Antagonizing cholecystokinin A receptor in the lung attenuates obesity-induced airway hyperresponsiveness.

Obesity increases asthma prevalence and severity. However, the underlying mechanisms are poorly understood, and consequently, therapeutic options for asthma patients with obesity remain limited. Here we report that cholecystokinin-a metabolic hormone best known for its role in signaling satiation and fat metabolism-is increased in the lungs of obese mice and that pharmacological blockade of cholecystokinin A receptor signaling reduces obesity-associated airway hyperresponsiveness. Activation of cholecystokinin A receptor by the hormone induces contraction of airway smooth muscle cells. In vivo, cholecystokinin level is elevated in the lungs of both genetically and diet-induced obese mice. Importantly, intranasal administration of cholecystokinin A receptor antagonists (proglumide and devazepide) suppresses the airway hyperresponsiveness in the obese mice. Together, our results reveal an unexpected role for cholecystokinin in the lung and support the repurposing of cholecystokinin A receptor antagonists as a potential therapy for asthma patients with obesity.

Captopril reduces lung inflammation and accelerated senescence in response to thoracic radiation in mice.

The lung is sensitive to radiation and exhibits several phases of injury, with an initial phase of radiation-induced pneumonitis followed by delayed and irreversible fibrosis. The angiotensin-converting enzyme inhibitor captopril has been demonstrated to mitigate radiation lung injury and to improve survival in animal models of thoracic irradiation, but the mechanism remains poorly understood. Here we investigated the effect of captopril on early inflammatory events in the lung in female CBA/J mice exposed to thoracic X-ray irradiation of 17-17.9 Gy (0.5-0.745 Gy min-1). For whole-body + thoracic irradiation, mice were exposed to 7.5 Gy (0.6 Gy min-1) total-body 60Co irradiation and 9.5 Gy thoracic irradiation. Captopril was administered orally (110 mg kg-1 day-1) in the drinking water, initiated 4 h through to150 days post-irradiation. Captopril treatment increased survival from thoracic irradiation to 75% at 150 days compared with 0% survival in vehicle-treated animals. Survival was characterized by a significant decrease in radiation-induced pneumonitis and fibrosis. Investigation of early inflammatory events showed that captopril significantly attenuated macrophage accumulation and decreased the synthesis of radiation-induced interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokines in the lungs of irradiated mice. Suppression of IL-1ß and TNF-α correlated with an increase of the anti-inflammatory cytokine IL-10 in the spleen with captopril treatment. We also found that captopril decreased markers for radiation-induced accelerated senescence in the lung tissue. Our data suggest that suppression of inflammation and senescence markers, combined with an increase of anti-inflammatory factors, are a part of the mechanism for captopril-induced survival in thoracic irradiated mice.

Accelerated senescence in skin in a murine model of radiation-induced multi-organ injury.

Accidental high-dose radiation exposures can lead to multi-organ injuries, including radiation dermatitis. The types of cellular damage leading to radiation dermatitis are not completely understood. To identify the cellular mechanisms that underlie radiation-induced skin injury in vivo, we evaluated the time-course of cellular effects of radiation (14, 16 or 17 Gy X-rays; 0.5 Gy/min) in the skin of C57BL/6 mice. Irradiation of 14 Gy induced mild inflammation, observed histologically, but no visible hair loss or erythema. However, 16 or 17 Gy radiation induced dry desquamation, erythema and mild ulceration, detectable within 14 days post-irradiation. Histological evaluation revealed inflammation with mast cell infiltration within 14 days. Fibrosis occurred 80 days following 17 Gy irradiation, with collagen deposition, admixed with neutrophilic dermatitis, and necrotic debris. We found that in cultures of normal human keratinocytes, exposure to 17.9 Gy irradiation caused the upregulation of p21/waf1, a marker of senescence. Using western blot analysis of 17.9 Gy-irradiated mice skin samples, we also detected a marker of accelerated senescence (p21/waf1) 7 days post-irradiation, and a marker of cellular apoptosis (activated caspase-3) at 30 days, both preceding histological evidence of inflammatory infiltrates. Immunohistochemistry revealed reduced epithelial stem cells from hair follicles 14-30 days post-irradiation. Furthermore, p21/waf1 expression was increased in the region of the hair follicle stem cells at 14 days post 17 Gy irradiation. These data indicate that radiation induces accelerated cellular senescence in the region of the stem cell population of the skin.

Inhibition of IGF-1R prevents ionizing radiation-induced primary endothelial cell senescence.

Accelerated senescence is a primary response to cellular stresses including DNA damaging agents (e.g., ionizing radiation) and is widely believed to be caused by continuous proliferative signaling in the presence of cell cycle arrest. Studies of signal transduction pathways leading to accelerated senescence have revealed that inhibition of mammalian target of rapamycin (mTOR) by rapamycin rescues cells from senescence. However, the molecular mechanisms upstream of mTOR following ionizing radiation (IR) are not well defined. We investigated signal transduction leading to IR-induced accelerated senescence in human pulmonary artery endothelial cells (HPAEC). Exposure of HPAEC to X-rays (10 Gy, 2.4 Gy/min) upregulated senescence markers including p53, p21/waf1, and senescence-associated beta galactosidase (SA-ß-gal). Ly294002 (a phosphatidylinositol-3-kinase [PI3K] inhibitor) or rapamycin (an mTOR inhibitor) blocked the induction of cellular senescence markers suggesting roles for PI3K and mTOR. Pathway-directed microarrays revealed increased transcription of insulin-like growth factor I (IGF-1), a modulator of cell growth and proliferation upstream of mTOR. qRT-PCR confirmed that both IGF-1 and IGF-2 mRNA were increased in response to X-rays, and ELISA showed increased secretion of IGF-1 protein into the medium of irradiated HPAEC. Consistent with upregulation of these ligands, we found that X-ray exposure led to hyperphosphorylation of IGF-1R, the receptor for IGF-1 and -2. Treatment with AG1024, an IGF-1R inhibitor, suppressed IR-induced upregulation of p53, p21/waf1, and SA-ß-gal. Together these findings suggest that IGF-1R is a key regulator of IR-induced accelerated senescence in a pathway that requires intact mTOR activity upstream of both p53 and p21/waf1.

Mechanisms of radiation toxicity in transformed and non-transformed cells.

Radiation damage to biological systems is determined by the type of radiation, the total dosage of exposure, the dose rate, and the region of the body exposed. Three modes of cell death-necrosis, apoptosis, and autophagy-as well as accelerated senescence have been demonstrated to occur in vitro and in vivo in response to radiation in cancer cells as well as in normal cells. The basis for cellular selection for each mode depends on various factors including the specific cell type involved, the dose of radiation absorbed by the cell, and whether it is proliferating and/or transformed. Here we review the signaling mechanisms activated by radiation for the induction of toxicity in transformed and normal cells. Understanding the molecular mechanisms of radiation toxicity is critical for the development of radiation countermeasures as well as for the improvement of clinical radiation in cancer treatment.

X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells.

PURPOSE: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells. METHODS: We evaluated necrosis, apoptosis, cellular senescence, and the contribution of endoplasmic reticulum (ER) stress in pulmonary artery endothelial cells (PAEC) irradiated with X-rays (2-50 Gray [Gy]). RESULTS: Clonogenic assays showed that 10 Gy induced ∼99.9% loss of cell viability. No necrosis was detected using lactate dehydrogenase assays, but a low population underwent extrinsic and intrinsic apoptosis, as indicated by the activation of caspases 3, 8, and 9 as well as by neutral comet assay. A majority of PAEC underwent accelerated senescence, as indicated by morphological changes, increased 21 kD cyclin-dependent kinase inhibitor (p21/waf1), decreased sirtuin 1 (SIRT1), and elevated senescence-associated ß-galactosidase (SA-ß-gal). ER stress was detected by assays for glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34) mRNA, and transient phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The ER stress inhibitor salubrinal blocked ∼50% of apoptosis with no effect on senescence. CONCLUSIONS: X-rays primarily induced cellular senescence with limited levels of apoptosis in endothelial cells. ER stress contributed to apoptosis but not to senescence.

Hepatocyte growth factor in lung repair and pulmonary fibrosis.

Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myofibroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.