Concern regarding accidental overexposure to radiation has been raised after the devastating Tohuku earthquake and tsunami which initiated the Fukushima Daiichi nuclear disaster in Japan, in March 2011. Radiation exposure is toxic and can be fatal depending on the dose received. Injury to the lung is often reported as part of multi-organ failure in victims of accidental exposures. Doses of radiation >8 Gray to the chest can induce pneumonitis with right ventricular hypertrophy starting after ~2 months. Higher doses may be followed by pulmonary fibrosis that presents months to years after exposure. Though the exact mechanisms of radiation lung damage are not known, experimental animal models have been widely used to study this injury. Rodent models for pneumonitis and fibrosis exhibit vascular, parenchymal and pleural injuries to the lung. Inflammation is a part of the injuries suggesting involvement of the immune system. Researchers world-wide have tested a number of interventions to prevent or mitigate radiation lung injury. One of the first and most successful class of mitigators are inhibitors of angiotensin converting enzyme (ACE), an enzyme that is abundant in the lung. These results offer hope that lung injury from radiation accidents may be mitigated, since the ACE inhibitor captopril was effective when started up to one week after irradiation.
captopril; lung fibrosis; mitigation; radiation pneumonitis; nuclear accident; radiological terrorism
We previously reported that the cytochrome P450 product 20-hydroxyeicosatetraenoic acid has prosurvival effects in pulmonary artery endothelial cells and ex vivo pulmonary arteries. We tested the potential of a 20-hydroxyeicosatetraenoic acid analog N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (20-5,14-HEDGE) to protect against lung ischemic reperfusion injury in rats. Furthermore, we examined activation of the innate immune system components high mobility group box 1 (HMGB1) and toll-like receptor 4 in this model as well as the effect of 20-5,14-HEDGE on this signaling pathway.
Sprague Dawley rats treated with 20-5,14-HEDGE or vehicle were subjected to surgically induced, unilateral lung ischemia for 60 minutes followed by reperfusion for two hours in vivo. Injury was assessed histologically by hematoxylin and eosin, and with identification of myeloperoxidase immunohistochemically. HMGB1 and toll-like receptor 4 proteins were identified by western blot. Caspase 3 activity or 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole incorporation were used to measure apoptosis and cell survival.
IR injury evoked atelectasis and hemorrhage, an influx of polymorphonuclear cells, and increased toll-like receptor 4 and HMGB1 expression. Caspase 3 activity was increased and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation was decreased. 20-5,14-HEDGE protected against each of these endpoints including infiltration of polymorphonuclear cells, with no changes in caspase 3 activity in other organs.
Lung IR produces apoptosis and activation of the innate immune system including HMGB1 and toll-like receptor 4 within two hours of reperfusion. Treatment with 20-5,14-HEDGE decreases activation of this response system, and salvages lung tissue.
ischemia reperfusion; myeloperoxidase; HMGB1; TLR4; caspase 3
To assess effects of a localized anastomosis between the aorta and left lower lobe pulmonary artery (LLLPA) on flows through central vessels, and on vascular reactivity of small PAs distal or contralateral to the shunt.
Flow rates in major vessels and tensions from small PAs from left and right lower lobes were determined 48 hours after creation of an end-to-side anastomosis of the LLLPA to the aorta.
Anastomoses increased flow through the LLLPA from 194 ± 6 to 452 ± 18 ml/min immediately after anastomosis and to 756 ± 19 ml/min by the time of harvest (n=88, p<0.05). Flow rates in main PAs from hosts with anastomoses were lower (557 ± 26 versus1033 ± 244 ml/min) while aortic root flows were not different from controls (1370 ± 53 compared to 1120 ± 111 ml/min; p = 0.07). Wet-to-dry weights of the both lungs and aortic flow rates were proportional to shunt flow rates. PA rings harvested from the right (unshunted) lobe of high flow hosts exhibited increased reactivity to the thromboxane agonist U46619 and phenylephrine relative to those of left PAs from the same animal or those of control hosts.
Our studies are the first to identify enhanced reactivity of PAs in a lung contralateral to a localized high output shunt between an aorta and pulmonary artery. These observations suggest that patients with localized systemic-to-pulmonary shunt could exhibit modified vascular tone in remote pulmonary arteries.
pulmonary vascular resistance; arteriovenous shunt; phenylephrine; U46619
Our model of a systemic-pulmonary shunt exhibits enhanced reactivity of pulmonary arteries contralateral to a localized shunt between the left lower lobe pulmonary artery (LLPA) and aorta relative to those of ipsilateral or control PAs 48 hours after anastomosis. We examined the contribution of nitric oxide (NO), cyclooxygenase (COX), lipoxygenase (LOX), or cytochrome P450 (CYP) production to mediating this enhanced reactivity.
We created a surgical end-to-side anastomosis of the left lower lobe PA to the aorta. 48 hours later we tested tension of PA rings from the right and left lower lobes for contraction to the thromboxane mimetic U46619 in the presence of vehicle or inhibitors of NO synthase (S), COX, CYP, or LOX. Western blots of PA homogenates were probed for eNOS or isoforms metabolizing arachidonic acid (AA). Eicosanoid products from intact PAs rings were detected using labeled AA and HPLC separation.
Enhanced reactivity of unshunted right PAs over that of left PAs from high flow hosts was not eliminated by inhibitors of NOS, COX, or CYP. Treatment with 2 different LOX inhibitors, nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-α-cyanocinnamate, closed the difference in contractility of shunted and unshunted PAs. PAs contralateral to shunts metabolized AA to 12-hydroxyeicosatetraenoic acid (HETE) in greater quantities than analogous PAs from the experimental left or control PAs.
48 hours after anastomosis enhanced reactivity of contralateral PAs is attributable in part to increased LOX products as opposed to NO or other eicosanoid products.
We examined the role of nitric oxide and eicosanoid products in mediating enhanced reactivity of PAs contralateral to a localized aorto-pulmonary shunt 48 hours after creation. Lipoxygense inhibitors eliminated hyperreactivity of unshunted PAs and these arteries produced more 12-HETE, supporting increased synthesis of proconstrictive lipoxygenase products in these vessels.
Lipoxygenase; pulmonary vascular resistance; arteriovenous shunt
Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog(214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog(214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog(214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog(214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.
8,9-EET; PASMCs; Rho-kinase; apoptosis; 8,9-EET analog
The goal of these studies was to characterize the infiltrating inflammatory cells during pneumonitis caused by moderate doses of radiation. Two groups of male rats (WAG/RijCmcr, 8 weeks old) were treated with single 10- or 15-Gy doses of thoracic X radiation; a third group of age-matched animals served as controls. Only 25% rats survived the 15-Gy dose. Bronchoalveolar lavage fluid and whole lung mounts were subjected to cytological and histological evaluation after 8 weeks for distribution of resident macrophages, neutrophils, lymphocytes and mast cells. There was a modest increase in airway and airspace-associated neutrophils in lungs from rats receiving 15 Gy. Mast cells (detected by immunohistochemistry for tryptase) increased over 70% with 10 Gy and over 13-fold after 15 Gy, with considerable leakage of tryptase into blood vessels and airways. Circulating levels of eight inflammatory cytokines were not altered after 10 Gy but appeared to decrease after 15 Gy. In summary, there were only modest increases in cellular inflammatory infiltrate during pneumonitis after a non-lethal dose of 10 Gy, but there was a dramatic rise in mast cell infiltration after 15 Gy, suggesting that circulating levels of mast cell products may be useful markers of severe pneumonitis.
To find mitigators of pneumonitis induced by moderate doses of thoracic radiation (10–15 Gy).
Materials and Methods
Unanesthetized WAG/RijCmcr female rats received single doses of X-irradiation (10, 12 or 15 Gy at 1.615 Gy/minute) to the thorax. Captopril (an angiotensin converting enzyme inhibitor) or losartan (an angiotensin receptor blocker) was administered in drinking water after irradiation. Pulmonary structure and function were assessed after 8 weeks in randomly selected rats by evaluating breathing rate, ex vivo vascular reactivity and histopathology. Survival analysis was undertaken on all animals except those scheduled for sacrifice.
Survival following a dose of 10 Gy to the thorax was not different from unirradiated rats up to one year. Survival decreased to less than 50%, by 45 weeks after 12 Gy and by 8–9 weeks after 15 Gy. Captopril (17–56 mg/kg/day) improved survival and reduced radiation-induced increases in breathing rate, changes in vascular reactivity and histopathological evidence of injury. Radiation-induced increases in breathing rate were prevented even if captopril was started 1 week following irradiation or if it was discontinued after 5 weeks. Losartan, though effective in reducing mortality was not as efficacious as captopril in mitigating radiation-induced increases in breathing rate or altered vasoreactivity.
In rats, a moderate thoracic dose of radiation induced pneumonitis and morbidity. These injuries were mitigated by captopril even when it was commenced 1 week after irradiation or if discontinued after 5 weeks following exposure. Losartan was less effective in protecting against radiation-induced changes in vascular reactivity or tachypnea.
Radiation injury; mitigation; lung function; angiotensin converting enzyme (ACE) inhibitors
The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or L-stereoisomer (L-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.
mitogen-activated protein kinase; extracellular signal-regulated kinase; mitogen-activated protein kinase phosphatase; dominant negative mitogen-activated protein kinase-extracellular signal-regulated kinase kinase; PD 184352
Epoxyeicosatrienoic acid(s) (EETs) have been shown to protect cardiovascular tissue against apoptosis dependent on activation of targets such as ATP-sensitive K+ (KATP) channels (sarcolemmal and mitochondrial), calcium-activated K+ channels, extracellular signal-regulated kinase or phosphoinositide 3-kinase (PI3K). We tested if EETs protect human atrial tissue ex vivo from hypoxia/reoxygenation (H/R) injury, and compared our results with myocardium from two rodent species, rats and mice. EETs reduced myocardial caspase 3 activity in all three species and protected against loss of mitochondrial membrane potential in primary cultures of neonatal rat ventricular myocytes submitted to H/R. In addition, EETs protected mouse pulmonary arteries ex vivo exposed to H/R. Myocardium and pulmonary arteries from genetically engineered mice having elevated plasma levels of EETs (Ephx2−/−) exhibited protection from H/R-induced injury over that of wild type controls, suggesting that endogenously produced EETs may have pro-survival effects. Electrophysiological studies in myocytes demonstrated that EETs can stimulate KATP currents in the absence of PI3K. Similarly, activation of PI3K/Akt occurred in the presence of the KATP channel blocker glibenclamide. Based upon loss of EETs protection in the presence of either wortmannin (a PI3K inhibitor) or glibenclamide, simultaneous activation of at least 2 pathways, PI3K and KATP channels respectively, appears to be required for protection. In conclusion, we demonstrate that exogenous and endogenous EETs have powerful pro-survival effects in cardiovascular tissues including diseased human myocardium, mediated by activation of not only one but at least two pathways, PI3K and KATP channels.
human myocardium; pulmonary artery; hypoxia/reoxygenation; mitochondrial membrane potential; epoxide hydrolase; lactate dehydrogenase release
Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.
To characterize structural and functional injuries following a single dose of whole-thorax irradiation that might be survivable after a nuclear attack/accident.
Rats were exposed to 5 or 10 Gy of X-rays to the whole thorax with other organs shielded. Non-invasive measurements of breathing rate and arterial oxygen saturation, and invasive evaluations of bronchoalveolar lavage fluid, (for total protein, Clara cell secretory protein), vascular reactivity and histology were conducted for at least 6 time points up to 52 wks after irradiation.
Irradiation with 10 Gy resulted in increased breathing rate, a reduction in oxygen saturation, an increase in bronchoalveolar lavage fluid protein and attenuation of vascular reactivity between 4–12 wks after irradiation. These changes were not observed with the lower dose of 5 Gy. Histological examination revealed perivascular edema at 4–8 wks after exposure to both doses, and mild fibrosis beyond 20 wks after 10 Gy.
Single-dose exposure of rat thorax to 10 but not 5 Gy X-irradiation resulted in a decrease in oxygen uptake and vasoreactivity and an increase in respiratory rate, which paralleled early pulmonary vascular pathology. Vascular edema resolved and was replaced by mild fibrosis beyond 20 wks after exposure, while lung function recovered.
Radiation injury; non invasive assays; lung injury markers; vascular reactivity
Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47phox and tyrosine phosphorylation of p47phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.
Superoxide; RAC1/2; Hydrogen Peroxide; Tempol; CYP4A; ROS
Epoxyeicosatrienoic acids (EETs) reduce infarction of the myocardium after ischemia/reperfusion injury to rodent and dog hearts mainly by opening sarcolemmal and mitochondrial potassium channels. Other mediators for the action of EET have been proposed though no definitive pathway or mechanism has yet been reported. Using cultured cells from two rodent species, immortalized myocytes from a mouse atrial lineage (HL-1) and primary myocytes derived from neonatal rat hearts, we observed that pretreatment with EETs (1 μM of 14,15-, 11,12-, or 8,9-EET) attenuates apoptosis after exposure to hypoxia/reoxygenation (HR). EETs also preserved functional beating of neonatal myocytes in culture after exposure to HR. We demonstrated that EETs increased activity of the pro-survival enzyme phosphoinositide 3 kinase (PI3K). In fact cardiomyocytes pretreated with EET and exposed to HR exhibited anti-apoptotic changes in at least five downstream effectors of PI3K: Akt, BAD (Bcl-xL/Bcl-2 associated death promoter), caspase 9 and 3 activities and expression of XIAP (X-linked inhibitor of apoptosis), when compared to vehicle-treated controls. The PI3K/Akt pathway is one of the strongest intracellular pro-survival signaling systems. Our studies show that EETs regulate multiple molecular effectors of this pathway. Understanding the targets of action of EET-mediated protection will promote development of these fatty acids as therapeutic agents against cardiac ischemia/reperfusion.
caspase; ischemia; rat; mouse; fatty acids
Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.
Objectives: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor–deficient mice.
Methods: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.
Measurements and Main Results: Estrogen receptor-α knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.
Conclusions: These data suggest that estrogen receptor-α is a critical regulator of airway hyperresponsiveness in mice.
lung function; asthma; hyperreactivity; M2 muscarinic receptor; estrogen receptor
Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.
To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor–deficient mice.
Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.
Measurements and Main Results
Estrogen receptor-α knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.
These data suggest that estrogen receptor-α is a critical regulator of airway hyperresponsiveness in mice.
lung function; asthma; hyperreactivity; M2 muscarinic receptor; estrogen receptor