Complement C1q reduces early atherosclerosis in low-density lipoprotein receptor-deficient mice

Abstract

We explored the role of the classic complement pathway in atherogenesis by intercrossing C1q-deficient mice (C1qa-/-) with low-density lipoprotein receptor knockout mice (Ldlr-/-). Mice were fed a normal rodent diet until 22 weeks of age. Aortic root lesions were threefold larger in C1qa-/-/Ldlr-/- mice compared with Ldlr-/- mice (3.72 +/- 1.0% aortic root versus 1.1 +/- 0.4%; mean +/- SEM, P < 0.001). Furthermore, the cellular composition of lesions in C1qa-/-/Ldlr-/- was more complex, with an increase in vascular smooth muscle cells. The greater aortic root lesion size in C1qa-/-/Ldlr-/- mice occurred despite a significant reduction in C5b-9 deposition per lesion unit area, suggesting the critical importance of proximal pathway activity. Apoptotic cells were readily detectable by cleaved caspase-3 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electron microscopy in C1qa-/-/Ldlr-/-, whereas apoptotic cells were not detected in Ldlr-/- mice. This is the first direct demonstration of a role for the classic complement pathway in atherogenesis. The greater lesion size in C1qa-/-/Ldlr-/- mice is consistent with the emerging homeostatic role for C1q in the disposal of dying cells. This study suggests the importance of effective apoptotic cell removal for containing the size and complexity of early lesions in atherosclerosis.

Figure 1

Increased aortic root lesion area in C1qa^−/−/Ldlr^−/− mice. Comparison of lesion areas in C1qa^−/−/Ldlr^−/− (n = 8) and Ldlr^−/− (n = 19) animals after 22 weeks: (A) area of lesions stained by Oil Red O and hematoxylin, (B) % of vessel stained by Oil Red O and hematoxylin, and (C) photomicrographs of aortic roots at low power (LP) and high power (HP). The bars show the group means + SEM.

Figure 2

Increased complexity of aortic root lesions in C1qa^−/−/Ldlr^−/− mice. Comparison of lesions in C1qa^−/−/Ldlr^−/− (n = 8) and Ldlr^−/− (n = 19) animals after 22 weeks: (A) percentage of vessel stained by Moma-2, (B) percentage of lesion cells stained by Moma-2, and (C) percentage of lesion cells staining positively for smooth muscle α-actin. Figure bars are group means + SEM. D: Confocal analysis of lesion complexity showing composite pseudocolored overlays. Aortic roots were double-immunostained with Moma-2 (Alexa488 detection) and anti-actin (Cy3 detection), together with a nuclear counterstain (TOPRO). The sections were mounted in PBS/glycerol and visualized using a Zeiss inverted confocal microscope. Purple, TOPRO (far red emission); red, Cy3 (orange/red emission); and green, Moma-2 (green emission). The fine white arrow in the right panel shows actin-positive VSMC (red) within a lesion surrounded by Moma-2-positive macrophages (green). This appearance was seen in lesions of C1qa^−/−/Ldlr^−/− (right) but not Ldlr^−/− (left) mice.

Figure 3

Deposition of C5b-9 in the aortic root. Photomicrograph of cryosections of aortic roots of (A) Ldlr^−/− and (B) C1qa^−/−/Ldlr^−/− mice stained with anti-C5b-9. C5b-9 deposition was seen diffusely around lesional foam cells; (C) shows a quantitative comparison between anti-C5b-9 staining of aortic roots of Ldlr^−/− and C1qa^−/−/Ldlr^−/− mice.

Figure 4

Increased apoptotic cells in aortic root lesions of C1qa^−/−/Ldlr^−/− mice. Comparison of lesions in C1qa^−/−/Ldlr^−/− (n = 8) and Ldlr^−/− (n = 19) animals after 22 weeks: (A) percentage of plaque cells staining positively for cleaved caspase 3, (B) percentage of plaque cells staining positively by TUNEL, and (C) photomicrographs with arrows showing the absence and presence of cells staining positively for cleaved caspase-3 in Ldlr^−/− mice (left panel) and C1qa^−/−/Ldlr^−/− (right panel) mice, respectively. Filled block arrows point to apoptotic cells immunopositive for cleaved capsase-3. Open arrowheads point to the endothelial layer. L, lumen. Open block arrows point to the internal elastic lamina delimiting deep boundary of intima.

Figure 5

Double staining of apoptotic cells assessed by confocal microscopy. A: Low-power composite of cleaved caspase-3 (Alexa488, green), Moma-2 (Alexa 568, red), and TOPRO-3 nuclear dye (purple, pseudocolored; far red, original); (B) single channels of boxed area in A, with composite at bottom right. White arrows show cells triple-positive for TOPRO-3, Moma-2, and cleaved caspase-3; (C) equivalent of B in parallel section stained with Moma-2 (Alex 568, red) and TUNEL (FITC, green), with composite at bottom right. White arrows show cells triple-positive for TOPRO-3, TUNEL, and Moma-2. No such colocalization was seen on actin double staining (not shown, the resolution was less because the actin-positive cells were distant from the cleaved caspase-3-positive cells).

Figure 6

Apoptotic cells in EM aortic root lesions of C1qa^−/−/Ldlr^−/− mice. A: Low magnification for orientation. L, lumen. White arrowhead points to endothelial layer. Fine arrows point to apoptotic cells, identified by characteristic electron dense peripheral chromatin condensation. B and C are higher magnifications. Gray-filled block arrows marked with F, foam cell(s). In B, the white block arrow points to a shrunken cell with characteristic electron dense peripheral chromatin condensation and cytoplasmic collapse, characteristic of apoptosis; in C, the white block arrow points to an early apoptotic cell with early electron dense peripheral chromatin condensation and characteristic blebbing, containing electron dense material (Bl, white script within filled black block arrow). Gray-filled block arrow with F, foam cell.