Chemokines, nitric oxide, and myocardial depression

浸润的中性粒细胞通过释放自由基、蛋白水解酶、促炎细胞因子和堵塞微血管加剧缺血再灌注损伤。某些趋化因子，如人类的IL-8，在吸引中性粒细胞到损伤或炎症部位中起关键作用。我们最近描述了三种中性粒细胞趋化因子CXC趋化因子- LIX（脂多糖诱导的CXC趋化因子），KC（细胞因子诱导的中性粒细胞趋化因子）和MIP-2（巨噬细胞炎症蛋白-2）-在大鼠心肌缺血-再灌注损伤期间的时间表达。此外，我们发现LIX可诱导NO的产生，抑制离体心肌细胞的收缩性能，并抑制心肌在体内的收缩功能。这些观察结果表明，lix诱导的心肌功能障碍可能是通过对NO和/或自由基产生的直接、不依赖中性粒细胞的影响介导的。本建议的具体目的如下。(1)我们将在体内确定LIX是否通过NO生成抑制心肌功能，以及用选择性iNOS抑制剂L-NIL预处理是否会减少NO生成并预防LIX诱导的左室功能障碍。LIX将在体内单独使用或在L-NIL预处理后使用，心肌抑制将通过超声心动图评估。一氧化氮合酶的表达和定位，活性氧和氮中间体和过氧亚硝酸盐的测量将被测量。(2)我们将确定lix诱导的心肌功能障碍是否与中性粒细胞浸润无关。在给予LIX之前，中性粒细胞会被抗中性粒细胞抗血清耗尽。(3)我们将在分离的心肌细胞中确定lix介导的iNOS诱导的细胞信号机制。iNOS启动子驱动的报告基因构建的活性将在体外转染的成年大鼠心肌细胞中进行评估。这些研究结果将进一步加深我们对趋化因子生物学的认识，并为预防或治疗缺血再灌注损伤心肌功能障碍提供新的治疗策略。

Infiltrating neutrophils exacerbate ischemia-reperfusion injury by releasing free radicals, proteolytic enzymes, proinflammatory cytokines, and by plugging microcapillaries. Certain chemokines, such as IL-8 in humans, play key roles in attracting neutrophils to sites of injury or inflammation. We have recently described the temporal expression of three neutrophil chemoattractant CXC chemokines - LIX (lipopolysachharide-induced CXC chemokine), KC (cytokine-induced neutrophil chemoattractant) and MIP-2 (macrophage inflammatory protein-2) - during myocardial ischemia-reperfusion injury in the rat. In addition, we found that administration of LIX induces NO production and depresses contractile performance in isolated cardiomyocytes, and depresses myocardial contractile function in vivo. These observations suggest that LIX-induced myocardial dysfunction may be mediated by a direct, neutrophil-independent, effect on myocardial production of NO and/or free radicals. The specific aims of this proposal are as follows. (1) We will determine whether LIX depresses myocardial function in vivo via NO generation, and whether pretreatment with L-NIL, a selective inhibitor of iNOS, will reduce NO generation and prevent LIX- induced LV dysfunction. LIX will be administered in vivo either alone or after pre-treatment with L-NIL, and myocardial depression will be assessed by echocardiography. Expression and localization of nitric oxide synthases, measurement of reactive oxygen and nitrogen intermediates and peroxynitrite will be measured. (2) We will determine whether LIX-induced myocardial dysfunction in vivo is independent on neutrophil infiltration. Neutrophils will be depleted by anti-neutrophil antiserum prior to administration of LIX. (3) We will determine the cellular signaling mechanisms responsible for LIX-mediated iNOS induction in isolated cardiomyocytes. The activity of iNOS promoter-driven reporter gene constructs will be assessed in vitro in transfected adult rat cardiomyocytes. The results from these studies will further our knowledge of chemokine biology and lead to new therapeutic strategies to prevent or treat myocardial dysfunction in ischemia-reperfusion injury.