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. 2002 Oct;161(4):1395-407.
doi: 10.1016/S0002-9440(10)64415-X.

Blockade of platelet-derived growth factor or its receptors transiently delays but does not prevent fibrous cap formation in ApoE null mice

Koichi Kozaki  1 , Wolfgang E Kaminski, Jingjing Tang, Stan Hollenbach, Per Lindahl, Carol Sullivan, Jin-Chen Yu, Keith Abe, Paul J Martin, Russell Ross, Christer Betsholtz, Neill A Giese, Elaine W Raines
Affiliations

Blockade of platelet-derived growth factor or its receptors transiently delays but does not prevent fibrous cap formation in ApoE null mice

Koichi Kozaki et al. Am J Pathol. 2002 Oct.

Abstract

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

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Figures

Figure 1.
Figure 1.
Two different approaches were used to test the role of PDGF in the formation of lesions of atherosclerosis in chow-fed ApoE −/− mice. Lesion formation was analyzed in two separate sets of experiments. 1) Bone marrow of lethally irradiated ApoE −/− mice was repopulated by transplant at 7 weeks with the bone marrow of PDGF-B hematopoietic chimeras generated by repopulation of first generation chimeras with fetal liver cells from PDGF-B +/+ and −/− embryos (top). 2) ApoE −/− mice were treated with a PDGF receptor antagonist or a vehicle control starting at 8 weeks. In both cases, treatments to remove or inhibit PDGF preceded the initiation of foam cell lesion formation as indicated by the bars at the bottom summarizing the normal kinetics of lesion progression. Lesion analysis was performed between 25 and 50 weeks to focus on the period of time during which fibrous caps are formed by the infiltrating SMC.
Figure 2.
Figure 2.
PDGF-B is primarily expressed in macrophages in developing lesions of ApoE −/− mice. To localize PDGF-B in lesions of 35-week ApoE −/−/PDGF-B chimeras, adjacent sections were stained with the PDGF-B monoclonal antibody PGF007 (A, D, and E) or with the macrophage-specific antibody Mac-2 (B and D) or normal mouse IgG1 (C). Fatty streaks (A–C) and advanced lesions (E and F) from PDGF-B +/+ chimeras contain primarily macrophages that stain positively for PDGF-B. No significant staining is observed with normal mouse IgG (C) or with the PDGF-B antibody in lesions from PDGF-B −/− chimeras (D). Arrows in A and E indicate some of the PDGF-B-positive cells. Magnification, ×40 for all panels. Lesions from chimeras at 35 weeks (+/+, n = 26; −/−, n = 29) and 45 weeks (+/+, n = 10; −/−, n = 10) showed similar immunostaining profiles.
Figure 3.
Figure 3.
At 35 weeks, no significant difference is observed between PDGF-B +/+ and −/− chimeras in atherosclerotic lesion volume in the brachiocephalic trunk, but lesion characteristics are altered. A: Mean lesion volume in PDGF-B +/+ and −/− chimeras was determined by evaluation of the entire length of the brachiocephalic trunk (innominate artery) at 75-μm intervals from a random start site within the first 75 μm of the vessel. The data shown are for 9 PDGF-B +/+ and 8 PDGF-B −/− chimeras and representative of an additional 17 PDGF-B +/+ and 16 PDGF-B −/− chimeras analyzed for all criteria shown in this figure. B: The frequency and extent (thin, intermediate, and thick) of fibrous cap formation was evaluated along the entire brachiocephalic trunk of all mice in this group (+/+, n = 9; −/−, n = 8) as described in A. The extent of fibrous cap formation was scored as thick (greater than 4 elastic layers), intermediate (2–4 elastic layers), and thin (a single elastic layer). The mean ± SEM is shown for 9 PDGF-B +/+ and 8 PDGF-B −/− chimeras, but P values are 0.2, considered not significant because of variability in fibrous cap formation. C: The occurrence of lesions with cholesterol clefts and necrotic cores was determined throughout the length of the brachiocephalic trunk at 75-μm intervals. The mean ± SEM is shown for 9 PDGF-B +/+ and 8 PDGF-B −/− chimeras (P = 0.5, considered not significant). D: The percentage of lesions occupied by macrophages (Mac-2-positive staining) was quantitated in lesions larger than 10,000 μm2 for PDGF-B +/+ (n = 14) and PDGF-B −/− (n = 18) chimeras (P = 0.07, considered not quite significant). E: The fibrous cap area was determined as a percentage of total lesion area for the most advanced lesion in each mouse. However, a significant number of animals in both group did not develop fibrous caps. F–I: Fibrous cap formation was evaluated by VVG staining. (F and H) PDGF-B +/+ chimeras show a more mature fibrous cap (F, thick; H, intermediate) than do (G and I) PDGF-B −/− chimeras (I, thin). Lesions in PDGF-B −/− chimeras are occupied primarily by macrophages (K) detected by staining with Mac-2 antibody, while lesions (J) in PDGF-B +/+ chimeras appear to have fewer macrophages and extensive deposits of extracellular matrix (F and H). Magnifications: F and G, ×40; H–K, ×20.
Figure 4.
Figure 4.
By 45 weeks, the fibrous caps of PDGF −/− chimeras are indistinguishable from those of PDGF-B +/+ chimeras. A: Mean lesion volume in PDGF-B +/+ and −/− chimeras was determined by evaluation of the entire length of the brachiocephalic trunk at 75-μm intervals using a random start site within the first 75 μm of the vessel. The mean ± SEM is shown for each PDGF-B +/+ and −/− chimera and representative of an additional 12 PDGF-B +/+ and 13 PDGF-B −/− chimeras analyzed for all criteria shown in this figure. B: The frequency and extent (thin and intermediate plus thick) of fibrous cap formation were evaluated along the entire brachiocephalic trunk of all mice in a second group (+/+, n = 10; −/−, n = 10) as described in A. The extracellular matrix content of the fibrous cap is highlighted for PDGF-B +/+ (C) and PDGF-B −/− (D) chimeras with GAF staining. Magnification, ×20.
Figure 5.
Figure 5.
The PDGF receptor antagonist inhibits all three combinations of receptor dimers and thus all possible ligand dimers. A: The lines drawn between the different PDGF ligand pairs and the PDGF receptor dimers indicate the demonstrated binding of the ligand pairs to the different receptor combinations. Only PDGF-BB is a universal ligand able to bind to all three receptor combinations, whereas the other ligand pairs are more restricted. Thus, in the PDGF-B null chimeras, the universal PDGF ligand has been deleted, but other forms of PDGF may be able to compensate for its absence. In contrast, the PDGF receptor antagonist blocks all three receptor combinations. B: Plasma concentration of CT52923 was determined by HPLC (detection limit 0.5 μg/ml), and inhibitory activity in an ex vivo phosphorylation assay was determined after a single oral dose of 60 mg/kg at each of the indicated time points for three ApoE −/− mice at each time point.
Figure 6.
Figure 6.
Daily administration of PDGF receptor antagonist CT52923 does not alter atherosclerotic lesion volume in the brachiocephalic trunk in ApoE −/− mice. A: Mean lesion volume in ApoE −/− mice that were untreated (control) or gavaged daily with vehicle or with the PDGF receptor antagonist CT52923 was determined by evaluation of the entire length of the brachiocephalic trunk at 75-μm intervals from a random start site. The number of mice evaluated at 25 weeks was: control, 8; vehicle, 10; CT52923, 15. At 35 weeks: control, = 10; vehicle, 10; CT52923, 14. At 50 weeks: control, 8; vehicle, 10; CT52923, 14. B–D: The lesion profile for each animal at 25 weeks (B), 35 weeks (C), and 45 weeks (D) is shown with the black and cross-hatched bars indicating varying extents of fibrous cap formation. As indicated in the key, the dotted box signifies foam cell lesions without any evidence of smooth muscle cells, and a white box has no evident lesion. Each box was scored from an H & E-stained section evaluated at 75-μm intervals using a random start site within the first 75 μm of the vessel.
Figure 7.
Figure 7.
At 35 weeks, in PDGF receptor antagonist-treated ApoE −/− mice, advanced fibrous cap formation is delayed but at 50 weeks is indistinguishable from vehicle and control mice. The frequency and extent (thin in A and intermediate plus thick in B) of fibrous cap formation were evaluated along the entire brachiocephalic trunk of all mice analyzed in Figure 6, B, C, and D ▶ . Comparisons between groups were made using Mann-Whitney test and P values are indicated on the graph for statistically significant differences. C–F: Fibrous cap formation was evaluated by Masson’s trichrome staining, and representative sections are shown. At 35 weeks, PDGF receptor antagonist-treated mice show less mature fibrous caps (D) than vehicle-treated (C) mice. Magnification, ×20. However, by 50 weeks, there is no difference in the complexity or extent of the fibrous caps of the vehicle-treated mice (E) and the PDGF receptor antagonist-treated mice (F). Magnification, ×10.
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