Chemokines, nitric oxide, and myocardial depression

渗入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而加重缺血-再灌注损伤。某些趋化因子，如人类的IL-8，在将中性粒细胞吸引到损伤或炎症部位发挥关键作用。最近我们研究了三种中性粒细胞趋化因子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(巨噬细胞炎性蛋白-2)在大鼠心肌缺血再灌注损伤中的瞬时表达。此外，我们还发现，给予Lix可诱导心肌细胞产生NO，抑制心肌细胞的收缩性能，并抑制在体心肌的收缩功能。这些观察结果表明，LIX诱导的心肌功能障碍可能是通过对心肌产生NO和/或自由基的直接、非中性粒细胞依赖性的影响而实现的。这项建议的具体目标如下。(1)我们将确定LIX是否通过产生NO而抑制在体心肌功能，以及预先给予诱导型一氧化氮合酶选择性抑制剂L-NIL是否能减少NO的产生，从而预防LIX所致的左心功能不全。LIX将单独或在L-Nil预治疗后在体内给予，心肌抑制将通过超声心动图进行评估。将测量一氧化氮合酶的表达和定位，活性氧和氮中间体以及过氧亚硝酸盐的测量。(2)我们将确定Lix所致的在体心肌功能障碍是否独立于中性粒细胞的浸润。在注射Lix之前，中性粒细胞将被抗中性粒细胞抗血清清除。(3)研究Lix诱导心肌细胞诱导型一氧化氮合酶的细胞信号机制。将在体外评估iNOS启动子驱动的报告基因构建体在转基因成年大鼠心肌细胞中的活性。这些研究的结果将进一步加深我们对趋化因子生物学的了解，并导致预防或治疗缺血再灌注损伤中的心肌功能障碍的新的治疗策略。

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.