Reactive Oxygen Species-Dependent Calpain Activation Contributes to Airway and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease.

Aims: Airway and pulmonary vascular remodeling is an important pathological feature in the pathogenesis of chronic obstructive pulmonary disease (COPD). Tobacco smoke (TS) induces the production of large amounts of reactive oxygen species (ROS) in COPD lungs. We investigated how ROS lead to airway and pulmonary vascular remodeling in COPD. Results: We used in vitro bronchial and pulmonary artery smooth muscle cells (BSMCs and PASMCs), in vivo TS-induced COPD rodent models, and lung tissues of COPD patients. We found that H2O2 and TS extract (TSE) induced calpain activation in BSMCs and PASMCs. Calpain activation was elevated in smooth muscle of bronchi and pulmonary arterioles in COPD patients and TS-induced COPD rodent models. Calpain inhibition attenuated H2O2- and TSE-induced collagen synthesis and proliferation of BSMCs and PASMCs. Exposure to TS causes increases in airway resistance, right ventricular systolic pressure (RVSP), and thickening of bronchi and pulmonary arteries. Calpain inhibition by smooth muscle-specific knockout of calpain and the calpain inhibitor MDL28170 attenuated increases in airway resistance, RVSP, and thickening of bronchi and pulmonary arteries. Moreover, smooth muscle-specific knockout of calpain did not reduce TS-induced emphysema in the mouse model, but MDL28170 did reduce TS-induced emphysema in the rat model. Innovation: This study provides the first evidence that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling in TS-induced COPD. Calpain might be a novel therapeutic target for the treatment of COPD. Conclusion: These results indicate that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling and pulmonary hypertension in COPD.

Functional impact of heterogeneous nuclear ribonucleoprotein A2/B1 in smooth muscle differentiation from stem cells and embryonic arteriogenesis.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play various roles in transcriptional and post-transcriptional modulation of gene expression. However, it remains unclear if hnRNPs are associated with smooth muscle cell (SMC) differentiation from stem cells and embryonic arteriogenesis. In this study, mouse embryonic stem (ES) cells were cultivated on collagen IV-coated plates and smooth muscle differentiation medium. We found that hnRNPA2/B1 gene and protein expression was significantly up-regulated following 3-7 days of cell differentiation. hnRNPA2/B1 knockdown resulted in down-regulation of specific smooth muscle markers and transcription factors, whereas enforced expression of hnRNPA2/B1 enhanced the expression of these genes. Moreover, we demonstrated by using luciferase and chromatin immunoprecipitation assays that hnRNPA2/B1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Smαa and Sm22α genes. We further demonstrated that chromobox protein homolog gene 3, a previously identified SMC differentiation regulatory nuclear protein, is required for hnRNPA2/B1-mediated SMC differentiation gene expression. Importantly, specifically designed Hnrnpa2/b1 morpholinos for in vivo knockdown could inhibit the migration and differentiation of neural crest cells into SMCs in chick embryos. This resulted in the maldevelopment of branchial arch arteries and increased embryo lethality at a later developmental stage. Our findings demonstrated that hnRNPA2/B1 plays a functional role in SMC differentiation from stem cells in vitro and embryonic branchial arch artery development. This indicates that hnRNPA2/B1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine.

Heterogeneity of bronchial endothelial cell permeability.

In vivo models of airway inflammation suggest that most protein transudation occurs from bronchial microcirculation. However, due to technical limitations in the isolation and culture of bronchial endothelial cells, most studies of lung vascular permeability have focused on pulmonary endothelium. Thus conditions for culture of sheep bronchial artery endothelial cells (BAEC) and bronchial microvascular endothelial cells (BMVEC) were established. The bronchial artery and the mainstem bronchi, stripped of epithelium, were dissected, and endothelial cells were isolated by enzymatic treatment. BAEC and BMVEC demonstrated positive staining for factor VIII-related antigen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein, and PECAM-1. Radioligand binding studies confirmed equivalent numbers of bradykinin B(2) receptors on BAEC and BMVEC. Permeability of BAEC and BMVEC was determined after treatment with bradykinin and thrombin by comparing the translocation of FITC-dextran (mol wt 9,500) across confluent monolayers (n = 10-12). Bradykinin caused a maximal increase in permeability in BAEC (165% increase) and BMVEC (144% increase) by 15 min compared with vehicle controls. Thrombin treatment altered BMVEC permeability only, reaching a maximal response at 60 min (109% increase). These results demonstrate bronchial endothelial cell heterogeneity and establish methods to determine intracellular mechanisms contributing to airway disease in relevant cell systems.

Membrane currents in canine bronchial artery and their regulation by excitatory agonists.

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.