Macrophage-derived microcalcificaitons

项目摘要 这项研究项目将检验假设，在糖尿病环境中，S100A9诱导钙化 巨噬细胞来源的细胞外小泡(EV)的潜力，微钙化的前体，有助于 脆弱动脉粥样硬化斑块的生物力学不稳定性。S100A9，一种新发现的 1型糖尿病患者血液中易损斑块的生物标记物表达为 巨噬细胞，是EV的一个组成部分。我们发表的研究将巨噬细胞和钙化联系在一起，以及 表明巨噬细胞可以释放具有很高钙化潜力的EV。本研究将探讨 此外，促炎巨噬细胞在糖尿病血管钙化中的作用。具体目标1将 在体外验证假设，糖尿病环境促进巨噬细胞激活并加速释放和 S100A9富集型EV的矿化作用这些实验将涉及创新的方法来检测 EV微钙化和巨噬细胞表型，包括密度依赖的扫描电子 显微镜(DDSEM)与元素分析、高分辨率显微镜、纳米粒子跟踪相结合 分析、3D-水凝胶系统、蛋白质组学、单细胞RNA测序和网络分析。特定的 目的2将在体内验证S100A9介导糖尿病诱导的微钙化的假说 动脉粥样硬化斑块。我们预计：1)S100A9基因缺失；2)巨噬细胞靶向siRNA S100A9的沉默，以及III)S100A9缺陷小鼠的骨髓移植将延缓 微钙化的进展和随后的破裂，由分子成像和 全面的组织病理学分析。具体目标3将定量评估 微钙化对动脉粥样硬化斑块生物力学不稳定性的影响 建模和有限元分析。这些互补性研究将通过确定 巨噬细胞S100A9在微钙化中的作用为了促进老鼠数据的临床翻译，我们将 使用人类原代巨噬细胞和糖尿病患者的动脉粥样硬化斑块标本。 该项目的发现将有助于开发急需的抗钙化疗法。

Project Summary This research project will test the hypothesis that, in the diabetic milieu, S100A9 induces the calcification potential of macrophage-derived extracellular vesicles (EV), precursors of microcalcifications, contributing to the biomechanical instability of the vulnerable atherosclerotic plaque. S100A9, a recently identified biomarker of vulnerable plaques, increases in the blood of patients with type 1 diabetes, is expressed by macrophages, and is a component of EV. Our published studies linked macrophages and calcification, and showed that macrophages can release EV with a high calcification potential. The present study will explore further the role of proinflammatory macrophages in vascular calcification in diabetes. Specific Aim 1 will test the hypothesis in vitro that diabetic milieu promotes macrophage activation and accelerates release and mineralization of S100A9–enriched EV. These experiments will involve innovative methods for detection of EV microcalcifications and macrophage phenotypes, including density dependent scanning electron microscopy (DDSEM) combined with elemental analysis, high-resolution microscopy, nanoparticle tracking analysis, 3D-hydrogel system, proteomics, single cell RNA sequencing, and network analyses. Specific Aim 2 will test the hypothesis in vivo that S100A9 mediates diabetes-induced microcalcifications in atherosclerotic plaques. We expect that i) genetic deletion of S100A9, ii) macrophage-targeted siRNA silencing of S100A9, and iii) bone marrow transplantation from S100A9-deficient mice will retard the progression of microcalcification and subsequent rupture, as determined by molecular imaging and comprehensive histopathological analyses. Specific Aim 3 will quantitatively evaluate the impact of microcalcification on the biomechanical instability of the atherosclerotic plaque, using mathematical modeling and finite element analysis. These complementary studies will advance the field by identifying the role of macrophage S100A9 in microcalcification. To facilitate clinical translation of mouse data, we will employ human primary macrophages and atherosclerotic plaque specimens from patients with diabetes. The findings from this project will help to develop much needed anti-calcification therapies.