Farley K S, Wang L F, Law C, Mehta S
Centre for Critical Illness Research, Division of Respirology, Lawson Health Research Institute, London Health Sciences Center, London, Ontario, Canada.
Microvasc Res. 2008 Nov;76(3):208-16. doi: 10.1016/j.mvr.2008.07.004. Epub 2008 Jul 29.
Inducible nitric oxide (NO) synthase (iNOS) from neutrophils and alveolar macrophages (AM) contributes to the pathophysiology of murine septic acute lung injury (ALI). It is not known if AM iNOS has a direct effect on septic pulmonary microvascular endothelial cell (PMVEC) permeability. We hypothesized that AM iNOS mediates PMVEC permeability in vitro under septic conditions through NO and peroxynitrite. 100,000 confluent PMVEC on cell-culture inserts were co-incubated with iNOS+/+ vs. iNOS-/- AM, in various ratios of AM to PMVEC. PMVEC injury was assessed by trans-PMVEC Evans Blue-labelled albumin flux in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta and IFN-gamma). Cytomix stimulation dose-dependently increased trans-PMVEC EB-albumin flux, which was exaggerated (1.4+/-0.1% vs. 0.4+/-0.1% in unstimulated PMVEC, p<0.05) in the presence of iNOS+/+, but not iNOS-/-, AM in the upper compartment. Similarly, iNOS+/+, but not iNOS-/-, AM in the lower compartment also enhanced septic trans-PMVEC albumin leak. The mechanism of iNOS-dependent septic PMVEC permeability was pursued through pharmacologic studies with inhibitors of NOS, and scavengers of NO, superoxide, and peroxynitrite, and treatment of PMVEC with the NO donor, DETA-NONOate. Septic iNOS+/+ AM-dependent trans-PMVEC albumin leak was significantly attenuated by pharmacologic iNOS inhibition (L-NAME and 1400W), and scavenging of either NO (oxyhemoglobin), superoxide (PEG-SOD), or peroxynitrite (FeTPPS). Exogenous NO (DETA-NONOate) had no effect on PMVEC permeability. These data are consistent with a direct role of AM iNOS in septic PMVEC barrier dysfunction, which is likely mediated, in part, through peroxynitrite.
中性粒细胞和肺泡巨噬细胞（AM）中的诱导型一氧化氮（NO）合酶（iNOS）参与了小鼠脓毒症急性肺损伤（ALI）的病理生理过程。目前尚不清楚AM中的iNOS是否对脓毒症肺微血管内皮细胞（PMVEC）的通透性有直接影响。我们推测，在脓毒症条件下，AM中的iNOS通过NO和过氧亚硝酸盐在体外介导PMVEC的通透性。将细胞培养插入物上100,000个汇合的PMVEC与iNOS+/+和iNOS-/-的AM以不同的AM与PMVEC比例共同孵育。在存在或不存在细胞混合液（等摩尔的TNF-α、IL-1β和IFN-γ）的情况下，通过跨PMVEC伊文思蓝标记白蛋白通量评估PMVEC损伤。细胞混合液刺激剂量依赖性地增加跨PMVEC伊文思蓝-白蛋白通量，在存在iNOS+/+而非iNOS-/-的AM时，这种增加更为明显（未刺激的PMVEC中为0.4±0.1%，而在有iNOS+/+的情况下为1.4±0.1%，p<0.05）。同样，下层隔室中iNOS+/+而非iNOS-/-的AM也增强了脓毒症时跨PMVEC的白蛋白渗漏。通过使用NOS抑制剂、NO清除剂、超氧化物清除剂和过氧亚硝酸盐清除剂进行药理学研究，以及用NO供体DETA-NO供体处理PMVEC，来探究iNOS依赖性脓毒症PMVEC通透性的机制。药理学抑制iNOS（L-NAME和1400W）以及清除NO（氧合血红蛋白）、超氧化物（聚乙二醇超氧化物歧化酶）或过氧亚硝酸盐（FeTPPS）可显著减轻脓毒症iNOS+/+ AM依赖性跨PMVEC白蛋白渗漏。外源性NO（DETA-NO供体）对PMVEC通透性无影响。这些数据与AM中的iNOS在脓毒症PMVEC屏障功能障碍中起直接作用一致，这可能部分是通过过氧亚硝酸盐介导的。
