Collagen fibril structure, surface charge and vascular calcification

点击翻译按钮获取中文摘要

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.