Collagen fibril structure, surface charge and vascular calcification

胶原纤维结构、表面电荷和血管钙化

• 批准号: 2000469
• 负责人: Gunjan Agarwal
• 金额: $ 33万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2000469&HistoricalAwards=false
• 关键词: Collagen; fibril; structure; surface; charge

Calcification of soft tissues (e.g. arteries) is a hallmark of several cardiovascular diseases like aortic aneurysms and atherosclerosis. Calcific deposits lead to adverse consequences such as stiffening of arteries and disruption of normal blood flow, often leading to morbidity and mortality. The goal of this project is to understand where calcific deposits are formed in soft tissues. In particular, the investigators will examine how collagen fibrils (present in the extracellular matrix surrounding the cells in the arterial wall) can facilitate pathological calcification. The insights gained will advance our understanding of the fundamental mechanisms governing soft-tissue calcification especially in its early stages. This can help improve cardiovascular outcomes by designing new strategies for early diagnosis and treatment and aid biomaterials development for cardiovascular bioprostheses. In addition to advancing the field of extracellular matrix and vascular biology, the project will help train the next generation of scientists and engineers by providing graduate and undergraduate student(s) with multidisciplinary hands-on research experiences and under-privileged K-8 students with enrichment activities. Furthermore, new education materials and microscopy approaches will be developed to enhance education and research infrastructure for the broader scientific community.The goal of this project is to understand the role of the collagen fibril in mediating vascular calcification. While it is accepted that pathological calcification is mediated by phenotypic switching of vascular smooth muscle cells into a ‘bone-like’ osteogenic phenotype, there is also evidence indicating that in abdominal aortic aneurysms (AAA), a subpopulation of collagen fibrils in the extracellular matrix undergoes a structural change as compared to the native (normal) fibrils. The project’s overall hypothesis is that structurally altered (abnormal) collagen fibrils in AAA serve as the major substrates for calcific deposits due to perturbations in their surface-charge distribution. To test this hypothesis, studies will be conducted at the single fibril level on human AAA tissue, beginning with mapping the micro and macro-calcifications in the excised AAA tissue using micro-computed tomography. The Research Plan is organized under two aims. The FIRST Aim is to test the hypothesis that structurally altered collagen fibrils in AAA are “hot-spots” for negative charges in the ECM. Surface potential and surface charge mapping of collagen fibrils in ambient air will be accomplished using Kelvin probe force microscopy (KPFM), a technique available on most commercial AFMs (Atomic Force Microscopes) that has been used for mapping surface potential and surface charge on metals and semiconductors, but is being uniquely applied in this project to the analysis of normal and abnormal fibrils in situ in tissue sections. Surface charge distribution of collagen fibrils in a fluid environment will be measured using another AFM based approach based on recording force-distance curves, which can map both surface charge and sample topography at high resolution in physiologically relevant fluid environments (e.g. different salt concentrations). The SECOND AIM is to test the hypothesis that both natural as well as bio-mimetically induced calcific deposits in AAA are localized on structurally altered fibrils. A biomimetic approach will be used to determine if abnormal fibrils can be a cause as well as an effect of vascular calcification. Biomimetic mineralization will be induced in a cell free manner (in mineral-poor regions) using ionic solutions (e.g. dental fluid) that mimic the native ion concentrations in plasma without its biological components and by incubating extracellular vesicles (EV) isolated from cultured cells with decellularized AAA tissue. Spatial distribution and characterization of calcific deposits will be determined using analytical transmission electron microscopy (TEM) approaches to characterize the size, composition and location of minerals with respect to the collagen fibril at the ultrastructural level. Abnormal fibrils are expected to be more prone to calcific deposits as compared to normal fibrils both with respect to percent of fibrils mineralized as well as number and size of calcific deposits present per fibril, and a similar trend is expected to be observed for biomimetic and naturally induced calcific deposits.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

软组织(如动脉)的钙化是几种心血管疾病的标志，如主动脉瘤和动脉粥样硬化。钙化沉积会导致动脉硬化和正常血流中断等不良后果，通常会导致发病率和死亡率。这个项目的目标是了解软组织中钙质沉积形成的位置。特别是，研究人员将研究胶原纤维(存在于动脉壁细胞周围的细胞外基质中)如何促进病理性钙化。所获得的见解将促进我们对软组织钙化的基本机制的理解，特别是在其早期阶段。这有助于通过设计新的早期诊断和治疗策略来改善心血管结果，并有助于心血管生物假体的生物材料开发。除了推进细胞外基质和血管生物学领域，该项目还将通过为研究生和本科生(S)提供多学科实践研究经验，并为K-8贫困学生提供丰富的活动，帮助培养下一代科学家和工程师。此外，将开发新的教育材料和显微镜方法，以加强更广泛的科学界的教育和研究基础设施。该项目的目标是了解胶原纤维在调节血管钙化中的作用。虽然公认病理性钙化是通过血管平滑肌细胞表型向骨样成骨表型转变所介导的，但也有证据表明，在腹主动脉瘤(AAA)中，与天然(正常)纤维相比，细胞外基质中的一种胶原纤维亚群经历了结构变化。该项目的总体假设是，由于其表面电荷分布的扰动，AAA中结构变化(异常)的胶原纤维是钙质沉积的主要底物。为了验证这一假设，将在人类AAA组织的单个纤维水平上进行研究，首先使用微型计算机断层扫描绘制切除的AAA组织中的微观和宏观钙化情况。《研究计划》有两个目标。第一个目的是验证AAA中结构改变的胶原纤维是ECM中负电荷的“热点”这一假说。环境空气中胶原原纤维的表面电位和表面电荷图谱将使用开尔文探针力显微镜(KPFM)完成，这是一种在大多数商用AFM(原子力显微镜)上可用的技术，已被用于绘制金属和半导体上的表面电位和表面电荷图，但在该项目中唯一应用于组织切片中的正常和异常纤维的原位分析。将使用另一种基于AFM的方法来测量流体环境中胶原纤维的表面电荷分布，该方法基于记录力-距离曲线，可以在生理相关的流体环境(例如不同的盐浓度)中以高分辨率绘制表面电荷和样品形貌图。第二个目的是验证这一假设，即AAA中自然和生物模拟诱导的钙化沉积都定位于结构改变的纤维上。一种仿生方法将被用来确定异常的纤维是否可能是血管钙化的原因和影响。仿生矿化将以无细胞的方式(在矿物质贫乏的地区)使用离子溶液(例如牙液)来模拟血浆中天然离子的浓度，而不包括其生物成分，并通过将从培养细胞中分离的细胞外小泡(EV)与去细胞的AAA组织孵育来诱导仿生矿化。钙质沉积物的空间分布和特征将使用分析透射式电子显微镜(TEM)方法来确定，以在超微结构水平上表征矿物相对于胶原纤维的大小、组成和位置。与正常纤维相比，异常纤维预计更容易发生钙化沉积，无论是就纤维矿化的百分比以及每个纤维存在的钙化沉积的数量和大小而言，预计在仿生和自然诱导的钙化沉积方面也会观察到类似的趋势。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Collagen fibril abnormalities in human and mice abdominal aortic aneurysm

• DOI: 10.1016/j.actbio.2020.04.022
• 发表时间: 2020-07-01
• 期刊: ACTA BIOMATERIALIA
• 影响因子: 9.7
• 作者: Jones, Blain;Tonniges, Jeffrey R.;Agarwal, Gunjan
• 通讯作者: Agarwal, Gunjan

Structurally abnormal collagen fibrils in abdominal aortic aneurysm resist platelet adhesion

腹主动脉瘤中结构异常的胶原纤维抵抗血小板粘附

• DOI: 10.1111/jth.15576
• 发表时间: 2022
• 期刊: Journal of Thrombosis and Haemostasis
• 影响因子: 10.4
• 作者: Jones, Blain;Debski, Anna;Hans, Chetan P.;Go, Michael R.;Agarwal, Gunjan
• 通讯作者: Agarwal, Gunjan