Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo.

Sean E Gill,Marta Rohan,Sanjay Mehta

doi:10.1186/s12931-015-0266-7

Abstract

BackgroundSepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Sepsis-induced ALI is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (PMVEC), resulting in enhanced pulmonary microvascular sequestration and pulmonary infiltration of polymorphonuclear leukocytes (PMN) as well as disruption of the normal alveolo-capillary permeability barrier with leak of albumin-rich edema fluid into pulmonary interstitium and alveoli. The role of PMVEC death and specifically apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established.MethodsIn a murine cecal ligation/perforation (CLP) model of sepsis, we quantified and correlated time-dependent changes in pulmonary microvascular Evans blue (EB)-labeled albumin permeability with (1) PMVEC death (propidium iodide [PI]-staining) by both fluorescent intravital videomicroscopy (IVVM) and histology, and (2) PMVEC apoptosis using histologic fluorescent microscopic assessment of a panel of 3 markers: cell surface phosphatidylserine (detected by Annexin V binding), caspase activation (detected by FLIVO labeling), and DNA fragmentation (TUNEL labeling).ResultsCompared to sham mice, CLP-sepsis resulted in pulmonary microvascular barrier dysfunction, quantified by increased EB-albumin leak, and PMVEC death (PI+ staining) as early as 2 h and more marked by 4 h after CLP. Septic PMVEC also exhibited increased presence of all 3 markers of apoptosis (Annexin V+, FLIVO+, TUNEL+) as early as 30 mins – 1 h after CLP-sepsis, which all similarly increased markedly until 4 h. The time-dependent changes in septic pulmonary microvascular albumin-permeability barrier dysfunction were highly correlated with PMVEC death (PI+; r = 0.976, p < 0.01) and PMVEC apoptosis (FLIVO+; r = 0.991, p < 0.01). Treatment with the pan-caspase inhibitor Q-VD prior to CLP reduced PMVEC death/apoptosis and attenuated septic pulmonary microvascular dysfunction, including both albumin-permeability barrier dysfunction and pulmonary microvascular PMN sequestration (p < 0.05). Septic PMVEC apoptosis and pulmonary microvascular dysfunction were also abrogated following CLP-sepsis in mice deficient in iNOS (Nos2−/−) or NADPH oxidase (p47phox−/− or gp91phox−/−) and in wild-type mice treated with the NADPH oxidase inhibitor, apocynin.ConclusionsSeptic murine pulmonary microvascular dysfunction in vivo is due to PMVEC death, which is mediated through caspase-dependent apoptosis and iNOS/NADPH-oxidase dependent signaling.

Highlights

Sepsis remains a common and serious clinical problem with significant morbidity and mortality
By 2 h post-cecal ligation/perforation (CLP), septic mice had evidence of significant pulmonary microvascular endothelial barrier dysfunction compared to sham-treated mice, and this difference was more marked at 2.7 ± 0.4 μg Evans blue (EB)/g lung/min by 4 h after CLP-sepsis (Fig. 1a)
Role of inducible NO synthase (iNOS) and NADPH oxidase in Sepsis-induced pulmonary microvascular endothelial cells (PMVEC) apoptosis We have previously reported that iNOS plays an important role in multiple features of septic acute lung injury (ALI) in the murine CLP-sepsis model, including pulmonary microvascular endothelial barrier dysfunction and the resulting albumin hyper-permeability, pulmonary oxidant stress, pulmonary microvascular polymorphonuclear leukocytes (PMN) sequestration and trans-PMVEC PMN migration [17, 18, 35, 47]

Summary

Introduction

Sepsis remains a common and serious clinical problem with significant morbidity and mortality. Morbidity/ mortality in sepsis are principally due to injury and dysfunction of multiple organs, most commonly acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) [2, 4,5,6]. Clinical trials of novel anti-inflammatory therapeutic approaches have been disappointingly negative, as none of these therapies has improved clinical outcomes in sepsis and septic ARDS [6, 9,10,11]. Sepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The role of PMVEC death and apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established

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