Atmospheric ultrafine particles promote vascular calcification via the NF-κB signaling pathway

Exposure to atmospheric fine particulate matter (PM2.5) is a modifiable risk factor of cardiovascular disease. Ultrafine particles (UFP, diameter <0.1 μm), a subfraction of PM2.5, promote vascular oxidative stress and inflammatory responses. Epidemiologic studies suggest that PM exposure promotes vascular calcification. Here, we assessed whether UFP exposure promotes vascular calcification via NF-κB signaling. UFP exposure at 50 μg/ml increased alkaline phosphatase (ALP) activity by 4.4 ± 0.2-fold on day 3 ( n = 3, P < 0.001) and matrix calcification by 3.5 ± 1.7-fold on day 10 ( n = 4, P < 0.05) in calcifying vascular cells (CVC), a subpopulation of vascular smooth muscle cells with osteoblastic potential. Treatment of CVC with conditioned media derived from UFP-treated macrophages (UFP-CM) also led to an increase in ALP activities and matrix calcification. Furthermore, both UFP and UFP-CM significantly increased NF-κB activity, and cotreatment with an NF-κB inhibitor, JSH23, attenuated both UFP- and UFP-CM-induced ALP activity and calcification. When low-density lipoprotein receptor-null mice were exposed to UFP at 359.5 μg/m3 for 10 wk, NF-κB activation and vascular calcification were detected in the regions of aortic roots compared with control filtered air-exposed mice. These findings suggest that UFP promotes vascular calcification via activating NF-κB signaling.

A dynamic model of calcific nodule destabilization in response to monocyte- and oxidized lipid-induced matrix metalloproteinases

Vulnerable plaque remains clinically undetectable, and there is no accepted in vitro model. We characterize the calcific nodules produced by calcifying vascular cells (CVC) in ApoE-null mice, demonstrating increased destabilization of cultured nodules in the presence of oxidized low-density lipoprotein (oxLDL) and monocytes under pulsatile shear stress. CVC implanted in the subcutaneous space of hyperlipidemic mice produced nodules revealing features of calcific atherosclerotic plaque including a fibrous cap, cholesterol clefts, thin shoulder, lipids, and calcium mineral deposits. CVC nodules seeded in the pulsatile flow channel ( τavg = 23 dyn/cm2, ∂ τ/∂ t = 71 dyn·cm−2·s−1) underwent deformation and destabilization. Computational fluid dynamics revealed distinct shear force profiles on the nodules. Presence of oxLDL or monocytic THP-1 cells significantly increased the numbers of nodules destabilized from the substrate. Both oxLDL and THP-1 increased matrix metalloproteinase (MMP) activity in CVC. The MMP inhibitor GM6001 significantly reversed oxLDL- and THP-1-induced nodule destabilization, whereas overexpression of MMP-9 increased destabilization. These findings demonstrate that CVC-derived nodules resembled calcific atherosclerotic plaque and were destabilized in the presence of active lipids and monocytes via induction of MMPs.

Increased Lipogenesis and Stearate Accelerate Vascular Calcification in Calcifying Vascular Cells

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRβ accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXRα and LXRβ reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.