脂肪因子CTRP9调控AMPK通路及平滑肌细胞表型转化抑制动脉钙化的机制研究

Atherosclerotic calcification is closely related plaque instability, and is an independent predictor of cardiovascular mortality. Phenotypic transition of smooth muscle cells was involved in arterial calcification. However, the intrinsic protective mechanisms remain unclear. We previously used in vivo intravascular optical coherence tomography and found early spotty calcification indicated high-risk plaque and was associated with decreased level of C1q/tumor necrosis factor-related protein 9 (CTRP9) in humans. The level of CTRP9 in aorta of mice decreased after high fat diet. In vitro studies showed CTRP9 inhibits osteogenic transition of smooth muscle cells. Recent studies showed that AMPK negatively modulated osteogenic differentiation, and CTRP9 protected the heart through upregulated AMPK. Thus, we hypothesized that CTRP9 might modulate AMPK pathway, and inhibit phenotypic transition of smooth muscle cells and atherosclerotic calcification. In this program, we aimed to validate the correlation of CTRP9 with spotty calcification in humans. Then, we established atherosclerotic calcification model in CTRP9 and ApoE double knockout mice, in combination with CTRP9 overexpression as well as genetic and drug intervention, to clarify the role of CTRP9 in modulating calcification through AMPK/PIAS1/Runx2 pathway by imaging and pathological analysis. It is expected that this program may explore new therapeutic targets to clinically intervene arterial calcification.

动脉粥样硬化钙化形成与斑块不稳定性密切相关，是心血管死亡的独立预测因素。平滑肌细胞表型转化参与动脉钙化形成，但内源性保护机制未明。我们前期通过人群在体血管内光学相干断层成像发现，早期点状钙化提示高危斑块，并伴随脂肪因子C1q肿瘤坏死因子相关蛋白9（CTRP9）水平下降；高脂饮食小鼠主动脉钙化组织CTRP9水平下降；体外实验显示CTRP9抑制平滑肌细胞成骨转化。新近研究提示AMPK负性调节成骨分化，而CTRP9可上调AMPK实现心脏保护。我们假设：CTRP9可能通过调控AMPK通路，抑制平滑肌细胞表型转化及动脉粥样硬化钙化形成。本项目拟在人群中验证CTRP9与点状钙化的相关性；在CTRP9和ApoE双基因敲除小鼠中建立钙化模型，结合CTRP9过表达和基因或药物干预，通过影像、病理等手段，阐明CTRP9通过AMPK/PIAS1/Runx2通路参与调控钙化形成。以期为临床干预动脉钙化提供靶点。

1. 研究背景.动脉粥样硬化钙化形成与斑块不稳定性密切相关，是心血管死亡的独立预测因素。平滑肌细胞表型转化参与动脉钙化形成，但内源性保护机制未明。.2. 研究内容.入选就诊的急性冠脉综合征（ACS）患者，行冠脉造影和OCT检查，并留取患者静脉血标本。.选择ApoE-/-小鼠，建立动脉粥样硬化钙化模型，观察相关细胞标记物的分布并定量分析。在小鼠主动脉外膜周给予Ad-CTRP9或Ad-β-gal后，建立CTRP9过表达模型，观察上述指标。.选择C57BL/6WT小鼠，建立CTRP9过表达模型，在慢性间歇性缺氧（CIH）或常氧（NOR）暴露4周后建立小鼠心肌梗死（MI）模型，观察左室功能、心肌细胞凋亡、肥大、心肌纤维化以及自噬的相关指标。.3. 研究结果.对于既往无心血管病史患者，呼吸暂停低通气指数（AHI）≥5事件/小时的患者的冠状动脉钙化（CAC）存在高于AHI<5事件/的患者(OR=1.896，95％CI=1.423-2.526，p<0.001)，且AHI越高CAC积分越高。.OCT亚组分析中，对于罪犯病变，OSA组的巨噬细胞浸润程度显著高于非OSA组（p＜0.05）；对于非罪犯病变，OSA组的巨噬细胞浸润总长度（p=0.04）、钙化病变严重程度（p=0.002）、点状钙化数量（p=0.01）显著高于非OSA组。.添加巨噬细胞上清的主动脉平滑肌细胞的表型变化：成骨表型标志物RUNX2表达水平上升同时收缩表型标志物SM22α表达水平下调；PCR检测显示：MOVAS炎症因子受体IL-1R、IL-6R的转录水平显著高于其他三组。.在CIH的条件下，CTRP9通过PI3K/AKT通路激活自噬保护心肌梗死后心脏损伤。免疫荧光检测CIH+MI组小鼠LC3B阳性点状表达降低（P＜0.01，CIH+MI vs. NOR+MI）。重组CTRP9给药后可预防CIH诱发的心脏损伤，减少间质纤维化面积（P=0.0418，MI+CIH+对照vs. MI+CIH+gCTRP9）。CTRP9可能部分激活了被CIH抑制的自噬，CIH+MI+AAV9-CTRP9组心脏中LC3B的点状表达增多（P＜0.05）。.在体外模型中，CTRP9的心肌保护作用部分通过PI3K/Akt通路，而不是AMPK通路，而3-MA处理可显著抵消CTRP9的抗凋亡作用(34.13% vs. 22.77%，P=0.0012)。