Collagen fibril structure, surface charge and vascular calcification

软组织（如动脉）的钙化是一些心血管疾病（如主动脉瘤和动脉粥样硬化）的标志。钙化沉积导致不良后果，如动脉硬化和正常血流中断，往往导致发病率和死亡率。该项目的目标是了解软组织中钙化沉积物的形成位置。特别是，研究人员将研究胶原原纤维（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。这可以通过设计早期诊断和治疗的新策略来帮助改善心血管预后，并有助于心血管生物假体生物材料的开发。除了推动细胞外基质和血管生物学领域的发展外，该项目还将为研究生和本科生提供多学科的实践研究经验，并为贫困的K-8学生提供丰富的活动，从而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。本项目的目的是了解胶原纤维在介导血管钙化中的作用。虽然人们普遍认为病理性钙化是由血管平滑肌细胞表型转换为“骨样”成骨表型介导的，但也有证据表明，在腹主动脉瘤（AAA）中，与天然（正常）原纤维相比，细胞外基质中的胶原原纤维亚群经历了结构变化。该项目的总体假设是，AAA中结构改变（异常）的胶原原纤维由于其表面电荷分布的扰动而成为钙化沉积的主要底物。为了验证这一假设，研究将在人类AAA组织的单纤维水平上进行，首先使用微型计算机断层扫描绘制切除AAA组织中的微观和宏观钙化。研究计划有两个目标。第一个目的是验证AAA结构改变的胶原原纤维是ECM中负电荷的“热点”这一假设。环境空气中胶原原纤维的表面电位和表面电荷映射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原子力显微镜（AFMs）上可用的技术，已用于绘制金属和半导体的表面电位和表面电荷，但在本项目中被独特地应用于分析组织切片中正常和异常原纤维的原位。胶原原纤维在流体环境中的表面电荷分布将使用另一种基于记录力-距离曲线的AFM方法来测量，该方法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地形。第二个目的是验证一个假设，即AAA中天然的和生物模拟诱导的钙沉积都定位于结构改变的原纤维上。仿生学方法将用于确定异常原纤维是否可能是血管钙化的原因以及结果。仿生矿化将以无细胞的方式（在缺乏矿物质的地区）诱导，使用离子溶液（例如牙液）模拟血浆中不含生物成分的天然离子浓度，并通过培养从具有去细胞化AAA组织的培养细胞中分离出的细胞外囊泡（EV）。钙化沉积物的空间分布和特征将通过分析透射电子显微镜（TEM）方法来确定，以表征超微结构水平上胶原纤维的大小，组成和矿物的位置。与正常原纤维相比，在矿化原纤维的百分比以及每个原纤维中存在的钙化沉积的数量和大小方面，异常原纤维预计更容易发生钙化沉积，并且在仿生和自然诱导的钙化沉积中也有望观察到类似的趋势。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.