Evaluation of inhaled carbon monoxide as an anti-inflammatory therapy in a nonhuman primate model of lung inflammation.

Carbon monoxide (CO) confers anti-inflammatory protection in rodent models of lung injury when applied at low concentration. Translation of these findings to clinical therapies for pulmonary inflammation requires validation in higher mammals. We have evaluated the efficacy of inhaled CO in reducing LPS-induced lung inflammation in cynomolgus macaques. LPS inhalation resulted in profound neutrophil influx and moderate increases in airway lymphocytes, which returned to baseline levels within 2 wk following exposure. CO exposure (500 ppm, 6 h) following LPS inhalation decreased TNF-α release in bronchoalveolar lavage fluid but did not affect IL-6 or IL-8 release. Lower concentrations of CO (250 ppm, 6 h) did not reduce pulmonary neutrophilia. Pretreatment with budesonide, a currently used inhaled corticosteroid, decreased LPS-induced expression of TNF-α, IL-6, and IL-8, and reduced LPS-induced neutrophilia by ∼84%. In comparison, CO inhalation (500 ppm, for 6 h after LPS exposure) reduced neutrophilia by ∼67%. Thus, inhaled CO was nearly as efficacious as pretreatment with an inhaled corticosteroid at reducing airway neutrophil influx in cynomolgus macaques. However, the therapeutic efficacy of CO required relatively high doses (500 ppm) that resulted in high carboxyhemoglobin (COHb) levels (>30%). Lower CO concentrations (250 ppm), associated with anti-inflammatory protection in rodents, were ineffective in cynomolgus macaques and also yielded relatively high COHb levels. These studies highlight the complexity of interspecies variation of dose-response relationships of CO to COHb levels and to the anti-inflammatory functions of CO. The findings of this study warrant further investigations for assessing the therapeutic application of CO in nonhuman primate models of tissue injury and in human diseases. The study also suggests that akin to many new therapies in human diseases, the translation of CO therapy to human disease will require additional extensive and rigorous proof-of-concept studies in humans in the future.

Circulating factors induce coronary endothelial cell activation following exposure to inhaled diesel exhaust and nitrogen dioxide in humans: evidence from a novel translational in vitro model.

The vascular toxicity of inhaled agents may be caused by soluble factors that are released into the systemic circulation. To confirm this in a straightforward manner, we obtained plasma from healthy human volunteers before and after exposure to diesel exhaust (DE) and nitrogen dioxide (NO(2)). Plasma samples were obtained from human volunteers exposed to 100 µg/m(3) DE or filtered air for 2 h. A second cohort was exposed to 500 ppb NO(2) or filtered air in an identical protocol. Primary human coronary artery endothelial cells (hCAECs) were grown to confluence and treated for 24 h with a 10 or 30% (in media) mixture of plasma obtained before, immediately post or 24 h postexposure to pollutant exposures. Messenger RNA (mRNA) was isolated from hCAECs following the incubation and probed for intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) expression. ICAM-1 mRNA expression was increased by plasma obtained at both timepoints following the NO(2) exposures. VCAM-1 was significantly elevated in cells treated with plasma obtained 24 h following diesel exposure and at both timepoints following NO(2) exposure. Interleukin-8 protein was elevated in the hCAEC supernatant when cells were incubated with plasma from NO(2) exposures. These data indicate that proinflammatory circulating factors are elevated acutely following exposure to both DE and a primary component thereof, NO(2). These functional translational assays offer novel approaches to assessing the cardiovascular risk associated with air pollution exposure.

Cardiac and vascular atrogin-1 mRNA expression is not associated with dexamethasone efficacy in the monocrotaline model of pulmonary hypertension.

Atrophic signaling elements of the ubiquitin-proteasome system (UPS) are involved in skeletal muscle wasting as well as pressure overload models of heart failure. In our prior experiments, we demonstrated a transcriptional downregulation of atrophy-inducing vascular E3 ubiquitin ligases in a toxic model of pulmonary hypertension where pulmonary artery and right ventricle (RV) hypertrophy are evident. Given the numerous reports of glucocorticoid activation of the UPS and the negative regulator of muscle mass, myostatin, we investigated the efficacy of dexamethasone to reverse monocrotaline (MCT)-induced pulmonary hypertension and augment atrogin-1 expression in both pulmonary arteries and myocardium. Dexamethasone caused significant reductions in body weight in combination with MCT. As predicted, MCT-induced pulmonary hypertension was evident by increases in RV systolic pressure, right ventricle to left ventricle plus septal weight ratios (RV/LVS) and arterial remodeling. MCT treatment significantly reduced both RV and PA atrogin-1 expression. Dexamethasone treatment reversed the MCT-induced pathological indices and restored RV atrogin-1 expression, but did not impact atrogin-1 expression in pulmonary arteries. Myostatin was poorly expressed in pulmonary arteries compared to the RV, and dexamethasone treatment increase RV myostatin in controls but not MCT-treated rats. These findings suggest that mechanisms independent of myostatin/atrogin-1 are responsible for glucocorticoid efficacy in this model of pulmonary hypertension.

Combination therapy with vidaza and entinostat suppresses tumor growth and reprograms the epigenome in an orthotopic lung cancer model.

Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of proapoptotic genes and the cyclin-dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the reexpression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings show the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs.