Lijie Zhen

Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase–deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema

Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress–dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke–induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress–dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-κB (NF-κB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-κB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke–induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke–induced lung injury.

Cigarette smoke-induced emphysema in A/J mice is associated with pulmonary oxidative stress, apoptosis of lung cells, and global alterations in gene expression

Cigarette smoking is the major risk factor for developing chronic obstructive pulmonary disease, the fourth leading cause of deaths in the United States. Despite recent advances, the molecular mechanisms involved in the initiation and progression of this disease remain elusive. We used Affymetrix Gene Chip arrays to determine the temporal alterations in global gene expression during the progression of pulmonary emphysema in A/J mice. Chronic cigarette smoke (CS) exposure caused pulmonary emphysema in A/J mice, which was associated with pronounced bronchoalveolar inflammation, enhanced oxidative stress, and increased apoptosis of alveolar septal cells. Microarray analysis revealed the upregulation of 1,190, 715, 260, and 246 genes and the downregulation of 1,840, 730, 442, and 236 genes in the lungs of mice exposed to CS for 5 h, 8 days, and 1.5 and 6 mo, respectively. Most of the genes belong to the functional categories of phase I genes, Nrf2-regulated antioxidant and phase II genes, phase III detoxification genes, and others including immune/inflammatory response genes. Induction of the genes encoding multiple phase I enzymes was markedly higher in the emphysematous lungs, whereas reduced expression of various cytoprotective genes constituting ubiquitin-proteasome complex, cell survival pathways, solute carriers and transporters, transcription factors, and Nrf2-regulated antioxidant and phase II-responsive genes was noted. Our data indicate that the progression of CS-induced emphysema is associated with a steady decline in the expression of various genes involved in multiple pathways in the lungs of A/J mice. Many of the genes discovered in this study could rationally play an important role in the susceptibility to CS-induced emphysema.

Targeted induction of lung endothelial cell apoptosis causes emphysema-like changes in the mouse

Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

Role of the TGF-β/Alk5 Signaling Pathway in Monocrotaline-induced Pulmonary Hypertension

RATIONALE Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-beta receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-beta family signaling in PH and pulmonary vascular remodeling remains unclear. OBJECTIVES To determine whether inhibition of TGF-beta signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). METHODS We have used an orally active small-molecule TGF-beta receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. MEASUREMENTS AND MAIN RESULTS The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-beta signaling as documented by psmad2 expression. Inhibition of TGF-beta signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-beta signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. CONCLUSIONS These findings provide evidence that increased TGF-beta signaling participates in the pathogenesis of experimental severe PH.

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer(12:0) or Cer(8:0); 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramides proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.