A miR-125 / Sirtuin-7 pathway drives pro-calcific potential of myeloid cells in diabetic vascular disease

Aims/hypothesis. Ectopic calcification is a typical feature of diabetic vascular disease and resembles an accelerated aging phenotype. We previously found an excess of myeloid calcifying cells (MCCs) in diabetic patients. We herein examined molecular and cellular pathways linking atherosclerotic calcification with calcification by myeloid cells in the diabetic milieu. Methods. We first examined the associations among coronary calcification, MCC levels, and mononuclear cell gene expression in a cross-sectional study of 87 type 2 diabetic patients undergoing elective coronary angiography. Then, we undertook in vitro studies on mesenchymal stem cells (MSCs) and on the THP-1 myeloid cells line to verify the causal relationships of the observed associations. Results. Coronary calcification was associated with 2.8-times higher MCC levels (p=0.037) and 50% elevated expression of the osteogenic gene RUNX2 in mononuclear cells, whereas expression of Sirtuin-7 (SIRT7) was inversely correlated with calcification. In standard differentiation assays of MSCs, SIRT7 knockdown activated the osteogenic program and worsened calcification, especially in the presence of high (20 mM) glucose. In the monocytic cell line THP-1, SIRT7 downregulation drove a pro-calcific phenotype, whereas SIRT7 overexpression prevented high-glucose induced calcification. Through the JAK/STAT pathway, high glucose induced miR-125b-5p, which in turn targeted SIRT7 in myeloid cells and was directly associated with coronary calcification. Conclusions/interpretation. We describe a new pathway elicited by high glucose trough the JAK/STAT cascade, involving regulation of SIRT7 by mir-125b-5p driving calcification by myeloid cells. This pathway is associated with coronary calcification in diabetic patients and may be a target to tackle diabetic vascular disease. mRNA profile on osteo-THP-1 cultured in high (20mM) glucose and normal (5 mM) glucose

研究目的与假说。异位钙化是糖尿病血管病变的典型特征，且呈现加速衰老表型。本团队此前在糖尿病患者体内发现了过量的髓系钙化细胞（myeloid calcifying cells, MCCs）。本研究旨在探索糖尿病微环境中，动脉粥样硬化钙化与髓系细胞钙化之间关联的分子与细胞通路。
研究方法。本研究首先对87名接受选择性冠状动脉造影的2型糖尿病患者开展横断面研究，分析冠状动脉钙化程度、髓系钙化细胞水平与外周血单核细胞基因表达三者间的关联。随后，本团队针对间充质干细胞（mesenchymal stem cells, MSCs）及THP-1髓系细胞系开展体外实验，以验证所观察到的关联的因果关系。
研究结果。冠状动脉钙化与髓系钙化细胞水平升高2.8倍（p=0.037）以及单核细胞中成骨基因RUNX2的表达量升高50%显著相关；而Sirtuin-7 (SIRT7)的表达与钙化程度呈负相关。在间充质干细胞的标准分化实验中，SIRT7基因敲低会激活成骨程序并加重钙化，尤其在高糖（20 mM）环境下更为显著。在单核细胞系THP-1中，SIRT7表达下调会促发钙化表型，而SIRT7过表达则可阻断高糖诱导的钙化。进一步机制研究显示，高糖可通过JAK/STAT通路上调miR-125b-5p，后者可靶向调控髓系细胞中的SIRT7，且其表达水平与冠状动脉钙化直接相关。
结论与阐释。本研究揭示了一条由高糖通过JAK/STAT级联反应介导的全新通路：miR-125b-5p通过调控SIRT7的表达，驱动髓系细胞发生钙化。该通路与糖尿病患者的冠状动脉钙化密切相关，或可成为治疗糖尿病血管病变的潜在干预靶点。于高糖（20 mM）及正常糖（5 mM）环境中培养的成骨分化THP-1细胞的mRNA表达谱