil Red O+ (lipidosis) and Sirius Red+ (collagen) staining (mean ± SEM; n = 6). Arrow indicated lumen surface. ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB

Figure 3

The effects of growth factors on intraplaque neovessels. A, Immunostaining of sections with the mouse anti‐CD31 antibody. Bar, 20 μm. B, Quantification of cells positive for mouse anti‐CD31 (circles indicate neovessels)/plaque area (mm²) (mean ± SEM; n = 6). Black dotted line indicates the boundaries between plaque and adventitia. ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB

Figure 4

Histologic analysis of intraplaque neovessels in each group. A, Quantification of Evans Blue dye leakage into plaques in the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups (mean ± SEM; n = 3). B, H&E staining demonstrating the presence of RBCs (black arrows indicate RBCs within the luminal wall of neovessels and yellow arrows indicate RBC leakage from neovessels) within atherosclerotic plaques. Bar, 20 μm. C, Plaque sections from rabbit abdominal aortas from each group were analysed on week 8 after transfection by IF double labelling for CD31 (an endothelial‐cell marker; red) and glycophorin A (an erythrocyte marker; green). Bar, 50 μm. D, Representative bright‐field microscopy analysis of Prussian Blue + hemosiderin (blue) counterstained with Nuclear Fast Red (pink) in each group. E, Graph demonstrating quantification of Prussian Blue + hemosiderin deposits from the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups (mean ± SEM; n = 3). ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB

Figure 5

Analysis of pericyte coverage in the intraplaque neovessels of each group. A, Plaque sections from the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups were analysed on week 8 after transfection by IF double labelling for CD31 (an endothelial‐cell marker), NG2 (a pericyte marker) and autofluorescence (reddish yellow) of RBCs. Bar, 20 μm. Arrow indicated neovessels. B, Pericyte coverage (%) as percentages of pericyte + vessels in different groups. (n = 6 rabbits/condition). ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB. C, Plaque specimens were obtained from the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups and assessed by TEM for microstructural changes in the above organizations. Yellow arrow indicated the endothelial‐cell cleft. Bar, 5 μm. MAC, macrophage; SMC, smooth muscle cells; EC, endothelial cells; RBC, red blood cells

Figure 6

IF staining and Western blot for growth factors in rabbit aortic plaques. A, Immunofluorescence of sections from rabbit atherosclerotic plaques 8 wk after lentiviral transfection. Bar, 20 μm. Test the expression of FGFR‐1+, FGFR‐2+ and PDGFRβ+ cells in the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups (mean ± SEM; n = 6). Arrow indicated lumen surface. B, Western blot indicating protein levels of FGFR‐1, FGFR‐2 and PDGFRβ in the sham, vector, VEGF‐A, FGF‐2, PDGF‐BB and FGF‐2 + PDGF‐BB groups (mean ± SEM; n = 3). All data as standardized values. ^* P < .05 vs Sham; ^# P < .05 vs Vector; ^{^} P < .05 vs VEGF‐A; ^$ P < .05 vs FGF‐2; ^& P < .05 vs PDGF‐BB

Figure 7

Effects of FGF‐2 + PDGF‐BB on pericyte migration, VEGFR2 degradation and epsin‐1/2 expression in vitro. A, Representative images and quantification of wound‐healing assay results at 0 and 6 h in FGF‐2‐, PDGF‐BB‐ or FGF‐2 + PDGF‐BB‐stimulated pericytes (n = 3 samples/group). Bar, 20 μm. B, Representative images and quantification of Transwell assays at 0 and 12 h in FGF‐2‐, PDGF‐BB‐ or FGF‐2 + PDGF‐BB‐stimulated pericytes (n = 3 samples/group). Bar, 20 μm. C, bEND.3 and 10T1/2 cells were labelled with carboxyfluorescein succinimidyl ester (green) and PKH26 (red), respectively and co‐cultured in Matrigel‐coated culture slides for 6 h under treatment with FGF‐2‐, PDGF‐BB‐ or FGF‐2 + PDGF‐BB. Bar, 20 μm. Quantification was performed by measuring the merged cells from 10 fields in three independent experiments. D, Time‐course analysis of total VEGFR‐2 and p‐VEGFR‐2 levels in FGF‐2‐, PDGF‐BB‐ or FGF‐2 + PDGF‐BB‐stimulated pericytes (n = 3 samples/group). E, Time‐course analysis of epsin‐1 and e‐2 protein levels in FGF‐2‐, PDGF‐BB‐ or FGF‐2 + PDGF‐BB‐stimulated pericytes (n = 3 samples/group). F, 10T1/2 cells transfected with either control siRNA or epsin‐2 siRNA were stimulated with FGF‐2 + PDGF‐BB and analysed by Western blot. Quantification of total PDGFR‐β, p‐PDGFR‐β, total VEGFR‐2 and p‐VEGFR2 levels (n = 3 samples/group). G, bEND.3 and 10T1/2 cells were labelled with carboxyfluorescein succinimidyl ester (green) and PKH26 (red), respectively, and co‐cultured in Matrigel‐coated culture slides for 6 h. Quantification was performed by measuring the merged cells from 10 fields in three independent experiments. Bar, 20 μm. All in vitro experiments were repeated at least twice. Data represent the mean ± SEM. All data as standardized values. *P < .05, **P < .01; and ***P < .001; analysis of variance. NT, no treatment