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: 8599053
• 负责人: DAVID LOMINADZE
• 金额: $ 55.69万
• 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/8599053
• 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）：阿尔茨海默氏病期间大脑中的斑块（AD）主要由淀粉样蛋白B（AB）肽组成。尽管AB，纤维蛋白原（FG）（FG）和同型半胱氨酸（HCY）的水平升高，即AD期间的高同性恋治疗（HHCY）之间存在牢固的关联，但尚不清楚淀粉样蛋白斑块形成的机理，但尚不清楚。该项目的长期目标是了解导致淀粉样菌斑形成的脑渗透性的机制。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，而FG可以交叉血管内皮细胞（EC）层，继激活基质金属蛋白酶-9（MMP-9）。此外，与AD相关的胶原蛋白酶-1（TIMP-1）的组织抑制剂水平升高，可增强胶原蛋白（SEM）中的胶原蛋白含量。在试点研究中，我们发现，尽管HHCY主要通过副细胞途径增强了血管通透性，但HFG主要激活跨细胞运输。我们还发现，HHCY期间FG的血液水平升高。因此，FG可以在HCY诱导的脑血管渗透性中具有加性作用。我们发现，HHCY在SEM和FG和AB沉积中增加了脑脉管系统的胶原蛋白水平。虽然HHCY增加了MMP-9活性并增强细胞细胞转运以及同胞素FG（使纤维蛋白血块更刚性），但FG通过Caveolin-1（CAV-1）信号（通过CAV-1的磷酸化）增强了细胞内的转运。但是，HCY和FG在HCY-FG-AB-Collagen复合物中的作用尚不清楚。该提案的假设是，HHCY主要通过主要影响内皮细胞（EC）连接蛋白并激活MMP-9来增加血管渗透性，并通过增加FG的血液含量而间接增加了血管渗透性，从而增强了跨细胞运输，从而增强了HCY-FG-FG-AB-AB-AB-AB-AB-AB-AB-COLLAGEN复合物的形成。 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增加了金属蛋白酶1的组织抑制剂继发于小鼠大脑中的纤维蛋白原-Ab-胶原蛋白酶复合物的积累。为了定义HCY，FG，MMP-9，CAV-1和TIMP-1在FG-AB-Collagen复合形成中的特定致病作用，研究将对野生型（WT），胱硫硫氨酸B-联合合酶Heterozygote（CBS +/--）小鼠（CBS +/-）小鼠（HHCY的模型，MMP-9 gene nock and genee congut（MMP-9 gene nock）和MMM9 gene nock和MMP-9 gene nock和MMP-9 gene nock and-cognee congut（MMM）（MMM 9 gene nctee nock and coct）进行研究。 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 运输。通过免疫组织化学分析，将评估脑皮质中FG-AB-胶原蛋白复合物的形成。拟议的研究的结果应揭示调节HCY-FG-AB-胶原复合物形成的分子机制，并为AD等疾病期间治疗脑血管并发症的有效策略。