Jeong-Ran Park

Cigarette smoke-mediated oxidative stress induces apoptosis via the MAPKs/STAT1 pathway in mouse lung fibroblasts.

Cigarette smoking is the major aetiologic factor in chronic obstructive pulmonary disease (COPD). Lung fibroblasts are key participants in the maintenance of the extracellular matrix within the lung parenchyma. However, it still remains unknown how pulmonary fibroblasts are affected by cigarette smoking. Therefore, in this study, we isolated lung fibroblasts from mice and determined the apoptotic mechanism in response to cigarette smoke extract (CSE). When the lung fibroblasts were exposed to CSE, the generation of ROS was increased as shown by H2-DCFDA staining and Flow Cytometry. By immunocytochemistry, Ki67 expressing cells gradually decreased in a dose-dependent manner. The nitrite concentration in the supernatants increased, while the SOD activity and GSH recycling decreased in response to CSE. CSE increased the mRNA levels of TNF-α and COX-2, and the secretory proteins TNF-α and IL-6 increased as measured by ELISA. We next determined whether this inflammatory process is associated with the Bax/Bcl-2 apoptosis pathway. The Bax/Bcl-2 mRNA ratio increased, and cleaved caspase-3 protein was activated in the lung fibroblasts treated with CSE. Moreover, CSE induced the phosphorylation of STAT1 at Tyr701/Ser727 and increased the activation of ERK1/2, p38, and JNK in the MAPK pathway. Taken together, these data suggest that CSE-mediated inflammation alters the redox regulation via the MAPK-STAT1 pathway, leading to intrinsic apoptosis of lung fibroblasts.

Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

The receptor for advanced glycan end products (RAGE) has been identified as a susceptibility gene for chronic obstructive pulmonary disease (COPD) in genome‐wide association studies (GWASs). However, less is known about how RAGE is involved in the pathogenesis of COPD. To determine the molecular mechanism by which RAGE influences COPD in experimental COPD models, we investigated the efficacy of the RAGE‐specific antagonist FPS‐ZM1 administration in in vivo and in vitro COPD models. We injected elastase intratracheally and the RAGE antagonist FPS‐ZM1 in mice, and the infiltrated inflammatory cells and cytokines were assessed by ELISA. Cellular expression of RAGE was determined in protein, serum, and bronchoalveolar lavage fluid of mice and lungs and serum of human donors and patients with COPD. Downstream damage‐associated molecular pattern (DAMP) pathway activation in vivo and in vitro and in patients with COPD was assessed by immunofluorescence staining, Western blot analysis, and ELISA. The expression of membrane RAGE in initiating the inflammatory response and of soluble RAGE acting as a decoy were associated with up‐regulation of the DAMP‐related signaling pathway via Nrf2. FPS‐ZM1 administration significantly reversed emphysema in the lung of mice. Moreover, FPS‐ZM1 treatment significantly reduced lung inflammation in Nrf2+/+, but not in Nrf2‒/‒ mice. Thus, our data indicate for the first time that RAGE inhibition has an essential protective role in COPD. Our observation of RAGE inhibition provided novel insight into its potential as a therapeutic target in emphysema/COPD.—Lee, H., Park, J.‐R., Kim, W. J., Sundar, I. K., Rahman, I., Park, S.‐M., Yang. S.‐R. Blockade of RAGE ameliorates elastase‐induced emphysema development and progression via RAGE‐DAMP signaling. FASEB J. 31, 2076–2089 (2017). www.fasebj.org

Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization

Recent studies have reported that stem cells can be isolated from a wide range of tissues including bone marrow, fatty tissue, adipose tissue and placenta. Moreover, several studies also suggest that skin dermis could serve as a source of stem cells, but are of unclear phenotype. Therefore, we isolated and investigated to determine the potential of stem cell within human skin dermis. We isolated cells from human dermis, termed here as human dermis-derived mesenchymal stem cells (hDMSCs) which is able to be isolated by using explants culture method. Our method has an advantage over the enzymatic method as it is easier, less expensive and less cell damage. hDMSCs were maintained in basal culture media and proliferation potential was measured. hDMSCs were highly proliferative and successfully expanded with no additional growth factor. In addition, hDMSCs revealed normal karyotype and expressed high levels of CD90, CD73 and CD105 while did not express the surface markers for CD34, CD45 and HLA-DR. Also, we confirmed that hDMSCs possess the capacity to differentiate into multiple lineage including adipocyte, osteocyte, chondrocyte and precursor of hepatocyte lineage. Considering these results, we suggest that hDMSCs might be a valuable source of stem cells and could potentially be a useful source of clinical application.

Γ-glutamyl transferase as an early and sensitive marker in ethanol-induced liver injury of rats.

γ-Glutamyl transferase (GGT) has been regarded as a biological marker of heavy alcohol consumption or hepatobiliary disease such as fatty liver. However, the role of GGT is unknown in the molecular pathway during alcohol-induced liver injury. To determine the role of GGT in alcohol-induced liver injury, Sprague-Dawley rats were administered 22% and 38% ethanol for 3 days as acute and 5 weeks as subchronic model. In serologic analysis, the level of GGT was significantly increased and the level of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were not changed at 3 days and 5 weeks. In histologic analysis, ethanol exposure induced granular deposit formation and sinusoidal dilation in the acute model for 3 days. In the subchronic model for 5 weeks, ethanol exposure further increased the granular deposit formation, sinusoidal congestion, and mild fatty liver change. To determine whether ethanol-exposed liver is associated with changes of antioxidants levels, we performed reverse-transcriptase polymerase chain reaction (RT-PCR) analysis on ethanol-exposed livers of rats. In RT-PCR analysis, the mRNA levels of GPX1 and SOD1 were significantly increased as well as up-regulation of CYP2E1. In the glutathione assay, the level of glutathione was significantly reduced in response to ethanol in rats. Therefore, in this study, ethanol increased the level of serum GGT but depleted the level of glutathione. Moreover, the CYP2E1 was rapidly reflected to ethanol in rats. Taken together, our findings suggest that the elevated GGT is associated with cellular antioxidant defense system, and the CYP2E1 can be used for early diagnosis in alcohol-related diseases.

Functional characteristics of mesenchymal stem cells derived from the adipose tissue of a patient with achondroplasia

Mesenchymal stem cells (MSCs) can be isolated from various tissues including bone marrow, adipose tissue, skin dermis, and umbilical Wharton’s jelly as well as injured tissues. MSCs possess the capacity for self-renewal and the potential for differentiation into adipogenic, osteogenic, and chondrogenic lineages. However, the characteristics of MSCs in injured tissues, such as achondroplasia (ACH), are not well known. In this study, we isolated MSCs from human subcutaneous adipose (ACH-SAMSCs) tissue and circumjacent human adipose tissue of the cartilage (ACH-CAMSCs) from a patient with ACH. We then analyzed the characterization of ACH-SAMSCs and ACH-CAMSCs, compared with normal human dermis-derived MSCs (hDMSCs). In flow cytometry analysis, the isolated ACH-MSCs expressed low levels of CD73, CD90, and CD105, compared with hDMSCs. Moreover, both ACH- SAMSCs and ACH-CAMSCs had constitutionally overactive fibroblast growth factor receptor 3 (FGFR3) and exhibited significantly reduced osteogenic differentiation, compared to enhanced adipogenic differentiation. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) was increased in ACH-MSCs. In addition, the efficacy of osteogenic differentiation was slightly restored in osteogenic differentiation medium with MAPKs inhibitors. These results suggest that they play essential roles in MSC differentiation toward adipogenesis in ACH pathology. In conclusion, the identification of the characteristics of ACH-MSCs and the favoring of adipogenic differentiation via the FGFR3/MAPK axis might help to elucidate the pathogenic mechanisms relevant to other skeletal diseases and could provide targets for therapeutic interventions.