Molecular Mechanism of Vascular Calcification

血管钙化是动脉粥样硬化的一个显著特征，几乎可以肯定与慢性血管炎症有关。然而，血管钙化的分子机制仍有待确定。人主动脉平滑肌细胞（HASMCs），人单核细胞系THP-1细胞，以及它们的共培养物生长在缺乏或存在的血管紧张素II，并测定其碱性磷酸酶（ALP）活性。虽然无论是HASMCs还是THP-1细胞单独显示ALP活性响应于Ang II的可测量的变化，但它们的共培养物通过Ang II刺激显示出显著更大的ALP活性。腺病毒介导的Notch胞内结构域（NICD）能显著增强HASMCs的ALP活性。HASMCs的RT-PCR分析表明，NICD过表达或Jagged-1刺激诱导Msx 2基因表达，这是成骨细胞分化的关键调节因子。荧光素酶检测结果表明，Jagged-1刺激可显著增强Msx 2启动子的转录活性，而选择性Notch信号抑制剂DAPT则可抑制Msx 2启动子的转录活性。DNA亲和沉淀分析证实了RBP-JK（Notch信号传导的主要介质）与Msx 2启动子内的序列元件的结合。因此，这些结果表明，血管紧张素II促进成骨细胞分化的HASMCs通过诱导Jagged-1在相邻的单核细胞/巨噬细胞，这反过来激活Notch信号转导及其下游靶Msx 2基因转录。

Vascular calcification is a prominent feature of atherosclerosis that is almost certainly associated with chronic vascular inflammation. However, molecular mechanisms of the vascular calcification remains to be determined. Human aortic smooth muscle cells (HASMCs), human monocytic cell line THP-1 cells, and their cocultures were grown in the absence or presence of Ang II, and their alkaline phosphatase (ALP) activity were measured. While neither HASMCs nor THP-1 cells alone showed measurable change in ALP activity in response to Ang II, their coculture exhibited significantly greater ALP activity by Ang II stimulation. Adenovirus expressing Notch intracellular domain (NICD) markedly augmented ALP activity in HASMCs. RT-PCR analysis of HASMCs showed that either NICD overexpression or stimulation by Jagged-1 induced Msx2 gene expression, a key regulator of osteoblastic differentiation. Luciferase assays showed that transcriptional activity of Msx2 promoter was significantly enhanced by Jagged-1stimulation, whereas it was abrogated by a selective Notch signaling inhibitor DAPT. DNA affinity precipitation assays confirmed the binding of RBP-JK, a major mediator of Notch signaling, to the sequence element within the Msx2 promoter. Thus, these results suggest that Ang II promotes osteoblastic differentiation of HASMCs through an induction of Jagged-1 in adjacent monocytes/macrophages, which in turn activates Notch signaling and its downstream target Msx2 gene transcription in HASMCs.