Yun-Song Lee

Bone marrow cells repair cigarette smoke-induced emphysema in rats

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.

Rosiglitazone attenuates hypoxia-induced pulmonary arterial hypertension in rats.

Background and objective:  Expression of peroxisome proliferator‐activated receptor gamma (PPARγ) is decreased in the lungs of patients with pulmonary hypertension, and PPARγ ligands have been associated with the release of vasoactive substances from vascular endothelial cells and prevention of vascular remodelling. We hypothesized that PPARγ may play a critical role in the development of pulmonary hypertension induced by chronic hypoxia.

Cigarette Smoke Induces Akt Protein Degradation by the Ubiquitin-Proteasome System

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.

Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract.

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IκB, and nuclear AP-1 or NF-κB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IκB-NF-κB are involved.

Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media.

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-α, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-γ. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-γ. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Green Tea Extract Inhibits Paraquat-Induced Pulmonary Fibrosis by Suppression of Oxidative Stress and Endothelin-l Expression

Paraquat-induced pulmonary fibrosis involves two factors, direct injury by oxygen free radicals and indirect injury by inflammatory cells and fibroblasts. Endothelin-1 (ET-1) has been shown to act as a mediator of pulmonary fibrosis, and its formation increases during oxidative stress. We investigated whether green tea extract (GTE), which has antioxidant properties, inhibits paraquat-induced pulmonary fibrosis and whether ET-1 is involved in this process. Paraquat (0.3 mg/kg) was instilled into the right lungs of rats, following which the rats were either not further treated (Group P, n = 7), or they were administered 1% GTE mixed with feed (Group PG; n = 7) or the ETA receptor antagonist ZD2574 (10 mg/kg through gavage; Group PZ; n = 7) for two weeks. As control, we used rats instilled with saline (Group N; n = 6). Two weeks after paraquat instillation, we assayed the degree of pulmonary fibrosis by light microscopic morphometry and hydroxyproline content; lipid peroxidation as a marker of oxidative stresses by measurement of malondialdehyde (MDA); ET-1 by immunohistochemistry; and prepro-ET-1 mRNA expression by reverse transcription-polymerase chain reaction. Compared with Group N, significant pulmonary fibrosis was observed in Group P, accompanied by increases in MDA, ET-1, and prepro-ET-1 mRNA expression. Compared with Group P, Group PG showed significant decreases in pulmonary fibrosis, along with decreases in MDA, ET-1, and prepro-ET-1 mRNA expression. We also observed significant decreases in pulmonary fibrosis in Group PZ compared with Group P. These findings suggest that GTE inhibits paraquat-induced pulmonary fibrosis by suppression of oxidative stress and ET-1 expression.

SP600125, a selective JNK inhibitor, aggravates hepatic ischemia-reperfusion injury.

c-Jun N-terminal kinase (JNK) is activated during hepatic reperfusion, and JNK inhibitors are known to protect other major organs from ischemia-reperfusion (I/R) injury. We attempted to determine the effect of SP600125, a JNK inhibitor, on hepatic I/R injury using a partial ischemia model in mice. Compared to a vehicle-treated group, the SP600125-treated group showed a greater increase in serum ALT levels 24 h after reperfusion with more severe parenchymal destruction and leukocyte infiltration. Similarly, tissue myeloperoxidase and malondialdehyde levels were higher in the SP600125-treated group, and chemokine expression was also higher in the SP600125-treated group. These data, which are contradictory to previous results, indicate that JNK inhibition by SP600125 may be harmful in hepatic I/R injury. Therefore, care must be taken when investigating the therapeutic use of JNK inhibitors in hepatic I/R injury, especially in the context of the effects of JNK inhibition on inflammatory infiltration.

Bortezomib alleviates experimental pulmonary arterial hypertension.

Vascular remodeling and endothelial dysfunction are important pathogenic features of pulmonary arterial hypertension (PAH). There is a growing body of evidence that proteasome inhibitors may be beneficial in vascular diseases by inhibiting proliferation of vascular smooth muscle cells (VSMCs) and ameliorating endothelial dysfunction. Here, we evaluated whether bortezomib (BTZ) could alleviate hypoxia- and monocrotaline (MCT)-induced PAH. BTZ (at doses from 1 to 100 μg/kg, or a dose of 100 μg/kg) was administered to mice every other day for the last 2 weeks of a 5-week hypoxia (10% O(2)) period, or to rats once daily from Day 22 to Day 34 after MCT challenge, respectively. BTZ treatment substantially suppressed elevation of right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling in hypoxia-exposed mice. Similarly, BTZ treatment inhibited RV hypertrophy and vascular remodeling in MCT-injected rats. Strikingly, BTZ rescued 70% of MCT-injected rats up to Day 60, along with a considerable reduction in RV systolic pressure and suppression of vascular remodeling, whereas, among MCT-injected rats not administered BTZ, there were no survivors by Day 41. BTZ significantly suppressed proliferation of pulmonary VSMCs in vivo and in vitro. Furthermore, BTZ increased not only endothelial nitric oxide (NO) synthase (eNOS), phosphorylated eNOS, and NO production in vitro, but also eNOS and p-eNOS in hypoxia-exposed mice and MCT-injected rats, respectively. In contrast to the beneficial effects, BTZ increased active caspase-3 in cardiac ventricles of MCT-injected rats. Taken together, with caution for cardiotoxicity, BTZ could be a potential therapeutic strategy in PAH, possibly acting by inhibition of VSMC proliferation and amelioration of endothelial dysfunction.

Eupatilin Blocks Mediator Release Via Tyrosine Kinase Inhibition in Activated Guinea Pig Lung Mast Cells

Eupatilin, an extract from Artemisia asiatica Nakai, is known to exert anti-gastric ulcer, anticancer, and anti-inflammatory effects. The aim of this study was to elucidate whether eupatilin has antiallergic reactions in activated guinea pig lung mast cells compared to apigenin and genistein. Mast cells were purified from guinea pig lung tissues by using enzyme digestion and rough and discontinuous density Percoll gradient. The purified mast cells were sensitized with immunoglobulin (Ig) G1 (anti-OVA antibody) and challenged with ovalbumin (OVA). Histamine was assayed using an automated fluorometric analyzer, leukotrienes by radioimmunoassay, and tyrosine phosphorylation by immunoblotting. Intracellular Ca2+ was analyzed by confocal laser scanning microscopy, protein kinase C (PKC) activity using protein phosphorylated with [γ-32P]ATP, and phopholipase D activity (PLD) and phosphatidic acid by using labeled phosphatidyl alcohol. Eupatilin, apigenin, or genistein reduced histamine release and leukotriene synthesis in a does-dependent manner. Eupatilin inhibited mediators to a greater extent than apigenin or genistein. Eupatilin, apigenin, and genistein initially blocked phosphorylation of Syk tyrosine and Ca2+ influx, PLD activity, phosphatidic acid, and Ca2+-dependent PKC α/βII activities during mast cell activation in a dose-dependent manner. Our data suggest that eupatilin initially inhibits Syk kinase, and then blocks downstream multisignal pathways and Ca2+ influx during mast cell activation triggered by a specific antigen–antibody reaction. Thus, eupatilin may have use clinically as a treatment for inflammatory disorders associated with allergic diseases including asthma.Eupatilin, an extract from Artemisia asiatica Nakai, is known to exert anti-gastric ulcer, anticancer, and anti-inflammatory effects. The aim of this study was to elucidate whether eupatilin has antiallergic reactions in activated guinea pig lung mast cells compared to apigenin and genistein. Mast cells were purified from guinea pig lung tissues by using enzyme digestion and rough and discontinuous density Percoll gradient. The purified mast cells were sensitized with immunoglobulin (Ig) G(1) (anti-OVA antibody) and challenged with ovalbumin (OVA). Histamine was assayed using an automated fluorometric analyzer, leukotrienes by radioimmunoassay, and tyrosine phosphorylation by immunoblotting. Intracellular Ca(2+) was analyzed by confocal laser scanning microscopy, protein kinase C (PKC) activity using protein phosphorylated with [gamma-(32)P]ATP, and phopholipase D activity (PLD) and phosphatidic acid by using labeled phosphatidyl alcohol. Eupatilin, apigenin, or genistein reduced histamine release and leukotriene synthesis in a does-dependent manner. Eupatilin inhibited mediators to a greater extent than apigenin or genistein. Eupatilin, apigenin, and genistein initially blocked phosphorylation of Syk tyrosine and Ca(2+) influx, PLD activity, phosphatidic acid, and Ca(2+)-dependent PKC alpha/betaII activities during mast cell activation in a dose-dependent manner. Our data suggest that eupatilin initially inhibits Syk kinase, and then blocks downstream multisignal pathways and Ca(2+) influx during mast cell activation triggered by a specific antigen-antibody reaction. Thus, eupatilin may have use clinically as a treatment for inflammatory disorders associated with allergic diseases including asthma.