Novel Approach to Stabilize Atherosclerotic Plaque in Carotid Artery

摘要 不稳定/易损斑块，特征为薄帽纤维粥样硬化，脂质池导致的坏死核心 可能导致严重的临床病症，包括短暂性脑缺血发作、中风、失语症， 和其他运动缺陷。炎症、斑块血管平滑肌细胞（VSMC）的凋亡和 细胞外基质降解的增加是斑块不稳定性的主要潜在原因。 尽管进行了广泛的研究，但易损斑块形成的致病因素尚不清楚。基于 根据我们的新发现，我们假设脂多糖（LPS）或最低限度的oxLDL诱导 脆弱/不稳定的颈动脉斑块和拮抗TREM-1和TLR 4稳定动脉粥样硬化 颈动脉斑块这些研究将在动脉粥样硬化尤卡坦微型猪模型中进行 与人类颈动脉闭塞性疾病相似的颈动脉闭塞。这种模式的 模拟颈动脉疾病患者的真实的情况，因此， 了解疾病过程的病理生理学，使我们能够开发更好的治疗方法， 稳定易损斑块的方法。目的1：该假设预测， 动脉粥样硬化尤卡坦小型猪颈动脉中的LPS或最低限度oxLDL诱导 颈动脉斑块样不稳定斑块组织学、形态学及生化特征 通过增加TREM-1、MMP-1、MMP-9、TLR 4和M1巨噬细胞的表达， 胶原、TREM-2和M2巨噬细胞。我们将探索LPS诱导的斑块不稳定性的影响， 灰阶超声、多普勒超声、光学相干断层扫描、组织学、免疫组化 免疫荧光法分析纤维帽变薄、炎症、坏死核心、内膜增生 泡沫状巨噬细胞、VSMCs凋亡、脂质核心、坏死、斑块内出血和血管 重塑这些发现将在体内和体外稳定和不稳定的颈动脉斑块中进行比较。 我们还将检查和比较TREM-1，TREM-2， MMP-1，MMP-9，Ⅰ型胶原（α1），Ⅲ型胶原（α1），M1-M2巨噬细胞，血管平滑肌细胞，表达及相互作用 除了TREM-1对泡沫细胞形成、炎性细胞释放和炎症介质释放的作用外， 细胞因子，以及VSMC和巨噬细胞之间的相互作用。目标2：假设预测， TREM-1和TLR 4拮抗剂的施用将防止不稳定斑块的发展 动脉粥样硬化猪的颈动脉。抑制性TREM-1肽，LR 12，乱序的 肽（安慰剂）和TAK-242（一种TLR 4拮抗剂）将在颈动脉中进行检查， LPS/oxLDL给药。此外，还将检查体内和体外功能/生化参数。 这项研究的结果将证实TREM-1是否是治疗干预的新靶点， 扩展知识以开发更好的分子来拮抗TREM-1并设计I期临床试验。

ABSTRACT Unstable/vulnerable plaques, that are characterized by a thin cap fibroatheroma, necrotic core due to lipid pools by macrophages, may result in serious clinical conditions, including transient ischemic attack, stroke, aphasia, and other motor defects. Inflammation, apoptosis of plaque vascular smooth muscle cells (VSMCs) and increased degradation of extracellular matrices are the primary underlying cause of plaque instability. Despite extensive research, the causative factor(s) in the initiation of vulnerable plaque is unknown. Based on our novel findings, we hypothesize that lipopolysaccharide (LPS) or minimally oxLDL induce vulnerable/unstable carotid plaque and antagonizing TREM-1 and TLR4 stabilizes atherosclerotic plaque in carotid arteries. These studies will be performed in atherosclerotic Yucatan microswine model with occlusion of carotid arteries that resemble to occlusive carotid artery disease in human. This model will mimic the real situation in patients with carotid artery disease, and thus, will make a direct contribution to understanding the pathophysiology of the disease process and allow us to develop better therapeutic approaches to stabilize vulnerable plaques. Aim 1: The hypothesis predicts that the administration of LPS or minimally oxLDL in the carotid artery of atherosclerotic Yucatan microswine induces histological, morphological and biochemical features of carotid plaque like human unstable plaque by increasing the expression of TREM-1, MMP-1, MMP-9, TLR4 and M1 macrophages and decreasing collagen, TREM-2, and M2 macrophages. We will probe the effect of LPS-induced plaque instability by gray scale and Doppler ultrasound, Optical coherence tomography and the histology, immunohistochemistry and immunofluorescence to analyze thinned fibrous cap, inflammation, necrotic core, intimal proliferation with foamy macrophages, VSMCs apoptosis, lipid core, necrosis, intraplaque hemorrhage, and vascular remodeling. These findings will be compared in-vivo and in-vitro in the stable and unstable carotid plaques. We will also examine and compare the mRNA transcripts and protein expression of TREM-1, TREM-2, MMP-1, MMP-9, collagen I(α1), collagen III(α1), M1-M2 macrophages, VSMCs, expression and interaction of TLRs with TREM-1, in addition to the effect of TREM-1 on foam cell formation, release of inflammatory cytokines, and in the cross-talk between VSMCs and macrophages. Aim 2: The hypothesis predicts that the administration of TREM-1 and TLR4 antagonists will prevent the development of unstable plaque in the carotid artery of atherosclerotic swine. Effect of an inhibitory TREM-1 peptide, LR12, a scrambled peptide (placebo), and TAK-242, a TLR4 antagonist, will be examined in carotid arteries following LPS/oxLDL administration. Also, in-vivo and in-vitro functional/biochemical parameters will be examined. The findings from this study will confirm if TREM-1 is a novel target for therapeutic intervention and extend the knowledge to develop better molecules to antagonize TREM-1 and design phase I clinical trials.