Alveolar heparan sulfate shedding impedes recovery from bleomycin-induced lung injury.

The pulmonary epithelial glycocalyx, an anionic cell surface layer enriched in glycosaminoglycans such as heparan sulfate and chondroitin sulfate, contributes to the alveolar barrier. Direct injury to the pulmonary epithelium induces shedding of heparan sulfate into the air space; the impact of this shedding on recovery after lung injury is unknown. Using mass spectrometry, we found that heparan sulfate was shed into the air space for up to 3 wk after intratracheal bleomycin-induced lung injury and coincided with induction of matrix metalloproteinases (MMPs), including MMP2. Delayed inhibition of metalloproteinases, beginning 7 days after bleomycin using the nonspecific MMP inhibitor doxycycline, attenuated heparan sulfate shedding and improved lung function, suggesting that heparan sulfate shedding may impair lung recovery. While we also observed an increase in air space heparanase activity after bleomycin, pharmacological and transgenic inhibition of heparanase in vivo failed to attenuate heparan sulfate shedding or protect against bleomycin-induced lung injury. However, experimental augmentation of airway heparanase activity significantly worsened post-bleomycin outcomes, confirming the importance of epithelial glycocalyx integrity to lung recovery. We hypothesized that MMP-associated heparan sulfate shedding contributed to delayed lung recovery, in part, by the release of large, highly sulfated fragments that sequestered lung-reparative growth factors such as hepatocyte growth factor. In vitro, heparan sulfate bound hepatocyte growth factor and attenuated growth factor signaling, suggesting that heparan sulfate shed into the air space after injury may directly impair lung repair. Accordingly, administration of exogenous heparan sulfate to mice after bleomycin injury increased the likelihood of death due to severe lung dysfunction. Together, our findings demonstrate that alveolar epithelial heparan sulfate shedding impedes lung recovery after bleomycin.

Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.

UNLABELLED: Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. SUMMARY: Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection.

Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples.

BACKGROUND: Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance. METHODS: In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples. RESULTS: We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations. CONCLUSIONS: In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.

Determining the aetiology of pulmonary oedema by the oedema fluid-to-plasma protein ratio.

We hypothesised that the oedema fluid-to-plasma protein (EF/PL) ratio, a noninvasive measure of alveolar capillary membrane permeability, can accurately determine the aetiology of acute pulmonary oedema. 390 mechanically ventilated patients with acute pulmonary oedema were enrolled. A clinical diagnosis of acute lung injury (ALI), cardiogenic pulmonary oedema or a mixed aetiology was based on expert medical record review at the end of hospitalisation. The EF/PL ratio was measured from pulmonary oedema fluid and plasma samples collected at intubation. 209 patients had a clinical diagnosis of ALI, 147 had a diagnosis of cardiogenic pulmonary oedema and 34 had a mixed aetiology. The EF/PL ratio had an area under the receiver-operating curve of 0.84 for differentiating ALI from cardiogenic pulmonary oedema. Using a predefined cut-off of 0.65, the EF/PL ratio had a sensitivity of 81% and a specificity of 81% for the diagnosis of ALI. An EF/PL ratio >/=0.65 was also associated with significantly higher mortality and fewer ventilator-free days. Noninvasive measurement of the EF/PL ratio is a safe and reliable bedside method for rapidly determining the aetiology of acute pulmonary oedema that can be used at the bedside in both developed and developing countries.