In the present study, we generated a novel genetic mouse model with severe HHcy and hypercholesterolemia, Tg-hCBS apoE−/−Cbs−/−
mice, and examined the effects of HHcy on atherosclerotic lesion formation and monocyte accumulation. The Tg-hCBS apoE−/−Cbs
mice described here is a useful model for HHcy related atherosclerosis research. This model has a survival advantage over our previous apoE−/−Cbs
The Tg-hCBS apoE−/−Cbs−/−
mice retained severe HHcy and survived normally. The control Tg-hCBSapoE−/−Cbs
mice fed a HF diet have plasma Hcy levels of 13.9 μM, which are similar to what we have observed in the apoE−/−Cbs−/+
mice fed the same diet (14.2 μM).16
Plasma Hcy levels in the Tg hCBS apoE−/−
mice on regular diet is 200μM, which is higher than 169 μM in the Tg hCBS Cbs−/−
These data support the notion that hyperlipidemia increases Hcy levels.
The Tg-hCBS apoE−/− Cbs−/−
mice were slightly smaller than their littermate controls. This was also found in Tg-S466L Cbs−/−
mice (data not shown). These results are in accordance with our previous reports.24
We have observed a trend in decreasing spleen weight in severe HHcy Tg-hCBS apoE−/− Cbs−/−
mice (), suggesting that HHcy causes growth retardation in general and reduces spleen weight, potentially creating a negative impact on immune system development.
We previously reported that HHcy accelerates atherosclerosis and increases LDL uptake by peritoneal macrophages in apoE−/−
mice with and without dietary perturbation.16
The current study is in agreement with our previous findings, and provides further evidence demonstrating that severe HHcy accelerates atherosclerosis, and increases Ly6C positive monocyte/macrophage inflammatory subset accumulation in the atherosclerotic lesion (). These findings led to the new hypothesis that severe HHcy contributes to atherosclerosis via promoting inflammatory monocytes.
It has been suggested that inflammatory processes play a pivotal role throughout atherosclerotic progression. We found that plasma TNFα and MCP-1 levels were significantly elevated in Tg-hCBS apoE−/− Cbs−/−
mice, and were positively correlated with plasma Hcy levels (). TNFα is a proinflammatory cytokine that activates the endothelium. MCP-1 is a chemokine that mediates monocyte adhesion and infiltration into inflammatory sites. It was suggested that Hcy-induced MCP-1 expression in aortic endothelial cells may also contribute to leukocyte recruitment.25
Our findings support the hypothesis that HHcy promotes systemic inflammation and monocyte infiltration into the vessel wall.
Clinical criteria usually consider >8% circulating MNC, based on their mononuclear morphological feature, as monocytosis in humans. The control Tg-S466LCbs mice animals have 3–5% MNC (gate ii) among the peripheral nucleated cells (gate i) (). Severe HHcy induced monocytosis (8.6% MNC) at 8 months of age. Monocytosis was also observed in the control Tg-hCBS apoE−/−Cbs mice (10.3% MNC) and exacerbated by HHcy in Tg-hCBS apoE−/−Cbs−/− mice (11.6% MNC) (). Using CD11b as a monocyte molecular marker, we found that severe HHcy significantly promoted monocyte expansion in peripheral blood, spleen and bone marrow in both mouse lines independent of hyperlipidemia. ().
Recent studies show that circulating monocytes display heterogeneity, which commit to specific functions in atherogenesis. Using Ly-6C as a marker, monocytes can be divided into three subsets: Ly-6Chi
monocytes are recognized as the inflammatory subset, which can differentiate into macrophages in atheromas of apoE−/−
monocytes are considered as the intermediate inflammatory subset based on their ability to uptake inflammatory particles and migrate to sites of inflammation.26
We found that severe HHcy was associated with increases in both Ly-6Chi
monocyte subsets in peripheral blood, spleen and BM in both mouse models, which were further increased with aging (). Therefore, we have demonstrated, for the first time, that HHcy selectively promotes the expansion of inflammatory monocyte subsets in the periphery and BM in mice independent of hyperlipidemia.
Our data indicate that mouse primary splenocytes are a relevant in vitro model of monocyte survival and differentiation.13
The mouse spleen is an hematopoietic organ and was used to study monocyte/macrophage heterogeneity and in vitro monocyte differentiation.13, 14
Here, we have demonstrated an Hcy specific effect on promoting Ly-6C monocyte differentiation that is not mimicked by Cys. The distinct effects of Hcy and Cys on monocyte differentiation may be related to the different biochemical activities of these amino acids. Our study is the first to demonstrate that HHcy induces monocyte expansion and selectively promotes differentiation of Ly-6Chi
inflammatory monocytes in the diseased models of severe HHcy. The modulation of HHcy on monocyte heterogeneity may enhance inflammatory responses during atherogenesis and contribute to the increased risk of CVD in HHcy.
Finally, we demonstrated that Hcy-induced inflammatory monocyte differentiaiton was prevented by SOD/CAT and NAD(P)H oxidase inhibitor apocynin and partially reduced by uric acid and L-NAME. These data suggest that oxidant stress, especially superoxide anion, mostly due to NAD(P)H oxidase activation, is the major mechanism mediating Hcy-induced Ly-6Cmid
inflammatory monocyte differentiation. Because Hcy did not induce the Ly-6Cmid
subset in the presence of adenosine, a condition we established to sensitize intracellular hypomethylation,6
we ruled out a hypomethylation mechanism. It have been reported that Hcy induced MCP-1 secretion in human monocytes via oxidant stress.27
Moreover, Hcy-stimulated superoxide anion production is regulated by protein kinase C activation dependent of NAD(P)H oxidase in the human monocytic cell line THP-1.28
Future studies examining NAD(P)H oxidase regulation should provide important insight into the understanding of Hcy-induced monocyte differentiation and atherosclerosis.
In summary, our studies provide the first evidence demonstrating that severe HHcy induces systematic inflammation and accelerates atherosclerosis in a novel model of severe HHcy and hypercholestorolemia. We found that HHcy induces inflammatory monocyte subset differentiation in mice independent of hyperlipidemia, and in cultured splenocytes mostly via NAD(P)H oxidase-mediated oxidant stress. Our studies indicate that HHcy-induced inflammatory monocyte subset differentiation may be responsible for the increased risk of CVD in HHcy.