RAGE/Gal-3调控基质小泡释放富集介导斑块内钙化转归的机制研究

Vascular calcification is an important feature of advanced plaques of diabetic atherosclerosis. Microcalcification accelerates the occurrence of plaque rupture, while macrocalcification promotes the stability of plaque. Our previous studies indicated that blocking RAGE or Gal-3, two major receptors of the advanced glycation end-products, had different effects on the release and fusion of matrix vesicles, so as to induce macrocalcification or microcalcification separately. This study intends to use computer 3D simulation, radioligand receptor binding assays and in vitro culture of smooth muscle cells (SMCs) / calcified aortic rings to clear the effect of RAGE / Gal-3 in the release and fusion of matrix vesicle and subsequent calcification evolution. Furthermore, our subsequent study utilizes microCT / microPET fusion imaging of plaque calcification animal model and performs 3D reconstruction via pathological staining of sequential sections to explore the temporal and spatial characteristics of matrix vesicles, macrocalcification and microcalcification evolution in aortic and coronary smooth muscle cells, thus comprehensively elaborating the signal transduction pathways of calcification evolution induced by RAGE / Gal-3 regulation of matrix vesicles in the process of diabetic atherosclerosis. Implementing the study without a hitch will provide a better experimental basis for further study in the calcification evolution mechanism of diabetic atherosclerosis plaques and offer a new perspective for targeting prevention and treatment.

血管钙化是糖尿病动脉粥样硬化晚期斑块的重要特征。点灶状微钙化促进斑块破裂的发生，线性大钙化促进斑块稳定性的形成。申请者前期研究发现：阻断糖基化终末产物两大受体RAGE、Gal-3，对基质小泡释放与富集的作用截然不同，并可因此诱发不同的大钙化或微钙化形成。为探明机制，本研究拟通过计算机三维模拟、放射性配体受体结合实验、平滑肌细胞/钙化主动脉环体外培养明确RAGE/Gal-3在影响基质小泡释放、富集及随后钙化转归中的作用；通过斑块内钙化动物模型的microCT、microPET融合显像及连续切片病理形态学染色三维重构，从在体主动脉和冠状动脉两个层面探索基质小泡、微钙化与大钙化三者间转归的时空特征，阐述RAGE/Gal-3调控基质小泡介导糖尿病斑块内钙化转归的信号传递路径。该研究的顺利实施将为深入挖掘糖尿病斑块内钙化的演进机制提供更为完整的实验依据，也将为靶向机制的防治策略提供新的切入点。

血管钙化是糖尿病动脉粥样硬化晚期斑块的重要特征。已有研究显示晚期糖基化终末产物（AGEs）的关键活性成分羧甲基赖氨酸（CML）可能通过糖基化终末产物两大受体RAGE、Gal-3诱发大钙化或微钙化形成，但具体机制尚不明确。本研究旨在探索CML所致斑块内钙化转归的时空特征，阐明RAGE /Gal-3通过触发分子开关Sortilin调控基质小泡释放富集介导斑块内钙化转归的相关机制，研究发现：在糖尿病临床样本中，在大钙化的血管平滑肌中RAGE表达明显少于Gal-3，而微钙化的血管平滑肌RAGE表达显著多于Gal-3。在糖尿病动脉粥样硬化模型下，Sortilin作为钙化转归的分子开关，在 RAGE/Gal-3 的下游介导微钙化/大钙化形成。RAGE抑制Sortilin表达促使微钙化的形成，Gal-3促进Sortilin表达，加速基质囊泡的聚集和释放并且增加钙的摄取和聚集，从而导致斑块内大钙化的形成。在体内和体外实验中，我们探讨了Sortilin在糖尿病动脉粥样硬化钙化转归中的作用机制，证实了CML-RAGE/Gal-3-Sortilin-MVs 钙化信号的传递途径。另外基于基础实验和临床数据，构建了基于螺旋CT的下肢血管钙化评价方法，并成功合成18F-CML探针并用于斑块内钙化的示踪，对显像剂不同富集程度动脉节段的vonKossa钙染色发现，18F-SFB-CML的富集程度能够比较准确的反映出血管壁钙化灶的分布和大小。该研究目前已获得五项授权专利，包括一项外国授权专利，为斑块内钙化的分子机理认识与钙化的检测评估提供了新的视角。