Vascular Tissue Invasion by Porphyromonas gingivalis

描述：牙龈卟啉单胞菌（卟啉单胞菌）是一种革兰氏阴性厌氧菌，已知在牙周炎的发展中起关键作用。一些证据也表明它在心血管疾病（CVD）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高。其次，发现p.g.的16S rDNA存在于动脉粥样斑块中。在体外，p.g.已被证明可以侵入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的长期系统性挑战可以加速载脂蛋白e缺乏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的存在。所有这些观察结果表明，p.g.可能侵入心血管组织，从而通过干扰宿主组织的细胞功能和引发炎症反应，促进动脉粥样硬化的发展。因此，中心假设是p.g.通过侵犯内膜和中内膜促进动脉粥样硬化的发展。本研究的具体目的是利用新的人体器官和细胞培养系统，探讨P.g.侵入和渗透心血管组织的能力和机制。将首次使用维持血管组织三维结构的人隐静脉器官培养来评估p.g.的侵袭和渗透能力。利用针对p.g.、内皮细胞和平滑肌细胞标记物的抗体，通过实时定量PCR和免疫荧光显微镜来确定p.g.在膜内膜和内膜介质中的浸润深度和数量。通过这个系统，我们将1)确定入侵是否会导致新原生岩的形成。2)比较损伤静脉与非损伤静脉的侵入能力。3)确定静脉供者的人口学和医学资料与入侵易感性的关系。为了探索p.g.g渗透组织的跨细胞或/和细胞旁途径，原代内皮细胞和平滑肌细胞系将在单层或双层transwell培养系统中共同培养，其中内皮细胞将保存在transwell插入物中，类似于体内布局。我们将评估P.g.脱离感染前内皮细胞层或穿透未感染内皮细胞层侵入平滑肌细胞的能力。同时，将通过扫描电镜监测内皮细胞层的形态和完整性，并通过测量跨内皮阻力来检测内皮细胞连接的状态。所提出的实验将确定P.g是否可以通过跨细胞或/和细胞旁途径侵入和穿透维管组织。这项研究的完成将有助于了解这种细菌诱导的血管炎症和确定新的治疗靶点的长期目标。

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.