Lanter Bernard B, Sauer Karin, Davies David G
Department of Biological Sciences, Binghamton University, Binghamton, New York, USA.
Department of Biological Sciences, Binghamton University, Binghamton, New York, USA
mBio. 2014 Jun 10;5(3):e01206-14. doi: 10.1128/mBio.01206-14.
Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin.
The association of bacteria with atherosclerosis has been only superficially studied, with little attention focused on the potential of bacteria to form biofilms within arterial plaques. In the current work, we show that bacteria form biofilm deposits within carotid arterial plaques, and we demonstrate that one species we have identified in plaques can be stimulated in vitro to undergo a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of transferrin. Biofilm dispersion is characterized by the release of bacterial enzymes into the surroundings of biofilm microcolonies, allowing bacteria to escape the biofilm matrix. We believe these enzymes may have the potential to damage surrounding tissues and facilitate plaque rupture if norepinephrine is able to stimulate biofilm dispersion in vivo. This research, therefore, suggests a potential mechanistic link between hormonal state and the potential for heart attack and stroke.
动脉粥样硬化是一种因动脉壁内脂肪斑块沉积而导致的疾病状态，是缺血（血液供应受限）的主要潜在原因，可导致外周动脉阻塞、充血性心力衰竭、心脏病发作和人类中风。新出现的研究表明，包括炎症和感染在内的因素可能在动脉粥样硬化的进展中起关键作用。在当前的研究中，来自15名患者的动脉粥样硬化颈动脉外植体均显示真细菌16S rRNA基因检测呈阳性。对其中5个样本进行密度梯度凝胶电泳分析发现，每个样本都包含10个或更多不同的16S rRNA基因序列。用真细菌特异性肽核酸探针分析5条病变颈动脉的横切面，直接显微镜观察显示这些部位形成了生物膜沉积物，每个分析的薄斑块切片中有1至6个沉积物。大部分（93%）沉积物位于内弹性膜近端并与纤维组织相关。在分析的15个斑块中的6个中，检测到了假单胞菌属的16S rRNA基因。在我们实验室中，已证明铜绿假单胞菌生物膜在体外受到游离铁刺激时会发生分散反应。已知转铁蛋白与儿茶酚胺激素（如去甲肾上腺素）相互作用后会将铁释放到血液中。体外实验表明，在生理水平的转铁蛋白存在但不存在时，添加生理相关水平的去甲肾上腺素会诱导铜绿假单胞菌生物膜在低铁条件下生长时发生分散。
细菌与动脉粥样硬化的关联仅得到了表面研究，很少有人关注细菌在动脉斑块内形成生物膜的可能性。在当前的研究中，我们表明细菌在颈动脉斑块内形成生物膜沉积物，并且我们证明，在转铁蛋白存在的情况下，当受到生理相关水平的去甲肾上腺素刺激时，我们在斑块中鉴定出的一种细菌在体外可发生生物膜分散反应。生物膜分散的特征是细菌酶释放到生物膜微菌落周围环境中，使细菌能够逃离生物膜基质。我们认为，如果去甲肾上腺素能够在体内刺激生物膜分散，这些酶可能有损害周围组织并促进斑块破裂的潜力。因此，这项研究表明激素状态与心脏病发作和中风可能性之间存在潜在的机制联系。
