血小板微粒促进动脉粥样硬化斑块内血管新生的作用及机制

Atherosclerotic intraplaque angiogenesis (IPA) is one of the main causes of plaque destabilization. We have recently found that platelet microparticles (PMPs) released by activated platelets provoke extensive inflammatory responses, and promote endothelial cell migration and tube formation, in which upregulated expression and activity of MMP-2 and MMP-9 by PMPs plays an important role. Whereas the exact mechanism responsible for MMP upregulation remains unclear, we hypothesize that the PMPs-derived high-mobility group box 1 (HMGB1) may play a crucial role in this process. In addition, PMPs are known to contain numerous pro-angiogenic miRNAs, such as miR-27b and miR-130a, but absence of anti-angiogenic microRNAs. Thus, we further hypothesize that PMPs promote IPA through orchestrated action of PMPs-derived HMGB1, miR-27b and miR-130a. The aim of this proposal is to explore this hypothesis. We will investigate: ① to establish the role of PMPs-derived HMGB1 in promoting angiogenesis via expression of a pro-angiogenic factor neutral sphingomyelinase-2 (nSMase2); ② to confirm that PMPs promote angiogenesis through transference of miR-27b and miR-130a into endothelial cells; ③ to evaluate the pro-angiogenic activity of PMPs in chicken embryo chorioallantoic membrane assay and a murine model of Matrigel plug subcutaneous implantation; ④ to test the effect of chronic administration of exogenous PMPs on distribution of atherosclerotic plaque, the IPA and the stability of plaques in apoE-gene-knockout mice as an animal model of atherosclerosis. Collectively, these studies are expected to establish the critical role of PMPs in plaque destabilization through promotion of IPA. Our findings would provide a clue for novel strategy to prevent plaque destabilization.

斑块内血管新生（IPA）是导致斑块失稳定的重要原因。我们前期研究表明，血小板微粒（PMPs）除了促炎症作用外，还可促进内皮细胞迁移和管腔形成，并与MMP-2和MMP-9的表达增加有关，但机制尚不清楚。PMPs来源的HMGB1可能发挥着关键作用。而且PMPs中具有促血管新生作用的microRNAs（miR-27b和miR-130a）可能也参与了IPA的形成。本项目拟研究：① PMPs来源的HMGB1对促血管新生的重要分子-中性鞘磷脂酶-2表达的影响和机制；②PMPs将microRNAs转移至内皮细胞后对血管新生的调控作用；③采用鸡胚绒毛尿囊膜和小鼠皮下基质胶塞模型，观察PMPs对血管新生的影响；④采用apoE基因敲除小鼠模型，通过定期静脉注射给予外源性PMPs，最后检测斑块的分布、稳定性和IPA的情况。本项目旨在进一步阐明PMPs促进斑块失稳定的作用和机制，并为其防治提供新的思路和实验依据。

血管新生也可发生于动脉粥样硬化斑块内，称之为斑块内血管新生（intraplaque angiogenesis, IPA）。但IPA不具有成熟的管腔，因而通透性高，极易导致局部出血，即斑块内出血（intraplaque haemorrhage, IPH）。IPH可使斑块内压力上升，同时伴有大量粒细胞进入斑块内部、加剧斑块内炎症反应，可共同导致斑块失稳定，最终发生斑块破裂、血栓形成，在临床上表现为急性心脑血管事件（急性心肌梗死、脑梗塞、主动脉夹层等）的发生。血小板微粒（PMPs），也称为血小板微囊泡（PMVs），是血小板激活后所产生和释放的微囊泡，其表面携带有血小板膜的多种糖蛋白和磷脂酰丝氨酸（PS），并含有丰富的细胞因子、生长因子和microRNA等生物活性物质，参与对内皮细胞功能和血管新生过程的调控。本项目进一步研究了PMPs对动脉粥样硬化斑块稳定性的影响和机制，并探讨了靶向性抗氧化纳米药物对动脉粥样硬化的防治效果。结果发现：①给予外源性的PMVs，可促进高脂饲养ApoE基因敲除小鼠动脉粥样硬化（AS）斑块的形成；通过增加动脉壁中巨噬细胞浸润、MMP-9表达、使胶原和平滑肌细胞数量下降，导致AS斑块的稳定性下降；此外，PMVs作用于内皮细胞后，除了可导致内皮细胞功能异常以外，还可导致内皮细胞损伤（详见第三军医大学学报, 2019;41:514-20&2020;42:758-64）；②血管紧张素II（AngII）可通过诱导STAT3/miR-21/PTEN信号通路活化，促进“人微血管内皮细胞株(HMECs)”的增殖、迁移和管腔形成，由于PMVs中富含miR-21，说明PMVs还可能通过其携带的miR-21参与血管新生过程的调节（详见J Cell Physiol,2019;234:19640-54）；③课题组采用改进的纳米沉淀-自组装方法，制备了广谱活性氧清除性纳米药物（TPCD NP），TPCD NP 能被巨噬细胞和平滑肌细胞吞噬，抑制细胞的炎症反应和和氧化应激损伤以及细胞凋亡，抑制细胞吞噬脂质能力和泡沫细胞的形成；动物实验证实，TPCD NP 具有良好的AS 斑块靶向性、以及抑制斑块形成和促进斑块稳定的作用（详见ACS Nano, 2018;12:8943-60）。