Mechanisms of Homocysteine-Induced Fibrinogen-Amyloid Plaque Formation

描述(申请人提供)：阿尔茨海默病(AD)期间大脑中的斑块主要由淀粉样蛋白b(Ab)多肽组成。虽然抗体、纤维蛋白原(Fg)与AD期间升高的同型半胱氨酸(Hcy)水平，即高同型半胱氨酸血症(HHcy)之间有很强的相关性，但淀粉样斑块的形成机制尚不清楚。该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。我们发现Hcy和Fg水平升高，即高纤维蛋白原血症(HFG)可增加脑血管通透性，Fg可通过继发于基质金属蛋白酶-9(MMP9)活化的血管内皮细胞(EC)层。此外，基质金属蛋白酶组织抑制因子-1(TIMP-1)水平升高，可增加内皮下基质(SEM)中的胶原含量，与AD有关。在初步研究中，我们发现HHcy主要通过细胞旁途径增强血管通透性，而HFG主要激活跨细胞转运。我们还发现，在HHcy过程中，Fg水平升高。因此，FG对HHcy诱导的脑血管通透性具有相加作用。我们发现，HHcy增加了扫描电子显微镜中的胶原水平和脑血管中Fg和Ab的沉积。虽然HHcy增加了基质金属蛋白酶-9的活性，增强了细胞旁的转运，以及同型半胱氨酸化的Fg(使纤维蛋白凝块变得更加坚硬)，但Fg通过小窝蛋白-1(Cav-1)信号(通过Cav-1的磷酸化)增强了跨细胞运输。然而，同型半胱氨酸和纤维蛋白原在同型半胱氨酸-纤维蛋白原-抗体-胶原复合体形成中的作用尚不清楚。这一假设认为HHcy主要通过影响内皮细胞连接蛋白和激活基质金属蛋白酶-9来增加血管通透性，间接地通过增加FG的血液含量来增加血管通透性，从而促进跨细胞转运，从而促进Hcy-FG-Ab-胶原复合体的形成。我们将通过三个具体的目标来验证这一假说：1)确定HHcy是否通过细胞旁转运而促进脑血管通透性，从而增强纤维蛋白原的脑血管通透性；2)确定纤维蛋白原沉积是否主要通过辅助MMP9激活的Cav-1信号通过空泡转运来增强脑血管通透性；3)确定HHcy是否增加了纤维蛋白原-抗体-胶原复合体在小鼠脑中的积累，继而增加了金属蛋白酶组织抑制物-1。为了明确Hcy、Fg、MMP9、Cav-1和TIMP-1在Fg-Ab-胶原复合体形成中的特定致病作用，我们将对野生型(WT)、胱硫醚b-合成酶杂合子(CBS+/-)小鼠(HHcy模型)、MMP9基因敲除(MMP9-/-)、CBS和MMP9双敲除(CBS+/-/MMP9-/-)、CaV-1基因敲除(Cav1-/-)、Fg链缺陷(Fg-/-)、CBS+/Fg-/-、CBS+/MMP9-/-、Cav-1基因敲除(Cav1-/-)、CBS+/Fg-/-、CBS+/Fg-/-、CBS+/MMP9-/-、CBS+/FG-/-、CBS+/MMP9-/-、CBS+/FG-/-、CBS+/FG-/-、CBS+/MMP9-/-、CBS+/MMP9-/-、CBS-1基因敲除(Cav1-/-)、FG链缺陷(FG-/-/-)、CBS+/FG-/-、CBS+/转基因(HFG)、HFG/MMP9-/-、CBS+/-/Cav1-//MMP9-/-、CBS+/-/HFG、CBS+/-/HFG/MMP9-/-、TIMP-1基因敲除(TIMP1-/-)、CBS+/-/TIMP1-/-双敲除、FG-/-/MMP1-/-和CBS+/-/FG-/-/TIMP1-/-小鼠。用免疫组织化学方法检测大脑皮层FG-Ab-胶原复合体的形成情况。拟议的研究结果将揭示调节Hcy-Fg-Ab-胶原复合体形成的分子机制，并为治疗AD等疾病中的脑血管并发症提供有效的策略。

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.