Atherosclerotic lesion-specific copper delivery suppresses atherosclerosis in high-cholesterol-fed rabbits

Abstract

Dietary cholesterol supplements cause hypercholesterolemia and atherosclerosis along with a reduction of copper concentrations in the atherosclerotic wall in animal models. This study was to determine if target-specific copper delivery to the copper-deficient atherosclerotic wall can block the pathogenesis of atherosclerosis. Male New Zealand white rabbits, 10-weeks-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as control. Twelve weeks after the feeding, the animals were injected with copper-albumin microbubbles and subjected to ultrasound sonication specifically directed at the atherosclerotic lesions (Cu-MB-US) for target-specific copper delivery, twice a week for four weeks. This regiment was repeated 3 times with a gap of two weeks in between. Two weeks after the last treatment, the animals were harvested for analyses of serum and aortic pathological changes. Compared to controls, rabbits fed cholesterol-rich diet developed atherosclerotic lesion with a reduction in copper concentrations in the lesion tissue. Cu-MB-US treatment significantly increased copper concentrations in the lesion, and reduced the size of the lesion. Furthermore, copper repletion reduced the number of apoptotic cells as well as the content of cholesterol and phospholipids in the atherosclerotic lesion without a disturbance of the stability of the lesion. The results thus demonstrate that target-specific copper supplementation suppresses the progression of atherosclerosis at least in part through preventing endothelial cell death, thus reducing lipid infiltration in the atherosclerotic lesion.

Keywords: Atherosclerosis; cholesterol; collagen; copper; phospholipid; rabbits.

Figure 1.

Cu-MB-US induced repression of atherosclerosis. (a) Representative ORO staining of abdominal aorta (left) and quantitative analysis of lesion aera (right). (b) The changes of copper concentrations in the aorta (intima-media membrane). (c) Linear correlation between copper concentrations and the severity of the atherosclerotic lesion, the regression coefficient r = –0.60. The blue line indicates the mean of prior-treated group. Data are presented as mean ± SD, *P < 0.05. n = 10–11. (A color version of this figure is available in the online journal.)

Figure 2.

Effect of Cu-MB-US treatment on lipid content in the atherosclerotic lesion. (a) The changes of cholesterol (CHOL) content in the aorta (intima-media membrane). (b) Linear correlation between CHOL content and the severity of the atherosclerotic lesion, the regression coefficient r = 0.69. (c) Linear correlation between copper concentrations and CHOL content, the regression coefficient r = –0.55. (d) The changes of phospholipid (PL) content in the aorta (intima-media membrane). (e) Linear correlation between PL content and the severity of the atherosclerotic lesion, the regression coefficient r = 0.54. (f) Linear correlation between copper concentrations and PL content, the regression coefficient r = –0.60. (g) Representative images of apoptotic cells in the atherosclerotic lesion, identified by co-staining with TUNEL (green) and DAPI (blue) (left) and quantification of apoptotic cells in the atherosclerotic lesion (right). Scale bar = 100 µm. The blue line indicates the mean of the prior-treated group. Data are presented as mean ± SD, *P < 0.05. n = 5–7. (A color version of this figure is available in the online journal.)

Figure 3.

Changes of collagen compositions, LOX activity, and mRNAs for MMPs in the atherosclerotic lesion. (a) Collagen (red) deposition in atherosclerotic lesion identified by Sirius red (SR) staining (left) and semi-quantitative analysis of total collagen content in the atherosclerotic lesion (right). Scale bar = 200 μm. (b) Polarization microphotography of the SR staining sections, yellow–red collagen fibers (type I collagen) and green collagen fibers (type III collagen) (left) and semi-quantitative analysis of the ratio of type I/III collagen (right). Scale bar = 150 μm. (c) Immunohistochemical staining (marked by HHF35) of SMCs in the atherosclerotic lesion (left) and semi-quantitative analysis of SMCs in the atherosclerotic lesion (right). Scale bar = 200 μm. (d) The change of LOX activity in the aorta (intima-media membrane). (e) The changes of mRNA levels for MMP2 (left), MMP3 (middle) and MMP9 (right) in the aorta (intima-media membrane). The blue line indicates the mean of prior-treated group. Data are presented as mean ± SD, *P < 0.05. n = 5–7. (A color version of this figure is available in the online journal.)