Inflammation is currently a well-documented component of atheroslcerosis pathogenesis, which plays a role at each stage of the disease development. Local activation of endothelial cells causing increase endothelial permeability, infiltration of intima with atherogenic low-density lipoprotein (LDL), and recruitment of circulating immune cells is regarded as the first step of atherosclerotic plaque development. Circulating modified LDL is immunogenic, and forms highly atherogenic aggregates with antibodies that are later accumulated in the arterial wall. In growing plaques, circulating monocytes are attracted to the lesions site by cytokine signaling. In the arterial wall, monocyte-derived macrophages play an active role in lipid accumulation, internalizing large associates of lipoprotein particles by means of phagocytosis. Phagocytic cells with cytoplasm filled by stored lipid droplets called foam cells can be found in developing plaques in large quantities. There is evidence that lipid accumulation in the arterial wall cells in its turn activates cytokine signaling leading to a vicious cycle and further aggravating the disease. However, the immune response in atherosclerosis is not limited to enhanced inflammation, since anti-inflammatory cytokines and alternatively-activated (M2) macrophages are also present in atherosclerotic plaques. Anti-inflammatory M2 macrophages are likely to be responsible for hte resolution of the inflammatory response and tissue remodeling observed in advancing plaques. At later stages of lesion development, lipofibrous plaquest with high lipid contents and cell counts give rise to fibrous plaques that contain less cells and lipids, but more extracellular matrix material.
Although the involvement of cytokines in atherosclerotic lesion development is currently beyond doubt, quantitative evaluation of the expression of pro- and anti-inflammatory cytokines in the plaque remains to be studied in detail. In this study, we analyzed the distribution of two cytokines, pro-inflammatory TNFα and anti-inflammatory CCL18, in sections of human carotid atherosclerotic plaques at different stages of development. Our results demonstrated that both pro- and anti-inflammatory cytokines were present in the plaques, although differently distributed and likely expressed by different cells, and appeared to be enriched as compared to grossly normal intima taken as a control. To test whether the expression of TNFα and CCL18 is increased in atherosclerotic lesions, we performed gene expression analysis by means of quantitative PCR. We found that the expression of both cytokines was indeed increased in different types of atherosclerotic lesions. Moreover, it followed a bell-shaped distribution across the 4 studied plaque stages, gradually increasing from the early initial lesions to fatty streaks, reaching maximum in lipofibrous plaques, and decreasing again in fibrous plaques. This distribution was consistent with our previously published observations of bell-shaped changes of atherosclerotic lesion cellularity, proliferative activity, collagen synthesis and lipid content at different stages of the development. For TNFα, the maximal increase in atherosclerotic lesions reached 2 folds as compared to normal tissue, while for CCL18, this number was 1.5 folds.
We next investigated the relationship between pro- and anti-inflammatory cytokine production and lipid accumulation in cells. To that end, we used cultured human monocyte-derived macrophages with lipid accumulation induced by incubation with atherogenic LDL obtained from atheroslcerosis patients' blood serum. Non-atherogenic LDL obtained from healthy donors, which did not cause cholesterol accumulation in cultured cells, was used as a control. We found that cholesterol accumulation in macrophages caused by atherogenic LDL treatment was associated with up-regulation of both TNFα and CCL18. The increase in relative gene expression was statistically significant (p=0.05 for TNFα and p=0.023 for CCL18) as compared to non-atherogenic LDL treatment.
In this work, we report the increased expression of pro-inflammatory cytokine TNFα and anti-inflammatory CCL18 in human atherosclerotic lesions, which could be observed microscopically and in gene expression analysis by means of quantitative PCR. Furthermore, we demonstrate that the increase of pro- and anti-inflammatory cytokines expression is associated with cholesterol accumulation caused by atherogenic LDL in cultured cells. It is likely that lipid accumulation is the trigger of cytokine expression in atherosclerotic lesions, since the maximum of expression is observed in atherosclerotic lesions most enriched in lipids. We discuss the implications of these findings for atheroslcerosis pathogenesis, postulating that a splash of cytokine signaling occurs in lesions with the highest lipid contents. We hypothesize that both pro- and anti-inflammatory responses take place in human atherosclerotic lesions, but are probably characterized by different dynamics. While pro-inflammatory signalling occurs rapidly in response to triggering stimuli and is transient, anti-inflammatory response is relatively slow and long-lasting. Under favorable conditions, resolution of inflammation should lead to a healing process and plaque stabilization, while chronic inflammation may aggravate the disease development.