Publications by authors named "Elizabeth R Jacobs"

76 Publications

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Am J Physiol Heart Circ Physiol 2021 Sep 24. Epub 2021 Sep 24.

Department of Medicine, grid.30760.32Medical College of Wisconsin, Milwaukee, Wisconsin, United States.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, TERT, plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 hours. Hyperoxia induced mitochondrial damage in rat lungs (TOMM20, MTT), which was paralleled by increased markers of inflammation (MPO, IL-1β, TLR9), impaired autophagy signaling (Beclin-1, LC3B-II/1, p62), and decreased the expression of TERT. Mitochondrial specific autophagy (mitophagy) was not altered as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT, and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, while activation of autophagy in vivo decreased mitochondrial damage (MTT) in both WT and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.
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http://dx.doi.org/10.1152/ajpheart.00166.2021DOI Listing
September 2021

Evaluation of Radiation-induced Pleural Effusions after Radiotherapy to Support Development of Animal Models of Radiation Pneumonitis.

Health Phys 2021 Oct;121(4):434-443

Department of Biostatistics, Medical College of Wisconsin, Milwaukee, WI.

Abstract: Not all animal models develop radiation-induced pleural effusions (RIPEs) as a form of radiation-induced lung injury (RILI). Such effusions are also not well characterized in humans. The purpose of this study is to identify occurrences of RIPE in humans, provide justification for development of relevant animal models, and further characterize its risk factors in cancer patients. We also aim to identify dose thresholds for cardiopulmonary toxicity in humans to shed light on possible pathogenic mechanisms for RIPEs. We carried out a retrospective review of medical records of 96 cancer patients receiving thoracic irradiation (TRT) at our institution. Fifty-three (53%) patients developed a new pleural effusion post TRT; 18 (19%) had RIPE; and 67% developed RIPE ipsilateral to the site irradiated. None developed "contralateral only" effusions. Median time to development was 6 mo (IQR; 4-8 mo). Of 18, 8 patients (44%) had concomitant asymptomatic (radiographic only) or symptomatic radiation pneumonitis and pericardial effusion. Dosimetric factors, including combined and ipsilateral mean lung dose (MLD), were significantly associated with increased risk of RIPE. Angiotensin converting enzyme inhibition, steroids, or concurrent chemotherapy did not modify incidence of RIPE. Our results substantiate the occurrence and incidence of RIPEs in humans. In cancer patients, a median time to development of effusions around 6 mo also supports the onset of RIPEs concurrent with radiation pneumonitis. Future work needs to include large populations of cancer survivors in whom delayed RIPEs can be tracked and correlated with cardiovascular changes in the context of injury to multiple organs.
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http://dx.doi.org/10.1097/HP.0000000000001462DOI Listing
October 2021

Corrigendum: Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

Front Pharmacol 2021 25;12:741485. Epub 2021 Aug 25.

Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, United States.

[This corrects the article DOI: 10.3389/fphar.2021.634477.].
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http://dx.doi.org/10.3389/fphar.2021.741485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424069PMC
August 2021

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies.

Cell Stress Chaperones 2021 Aug 12. Epub 2021 Aug 12.

Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA.

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.
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http://dx.doi.org/10.1007/s12192-021-01230-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357488PMC
August 2021

Molecular Changes in miRNA in Irradiated Rat Kidneys: Role of miR-34a and its Vascular Targets in the Notch Pathway.

Radiat Res 2021 Jul 30. Epub 2021 Jul 30.

Department of Radiation Oncology, Medical College of Wisconsin, Wauwatosa, Wisconsin.

The mechanism(s) of vascular regression in adult organs remains an unexplored gap. Irradiation to the kidney results in vascular regression and renal failure. The goal of this work was to determine molecular mechanism(s) of radiation-induced vascular regression and its mitigation by the drug lisinopril. Female WAG/RijCmcr rats received either 13 Gy X-ray irradiation, sparing one leg, or no irradiation, the latter serving as age-matched controls. Some irradiated animals received lisinopril. Kidney miRNA-seq was performed 35 days postirradiation, before symptoms of nephropathy. MicroRNA expression profiles were compared with data from humans. MicroRNA targets were predicted using TargetScan and confirmed by qRT-PCR and Western blot. Renal vascular endothelial cell density was evaluated at 100 days to confirm vascular regression. The normal rat kidney microRNA profile resembled that of humans. MiR-34a was increased >7-fold and emerged as the predominant rat microRNA altered by radiation. Expression of Jagged1, a ligand in the Notch pathway of vascular development and a target of miR-34a-5p was decreased by radiation but not in irradiated rats receiving lisinopril. Radiation decreased endothelial cells in the kidneys at 100 days, confirming vascular regression. In conclusion, the results of this study showed that radiation greatly increased miRNA34-a in rat kidneys, while lisinopril mitigated radiation-induced decrease of the Notch ligand, Jagged1, a molecular target of miRNA34-a.
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http://dx.doi.org/10.1667/RADE-20-00078.1DOI Listing
July 2021

Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

Front Pharmacol 2021 17;12:634477. Epub 2021 May 17.

Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, United States.

There is a need for countermeasures to mitigate lethal acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE). In WAG/RijCmcr rats, ARS occurs by 30-days following total body irradiation (TBI), and manifests as potentially lethal gastrointestinal (GI) and hematopoietic (H-ARS) toxicities after >12.5 and >7 Gy, respectively. DEARE, which includes potentially lethal lung and kidney injuries, is observed after partial body irradiation >12.5 Gy, with one hind limb shielded (leg-out PBI). The goal of this study is to enhance survival from ARS and DEARE by polypharmacy, since no monotherapy has demonstrated efficacy to mitigate both sets of injuries. For mitigation of ARS following 7.5 Gy TBI, a combination of three hematopoietic growth factors (polyethylene glycol (PEG) human granulocyte colony-stimulating factor (hG-CSF), PEG murine granulocyte-macrophage-CSF (mGM-CSF), and PEG human Interleukin (hIL)-11), which have shown survival efficacy in murine models of H-ARS were tested. This triple combination (TC) enhanced survival by 30-days from ∼25% to >60%. The TC was then combined with proven medical countermeasures for GI-ARS and DEARE, namely enrofloxacin, saline and the angiotensin converting enzyme inhibitor, lisinopril. This combination of ARS and DEARE mitigators improved survival from GI-ARS, H-ARS, and DEARE after 7.5 Gy TBI or 13 Gy PBI. Circulating blood cell recovery as well as lung and kidney function were also improved by TC + lisinopril. Taken together these results demonstrate an efficacious polypharmacy to mitigate radiation-induced ARS and DEARE in rats.
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http://dx.doi.org/10.3389/fphar.2021.634477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165380PMC
May 2021

Wound Trauma Exacerbates Acute, but not Delayed, Effects of Radiation in Rats: Mitigation by Lisinopril.

Int J Mol Sci 2020 May 30;21(11). Epub 2020 May 30.

Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

The goal of this study is to understand and mitigate the effects of wounds on acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), for preparedness against a radiological attack or accident. Combined injuries from concomitant trauma and radiation are likely in these scenarios. Either exacerbation or mitigation of radiation damage by wound trauma has been previously reported in preclinical studies. Female WAG/RijCmcr rats received 13 Gy X-rays, with partial-body shielding of one leg. Within 2 h, irradiated rats and non-irradiated controls were given full-thickness skin wounds with or without lisinopril, started orally 7 days after irradiation. Morbidity, skin wound area, breathing interval and blood urea nitrogen were measured up to 160 days post-irradiation to independently evaluate wound trauma and DEARE. Wounding exacerbated morbidity in irradiated rats between 5 and 14 days post-irradiation (during the ARS phase), and irradiation delayed wound healing. Wounding did not alter delayed morbidities from radiation pneumonitis or nephropathy after 30 days post-irradiation. Lisinopril did not mitigate wound healing, but markedly decreased morbidity during DEARE from 31 through 160 days. The results derived from this unique model of combined injuries suggest different molecular mechanisms of injury and healing of ARS and DEARE after radiation exposure.
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http://dx.doi.org/10.3390/ijms21113908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312718PMC
May 2020

Quantification of mitochondrial membrane potential in the isolated rat lung using rhodamine 6G.

J Appl Physiol (1985) 2020 04 5;128(4):892-906. Epub 2020 Mar 5.

Clement J. Zablocki Department of Veterans Affairs Medical Center, Milwaukee, Wisconsin.

Mitochondrial membrane potential (Δψ) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction. The objective of this study was to develop an experimental and computational approach for estimating Δψ in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of three single-pass phases, loading, washing, and uncoupling, in which the lungs were perfused with R6G-containing perfusate, fresh R6G-free perfusate, or R6G-free perfusate containing the mitochondrial uncoupler FCCP, respectively. This protocol was carried out with lung perfusate containing verapamil vehicle or verapamil, an inhibitor of the multidrug efflux pump P-glycoprotein (Pgp). Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration and that this increase was larger in the presence of verapamil than in its absence. A physiologically based pharmacokinetic (PBPK) model for the pulmonary disposition of R6G was developed and used for quantitative interpretation of the kinetic data, including estimating Δψ. The estimated value of Δψ [-144 ± 24 (SD) mV] was not significantly altered by inhibiting Pgp with verapamil and is comparable with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying Δψ in intact functioning lungs. This approach has potential to provide quantitative assessment of the effect of injurious conditions on lung mitochondrial function and to evaluate the impact of therapies that target mitochondria. A novel experimental and computational approach for estimating mitochondrial membrane potential (Δψ) in intact functioning lungs is presented. The isolated rat lung inlet-outlet concentrations of the fluorescent cationic dye rhodamine 6G were measured and analyzed by using a computational model of its pulmonary disposition to determine Δψ. The approach has the potential to provide quantitative assessment of the effect of injurious conditions and their therapies on lung mitochondrial function.
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http://dx.doi.org/10.1152/japplphysiol.00789.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191506PMC
April 2020

Prophylactic supplementation of 20-HETE ameliorates hypoxia/reoxygenation injury in pulmonary vascular endothelial cells by inhibiting apoptosis.

Acta Histochem 2020 Jan 6;122(1):151461. Epub 2019 Nov 6.

Center for Biotechnology, Anna University, Chennai, 600 025, India. Electronic address:

Hypoxia reoxygenation (HR) injury perturbs structural and functional syncytium in lung tissues. It is commonly implicated in conditions such as stroke, lung transplant or severe pneumonia. In the present study, we investigated the cytoprotective action of 20-hydroxyeicosatetraenoic acid (20-HETE) on pulmonary vascular endothelial cells (PMVECs) under normoxic and hypoxic niche followed by HR. 20-HETE pretreatment showed a protective effect at a concentration of 1μM as there was a marked increase (20%) in the cell viability compared to control and HR groups. Pretreatment of 20-HETE in HR induced injury decreased ROS production dictated its antioxidant property. Similarly, SOD and ATP levels were also downregulated by 20-HETE pretreatment. Cell apoptosis was detected by TUNEL assay, Acridine orange, and procaspase-3 cleavage, caspase-3 activity assay, respectively. JC-1 mitochondrial membrane potential assay and protein expression pattern of BCL-2, and BAD phosphorylation status were examined. The results showed that HR induced significant increase of apoptotic PMVECs, while 20-HETE pretreatment attenuated the effects. Further, 20-HETE pretreatment activated PI3K/Akt and HIF-1α signaling pathway to exhibit its protective effects against HR-induced oxidative stress and apoptosis. Overall, the results concluded the potent antioxidant role of 20-HETE in aiding cytoprotection upon HR injury.
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http://dx.doi.org/10.1016/j.acthis.2019.151461DOI Listing
January 2020

Pharmacokinetics of Tc-HMPAO in isolated perfused rat lungs.

J Appl Physiol (1985) 2019 11 15;127(5):1317-1327. Epub 2019 Aug 15.

Milwaukee Veterans Affairs Medical Center, Milwaukee, Wisconsin.

Lung uptake of technetium-labeled hexamethylpropyleneamine oxime (HMPAO) increases in rat models of human acute lung injury, consistent with increases in lung tissue glutathione (GSH). Since Tc-HMPAO uptake is the net result of multiple cellular and vascular processes, the objective was to develop an approach to investigate the pharmacokinetics of Tc-HMPAO uptake in isolated perfused rat lungs. Lungs of anesthetized rats were excised and connected to a ventilation-perfusion system. Tc-HMPAO (56 MBq) was injected into the pulmonary arterial cannula, a time sequence of images was acquired, and lung time-activity curves were constructed. Imaging was repeated with a range of pump flows and perfusate albumin concentrations and before and after depletion of GSH with diethyl maleate (DEM). A pharmacokinetic model of Tc-HMPAO pulmonary disposition was developed and used for quantitative interpretation of the time-activity curves. Experimental results reveal that Tc-HMPAO lung uptake, defined as the steady-state value of the Tc-HMPAO lung time-activity curve, was inversely related to pump flow. Also, Tc-HMPAO lung uptake decreased by ~65% after addition of DEM to the perfusate. Increased perfusate albumin concentration also resulted in decreased Tc-HMPAO lung uptake. Model simulations under in vivo flow conditions indicate that lung tissue GSH is the dominant factor in Tc-HMPAO retention in lung tissue. The approach allows for evaluation of the dominant factors that determine imaging biomarker uptake, separation of the contributions of pulmonary versus systemic processes, and application of this knowledge to in vivo studies. We developed an approach for studying the pharmacokinetics of technetium-labeled hexamethylpropyleneamine oxime (Tc-HMPAO) in isolated perfused lungs. A distributed-in-space-and-time computational model was fit to data and used to investigate questions that cannot readily be addressed in vivo. Experimental and modeling results indicate that tissue GSH is the dominant factor in Tc-HMPAO retention in lung tissue. This modeling approach can be readily extended to investigate the lung pharmacokinetics of other biomarkers and models of lung injury and treatment thereof.
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http://dx.doi.org/10.1152/japplphysiol.00717.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957362PMC
November 2019

Integrated Computational Model of Lung Tissue Bioenergetics.

Front Physiol 2019 8;10:191. Epub 2019 Mar 8.

Department of Biomedical Engineering, Marquette University, Milwaukee, WI, United States.

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics.
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http://dx.doi.org/10.3389/fphys.2019.00191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418344PMC
March 2019

Delayed Effects of Acute Radiation Exposure (Deare) in Juvenile and Old Rats: Mitigation by Lisinopril.

Health Phys 2019 04;116(4):529-545

Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI.

Our goal is to develop lisinopril as a mitigator of delayed effects of acute radiation exposure in the National Institute of Allergy and Infectious Diseases program for radiation countermeasures. Published studies demonstrated mitigation of delayed effects of acute radiation exposure by lisinopril in adult rats. However, juvenile or old rats beyond their reproductive lifespans have never been tested. Since no preclinical models of delayed effects of acute radiation exposure were available in these special populations, appropriate rat models were developed to test lisinopril after irradiation. Juvenile (42-d-old, prepubertal) female and male WAG/RijCmcr (Wistar) rats were given 13-Gy partial-body irradiation with only part of one hind limb shielded. Lethality from lung injury between 39-58 d and radiation nephropathy between 106-114 d were recorded. All irradiated-only juvenile rats were morbid from delayed effects of acute radiation exposure by 114 d, while lisinopril (24 mg m d) started 7 d after irradiation and continued improved survival to 88% (p = 0.0015, n ≥ 8/group). Old rats (>483-d-old, reproductively senescent) were irradiated with 13-Gy partial-body irradiation keeping part of one leg shielded and additionally shielding the head in some animals. Irradiated old females developed lethal nephropathy, and all became morbid by 170 d after irradiation, though no rats displayed lethal radiation pneumonitis. Similar results were observed for irradiated geriatric males, though 33% of rats remained alive at 180 d after irradiation. Lisinopril mitigated radiation nephropathy in old rats of both sexes. Finally, comparison of delayed effects of acute radiation exposure between irradiated juvenile, adult, and old rats showed younger rats were more sensitive to delayed effects of acute radiation exposure with earlier manifestation of injuries to some organs.
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http://dx.doi.org/10.1097/HP.0000000000000920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384142PMC
April 2019

Cardiac Remodeling and Reversible Pulmonary Hypertension During Pneumonitis in Rats after 13-Gy Partial-Body Irradiation with Minimal Bone Marrow Sparing: Effect of Lisinopril.

Health Phys 2019 04;116(4):558-565

Department of Pulmonary Medicine, Medical College of Wisconsin, Milwaukee, WI.

Total-body irradiation causes acute and delayed toxicity to hematopoietic, pulmonary, cardiac, gastrointestinal, renal, and other organ systems. Angiotensin-converting enzyme inhibitors mitigate many of the delayed injuries to these systems. The purpose of this study was to define echocardiographic features in rats at two times after irradiation, the first before lethal radiation pneumonitis (50 d) and the second after recovery from pneumonitis but before lethal radiation nephropathy (100 d), and to determine the actions of the angiotensin-converting enzyme inhibitor lisinopril. Four groups of female WAG/RijCmcr rats at 11-12 wk of age were studied: nonirradiated, nonirradiated plus lisinopril, 13-Gy partial-body irradiation sparing one hind leg (leg-out partial-body irradiation), and 13-Gy leg-out partial-body irradiation plus lisinopril. Lisinopril was started 7 d after radiation. Echocardiograms were obtained at 50 and 100 d, and cardiac histology was assessed after 100 d. Irradiation without lisinopril demonstrated echocardiographic transient pulmonary hypertension by 50 d which was largely resolved by 100 d in survivors. Irradiated rats given lisinopril showed no increase in pulmonary artery pressures at 50 d but exhibited left ventricular remodeling. By 100 d these rats showed some signs of pulmonary hypertension. Lisinopril alone had no impact on echocardiographic end points at either time point in nonirradiated rats. Mild increases in mast cells and fibrosis in the heart were observed after 100 d following 13-Gy leg-out partial-body irradiation. These data demonstrate irradiation-induced pulmonary hypertension which was reversed in survivors of pneumonitis. Lisinopril modified cardiovascular remodeling to enhance survival in this model from 41% to 86% (p = 0.0013).
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http://dx.doi.org/10.1097/HP.0000000000000919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384144PMC
April 2019

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red.

Free Radic Res 2018 Sep 28;52(9):1052-1062. Epub 2018 Sep 28.

b Zablocki VA Medical Center , Milwaukee, WI , USA.

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (HO) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured HO using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR's highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O) concentration of ventilation gas on lung rate of HO release into the perfusate. Baseline lung rate of HO release was 8.45 ± 0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial HO, and that most of acute hyperoxia-enhanced lung rate of HO release is from nonmitochondrial rather than mitochondrial sources.
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http://dx.doi.org/10.1080/10715762.2018.1511051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298832PMC
September 2018

Integrated computational model of the bioenergetics of isolated lung mitochondria.

PLoS One 2018 11;13(6):e0197921. Epub 2018 Jun 11.

Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin, United States of America.

Integrated computational modeling provides a mechanistic and quantitative framework for describing lung mitochondrial bioenergetics. Thus, the objective of this study was to develop and validate a thermodynamically-constrained integrated computational model of the bioenergetics of isolated lung mitochondria. The model incorporates the major biochemical reactions and transport processes in lung mitochondria. A general framework was developed to model those biochemical reactions and transport processes. Intrinsic model parameters such as binding constants were estimated using previously published isolated enzymes and transporters kinetic data. Extrinsic model parameters such as maximal reaction and transport velocities were estimated by fitting the integrated bioenergetics model to published and new tricarboxylic acid cycle and respirometry data measured in isolated rat lung mitochondria. The integrated model was then validated by assessing its ability to predict experimental data not used for the estimation of the extrinsic model parameters. For example, the model was able to predict reasonably well the substrate and temperature dependency of mitochondrial oxygen consumption, kinetics of NADH redox status, and the kinetics of mitochondrial accumulation of the cationic dye rhodamine 123, driven by mitochondrial membrane potential, under different respiratory states. The latter required the coupling of the integrated bioenergetics model to a pharmacokinetic model for the mitochondrial uptake of rhodamine 123 from buffer. The integrated bioenergetics model provides a mechanistic and quantitative framework for 1) integrating experimental data from isolated lung mitochondria under diverse experimental conditions, and 2) assessing the impact of a change in one or more mitochondrial processes on overall lung mitochondrial bioenergetics. In addition, the model provides important insights into the bioenergetics and respiration of lung mitochondria and how they differ from those of mitochondria from other organs. To the best of our knowledge, this model is the first for the bioenergetics of isolated lung mitochondria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197921PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995348PMC
December 2018

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

Arterioscler Thromb Vasc Biol 2018 03 1;38(3):622-635. Epub 2018 Feb 1.

From the College of Medical Laboratory Science and Technology, Harbin Medical University, Daqing, China (C.M., D.Z., M.M., E.R.J.); Department of Medicine (C.M., A.M.B., A.C., L.N., J.E., M.M., E.R.J.), Department of Physical Medicine and Rehabilitation (M.D.), Department of Physiology (A.M.B., M.M., E.R.J.), Department of Biochemistry (B.H.), Department of Radiation Oncology (M.M.), Department of Biophysics (N.H.), and Cardiovascular Center (M.T., C.M., A.B., M.D., M.M., E.R.J.), Medical College of Wisconsin, Milwaukee; Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee (A.C., S.H.A., M.M., E.R.J.); Department of Biomedical Engineering, Marquette University, Milwaukee (S.H.A.); and Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee (A.C.).

Objective: We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia.

Approach And Results: Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1.

Conclusions: Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction.
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http://dx.doi.org/10.1161/ATVBAHA.117.310605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823793PMC
March 2018

Protection by Inhaled Hydrogen Therapy in a Rat Model of Acute Lung Injury can be Tracked in vivo Using Molecular Imaging.

Shock 2017 10;48(4):467-476

*Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin †Zablocki V.A. Medical Center, Milwaukee, Wisconsin ‡Division of Pulmonary and Critical Care Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin §Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin ||Division of Cardiology, Northwestern University, Evanston, Illinois ¶Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin #Department of Mathematics, Statistics, and Computer Science, Marquette University, Milwaukee, Wisconsin.

Inhaled hydrogen gas (H2) provides protection in rat models of human acute lung injury (ALI). We previously reported that biomarker imaging can detect oxidative stress and endothelial cell death in vivo in a rat model of ALI. Our objective was to evaluate the ability of Tc-hexamethylpropyleneamineoxime (HMPAO) and Tc-duramycin to track the effectiveness of H2 therapy in vivo in the hyperoxia rat model of ALI. Rats were exposed to room air (normoxia), 98% O2 + 2% N2 (hyperoxia) or 98% O2 + 2% H2 (hyperoxia+H2) for up to 60 h. In vivo scintigraphy images were acquired following injection of Tc-HMPAO or Tc-duramycin. For hyperoxia rats, Tc-HMPAO and Tc-duramycin lung uptake increased in a time-dependent manner, reaching a maximum increase of 270% and 150% at 60 h, respectively. These increases were reduced to 120% and 70%, respectively, in hyperoxia+H2 rats. Hyperoxia exposure increased glutathione content in lung homogenate (36%) more than hyperoxia+H2 (21%), consistent with increases measured in Tc-HMPAO lung uptake. In 60-h hyperoxia rats, pleural effusion, which was undetectable in normoxia rats, averaged 9.3 gram/rat, and lung tissue 3-nitrotyrosine expression increased by 790%. Increases were reduced by 69% and 59%, respectively, in 60-h hyperoxia+H2 rats. This study detects and tracks the anti-oxidant and anti-apoptotic properties of H2 therapy in vivo after as early as 24 h of hyperoxia exposure. The results suggest the potential utility of these SPECT biomarkers for in vivo assessment of key cellular pathways in the pathogenesis of ALI and for monitoring responses to therapies.
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http://dx.doi.org/10.1097/SHK.0000000000000872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636640PMC
October 2017

Impact of duration of hypotension prior to norepinephrine initiation in medical intensive care unit patients with septic shock: A prospective observational study.

J Crit Care 2017 08 9;40:178-183. Epub 2017 Apr 9.

Department of Medicine, Division of Pulmonary & Critical Care Medicine, Medical College of Wisconsin, Milwaukee, WI, USA.

Purpose: To determine the impact of duration of hypotension prior to norepinephrine initiation on outcomes in MICU patients with septic shock. We hypothesized increased duration of hypotension prior to norepinephrine initiation would be associated with an increased risk for ICU mortality.

Materials And Methods: We conducted a prospective-observational study in the MICU of a single-center tertiary academic medical center. We enrolled 160 adults ≥18years old with septic shock. Descriptive statistics were computed for demographic and outcome variables. Primary logistic regression analysis was adjusted for severity of illness.

Results: The mean age of our patients was 59years (±17); 42% were female; the mean APACHE II score was 24.1 (±8.0), and the mean SOFA score was 9.6 (±4.0). Median duration of hypotension prior to norepinephrine initiation was 3.6h (IQR 1.6-9.9). Duration of hypotension prior to norepinephrine did not increase the risk for ICU mortality (OR 1.03 per hour after hypotension, 95% CI: 0.98-1.09, p=0.20).

Conclusion: Duration of hypotension less than one hour and greater than one hour prior to norepinephrine initiation in MICU patients with septic shock is not associated with an increased risk for ICU mortality.
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http://dx.doi.org/10.1016/j.jcrc.2017.04.013DOI Listing
August 2017

Changes in miRNA in the lung and whole blood after whole thorax irradiation in rats.

Sci Rep 2017 03 17;7:44132. Epub 2017 Mar 17.

Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, USA.

We used a rat model of whole thorax x-ray irradiation to profile the microRNA (miRNA) in lung and blood up to 4 weeks after radiation. MiRNA from normal and irradiated Wistar rat lungs and whole blood were analyzed by next-generation sequencing and the changes by radiation were identified by differential deRNA-seq 1, 2, 3 and 4 weeks after irradiation. The average total reads/library was 2,703,137 with a mean of 88% mapping to the rat genome. Detailed profiles of 100 of the most abundant miRNA in rat blood and lung are described. We identified upregulation of 4 miRNA, miR-144-5p, miR-144-3p, miR-142-5p and miR-19a-3p in rat blood 2 weeks after radiation that have not previously been shown to be altered after radiation to the lung. Ingenuity Pathway Analysis identified signaling of inflammatory response pathways. These findings will support development of early detection methods, as well as mechanism(s) of injury and mitigation in patients after radiotherapy or radiological accidents.
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http://dx.doi.org/10.1038/srep44132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355888PMC
March 2017

Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia.

Exp Lung Res 2017 02 7;43(1):38-48. Epub 2017 Mar 7.

b Clinical and Translational Science Institute , Milwaukee , Wisconsin , USA.

Purpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia.

Materials And Methods: Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague-Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A (AAR) and A adenosine receptor (AAR) mRNA and protein were quantified.

Results: Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. AAR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. AAR mRNA and protein concentrations remained unchanged following ischemia. AAR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes.

Conclusion: Increased AAR and adenosine following unilateral lung ischemia as well as more BOOP in AAR mutant rats implicate a protective role for AAR signaling in countering ischemic lung injury.
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http://dx.doi.org/10.1080/01902148.2017.1286697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831175PMC
February 2017

Combined Hydration and Antibiotics with Lisinopril to Mitigate Acute and Delayed High-dose Radiation Injuries to Multiple Organs.

Health Phys 2016 11;111(5):410-9

*Department of Radiation Oncology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226; †Department of Medicine, Department of Physiology, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226; Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, WI 53295; ‡Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202.

The NIAID Radiation and Nuclear Countermeasures Program is developing medical agents to mitigate the acute and delayed effects of radiation that may occur from a radionuclear attack or accident. To date, most such medical countermeasures have been developed for single organ injuries. Angiotensin converting enzyme (ACE) inhibitors have been used to mitigate radiation-induced lung, skin, brain, and renal injuries in rats. ACE inhibitors have also been reported to decrease normal tissue complication in radiation oncology patients. In the current study, the authors have developed a rat partial-body irradiation (leg-out PBI) model with minimal bone marrow sparing (one leg shielded) that results in acute and late injuries to multiple organs. In this model, the ACE inhibitor lisinopril (at ~24 mg m d started orally in the drinking water at 7 d after irradiation and continued to ≥150 d) mitigated late effects in the lungs and kidneys after 12.5-Gy leg-out PBI. Also in this model, a short course of saline hydration and antibiotics mitigated acute radiation syndrome following doses as high as 13 Gy. Combining this supportive care with the lisinopril regimen mitigated overall morbidity for up to 150 d after 13-Gy leg-out PBI. Furthermore, lisinopril was an effective mitigator in the presence of the growth factor G-CSF (100 μg kg d from days 1-14), which is FDA-approved for use in a radionuclear event. In summary, by combining lisinopril (FDA-approved for other indications) with hydration and antibiotics, acute and delayed radiation injuries in multiple organs were mitigated.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065284PMC
http://dx.doi.org/10.1097/HP.0000000000000554DOI Listing
November 2016

Biomarkers for Radiation Pneumonitis Using Noninvasive Molecular Imaging.

J Nucl Med 2016 08 31;57(8):1296-301. Epub 2016 Mar 31.

Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee, Wisconsin.

Unlabelled: Our goal is to develop minimally invasive biomarkers for predicting radiation-induced lung injury before symptoms develop. Currently, there are no biomarkers that can predict radiation pneumonitis. Radiation damage to the whole lung is a serious risk in nuclear accidents or in radiologic terrorism. Our previous studies have shown that a single dose of 15 Gy of x-rays to the thorax causes severe pneumonitis in rats by 6-8 wk. We have also developed a mitigator for radiation pneumonitis and fibrosis that can be started as late as 5 wk after radiation.

Methods: We used 2 functional SPECT probes in vivo in irradiated rat lungs. Regional pulmonary perfusion was measured by injection of (99m)Tc-macroaggregated albumin. Perfused volume was determined by comparing the volume of distribution of (99m)Tc-macroaggregated albumin to the anatomic lung volume obtained by small-animal CT. A second probe, (99m)Tc-labeled Duramycin, which binds to apoptotic cells, was used to measure pulmonary cell death in the same rat model.

Results: The perfused volume of lung was decreased by about 25% at 1, 2, and 3 wk after receipt of 15 Gy, and (99m)Tc-Duramycin uptake was more than doubled at 2 and 3 wk. There was no change in body weight, breathing rate, or lung histology between irradiated and nonirradiated rats at these times. Pulmonary vascular resistance and vascular permeability measured in isolated perfused lungs ex vivo increased at 2 wk after 15 Gy of irradiation.

Conclusion: Our results suggest that SPECT biomarkers have the potential to predict radiation injury to the lungs before substantial functional or histologic damage is observed. Early prediction of radiation pneumonitis in time to initiate mitigation will benefit those exposed to radiation in the context of therapy, accidents, or terrorism.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053021PMC
http://dx.doi.org/10.2967/jnumed.115.160291DOI Listing
August 2016

99MTc-Hexamethylpropyleneamine Oxime Imaging for Early Detection of Acute Lung Injury in Rats Exposed to Hyperoxia or Lipopolysaccharide Treatment.

Shock 2016 10;46(4):420-30

*Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin †Division of Pulmonary and Critical Care Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin ‡Zablocki V.A. Medical Center, Milwaukee, Wisconsin §Department of Mathematics, Statistics, and Computer Science, Marquette University, Milwaukee, Wisconsin ||Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin ¶Department of Electrical Engineering, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin **Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin.

Tc-Hexamethylpropyleneamine oxime (HMPAO) is a clinical single-photon emission computed tomography biomarker of tissue oxidoreductive state. Our objective was to investigate whether HMPAO lung uptake can serve as a preclinical marker of lung injury in two well-established rat models of human acute lung injury (ALI).Rats were exposed to >95% O2 (hyperoxia) or treated with intratracheal lipopolysaccharide (LPS), with first endpoints obtained 24 h later. HMPAO was administered intravenously before and after treatment with the glutathione-depleting agent diethyl maleate (DEM), scintigraphy images were acquired, and HMPAO lung uptake was quantified from the images. We also measured breathing rates, heart rates, oxygen saturation, bronchoalveolar lavage (BAL) cell counts and protein, lung homogenate glutathione (GSH) content, and pulmonary vascular endothelial filtration coefficient (Kf).For hyperoxia rats, HMPAO lung uptake increased after 24 h (134%) and 48 h (172%) of exposure. For LPS-treated rats, HMPAO lung uptake increased (188%) 24 h after injury and fell with resolution of injury. DEM reduced HMPAO uptake in hyperoxia and LPS rats by a greater fraction than in normoxia rats. Both hyperoxia exposure (18%) and LPS treatment (26%) increased lung homogenate GSH content, which correlated strongly with HMPAO uptake. Neither of the treatments had an effect on Kf at 24 h. LPS-treated rats appeared healthy but exhibited mild tachypnea, BAL, and histological evidence of inflammation, and increased wet and dry lung weights. These results suggest the potential utility of HMPAO as a tool for detecting ALI at a phase likely to exhibit minimal clinical evidence of injury.
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http://dx.doi.org/10.1097/SHK.0000000000000605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014734PMC
October 2016

Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

Am J Physiol Lung Cell Mol Physiol 2016 04 19;310(8):L772-83. Epub 2016 Feb 19.

Department of Pharmacology, New York Medical College, Valhalla, New York;

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18β-glycyrrhetinic acid (18β-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18β-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18β-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18β-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.
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http://dx.doi.org/10.1152/ajplung.00377.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836112PMC
April 2016

Whole-thorax irradiation induces hypoxic respiratory failure, pleural effusions and cardiac remodeling.

J Radiat Res 2015 Mar 3;56(2):248-60. Epub 2014 Nov 3.

Cardiovascular Center, Medical College of Wisconsin, 8701, Watertown Plank Road, Milwaukee, WI 53226, USA Division of Pulmonary Medicine, Medical College of Wisconsin, 8701, Watertown Plank Road, Milwaukee, WI 53226, USA Research Service, Department of Veteran's Affairs, Clement J. Zablocki VA Medical Center, Milwaukee, Wisconsin, USA.

To study the mechanisms of death following a single lethal dose of thoracic radiation, WAG/RijCmcr (Wistar) rats were treated with 15 Gy to the whole thorax and followed until they were morbid or sacrificed for invasive assays at 6 weeks. Lung function was assessed by breathing rate and arterial oxygen saturation. Lung structure was evaluated histologically. Cardiac structure and function were examined by echocardiography. The frequency and characteristics of pleural effusions were determined. Morbidity from 15 Gy radiation occurred in all rats 5 to 8 weeks after exposure, coincident with histological pneumonitis. Increases in breathing frequencies peaked at 6 weeks, when profound arterial hypoxia was also recorded. Echocardiography analysis at 6 weeks showed pulmonary hypertension and severe right ventricular enlargement with impaired left ventricular function and cardiac output. Histologic sections of the heart revealed only rare foci of lymphocytic infiltration. Total lung weight more than doubled. Pleural effusions were present in the majority of the irradiated rats and contained elevated protein, but low lactate dehydrogenase, when compared with serum from the same animal. Pleural effusions had a higher percentage of macrophages and large monocytes than neutrophils and contained mast cells that are rarely present in other pathological states. Lethal irradiation to rat lungs leads to hypoxia with infiltration of immune cells, edema and pleural effusion. These changes may contribute to pulmonary vascular and parenchymal injury that result in secondary changes in heart structure and function. We report that conditions resembling congestive heart failure contribute to death during radiation pneumonitis, which indicates new targets for therapy.
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http://dx.doi.org/10.1093/jrr/rru095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380043PMC
March 2015

Model development and use of ACE inhibitors for preclinical mitigation of radiation-induced injury to multiple organs.

Radiat Res 2014 Nov 31;182(5):545-55. Epub 2014 Oct 31.

a  Department of Radiation Oncology.

The NIH/NIAID initiated a countermeasure program to develop mitigators for radiation-induced injuries from a radiological attack or nuclear accident. We have previously characterized and demonstrated mitigation of single organ injuries, such as radiation pneumonitis, pulmonary fibrosis or nephropathy by angiotensin converting enzyme (ACE) inhibitors. Our current work extends this research to examine the potential for mitigating multiple organ dysfunctions occurring in the same irradiated rats. Using total body irradiation (TBI) followed by bone marrow transplant, we tested four doses of X radiation (11, 11.25, 11.5 and 12 Gy) to develop lethal late effects. We identified three of these doses (11, 11.25 and 11.5 Gy TBI) that were lethal to all irradiated rats by 160 days to test mitigation by ACE inhibitors of injury to the lungs and kidneys. In this study we tested three ACE inhibitors at doses: captopril (88 and 176 mg/m(2)/day), enalapril (18, 24 and 36 mg/m(2)/day) and fosinopril (60 mg/m(2)/day) for mitigation. Our primary end point was survival or criteria for euthanization of morbid animals. Secondary end points included breathing intervals, other assays for lung structure and function and blood urea nitrogen (BUN) to assess renal damage. We found that captopril at 176 mg/m(2)/day increased survival after 11 or 11.5 Gy TBI. Enalapril at 18-36 mg/m(2)/day improved survival at all three doses (TBI). Fosinopril at 60 mg/m(2)/day enhanced survival at a dose of 11 Gy, although no improvement was observed for pneumonitis. These results demonstrate the use of a single countermeasure to mitigate the lethal late effects in the same animal after TBI.
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http://dx.doi.org/10.1667/RR13425.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261643PMC
November 2014

Early Trophic Enteral Nutrition Is Associated With Improved Outcomes in Mechanically Ventilated Patients With Septic Shock: A Retrospective Review.

J Intensive Care Med 2016 Aug 14;31(7):471-7. Epub 2014 Oct 14.

Division of Pulmonary & Critical Care Medicine, Medical College of Wisconsin, Milwaukee, WI, USA Clement J. Zablocki VA Medical Center, Medical College of Wisconsin, Milwaukee, WI, USA.

Purpose: Current guidelines provide weak recommendations for starting enteral nutrition (EN) in patients with septic shock (on vasopressor support). Outcomes of patients receiving EN in septic shock on vasopressor support have not been well studied. We hypothesize that early trophic EN in mechanically ventilated patients with septic shock is associated with improved outcomes.

Methods: Single-center retrospective study of mechanically ventilated patients admitted with septic shock to identify patients receiving (1) no EN, (2) <600 kcal/d within 48 hours, and (3) ≥600 kcal/d within 48 hours. Outcomes studied included in-hospital mortality, length of intensive care unit stay (LOS), duration of mechanical ventilation (DOMV), and complications of feeding intolerance.

Results: Sixty-six patients were identified. In all, 15 received no EN, 37 received <600 kcal/d, and 14 received ≥600 kcal/d EN daily. Median LOS was 12, 5, and 13 days, respectively. The LOS was lower in patients receiving <600 kcal/d when compared to either no EN (P < .001) or those receiving ≥600 kcal/d (P < .001). Median DOMV was lower in patients receiving <600 kcal/d (median 3, P < .001) as compared to no EN (median 7, P < .001) or those receiving ≥600 kcal/d (median 7.5, P < .001). Mortality was not different. There were no significant complications among groups.

Conclusion: In patients with septic shock, those receiving <600 kcal/d EN within 48 hours had lower DOMV and LOS when compared to those who did not receive EN or those who received ≥600 kcal/d. These observations provide strong justification for prospective evaluation of the effect of early trophic EN in patients with septic shock.
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http://dx.doi.org/10.1177/0885066614554887DOI Listing
August 2016

In vivo detection of hyperoxia-induced pulmonary endothelial cell death using (99m)Tc-duramycin.

Nucl Med Biol 2015 Jan 19;42(1):46-52. Epub 2014 Aug 19.

Department of Biomedical Engineering, Marquette University; Division of Pulmonary and Critical Care Medicine, Medical College of Wisconsin; Department of Mathematics, Statistics, and Computer Science, Marquette University. Electronic address:

Introduction: (99m)Tc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury.

Methods: Rats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells.

Results: Lung DU uptake increased significantly (p<0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r(2)=0.82, p=0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate.

Conclusions: Rat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo.
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http://dx.doi.org/10.1016/j.nucmedbio.2014.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383294PMC
January 2015

Enhanced survival from radiation pneumonitis by combined irradiation to the skin.

Int J Radiat Biol 2014 Sep 25;90(9):753-61. Epub 2014 Jun 25.

Department of Radiation Oncology.

Purpose: To develop mitigators for combined irradiation to the lung and skin.

Methods: Rats were treated with X-rays as follows: (1) 12.5 or 13 Gy whole thorax irradiation (WTI); (2) 30 Gy soft X-rays to 10% area of the skin only; (3) 12.5 or 13 Gy WTI + 30 Gy skin irradiation after 3 hours; (4) 12.5 Gy WTI + skin irradiation and treated with captopril (160 mg/m(2)/day) started after 7 days. Our end points were survival (primary) based on IACUC euthanization criteria and secondary measurements of breathing intervals and skin injury. Lung collagen at 210 days was measured in rats surviving 13 Gy WTI.

Results: After 12.5 Gy WTI with or without skin irradiation, one rat (12.5 Gy WTI) was euthanized. Survival was less than 10% in rats receiving 13 Gy WTI, but was enhanced when combined with skin irradiation (p < 0.0001). Collagen content was increased at 210 days after 13 Gy WTI vs. 13 Gy WTI + 30 Gy skin irradiation (p < 0.05). Captopril improved radiation-dermatitis after 12.5 Gy WTI + 30 Gy skin irradiation (p = 0.008).

Conclusions: Radiation to the skin given 3 h after WTI mitigated morbidity during pneumonitis in rats. Captopril enhanced the rate of healing of radiation-dermatitis after combined irradiations to the thorax and skin.
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http://dx.doi.org/10.3109/09553002.2014.922722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380176PMC
September 2014

Hypoxia preconditioning increases survival and decreases expression of Toll-like receptor 4 in pulmonary artery endothelial cells exposed to lipopolysaccharide.

Pulm Circ 2013 Sep 4;3(3):578-88. Epub 2013 Dec 4.

1 Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

Abstract Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo.
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http://dx.doi.org/10.1086/674337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070795PMC
September 2013
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