Proteomic analysis and comparison of intra‑ and extracranial cerebral atherosclerosis responses to hyperlipidemia in rabbits

Abstract

The present study aimed to investigate protein expression levels of intra‑ and extracranial atherosclerosis in rabbits following administration of a high‑fat diet. Rabbits were randomly divided into control (group A; n=9) and high‑fat diet (group B; n=9) groups. At week 12, tissues were sectioned from the common carotid artery (CCA) and middle cerebral artery (MCA). Pathological analysis was performed. Differential protein expression levels were examined by 2‑D gel electrophoresis (2‑DE) and mass spectrometry (MS) analysis and validated by western blotting. Serum lipid levels, the intima‑media thickness (IMT) and degree of atherosclerosis of the CCA and MCA were increased at week 12 in the high‑fat diet group compared with rabbits that received a normal diet. 2‑DE and MS analysis of the protein extracted from CCA and MCA detected >439 different proteins; the expression of 25 proteins was altered, and 8 proteins [albumin A chain, tropomyosin α‑1 chain (TPM1), heat shock protein 70 (HSP70), α‑smooth muscle actin, β‑galactose binding agglutinin, TPM4 isoform 2, cell keratin 9, single octylic acid glyceride β‑2) demonstrated significant alterations in expression levels. Due to limited antibody sources, only three differentially expressed proteins (TPM1, HSP70 and α‑smooth muscle actin) were examined by western blotting. The results of our previous study demonstrated that hyperlipidemia affected the IMT of intracranial and extracranial cerebral arteries. In the present study, protein expression levels of TPM1 and α‑smooth muscle actin from extracranial cerebral arteries were significantly increased compared with intracranial cerebral arteries; however, protein expression levels of HSP70 from intracranial cerebral arteries was increased compared with extracranial cerebral arteries. The differences may be closely associated with cell proliferation and metastasis, and oxidoreduction, in intra‑ and extracranial cerebral atherosclerosis. HSP70 may have protective properties against atherosclerosis via underlying anti‑inflammatory mechanisms, furthermore, differential protein expression levels (TPM1, HSP70 and α‑smooth muscle actin) between intra‑ and extracranial cerebral arteries may facilitate the identification of novel biological markers for the diagnosis and treatment of cerebral arteriosclerosis.

Figure 1.

Hematoxylin-eosin-stained sections of arteries at week 12. Common carotid artery sections from (A) group A demonstrated smooth endomembrane and (B) in group B fatty streak formation plaque could be observed (magnification, ×4). Middle cerebral artery sections from (C) group A had smooth endomembrane and (D) in group B the inhomogeneous intima-media was thickened (magnification, ×20). Scale bar, 100 µm. Group A, control group; group B, high-fat diet group.

Figure 2.

A representative 2-D gel map of CCA and MCA sections from groups A and B. Total protein extracts from (A) CCA in group A, (B) CCA in group B, (C) MCA in group A and (D) MCA in group B were separated on pH 3–10 nonlinear immobilized pH gradient strips in the first dimension followed by SDS-PAGE in the second dimension. The gels were visualized. In total, 8 altered spots were identified by 2-D gel electrophoresis and mass spectrometry analyses. Data on each of these protein species is reported in Table III. CCA, common carotid artery; MCA, middle cerebral artery; group A, control group; group B, high-fat diet group.

Figure 3.

Protein expression differences in groups A and B, as analyzed by mass spectrometry. (A) CCA in group A, (B) CCA in group B, (C) MCA in group A and (D) MCA in group B. Black arrows indicate proteins that were significantly altered following a high-fat diet; >439 protein spots were detected per gel. Of the altered proteins, 25 protein expression levels were different and 8 spots (marked with arrow and number) demonstrated significant alterations and were quantitatively high enough to be identified by Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry. CCA, common carotid artery; MCA, middle cerebral artery; group A, control group; group B, high-fat diet group.

Figure 4.

Western blot analysis. (A) Representative western blot images and (B) quantification of TPM1, α-smooth muscle actin and HSP70 protein expression levels in the CCA and MCA of groups A and B. GAPDH served as an internal control. Data are expressed as the mean ± standard deviation from two experiments. *P<0.05 vs. group A CCA and ^#P<0.05 vs. group B CCA. TPM1, tropomyosin α-1 chain; HSP70, heat shock protein 70; α-actin, α-smooth muscle actin; CCA, common carotid artery; MCA, middle cerebral artery; group A, control group; group B, high-fat diet group; A/G, α-smooth muscle actin/GAPDH; T/G, TPM1/GAPDH; H/G, HSP70/GAPDH.