Abstract
Sepsis is a systemic inflammatory response that targets multiple components of the cardiovascular system including the microvasculature. Microvascular endothelial cells (MVEC) are central to normal microvascular function, including maintenance of the microvascular permeability barrier. Microvascular/MVEC dysfunction during sepsis is associated with barrier dysfunction, resulting in the leak of protein-rich edema fluid into organs, especially the lung. The specific role of MVEC apoptosis in septic microvascular/MVEC dysfunction in vivo remains to be determined. To examine pulmonary MVEC death in vivo under septic conditions, we used a murine cecal ligation/perforation (CLP) model of sepsis and identified non-viable pulmonary cells with propidium iodide (PI) by intravital videomicroscopy (IVVM), and confirmed this by histology. Septic pulmonary microvascular Evans blue (EB)-labeled albumin leak was associated with an increased number of PI-positive cells, which were confirmed to be predominantly MVEC based on specific labeling with three markers, anti-CD31 (PECAM), anti-CD34, and lectin binding. Furthermore, this septic death of pulmonary MVEC was markedly attenuated by cyclophosphamide-mediated depletion of neutrophils (PMN) or use of an anti-CD18 antibody developed for immunohistochemistry but shown to block CD18-dependent signaling. Additionally, septic pulmonary MVEC death was iNOS-dependent as mice lacking iNOS had markedly fewer PI-positive MVEC. Septic PI-positive pulmonary cell death was confirmed to be due to apoptosis by three independent markers: caspase activation by FLIVO, translocation of phosphatidylserine to the cell surface by Annexin V binding, and DNA fragmentation by TUNEL. Collectively, these findings indicate that septic pulmonary MVEC death, putatively apoptosis, is a result of leukocyte activation and iNOS-dependent signaling, and in turn, may contribute to pulmonary microvascular barrier dysfunction and albumin hyper-permeability during sepsis.
Highlights
Sepsis remains a common and important clinical problem with significant morbidity and mortality
Cyclophosphamide-treated mice had markedly reduced circulating leukocyte levels at 72 h (Table 1), which was associated with almost complete inhibition of the cecal ligation/perforation (CLP)-induced increase in pulmonary Evans blue (EB)-albumin leak (Fig. 1)
Examination of Rhodamine-6-G-labeled PMNs sequestered in the pulmonary microvasculature in vivo by intravital videomicroscopy (IVVM) and ex vivo in lung histologic sections by immunofluorescence indicated that CLP/sepsis caused significantly more PMN sequestration compared to sham surgery (Fig. 2)
Summary
Sepsis remains a common and important clinical problem with significant morbidity and mortality. In North America, ,one million cases of sepsis occur annually, leading to severe sepsis 40% of the time and 300,000 deaths. This consumes up to 45% of total ICU costs [2,3]. Morbidity/mortality in sepsis are largely due to multiple organ dysfunction/failure, most commonly lung injury, as well as renal and cardiac dysfunction [2,3,4,5,6]. Despite intensive basic and clinical research, treatment of sepsis and related organ dysfunction consists largely of supportive care, as all novel antiinflammatory therapeutic approaches, including the recently withdrawn activated protein C, have failed to improve the outcome of patients with sepsis and multiple organ dysfunction [6,7,8]
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