In vivo detection of hyperoxia-induced pulmonary endothelial cell death using 99mTc-Duramycin
Introduction
Acute lung injury (ALI) is characterized by rapidly progressing hypoxic lung failure following a direct or indirect injury to the pulmonary parenchyma or vasculature [1]. This condition is one of the most frequent causes of admission to medical intensive care units, and of the approximately one million patients receiving invasive mechanical ventilation per year, 10% develop new ALI [2]. The most serious form of ALI is acute respiratory distress syndrome (ARDS) which occurs in ~ 200,000 patients in the U.S. per year and carries a mortality rate of nearly 40% despite the best supportive care [3].
Recent studies suggest that ~ 70% of ALI patients are not recognized as such by bedside providers, and stress the importance of early detection of ALI (i.e. prior to clinical evidence) for enhancing the efficacy of existing therapies and improving outcomes of ALI/ARDS patients [3], [4]. Beyond antibiotics, patients at risk for ALI development would be strong candidates for strategies including limited blood transfusions (to avoid transfusion related lung injury), closer clinical observation (such as in an intermediate care unit), limitation of inhaled oxygen fractions to levels needed to support vital organ function, but not more, and strict attention to intake and output measurements to detect fluid retention and pulmonary edema. Each of these interventions is not universally implemented because of risk to benefit ratios. Thus, our long-term goal is to develop a clinical means for early detection and monitoring of ALI in individual patients.
Rat exposure to lethal (> 95%) concentrations of oxygen (hyperoxia) is a well-documented model of human ALI/ARDS [5], [6], [7], [8]. Crapo et al. [7] provide a detailed description of histological and morphometric changes in lungs of rats exposed to 100% O2. No structural changes are observed in lungs of rats exposed to 100% O2 for up to 40 hours. However, by 60 hours, there is a 30% loss in capillary endothelial cells due to cell death, infiltration of phagocytic cells, thickening of the air–blood barrier, and pleural effusion. This is followed by further loss in endothelial cells and edema, pleural effusion, severe hypoxemia, and death within 72 hours 7. There is strong evidence that the pulmonary capillary endothelium is a primary and early site of oxygen toxicity injury [7], [9], [10]. This and the large pulmonary capillary surface area in direct apposition with blood-borne compounds suggest the utility of biomarker imaging for detecting early lung endothelial injury due to hyperoxic ALI [11], [12].
99mTc-duramycin, DU, is a new SPECT biomarker sensing cell death via apoptosis and/or necrosis [12], [13], [14]. DU serves as a molecular probe that binds to phosphatidylethanolamine (PE), which has little presence on the surface of normal viable cells, but becomes exposed onto the cell surface and/or accessible to the extracellular milieu with apoptosis and necrosis, respectively [13], [14]. Recently we reported an increase in the lung uptake of DU in rats exposed to sublethal 85% O2 [12]. The objective of the present study is to determine the extent of DU lung uptake in rats injured with > 98% O2 exposure for up to 60 hours, and to correlate that uptake with capillary endothelial cell apoptosis detected histologically.
Section snippets
Materials and methods
Duramycin (3,035 g/mole MW) kits were prepared as previously described [12], and technetium-labeled macroaggregated albumin (99mTc-MAA, particle sizes 20–40 μm) was purchased from Cardinal Health (Wauwatosa, WI). Antibodies to cleaved caspase 3 (Biocare, #CP229B), CD31 (Acris, #AP15436PU-N), keratin 7 (Abcam, #9021), and myeloperoxidase (Abcam, #45977) were used with appropriate secondary antibodies (Jackson Immuno #711-066-152 for CD31, Jackson Immuno #715-066-151 for keratin 7, and Abcam #6829
Results
Rats did not display any sign of distress after 48 hrs of hyperoxic exposure. However by 60 hrs, rats exhibited decreased intake of food and water, and some demonstrated respiratory distress, especially upon removal from the chamber. Pleural effusion weight, which was negligible at 0 and 48 hours of hyperoxic exposure, measured 7.6 ± 1.6 g (p < 0.001) at 60 hrs (Table 1).
Body weight was measured as an index of general health status. Rats lost 6 ± 2 g (paired t-test, p = 0.003) and 18 ± 2 g (p < 0.001) of their
Discussion
We report an increase in lung DU uptake measured in vivo after just 48 hours of > 98% O2 exposure, prior to the onset of significant histological or functional evidence of lung injury. Moreover, we identified a strong correlation between DU lung uptake and the number of CC3 positive cells. Since more than 50% of CC3 positive cells were endothelial cells, apoptotic endothelial cells appear to contribute more to the enhanced DU lung uptake signal in hyperoxic rats than other cell types.
In this
Conflict of interest
Nothing to disclose.
Acknowledgments
This work was supported by NIH grants 8UL1TR000055 (CTSI) (Audi, Clough), 1R01HL116530 (Jacobs, Audi, Clough), and 5R01HL102085 (Zhao), VA Merit Review Award BX001681 (Jacobs, Audi, Clough), The Alvin and Marion Birnschein Foundation, and Marquette University Graduate School (Audi, Clough). Statistical analysis was supported by the National Center for Advancing Translational Sciences, NIH grant 8UL1TR000055. The authors gratefully acknowledge the contributions of Dr. Naveen Bansal (Marquette
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