CVD is a leading cause of the mortality in diabetic patients, and its remarkable pathological change is AS. Given the recent study of β2GPI complexes, the present study explored the role of β2GPI complexes on AS in the STZ-induced diabetic AS model.
This study successfully developed an animal model that acquired the properties of diabetic AS, that was higher blood sugar level, lipid level and intuitive lesion staining with Sudan IV, after atherogenic diet for 19 weeks and STZ injection.
Increased intima-media thickness (IMT) reflects cardiovascular risk, and its increasing value (at or above 1 mm) represents increased risk of myocardial infarction and/or cerebrovascular disease . Our results, demonstrated that CRP/oxLDL/β2GPI treatment aggravated lipid deposition, increased IMT value in STZ-induced diabetic AS, promoted AS development and the risk of CVD.
Macrophages, SMCs and T cells have been considered to be the key cells involved in AS lesion development. Macrophages evolve into foam cells after amounts of lipid uptake and finally leading to early fatty streak formation. T cells, the main cells in cellular immunity, reach intima at an early stage of AS formation. Activated T cells produce cytokines, taking part in regulating lesion formation . Meanwhile, As atherosclerosis progresses, vascular SMCs are in proximity to and physically interact with inflammatory cell types, e.g. monocytes and macrophages, which play a very important role in further exacerbating the disease . Furthermore, histological images have shown that prior to the onset of complex plaque development in human tissues [11, 12] and experimental models of atherosclerosis, markers of inflammation are observed in SMCs in the media of the vessel wall, such as VCAM-1 and inflammatory transcriptional mediators, such as activated NF-κB [13, 14].
Immunohistochemistry results confirmed stronger positive expressions and more infiltration of SMCs, macrophages and T cells along the intima of aortas in DM groups, especially after being treated with CRP/oxLDL/β2GPI.
Although much of the researches on atherosclerosis has focused on the intimal accumulation of lipids and inflammatory cells, there is an increasing amount of interest in the role of the adventitia in coordinating the immune response in atherosclerosis . From the T cells and macrophage stain results (Figure 4), we found parts of positive expressions in the intima and in the adventitia in CRP/oxLDL/β2GPI group, but not in the other groups. Research has found that adventitia inflammatory cell infiltration occurs earlier than AS lesion formation , which indicating CRP/oxLDL/β2GPI could cause an early changes detected in diabetic AS.
ABCA1 and ABCG1 belong to cholesterol transporters in human pancreatic β cells, that play an important role in protecting against AS by facilitating cholesterol extracellular efflux from macrophages or foam cells . Moreover, recent studies have found that the effect of ABCG1 on AS lesion development in LDLR-/- mice depends on the stage of AS, that is, ABCG1 is atheroprotective in early lesions. As the lesions advance, ABCG1 enhances apoptosis and the compensatory mechanism, thus promoting further AS progression . In addition, ABCA1 high expression was associated with plaque complexity and oxLDL high level in plaques. ABCA1 mRNA and protein expressions exhibited an increasing trend before AS formation. oxLDL induced ABCA1 expression, and the foam cells induced by oxLDL could also enhance ABCA1 expression . Our study found that mRNA expressions of ABCA1 and ABCG1 in aortas of diabetic Balb/c mice were elevated in the CRP/oxLDL/β2GPI group. Therefore, the capability of CRP/oxLDL/β2GPI complex to increase ABCA1 and ABCG1 mRNA expression may be one of the important mechanisms of pro-AS.
Moreover, not only cholesterol metabolism is involved in the pathological change of AS, lipoprotein receptors, especially scavenger receptors, participate in AS process directly.
Scavenger receptor-B includes two subtypes, CD36 and SR-BI, which ligands are oxLDL and HDL, respectively. Combined with CD36, oxLDL, subsequently stimulates macrophage infiltration and SMC migration and proliferation, leading to vascular endothelial cells apoptosis, erosion of vessel walls, destroyed coagulation mechanism, and finally macrophage apoptosis, resulting in AS plaques . CD36 inhibition of CRP/oxLDL/β2GPI may be a result of compensatory mechanism at the early stage of AS lesion. On the other hand, SR-BI is the only membrane receptor that mediates the interaction between cells and HDL, preventing free cholesterol from accumulating at the artery walls, thus reducing AS incidence . Lowering SR-BI expression through the CRP/oxLDL/β2GPI complex consequently leads to cholesterol accumulation in the macrophages and foam cell formation, both of which may take part in the pro-AS process.
It is known that β2GPI interacts with oxLDL via 7-ketocholesterol having an w-carboxyl acyl chain, producing stable and nondissociable oxLDL/β2GPI complexes [5, 21, 22].
Once β2GPI binds with oxLDL, specific epitope of oxLDL was covered, distrubing its binding with antibodies, thus also ameliorating the pro-atherosclerosis of oxLDL . Furthermore, β2GPI decreased cellular accumulation of cholesterol via a reduction in cholesterol influx and an increase in cholesterol efflux, suggesting that β2GPI might play an important role in the prevention of atherosclerosis , and that may explain β2GPI and oxLDL/β2GPI made less lipid accumulation than oxLDL. However, when oxLDL/β2GPI further interacts with CRP through another site named oxPC , the pro-AS function of CRP/oxLDL/β2GPI complex was enhanced as our results showed, maybe due to the strong pro-inflammation function of CRP in AS of both cardiovascular and cerebrovascular diseases .
When refering to the signaling pathway potentially involved in AS pathogenesis, hyperglycemia AS causes activation of MAPK in the aortas of BALB/c mice. The major members of MAPKs found in aortas include p38 MAPK and are most strongly activated by oxidative stress, hyperglycemia and proinflammatory cytokines .
Our results demonstrated enhanced activation p38MAPK and its upstream protein p-MKK3/6 with the treatment of CRP/oxLDL/β2GPI complex in diabetic Balb/c mice. Hence, the p38MAPK pathway activated by the CRP/oxLDL/β2GPI complex may be the one of the key molecule mechanisms of pro-AS.
The protein chip was also applied in the investigation to quickly detect cytokines associated with AS development. We found that the expression level of five cytokines, namely, bFGF, IGF-II, IL-1α, IL-9, and PF-4 were significantly elevated. The five specific cytokines were expressed highest in the CRP/oxLDL/β2GPI group. All of them are participating in the regulation of AS onset and drive AS development through different ways [28–31], which prove that the CRP/oxLDL/β2GPI complex increases the inflammation level in diabetic Balb/c mice by increasing pro-inflammatory cytokine expression, leading to further AS development. However, the level of cytokines with elevated expression needs further verification by Western blot or enzyme-linked immunosorbent assay.
This study focused on the CRP/oxLDL/β2GPI complex, and explored and compared its effects with those of oxLDL, β2GPI, and oxLDL/β2GPI complexes on the genesis and progression of diabetic atherosclerosis. After the invention of the CRP/oxLDL/β2GPI complex, more AS plaques and lesions were elicited in diabetic Balb/c mice, which indicated the importance of CRP/oxLDL/β2GPI in the development of diabetic atherosclerosis and provided new evidence that p38MAPK is a principal regulator of inflammatory response.