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进一步研究。人主动脉平滑肌细胞(HASMCs)、人单核细胞系THP-1细胞及其与Ang II共培养，测定其碱性磷酸酶(ALP)活性。单独培养的HASMC和THP-1细胞在Ang II刺激下ALP活性均无明显变化，但经Ang II刺激后，HASMC和THP-1细胞的ALP活性显著升高。表达Notch胞内区(NICD)的腺病毒可显著增强HASMCs的ALP活性。RT-PCR分析表明，NICD过表达或Jagge-1刺激均可诱导MSX2基因表达，而MSX2基因是成骨细胞分化的关键调控因子。荧光素酶分析表明，Jagge-1刺激显著增强了MSX2启动子的转录活性，而选择性Notch信号抑制剂DAPT则抑制了其转录活性。DNA亲和沉淀分析证实了Notch信号的主要介导物RBP-JK与MSX2启动子内的序列元件的结合。因此，这些结果表明，Ang II通过在相邻的单核/巨噬细胞中诱导Jagge-1，进而激活HASMCs中的Notch信号及其下游靶基因MSX2基因转录，从而促进HASMCs的成骨分化。

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.