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Elucidating the Mechanisms Behind Basement Membrane Stretching

阐明基底膜拉伸背后的机制

基本信息

批准号：
10516008
负责人：
Claire Gianakas
金额：
$ 1.16万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10516008
关键词：
Affect Aneurysm Animal Model Animals Atomic Force Microscopy Basement membrane Bioinformatics Biological Assay Blood Blood Vessels Brain hemorrhage CRISPR/Cas technology Caenorhabditis elegans Cardiovascular Physiology Cardiovascular system Cells Collagen Collagen Type IV Contracts Defect Diabetes Mellitus Disease Elderly Embryonic Development Environment Enzymes Equilibrium Exhibits Extracellular Matrix Fertilization Fluorescence Genes Genetic Screening Glycoproteins Gonadal structure Hemorrhage Human Image Knockout Mice Laminin Lead Link Location Mechanical Stress Mechanics Mediating Membrane Proteins Modeling Molecular Mutation Nematoda Optics Ovulation Pathology Patients Photobleaching Play Polymers Protein Isoforms Proteins RNA interference screen Regulation Role Rupture Stretching Testing Tissue membrane Tissues Vascular Diseases Work base cerebral microbleeds crosslink diabetic effective therapy fibulin genetic analysis human disease improved in vivo Model knock-down overexpression peroxidasin recruit

项目摘要

Abstract Basement membranes (BMs) are dense extracellular matrices that surround and structurally support tissues. A primary component of BMs is type IV collagen, which forms a cross-linked network that protects BMs and tissues from mechanical stress. Although the key role of type IV collagen in structural support is established, how this network accommodates BM stretching in mechanically active tissues is unknown. BM stretching is critically important in the cardiovascular system, where vessels must expand and contract to accommodate pulsatile forces from the blood. Consistent with this important role, disruptions in the type IV collagen network can lead to vascular diseases such as vascular thickening in diabetes, cerebral microbleeds, and hemorrhagic stroke. Despite its significance, our understanding of BM stretching has been limited by a lack of in vivo models to study how BMs dynamically expand. To fill this need, I have established the gonadal BM of the visually and genetically tractable model organism C. elegans as a new in vivo model for BM stretching. I discovered that the region of the gonadal BM specialized for fertilization/ovulation, the spermathecal BM, is stretched dramatically (~2-fold) during ovulation. Using atomic force microscopy (AFM), I found that there is a stiffness gradient within the gonad with the spermatheca being the least stiff. Furthermore, 20 BM components have been tagged with mNeonGreen or mRuby using CRISPR/Cas-9, making C. elegans the only animal where all major BM components are endogenously tagged. Using these strains, I found: (1) Increased levels of type IV collagen limit stretching during ovulation; (2) there are high levels of peroxidasin-1, a protein that negatively regulates type IV collagen cross- linking, in the spermathecal BM; and (3) fibulin, a BM protein thought to maintain type IV collagen, is enriched in the spermatheca. My overall hypothesis is that levels and cross-linking of type IV collagen are precisely regulated to allow the collagen network to maintain BM/tissue integrity while enabling BM/tissue stretching. In Aim 1, I will use genetic analysis, conditional knockdown, live imaging, and AFM to test the hypothesis that type IV collagen levels play a crucial role in BM stretching (low levels promote decreased stiffness, increased stretching and eventual rupture; high levels increase stiffness and restrict stretching) and determine whether the effects of type IV collagen α1 and α2 human disease mutations in C. elegans are due to altered levels or location in unique domains associated with BM stretching. Aim 2 will test the hypothesis that low type IV collagen cross-linking, mediated by peroxidasins, promotes BM stretching by reducing BM stiffness and that fibulin maintains type IV collagen levels in this dynamic environment. In Aim 3, I will determine if additional components regulate BM stretching by conducting a bioinformatics-driven RNAi screen to identify vascular disease associated genes that affect BM stretching. Ultimately, I expect my work will show that precise type IV collagen levels and cross-linking are critical for balancing tissue support and BM stretching and that these mechanisms are perturbed in human vascular disease, making them ideal targets for more effective therapies.

摘要基底膜（BM）是围绕组织并在结构上支持组织的致密细胞外基质。一 BM的主要成分是IV型胶原，其形成保护BM和组织的交联网络来自机械应力。虽然IV型胶原蛋白在结构支持中的关键作用已经确立，但这是如何实现的呢？网络适应BM拉伸机械活性组织是未知的。BM拉伸是至关重要的在心血管系统中很重要，其中血管必须扩张和收缩以适应脉动来自血液的力量与这一重要作用相一致，IV型胶原蛋白网络的破坏可导致血管疾病，如糖尿病中的血管增厚、脑微出血和出血性中风。尽管其意义重大，但我们对BM拉伸的理解受到缺乏体内模型研究的限制 BM如何动态扩展。为了满足这一需要，我已经建立了视觉和遗传的性腺BM 易处理模式生物C. elegans作为BM拉伸的新体内模型。我发现，专门用于受精/排卵的性腺BM，受精囊BM，被显著拉伸（~2倍）在排卵期使用原子力显微镜（AFM），我发现有一个刚度梯度内的性腺受精囊最不僵硬。此外，20个BM组件已标记mNeonGreen 或mRuby使用CRISPR/Cas-9，使C.线虫是唯一一种所有主要的BM成分都内源性标记。使用这些菌株，我发现：（1）IV型胶原蛋白水平的增加限制了拉伸，排卵;（2）有高水平的过氧化物酶-1，一种负调节IV型胶原交叉的蛋白质，连接，在受精囊BM中;和（3）fibulin，一种被认为维持IV型胶原的BM蛋白，在受精囊BM中富集，受精囊我的总体假设是，IV型胶原蛋白的水平和交联正是调节以允许胶原网络维持BM/组织完整性，同时使BM/组织伸展在目标1中，我将使用遗传分析，条件敲除，活体成像和AFM来测试假设IV型胶原蛋白水平在BM拉伸中起关键作用（低水平促进降低硬度增加，拉伸增加，最终破裂;高水平增加硬度，限制拉伸），确定IV型胶原α1和α2人类疾病突变是否对C.优雅是由于与BM拉伸相关的独特结构域中的改变的水平或位置。目标2将检验以下假设：由过氧化物酶介导的低IV型胶原交联通过降低BM刚度促进BM拉伸而纤蛋白在这种动态环境中维持IV型胶原蛋白水平。在目标3中，我将确定其他成分通过进行生物信息学驱动的RNAi筛选来调节BM拉伸，血管疾病相关基因影响BM拉伸。最终，我希望我的工作能证明 IV型胶原蛋白水平和交联对于平衡组织支撑和BM拉伸是至关重要的，在人类血管疾病中，这些机制受到干扰，使其成为更有效治疗的理想靶点。