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C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway.

Zhang R, Zhou SJ, Li CJ, Wang XN, Tang YZ, Chen R, Lv L, Zhao Q, Xing QL, Yu DM, Yu P - Lipids Health Dis (2013)

Bottom Line: The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR.CRP/oxLDL/β2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05).The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/β2GPI group (P < 0.05).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diabetic Nephropathy Hemodialysis, Key Laboratory of Hormones and Development, Ministry of Health, Metabolic Diseases Hospital & Tianjin Institute of Endocrinology Tianjin Medical University, Tongan Street, Tianjin, Heping District 300070, China.

ABSTRACT

Background: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I (CRP/oxLDL/β2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice.

Methods: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 μg oxLDL, 20 μg β2GPI, 40 μg oxLDL/β2GPI complex, 44 μg CRP/oxLDL/β2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip.

Results: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/β2GPI groups (P < 0.05). CRP/oxLDL/β2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/β2GPI group (P < 0.05).

Conclusions: CRP/oxLDL/β2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

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Effect of CRP/oxLDL/β2-GPI on infiltration of inflammatory cells in the aortas. Aortas were removed, paraffin-embedded, deparaffinated, underwent antigen retrieval, blocked, and then incubated with primary antibodies (Mac 1:200 or α- SMA 1:100 or CD3 1:100) at 4°C overnight. Biotinylated secondary antibody (1:200) was added, followed by HRP-labeled streptavidin incubation. Infiltrations of macrophages, SMCs and T cells in the aortas were quantified as the percentage of the luminal surface covered by positive cells to the plaque area using Image Pro-Plus 6.0. Results are shown as means ± SEM. A: Infiltration of SMCs in the aortas, 400× magnification. B: Infiltration of macrophages in the aortas, 400× magnification. C: Infiltration of T cells in aortas, 400× magnification. D: Positive expressions of SMA, MAC and CD3 (n=4), expressed as the percentage to plaque area. *P <0.05, compared with the CRP/oxLDL/β2GPI group, †P < 0.05, compared with the PBS group.
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Figure 4: Effect of CRP/oxLDL/β2-GPI on infiltration of inflammatory cells in the aortas. Aortas were removed, paraffin-embedded, deparaffinated, underwent antigen retrieval, blocked, and then incubated with primary antibodies (Mac 1:200 or α- SMA 1:100 or CD3 1:100) at 4°C overnight. Biotinylated secondary antibody (1:200) was added, followed by HRP-labeled streptavidin incubation. Infiltrations of macrophages, SMCs and T cells in the aortas were quantified as the percentage of the luminal surface covered by positive cells to the plaque area using Image Pro-Plus 6.0. Results are shown as means ± SEM. A: Infiltration of SMCs in the aortas, 400× magnification. B: Infiltration of macrophages in the aortas, 400× magnification. C: Infiltration of T cells in aortas, 400× magnification. D: Positive expressions of SMA, MAC and CD3 (n=4), expressed as the percentage to plaque area. *P <0.05, compared with the CRP/oxLDL/β2GPI group, †P < 0.05, compared with the PBS group.

Mentions: CRP/oxLDL/β2GPI group showed strong positive expression of α-SMA in each layer of aorta. The other DM groups only displayed positive expression on the surface of the intima, not on the shrink media or not as strong as that in the CRP/oxLDL/β2GPI group (Figure 4A). CRP/oxLDL/β2GPI group showed remarkable positive areas of macrophages (Figure 4B). T cell expression was not as obvious as those of SMC and macrophages, but certain amounts of T cells were found in the lumen in the CRP/oxLDL/β2GPI group. Moreover, positive CD3 expression was not limited to the intima, but was also found on the adventitia (Figure 4C). Using Image Pro-Plus 6.0, the α-SMA, MAC, and CD3 positive expressions of AS lesion in the CRP/oxLDL/β2GPI group were found to be 1.925, 7.025, and 0.8025, respectively, which were higher than those in other DM groups (P<0.05). By contrast, the expressions of the three kinds of cells in the PBS group were much less than those of the others (Figure 4D).


C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway.

Zhang R, Zhou SJ, Li CJ, Wang XN, Tang YZ, Chen R, Lv L, Zhao Q, Xing QL, Yu DM, Yu P - Lipids Health Dis (2013)

Effect of CRP/oxLDL/β2-GPI on infiltration of inflammatory cells in the aortas. Aortas were removed, paraffin-embedded, deparaffinated, underwent antigen retrieval, blocked, and then incubated with primary antibodies (Mac 1:200 or α- SMA 1:100 or CD3 1:100) at 4°C overnight. Biotinylated secondary antibody (1:200) was added, followed by HRP-labeled streptavidin incubation. Infiltrations of macrophages, SMCs and T cells in the aortas were quantified as the percentage of the luminal surface covered by positive cells to the plaque area using Image Pro-Plus 6.0. Results are shown as means ± SEM. A: Infiltration of SMCs in the aortas, 400× magnification. B: Infiltration of macrophages in the aortas, 400× magnification. C: Infiltration of T cells in aortas, 400× magnification. D: Positive expressions of SMA, MAC and CD3 (n=4), expressed as the percentage to plaque area. *P <0.05, compared with the CRP/oxLDL/β2GPI group, †P < 0.05, compared with the PBS group.
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Related In: Results  -  Collection

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Figure 4: Effect of CRP/oxLDL/β2-GPI on infiltration of inflammatory cells in the aortas. Aortas were removed, paraffin-embedded, deparaffinated, underwent antigen retrieval, blocked, and then incubated with primary antibodies (Mac 1:200 or α- SMA 1:100 or CD3 1:100) at 4°C overnight. Biotinylated secondary antibody (1:200) was added, followed by HRP-labeled streptavidin incubation. Infiltrations of macrophages, SMCs and T cells in the aortas were quantified as the percentage of the luminal surface covered by positive cells to the plaque area using Image Pro-Plus 6.0. Results are shown as means ± SEM. A: Infiltration of SMCs in the aortas, 400× magnification. B: Infiltration of macrophages in the aortas, 400× magnification. C: Infiltration of T cells in aortas, 400× magnification. D: Positive expressions of SMA, MAC and CD3 (n=4), expressed as the percentage to plaque area. *P <0.05, compared with the CRP/oxLDL/β2GPI group, †P < 0.05, compared with the PBS group.
Mentions: CRP/oxLDL/β2GPI group showed strong positive expression of α-SMA in each layer of aorta. The other DM groups only displayed positive expression on the surface of the intima, not on the shrink media or not as strong as that in the CRP/oxLDL/β2GPI group (Figure 4A). CRP/oxLDL/β2GPI group showed remarkable positive areas of macrophages (Figure 4B). T cell expression was not as obvious as those of SMC and macrophages, but certain amounts of T cells were found in the lumen in the CRP/oxLDL/β2GPI group. Moreover, positive CD3 expression was not limited to the intima, but was also found on the adventitia (Figure 4C). Using Image Pro-Plus 6.0, the α-SMA, MAC, and CD3 positive expressions of AS lesion in the CRP/oxLDL/β2GPI group were found to be 1.925, 7.025, and 0.8025, respectively, which were higher than those in other DM groups (P<0.05). By contrast, the expressions of the three kinds of cells in the PBS group were much less than those of the others (Figure 4D).

Bottom Line: The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR.CRP/oxLDL/β2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05).The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/β2GPI group (P < 0.05).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diabetic Nephropathy Hemodialysis, Key Laboratory of Hormones and Development, Ministry of Health, Metabolic Diseases Hospital & Tianjin Institute of Endocrinology Tianjin Medical University, Tongan Street, Tianjin, Heping District 300070, China.

ABSTRACT

Background: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I (CRP/oxLDL/β2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice.

Methods: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 μg oxLDL, 20 μg β2GPI, 40 μg oxLDL/β2GPI complex, 44 μg CRP/oxLDL/β2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip.

Results: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/β2GPI groups (P < 0.05). CRP/oxLDL/β2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/β2GPI group (P < 0.05).

Conclusions: CRP/oxLDL/β2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

Show MeSH
Related in: MedlinePlus