Mechanisms of Homocysteine-Induced Fibrinogen-Amyloid Plaque Formation

同型半胱氨酸诱导纤维蛋白原淀粉样蛋白斑形成的机制

• 批准号: 8689198
• 负责人: DAVID LOMINADZE
• 金额: $ 53.94万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689198
• 关键词: Affect; Aging; Alzheimer' s Disease; Amyloid; Binding; Blood; Brain; CAV1 gene; Caveolae; Coagulation Process; Collagen; Complex; Cryoultramicrotomy; Cystathionine; Dementia; Deposition; Development; Disease; Endothelial Cells; Extravasation; Fibrin; Fibrinogen; Gelatinase B; Goals; Heterozygote; Homocysteine; Homocystine; Hyperhomocysteinemia; Immunohistochemistry; Intercellular Junctions; Interstitial Collagenase; Knock-out; Knowledge; Lead; MMP9 gene; Matrix Metalloproteinases; Measures; Methods; Mission; Modeling; Molecular; Mus; Neurodegenerative Disorders; Pathway interactions; Peptides; Permeability; Phosphorylation; Pilot Projects; Presenile Alzheimer Dementia; Proteins; Public Health; Research; Role; Sampling; Secondary to; Senile Plaques; Signal Transduction; TIMP1 gene; Testing; Therapeutic; Tissue Inhibitor of Metalloproteinase-1; Tracer; Transgenic Organisms; United States National Institutes of Health; Vascular Endothelial Cell; Vascular Permeabilities; caveolin 1; cerebrovascular; design; knockout gene; novel; occludin; public health relevance; transcytosis; treatment strategy; vascular endothelial cadherin-2; venule

DESCRIPTION (provided by applicant): The plaques in the brain during Alzheimer's diseases (AD) are primarily composed of amyloid-b (Ab) peptide. Although a strong association between Ab, fibrinogen (Fg), and elevated level of homocysteine (Hcy), i.e. hyperhomocysteinemia (HHcy) during AD is well- documented, the mechanism of amyloid plaque formation is unclear. The long-term goal of this project is to understand the mechanisms of cerebrovascular permeability leading to amyloid plaque formation. We have shown that elevated levels of Hcy and Fg i.e. hyperfibrinogenemia (HFg) increase cerebrovascular permeability and Fg can cross vascular endothelial cell (EC) layer secondary to activation of matrix metalloproteinase-9 (MMP-9). In addition, increased levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), which enhances collagen content in subendothelial matrix (SEM), are associated with AD. In pilot studies, we found that while HHcy enhances vascular permeability through mainly the paracellular pathway, HFg activates mainly the transcellular transport. We also found that blood level of Fg is increased during HHcy. Therefore, Fg can have an additive effect in HHcy-induced cerebrovascular permeability. We found that HHcy increases collagen level in SEM and Fg and Ab depositions in brain vasculature. While HHcy increases MMP-9 activity and enhances the paracellular transport as well as homocyteinylates Fg (making fibrin clots more rigid), Fg enhances the transcellular transport through caveolin-1 (Cav-1) signaling (via phosphorylation of Cav-1). However, the role of Hcy and Fg in Hcy-Fg-Ab-collagen complex formation is unclear. The hypothesis of this proposal is that HHcy increases vascular permeability by primarily affecting the endothelial cell (EC) junction proteins and activating MMP-9, and indirectly by increasing blood content of Fg, which enhances transcellular transport leading to an enhanced Hcy- Fg-Ab-collagen complex formation. We will test this hypothesis with three specific aims: 1) To determine whether the HHcy instigates cerebrovascular permeability via paracellular transport leading to enhanced cerebrovascular crossing of fibrinogen, 2) To determine whether the fibrinogen deposition enhances cerebrovascular permeability mainly through caveolar transcytosis via Cav-1 signaling secondary to MMP-9 activation, 3) To determine whether the HHcy increases fibrinogen-Ab-collagen complex accumulation in mouse brains secondary to increasing tissue inhibitor of metalloproteinase-1. To define specific causative effects of Hcy, Fg, MMP-9, Cav-1, and TIMP-1 in Fg-Ab-collagen complex formation, the studies will be done on pial vessels of wild type (WT), Cystathionine b-Synthase heterozygote (CBS+/-) mice (a model of HHcy), MMP-9 gene knockout (MMP9-/-), CBS and MMP-9 double knockout (CBS+/-/MMP9-/-), Cav-1 gene knockout (Cav1-/-), Fg g-chain-deficient (Fg-/-), CBS+/-/Fg-/-, HFg transgenic (HFg), HFg/MMP9-/-, CBS+/-/Cav1-/-/MMP9-/-, CBS+/- /HFg, CBS+/-/HFg/MMP9-/-, TIMP-1 gene knockout (TIMP1-/-), CBS+/-/TIMP1-/- double knockout, Fg-/-/MMP1-/-, and CBS+/-/Fg-/-/TIMP1-/- mice using a newly developed dual-tracer probing method that allows separation of paracellular from transcellular transport. Formation of Fg-Ab-collagen complex in brain cortex will be assessed with immunohistochemical analysis. The results of the proposed research should uncover the molecular mechanisms regulating Hcy-Fg-Ab-collagen complex formation and lead to effective strategies for the treatment of cerebrovascular complications during diseases such as AD.

描述（由申请人提供）：阿尔茨海默病（AD）期间脑中的斑块主要由淀粉样蛋白-b（Ab）肽组成。虽然在AD期间Ab、纤维蛋白原（Fg）和高半胱氨酸（Hcy）水平升高（即高同型半胱氨酸血症（HHcy））之间的强关联已被充分证明，但淀粉样蛋白斑块形成的机制尚不清楚。本项目的长期目标是了解脑血管通透性导致淀粉样斑块形成的机制。我们已经表明，Hcy和Fg水平升高，即高纤维蛋白原血症（HFg）增加脑血管通透性，并且Fg可以穿过血管内皮细胞（EC）层，继发于基质金属蛋白酶-9（MMP-9）的活化。此外，增加的金属蛋白酶组织抑制剂-1（TIMP-1）水平增加，增加了内皮下基质（SEM）中的胶原含量，与AD相关。在初步研究中，我们发现虽然HHcy主要通过细胞旁途径增强血管通透性，但HFg主要激活跨细胞转运。我们还发现在HHcy期间血Fg水平升高。因此，Fg可以在HHcy诱导的脑血管通透性中具有累加效应。我们发现HHCy增加了SEM中的胶原蛋白水平以及脑血管中的Fg和Ab沉积。虽然HHcy增加MMP-9活性并增强细胞旁转运以及使Fg同型半胱氨酸化（使纤维蛋白凝块更坚硬），但Fg通过小窝蛋白-1（Cav-1）信号传导（通过Cav-1的磷酸化）增强跨细胞转运。然而，Hcy和Fg在Hcy-Fg-Ab-胶原复合物形成中的作用尚不清楚。该提议的假设是HHcy主要通过影响内皮细胞（EC）连接蛋白和激活MMP-9来增加血管通透性，并且间接地通过增加Fg的血液含量来增加血管通透性，这增强了跨细胞转运，导致增强的Hcy-Fg-Ab-胶原蛋白复合物形成。我们将通过三个具体目标来检验这一假设：1）确定HHcy是否通过细胞旁转运引起脑血管通透性增加，导致纤维蛋白原的脑血管穿越增强，2）确定纤维蛋白原沉积是否主要通过继发于MMP-9活化的Cav-1信号转导通过小窝胞吞转运增强脑血管通透性，3）确定HHcy是否增加小鼠脑中继发于增加金属蛋白酶组织抑制剂-1的纤维蛋白原-Ab-胶原复合物的积累。为了确定Hcy、Fg、MMP-9、Cav-1和TIMP-1在Fg-Ab-胶原蛋白复合物形成中的特异性致病作用，将对野生型（WT）、胱硫醚b-合酶杂合子（CBS+/-）小鼠的软脑膜血管进行研究MMP-9基因敲除（MMP 9-/-）、CBS和MMP-9双敲除（CBS+/-/MMP 9-/-），Cav-1基因敲除（Cav 1-/-），Fg g-链缺陷（Fg-/-），CBS+/-/Fg-/-，HFg转基因（HFg），HFg/MMP 9-/-，CBS+/-/Cav 1-/-/MMP 9-/-，CBS+/-/HFg，CBS+/-/HFg/MMP 9-/-，TIMP-1基因敲除（TIMP 1-/-）、CBS+/-/TIMP 1-/-双敲除、Fg-/-/MMP 1-/-和CBS+/-/Fg-/-/TIMP 1-/-小鼠使用新开发的双示踪剂探测方法，该方法允许将细胞旁转运与跨细胞转运分离。将采用免疫组织化学分析评估脑皮质中Fg-Ab-胶原蛋白复合物的形成。拟议的研究结果应揭示的分子机制，调节HCY-Fg-Ab-胶原蛋白复合物的形成，并导致有效的策略，治疗脑血管并发症的疾病，如AD。