Joel Michalski

Reduced miR-146a Increases Prostaglandin E2 in Chronic Obstructive Pulmonary Disease Fibroblasts

RATIONALE Persistent inflammation plays a major role in chronic obstructive pulmonary disease (COPD) pathogenesis, but its mechanisms are incompletely defined. Overproduction of the inflammatory mediator prostaglandin (PG) E₂ by COPD fibroblasts contributes to reduced repair function. OBJECTIVES The present study determined if fibroblasts from subjects with COPD overproduce PGE₂ after stimulation with the inflammatory cytokines IL-1β and tumor necrosis factor-α, and further defined the mechanism for overproduction. METHODS Fibroblasts were isolated from parenchymal tissue obtained from smokers with and without COPD undergoing lung surgery. PGE₂, cyclooxygenases (COX), and miR-146a in these cells were evaluated by in vitro studies. MEASUREMENTS AND MAIN RESULTS After stimulation with inflammatory cytokines, COPD fibroblasts produced 2.7-fold more PGE₂ compared with controls with similar smoking history. The increase in PGE₂ depended on induction of COX-2, which increased to a greater degree in fibroblasts from subjects with COPD. Cytokines also induced microRNA miR-146a expression in both fibroblasts, but significantly less in COPD fibroblasts. miR-146a caused degradation of COX-2 mRNA; reduced expression prolonged COX-2 mRNA half-life in fibroblasts from subjects with COPD. Cytokine-stimulated PGE₂ production and miR-146a expression in cultured fibroblasts correlated with clinical severity assessed by expiratory airflow and diffusion capacity. CONCLUSIONS miR-146a seems to play a pathogenetic role in the abnormal inflammatory response in COPD. Increased half-life of inflammatory mRNAs is a mechanism of abnormal inflammation in this disease.

PDE4: A Novel Target in the Treatment of Chronic Obstructive Pulmonary Disease

Phosphodiesterases (PDEs) are important modulators of inflammation and wound healing. In this capacity, specific targeting of PDEs for the treatment of many diseases, including chronic obstructive pulmonary disease (COPD), has been investigated. Currently, treatment of COPD is suboptimal. PDE4 modulates the inflammatory response of the lung, and inhibition of PDE4 may be a novel, COPD‐specific approach toward more effective treatment strategies. This review describes the state of PDE4‐inhibitor therapy for use in COPD treatment.

MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis.

MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-beta1 plus cytomix (a mixture of IL-1beta, IFN-gamma and TNF-alpha). TGF-beta1 plus cytomix (TCM) induced apoptosis in HBECs (3.4+/-0.6% of control vs 83.1+/-4.0% of TCM treated cells, p<0.01), and this was significantly blocked by the miRNA-146a mimic (8.8+/-1.5%, p<0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.

Prostaglandin E2 Inhibits Human Lung Fibroblast Chemotaxis through Disparate Actions on Different E-Prostanoid Receptors

The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbeccos modified Eagles medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.

Differential roles of JNK, ERK1/2, and p38 mitogen-activated protein kinases on endothelial cell tissue repair functions in response to tumor necrosis factor-α

Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µm) or ERK1/2 (PD98059, 5 µm) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µm) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.

Fibroblasts that resist cigarette smoke-induced senescence acquire profibrotic phenotypes

This study assessed the effect of extended exposure to cigarette smoke extract (CSE) on tissue repair functions in lung fibroblasts. Human fetal (HFL-1) and adult lung fibroblasts were exposed to CSE for 14 days. Senescence-associated β-galactosidase (SA β-gal) expression, cell proliferation, and tissue repair functions including chemotaxis and gel contraction were assessed. HFL-1 proliferation was inhibited by CSE and nearly half of the CSE-exposed cells were SA β-gal positive after 14 days exposure, whereas 33% of adult lung fibroblasts were SA β-gal positive in response to 10% CSE exposure. The SA β-gal-positive cells did not proliferate as indicated by bromodeoxyuridine incorporation. In contrast, cells negative for SA β-gal after CSE exposure proliferated faster than cells never exposed to CSE. These nonsenescent cells migrated more and contracted collagen gels more than control cells. CSE exposure stimulated TGF-β1 production, and both inhibition of TGF-β receptor kinase and TGF-β1 siRNA blocked CSE modulation of fibroblast function. Extended exposure to CSE might induce two different fibroblast phenotypes, a senescent and a profibrotic phenotype. The fibroblasts that resist CSE-induced cellular senescence may contribute to the pathogenesis of idiopathic pulmonary fibrosis and could contribute to fibrotic lesions in chronic obstructive pulmonary disease acting through a TGF-β1-mediated pathway. In contrast, the senescent cells may contribute to the pathogenesis of emphysema.

Inflammatory cytokines regulate endothelial cell survival and tissue repair functions via NF-κB signaling

Inflammation contributes to the development of fibrotic and malignant diseases. We assessed the ability of inflammatory cytokines to modulate endothelial cell survival and functions related to tissue repair/remodeling. Treatment with interleukin (IL)-1β or tumor necrosis factor (TNF)-α (2 ng/mL) led to human pulmonary artery endothelial cells becoming spindle-shaped fibroblast-like cells. However, immunoblot and DNA microarray showed no change in most endothelial and mesenchymal markers. In the presence of IL-1β or TNF-α, cells were resistant to apoptosis induced by deprivation of serum and growth factor, and were more migratory. In addition, cells treated with IL-1β or TNF-α contracted collagen gels more robustly. In contrast, transforming growth factor-β1 did not induce these responses. RNA interference targeting nuclear factor (NF)-κB p65 blocked the effects of IL-1β or TNF-α on cell morphologic change, survival, migration, and collagen gel contraction. These results suggest that endothelial cells may contribute to tissue repair/remodeling via the NF-κB signaling in a milieu of airway inflammation.

Smad3 mediates cigarette smoke extract (CSE) induction of VEGF release by human fetal lung fibroblasts.

Cigarette smoke is the major cause of chronic obstructive pulmonary disease (COPD), yet pathogenic mechanisms are not fully understood. Vascular endothelial growth factor (VEGF) is one of the major regulators of endothelial cell survival and is believed to play a role in the pathogenesis of COPD. Fibroblasts are a significant source of VEGF in the lungs; however the effect of cigarette smoke exposure on VEGF release by fibroblasts is not fully understood. We hypothesized that cigarette smoke-induced disturbed VEGF release by human lung fibroblasts is a potential pathogenic mechanism that could contribute to COPD. Cigarette smoke extract (CSE) was prepared by modification of the methods of Carp and Janoff (American Review of Respiratory Disease, 1978). Human fetal lung fibroblasts (HFL-1) were exposed to different concentrations of CSE and for different durations. VEGF release into the media was measured using ELISA. TGF-β1 receptor (TβR1)/Smad3 as a potential pathway for CSE modulated VEGF release was also investigated using biochemical analyses and siRNA inhibition of Smad3 and siRNA and pharmacologic inhibition of TβR1. CSE induced VEGF release by HFL-1 in concentration and time dependent manner. This was confirmed in two additional types of primary human fetal lung fibroblasts. CSE induced Smad3 phosphorylation and nuclear translocation in HFL-1 cells. Silencing of Smad3 by siRNA not only eliminated the stimulatory effect of CSE on VEGF release but also inhibited baseline VEGF production. Suppression of TβR1 by the pharmacological inhibitor (SB431542) markedly reduced VEGF release by HFL-1 in response to CSE and this effect was confirmed by TβR1 siRNA. In contrast, nicotine inhibited VEGF release by HFL-1 in a dose and time dependent manner. Our findings indicate that CSE stimulates Smad3-mediated VEGF release by lung fibroblasts. Nicotine does not account for the CSE stimulation of VEGF in HFL-1. The ability of lung fibroblasts to produce VEGF may play a role in pathogenesis of cigarette smoke induced lung disease.

Phosphodiesterase-4 inhibition augments human lung fibroblast vascular endothelial growth factor production induced by prostaglandin E2.

Lung fibroblasts are believed to be a major source of vascular endothelial growth factor (VEGF), which supports the survival of lung endothelial cells and modulates the maintenance of the pulmonary microvasculature. VEGF has been related to the pathogenesis of lung diseases, including chronic obstructive pulmonary disease (COPD). Prostaglandin E2 (PGE2) stimulates VEGF production from lung fibroblasts via the E-prostanoid (EP)-2 receptor. The EP2 signaling pathway uses cyclic adenosine monophosphate (cAMP) as a second messenger, and cAMP is degraded by phosphodiesterases (PDEs). This study investigates whether phosphodiesterase inhibition modulates the human lung fibroblast VEGF production induced by PGE2. Human fetal lung fibroblasts were cultured with PGE2 and PDE inhibitors. The PDE4 inhibitors roflumilast, roflumilast N-oxide, and rolipram with PGE2 increased VEGF release, as quantified in supernatant media by ELISA. In contrast, PDE3, PDE5, and PDE7 inhibitors did not affect VEGF release. Roflumilast increased VEGF release with either an EP2 or an EP4 agonist. Roflumilast augmented the cytosolic cAMP levels induced by PGE2 and VEGF release with other agents that use the cAMP signaling pathway. Roflumilast-augmented VEGF release was completely inhibited by a protein kinase A (PKA) inhibitor. Roflumilast with PGE2 increased VEGF mRNA levels, and the blockade of mRNA synthesis inhibited the augmented VEGF release. The stimulatory effect of roflumilast on VEGF release was replicated using primary healthy and COPD lung fibroblasts. These findings demonstrate that PDE4 inhibition can modulate human lung fibroblast VEGF release by PGE2 acting through the EP2 and EP4 receptor-cAMP/PKA signaling pathway. Through this action, PDE4 inhibitors such as roflumilast could contribute to the survival of lung endothelial cells.