Vascular Tissue Invasion by Porphyromonas gingivalis

牙龈卟啉单胞菌侵入血管组织

• 批准号: 6836569
• 负责人: LING LI
• 金额: $ 12.52万
• 依托单位: NOVA SOUTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2006-01-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6836569
• 关键词: Bacteroides gingivalis; antibacterial antibody; atherosclerosis; bacteria infection mechanism; cardiovascular disorder risk; cardiovascular system; cell morphology; clinical research; human subject; immunofluorescence technique; mixed tissue /cell culture; organ culture; polymerase chain reaction; scanning electron microscopy; smooth muscle; tissue donors; vascular endothelium

DESCRIPTION: Porphyromonas gingivalis (P. g.), a Gram-negative anaerobe is known to play a critical role in the development of periodontitis. Several lines of evidence had also indicated its role in the development of cardiovascular disease (CVD). First, epidemiological studies have concluded that individuals with periodontitis were at greater risk for CVD. Second, 16S rDNA of P. g. was found to be present in atheromatous plaques. In vitro, P. g. has been shown to invade human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC). In vivo, the long-term systemic challenge of Pg can accelerate atherogenic plaque progression in apolipoprotein E-deficient mice. Recently, we have found the presence of live bacteria in atherosclerotic tissue from patients. All these observations suggest that P. g. may invade cardiovascular tissues, thus contributing to the development of atherosclerosis by interfering with cellular function of host tissues and by eliciting an inflammatory response. Therefore, the central hypothesis is that P. g. contributes to the development of atherosclerosis by invasion of tunica intima and intima media. The Specific Aims of this study are to probe the ability and mechanism of cardiovascular tissue invasion and penetration by P.g. using novel human organ and cell culture systems. Human saphenous vein organ culture that maintains 3-D structure of vascular tissue will be used for the first time to evaluate invasion and penetration ability of P.g. The depth and amount of invaded P.g in tunica intima and intima media will be determined by real time quantitative PCR and immunofluorescent microscopy using antibodies against P.g., endothelial and smooth muscle cell markers. By this system, we will 1) determine whether invasion can lead to neoinitima formation. 2) compare the invasion ability in injured and non-injured veins. 3) determine the relationship of demographic and medical data of vein donors with the susceptibility of the invasion. To probe the transcellular or/and paracellular route(s) of tissue penetration by P.g., primary endothelial and smooth muscle cell lines will be co-cultured in a mono-layer or bi-layer transwell culture system in which endothelial cells will be kept in transwell inserts, resembling in vivo layout. The ability of P.g. to either exit the pre-infected endothelial cell layer or penetrate the un-infected endothelial cell layer to invade smooth muscle cells will be evaluated. In parallel, the endothelial cell layer morphology and integrity will be monitored by scanning electronic microscope and the status of endothelial cell junctions will be detected by measuring transendothelial resistance. The proposed experiments will determine whether P.g can invade and penetration vascular tissue through transcellular or/and paracellular pathway(s). The completion of this study will contribute to the long-term goal of understanding this bacterially induced vascular inflammation and identification of new therapeutic targets.

牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，已知在牙周炎的发生发展中起关键作用。一些证据也表明它在心血管疾病(CVD)的发展中的作用。首先，流行病学研究得出结论，患有牙周炎的人患心血管疾病的风险更大。其次，在动脉粥样硬化斑块中发现了P.G.的16S rDNA。在体外，P.G.已被证明侵袭人冠状动脉内皮细胞(HCAEC)和冠状动脉平滑肌细胞(CASMC)。在体内，PG的长期全身挑战可以加速载脂蛋白E缺陷小鼠的动脉粥样硬化斑块进展。最近，我们在患者的动脉粥样硬化组织中发现了活细菌。所有这些观察表明，P.G.可能通过干扰宿主组织的细胞功能和引发炎症反应而侵袭心血管组织，从而促进动脉粥样硬化的发展。因此，中心假说是P.G.通过侵袭内膜和中膜而参与动脉粥样硬化的发展。本研究的具体目的是探讨P.G.侵袭和穿透心血管组织的能力和机制。使用新的人体器官和细胞培养系统。首次将维持血管组织三维结构的人体大隐静脉器官培养用于评价P.G.通过实时定量聚合酶链式反应和免疫荧光显微镜，利用P.G.、内皮细胞和平滑肌细胞标志物的抗体，将确定P.G.在内膜和中膜的侵袭深度和数量。通过这个系统，我们将1)确定入侵是否会导致新的肿瘤的形成。2)比较损伤静脉和非损伤静脉的侵袭能力。3)确定静脉献血者的人口学和医学资料与侵袭易感性的关系。为了探索P.G.组织穿透的跨细胞或/和细胞旁途径(S)，将原代血管内皮细胞与血管内皮细胞系在单层或双层Transwell培养系统中共培养，内皮细胞将保留在Transwell内，类似于体内的布局。P.G.的能力。无论是离开感染前的内皮细胞层，还是穿透未感染的内皮细胞层，以侵袭血管内皮细胞，都将被评估。同时，将通过扫描电子显微镜监测内皮细胞层的形态和完整性，并通过测量跨内皮细胞阻力来检测内皮细胞连接的状态。拟议的实验将确定P.G是否可以通过跨细胞或/和旁细胞途径侵入和穿透血管组织(S)。这项研究的完成将有助于了解这种细菌引起的血管炎症的长期目标，并确定新的治疗靶点。