Regulation of JAM2 Expression in the Lungs of Streptozotocin-Induced Diabetic Mice and Human Pluripotent Stem Cell-Derived Alveolar Organoids
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biomedicines Article Regulation of JAM2 Expression in the Lungs of Streptozotocin-Induced Diabetic Mice and Human Pluripotent Stem Cell-Derived Alveolar Organoids 1, 1, 1 2 1 Roya Rasaei y, Eunbi Kim y, Ji-Young Kim , Sunghun Na , Jung-Hyun Kim , Jinbeom Heo 3 , Dong-Myung Shin 3 , Sun Shim Choi 4 and Seok-Ho Hong 1,* 1 Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 24341, Korea; [email protected] (R.R.); [email protected] (E.K.); [email protected] (J.-Y.K.); [email protected] (J.-H.K.) 2 Department of Obstetrics and Gynecology, School of Medicine, Kangwon National University, Chuncheon 24341, Korea; [email protected] 3 Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea; [email protected] (J.H.); [email protected] (D.-M.S.) 4 Division of Biomedical Convergence, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 24341, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-33-250-7819; Fax: +82-33-244-2367 These authors equally contributed to this work. y Received: 11 August 2020; Accepted: 9 September 2020; Published: 11 September 2020 Abstract: Hyperglycemia is a causative factor in the pathogenesis of respiratory diseases, known to induce fibrosis and inflammation in the lung. However, little attention has been paid to genes related to hyperglycemic-induced lung alterations and stem cell applications for therapeutic use. In this study, our microarray data revealed significantly increased levels of junctional adhesion molecule 2 (JAM2) in the high glucose (HG)-induced transcriptional profile in human perivascular cells (hPVCs). The elevated level of JAM2 in HG-treated hPVCs was transcriptionally and epigenetically reversible when HG treatment was removed. We further investigated the expression of JAM2 using in vivo and in vitro hyperglycemic models. Our results showed significant upregulation of JAM2 in the lungs of streptozotocin (STZ)-induced diabetic mice, which was greatly suppressed by the administration of conditioned medium obtained from human mesenchymal stem cell cultures. Furthermore, JAM2 was found to be significantly upregulated in human pluripotent stem cell-derived multicellular alveolar organoids by exposure to HG. Our results suggest that JAM2 may play an important role in STZ-induced lung alterations and could be a potential indicator for predicting the therapeutic effects of stem cells and drugs in diabetic lung complications. Keywords: hyperglycemia; JAM2; lung; hPSC; alveolar organoid 1. Introduction A chronic hyperglycemic condition has widespread adverse effects on various tissues, including the heart, kidney, retina, muscle, liver, and vasculature [1]. Hyperglycemia triggers the infiltration of immune cells in multiple organ tissues and the production of reactive oxygen species, which result in an increased proinflammatory process and oxidative stress [2,3]. The persistence of inflammatory states and oxidative stress lead to the inflexibility of vessels and vascular leakage and induce fibrotic changes by promoting epithelial to mesenchymal transition (EMT) in multiple tissues [4] The vascular dysfunction and EMT process play a central role in developing diabetic complications, suggesting that the functions and structure of the lung are also affected by diabetes mellitus (DM) because the Biomedicines 2020, 8, 346; doi:10.3390/biomedicines8090346 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 346 2 of 13 alveolar–capillary system is characterized by a great microvascular and epithelial reserve. However, little attention has been paid to pulmonary pathologies in the context of DM. Recent studies on streptozotocin (STZ)-induced diabetic animal models reported that DM induces fibrotic changes in the lung, which is mediated through TGF (Transforming growth factor)-β-activated EMT signaling pathways [5]. The antioxidant activity in the lungs of diabetic rats was significantly reduced, suggesting that STZ-induced oxidative stress plays a key role in the pathogenesis of diabetic lung tissue. In fact, the treatment of antioxidants such as N-acetylcystein and Expendin-4 in diabetic animals was found to contribute to the attenuation of STZ-induced lung damage [6,7]. In humans, the pulmonary function test suggested a reduction in the lung functions, including total lung capacity, forced vital capacity, diffusing capacity and respiratory muscle strength in type 1 and/or 2 DM patients [8]. These results link the functional abnormalities in the respiratory tract of DM patients with the development of diverse pulmonary diseases. Meta-analyses, retrospective and cohort studies have shown that DM patients have a higher risk of developing pulmonary diseases including lung cancer, tuberculosis, asthma, pulmonary fibrosis (PF) and chronic obstructive pulmonary disease (COPD) [9,10]. While all these findings indicate a positive correlation between DM and lung disorders, the underlying pathophysiological mechanisms for lung dysfunction in patients with DM and therapeutic strategies are not completely understood due to the lack of accessibility of human diabetic lung tissues and potential indicators of dysfunction in diabetic lung tissue. In the present study, we defined a set of genes with hyperglycemia-upregulated expression in human perivascular cells (hPVCs), as the primary target of the hyperglycemia-induced adverse effects, using DNA microarrays. Among the genes, junctional adhesion molecule 2 (JAM2) was found to be transcriptionally and epigenetically regulated by hyperglycemia. We further investigated the regulation of JAM2 expression using in vivo and in vitro hyperglycemic models and evaluated it as a potential indicator for predicting diabetic lung complications and therapeutic effects of multipotent stem cells. 2. Materials and Methods 2.1. hPVC Cultures and HG Treatment PVCs were isolated from human umbilical cords and cultured as previously described [11]. hPVCs were obtained from full-term deliveries by cesarean section with donors’ written consent (Institutional Review Board No. 2014-06-003-010; Kangwon National University Hospital). hPVCs were treated with normal glucose (NG, 5 mM) and high glucose (HG) (25 and 50 mM) for the indicated periods. 2.2. Cell Proliferation Assay The short- and long-term growth properties of hPVCs under hyperglycemic conditions were analyzed as previously described [12]. 2.3. hPVC Differentiation The multilineage differentiation potential of hPVCs cultured in NG and HG conditions was evaluated at passages 3 and 4 as previously described [13]. Briefly, for adipogenic differentiation, hPVCs (4 104 cells/well) were plated in 12-well tissue culture plates and cultured in adipogensis × differentiation medium (A10070-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. After 21 days, the cultures were stained with Oil Red O (CM-005; LifeLine Cell Technology, CA, USA) to visualize the intracellular accumulation of lipid vacuoles. For osteogenic differentiation, hPVCs (4 104 cells/well) were seeded in 12-well tissue culture plates and cultured in osteogenesis × differentiation medium (A100-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. On day 21, the cells were stained with Alizarin Red S (Lifeline Cell Technology) to visualize the mineralization in osteogenic cell cultures. Biomedicines 2020, 8, 346 3 of 13 2.4. Preparation of MSC-CM Conditioned medium (CM) from human mesenchymal stem cells (MSCs) was prepared as previously described [14]. Briefly, MSCs were plated on 100-mm dishes and cultured until they reached 80–90% confluence. The cells were incubated with serum-free α-MEM for 24 h at 37 ◦C in a humidified atmosphere with 5% CO2. The conditioned medium was harvested and passed through a 0.22-µm filter. The filtered medium was concentrated using an Amicon Ultra-15 Centrifugal Filter Units (3-kDa cutoff membrane, Millipore, Burlington, MA, USA) and stored at 80 C until use. − ◦ 2.5. Mice and CM Administration Male C57BL/6J mice were purchased from Dooyeol Biotech (Seoul, Korea) and housed in a specific pathogen-free facility. All animal studies were approved by the Institutional Animal Care and Use Commitment of Kangwon National University (KW-180809-2, approved on 9 August 2018). Mice (18–20 g, 8 weeks) were intraperitoneally injected with 50 mg/kg STZ (S0130, Sigma-Aldrich, Burlington, MA, USA) daily for 5 consecutive days to induce type 1 DM [2]. After 5 days, glucose levels were evaluated using Accu-CHECK Active (Roche Diagnostic Inc., Korea). Mice with a basal blood glucose level of more than 250 mg/dL were considered diabetic and included in experiments. After induction of diabetes, mice were intravenously administered with MSC-CM (40 µg/100 µL) for 6 weeks, whereas control animals received intravenous saline. 2.6. Morphological Analysis of the Mouse Lung Mouse lung tissue was stained with hematoxylin and eosin (H&E) to analyze the histological structure following the induction of hyperglycemia. Eight random fields were taken to evaluate alveolar wall thickness and alveolar size using digital imaging software (NIH Image J) as previously described [15]. 2.7. Immunofluorescence Staining For immunofluorescence staining, 4-µm-thick lung sections were dewaxed with xylene and rehydrated with a gradient of ethanol. The sections were subjected to antigen retrieval with a citrate buffer bath (pH 6) at boiling temperature and then blocked for endogenous peroxidase