UKY DENTAL COBRE: ORAL INFECTIONS: COX-2

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 具体目标1。确定考克斯-2在牙龈卟啉单胞菌诱导的牙周病和动脉粥样硬化中的作用。 牙周炎是一种由口腔微生物感染引起的慢性炎症性疾病。该疾病是由革兰氏阴性厌氧物种，包括牙龈卟啉单胞菌。牙龈卟啉单胞菌感染可在口腔内产生局部炎症，导致牙槽骨吸收，并可产生由菌血症引起的全身炎症反应。全身炎症反应被认为是牙周炎患者发生心血管疾病并发症的风险增加的原因。前列腺素是一类炎症介质，在患病的牙周组织中显著增加。洋地黄素的合成需要环氧合酶的活性，而环氧合酶-2（考克斯-2）是由革兰氏阴性细菌产物高度诱导并在动脉粥样硬化病变中增加的同种型。 目前的研究利用建立的牙龈卟啉单胞菌诱导的牙周炎小鼠模型来检查考克斯-2衍生的洋地黄素在牙槽骨吸收和动脉粥样硬化中的作用。由牙龈卟啉单胞菌引起的牙槽骨吸收和心血管疾病需要在小鼠口腔中建立慢性感染。因此，在12周的过程中，每周4天，每3周用牙龈卟啉单胞菌接种小鼠。我们已经优化了PCR技术，以确认在小鼠中建立牙龈卟啉单胞菌的慢性感染。我们还利用高灵敏度的实时PCR技术检测了主动脉组织中考克斯-2和下游前列腺素酶的表达。此外，我们还优化了免疫组化方法，用于检测小鼠睾丸组织中考克斯-2蛋白的表达。这些程序将使我们能够检查牙龈卟啉单胞菌感染对考克斯-2和下游前列腺素酶表达的影响。研究正在进行中，以检查考克斯-2的遗传或药理学失活在减少牙龈卟啉单胞菌诱导的小鼠牙槽骨吸收和动脉粥样硬化中的有效性。 具体目标2。 明确12/15-脂氧合酶在牙龈卟啉单胞菌引起的牙周病和动脉粥样硬化中的作用。 血管紧张素II（AngII）可明显促进高脂血症小鼠动脉粥样硬化的形成。 此外，AngII增加12/15-脂氧合酶（12/15-LO）的表达。 12/15-LO缺乏显著减轻高脂血症小鼠动脉粥样硬化。 因此，我们试图确定12/15-LO是否在AngII诱导的血管疾病中起作用。 年龄匹配的雄性载脂蛋白E缺陷（apoE-/-）小鼠或12/15-LO缺陷apoE-/-小鼠用AngII（1，000 ng/kg/min）输注28天。 与对照组相比，AngII输注后12/15-LO缺陷小鼠的血清总胆固醇或脂蛋白谱没有改变。12/15-LO适度降低了主动脉弓内膜表面上AngII诱导的动脉粥样硬化病变形成（12/15-LO+/+：1.26% vs 12/15-LO-/-：1.99%; P = 0.12），然而，这种降低未达到统计学显著性。 此外，12/15-LO缺乏降低了AngII诱导的腹主动脉瘤形成的发生率（12/15-LO+/+：42% vs 12/15-LO-/-：17%; P = 0.12）和腹主动脉直径（12/15-LO+/+：1.6 mm vs 12/15-LO-/-：1.1 mm; P = 0.11），然而，这两个参数的降低均未达到统计学显著性。 虽然这些结果表明12/15-LO不会显著改变AngII诱导的动脉粥样硬化和AAA形成，但这些研究中使用的小鼠数量没有产生0.08的功效。 这与动脉粥样硬化和AAA形成减少的趋势相结合，表明需要进行额外的研究来确定12/15-LO对AngII诱导的血管疾病的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aim 1. Determine the role of COX-2 in P. gingivalis-induced periodontal disease and atherosclerosis. Periodontitis is a chronic inflammatory disease resulting from microbial infection of the oral cavity. The disease is initiated by gram-negative anaerobic species including Porphyromonas gingivalis. P. gingivalis infection may produce inflammation localized within the oral cavity which results in alveolar bone resorption and may produce a systemic inflammatory response resulting from bacteremia. The systemic inflammatory response is thought to account for the increased risk of developing complications of cardiovascular disease in patients with periodontitis. Prostaglandins are a class of inflammatory mediators that are significantly increased in diseased periodontal tissues. The synthesis of prostaglandins requires the activity of a cyclooxygenase, and cyclooxygenase-2 (COX-2) is the isoform that is highly induced by gram-negative bacterial products and is increased in atherosclerotic lesions. The current studies utilized an established mouse model of P. gingivalis-induced periodontitis to examine the role of COX-2-derived prostaglandins in alveolar bone resorption and atherosclerosis. Alveolar bone resorption and cardiovascular disease resulting from P. gingivalis requires chronic infection to be established in the oral cavity of the mice. Therefore, the mice are inoculated with P. gingivalis 4 days a week, every 3rd week, over a 12 week course. We have optimized the PCR technique to confirm that chronic infection with P. gingivalis is established in the mice. We have also utilized the highly sensitive technique, real-time PCR, to examine the expression of COX-2, as well as downstream prostaglandin synthases in aortic tissue. Furthermore, we have optimized immunohistochemical analysis for detection of COX-2 protein expression in the aortas of mice. These procedures will allow us to examine the effects of P. gingivalis infection on the expression of COX-2 and downstream prostaglandin synthases. Studies are in progress to examine the effectiveness of genetic or pharmacological inactivation of COX-2 in reducing P. gingivalis-induced alveolar bone resorption and atherosclerosis in mice. Specific Aim 2. Define the role of 12/15-lipoxygenase in P. gingivalis-induced periodontal disease and atherosclerosis. Angiotensin II (AngII) markedly accelerates atherogenesis in hyperlipidemic mice. Moreover, AngII increases the expression of 12/15-lipoxygenase (12/15-LO). Deficiency of 12/15-LO markedly attenuates atherosclerosis in hyperlipidemic mice. Therefore, we sought to determine if 12/15-LO plays a role in AngII-induced vascular disease. Age matched male apolipoproteinE deficient (apoE-/-) mice or 12/15-LO deficient apoE-/- mice were infused with AngII (1,000 ng/kg/min) for 28 days. Total serum cholesterol or lipoprotein profile was not altered in 12/15-LO deficient mice following AngII infusion compared to control. 12/15-LO modestly decreased AngII-induced atherosclerotic lesion formation (12/15-LO+/+: 1.26% vs 12/15-LO-/-: 1.99%; P = 0.12) on the intimal surface of the aortic arch however, this decrease did not reach statistical significance. Additionally, 12/15-LO deficiency decreased the incidence of AngII-induced abdominal aortic aneurysm formation (12/15-LO+/+: 42% vs 12/15-LO-/-: 17%; P = 0.12) and the abdominal aortic diameter (12/15-LO+/+: 1.6 mm vs 12/15-LO-/-: 1.1 mm; P = 0.11), however, the reduction in both of these parameters did not reach statistical significance. While these results suggest 12/15-LO does not significantly alter AngII-induced atherosclerosis and AAA formation, the number of mice utilized in these studies did not result in a power of 0.08. This coupled with the trend toward a decrease in both atherosclerosis and AAA formation suggest that additional studies are required to define the effect of 12/15-LO on AngII-induced vascular disease.