Diesel exhaust particle exposure exacerbates ciliary and epithelial barrier dysfunction in the multiciliated bronchial epithelium models.

Airway epithelium, the first defense barrier of the respiratory system, facilitates mucociliary clearance against inflammatory stimuli, such as pathogens and particulates inhaled into the airway and lung. Inhaled particulate matter 2.5 (PM2.5) can penetrate the alveolar region of the lung, and it can develop and exacerbate respiratory diseases. Although the pathophysiological effects of PM2.5 in the respiratory system are well known, its impact on mucociliary clearance of airway epithelium has yet to be clearly defined. In this study, we used two different 3D in vitro airway models, namely the EpiAirway-full-thickness (FT) model and a normal human bronchial epithelial cell (NHBE)-based air-liquid interface (ALI) system, to investigate the effect of diesel exhaust particles (DEPs) belonging to PM2.5 on mucociliary clearance. RNA-sequencing (RNA-Seq) analyses of EpiAirway-FT exposed to DEPs indicated that DEP-induced differentially expressed genes (DEGs) are related to ciliary and microtubule function and inflammatory-related pathways. The exposure to DEPs significantly decreased the number of ciliated cells and shortened ciliary length. It reduced the expression of cilium-related genes such as acetylated α-tubulin, ARL13B, DNAH5, and DNAL1 in the NHBEs cultured in the ALI system. Furthermore, DEPs significantly increased the expression of MUC5AC, whereas they decreased the expression of epithelial junction proteins, namely, ZO1, Occludin, and E-cadherin. Impairment of mucociliary clearance by DEPs significantly improved the release of epithelial-derived inflammatory and fibrotic mediators such as IL-1ß, IL-6, IL-8, GM-CSF, MMP-1, VEGF, and S100A9. Taken together, it can be speculated that DEPs can cause ciliary dysfunction, hyperplasia of goblet cells, and the disruption of the epithelial barrier, resulting in the hyperproduction of lung injury mediators. Our data strongly suggest that PM2.5 exposure is directly associated with ciliary and epithelial barrier dysfunction and may exacerbate lung injury.

Enhanced Osteocyte Differentiation: Cathepsin D and L Secretion by Human Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases.

The Role of Epicardial Adipose Tissue-Derived MicroRNAs in the Regulation of Cardiovascular Disease: A Narrative Review.

Cardiovascular diseases have been leading cause of death worldwide for many decades, and obesity has been acknowledged as a risk factor for cardiovascular diseases. In the present review, human epicardial adipose tissue-derived miRNAs reported to be differentially expressed under pathologic conditions are discussed and summarized. The results of the literature review indicate that some of the epicardial adipose tissue-derived miRNAs are believed to be cardioprotective, while some others show quite the opposite effects depending on the underlying pathologic conditions. Furthermore, they suggest that that the epicardial adipose tissue-derived miRNAs have great potential as both a diagnostic and therapeutic modality. Nevertheless, mainly due to highly limited availability of human samples, it is very difficult to make any generalized claims on a given miRNA in terms of its overall impact on the cardiovascular system. Therefore, further functional investigation of a given miRNA including, but not limited to, the study of its dose effect, off-target effects, and potential toxicity is required. We hope that this review can provide novel insights to transform our current knowledge on epicardial adipose tissue-derived miRNAs into clinically viable therapeutic strategies for preventing and treating cardiovascular diseases.

A Low Concentration of Citreoviridin Prevents Both Intracellular Calcium Deposition in Vascular Smooth Muscle Cell and Osteoclast Activation In Vitro.

Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.

Organ-on-a-chip: Its use in cardiovascular research.

Organ-on-a-chip (OOAC) has attracted great attention during the last decade as a revolutionary alternative to conventional animal models. This cutting-edge technology has also brought constructive changes to the field of cardiovascular research. The cardiovascular system, especially the heart as a well-protected vital organ, is virtually impossible to replicate in vitro with conventional approaches. This made scientists assume that they needed to use animal models for cardiovascular research. However, the frequent failure of animal models to correctly reflect the native cardiovascular system necessitated a search for alternative platforms for preclinical studies. Hence, as a promising alternative to conventional animal models, OOAC technology is being actively developed and tested in a wide range of biomedical fields, including cardiovascular research. Therefore, in this review, the current literature on the use of OOACs for cardiovascular research is presented with a focus on the basis for using OOACs, and what has been specifically achieved by using OOACs is also discussed. By providing an overview of the current status of OOACs in cardiovascular research and its future perspectives, we hope that this review can help to develop better and optimized research strategies for cardiovascular diseases (CVDs) as well as identify novel applications of OOACs in the near future.

Brite Adipocyte FGF21 Attenuates Cardiac Ischemia/Reperfusion Injury in Rat Hearts by Modulating NRF2.

Although the optimal therapy for myocardial infarction includes reperfusion to restore blood flow to the ischemic area, myocardial injury after ischemia/reperfusion usually leads to an inflammatory response, oxidative stress, and cardiomyocyte apoptosis. In this study, rat adipose-derived stem cells were differentiated into low-thermogenic beige adipocytes (LBACs) and high-thermogenic beige adipocytes (HBACs) to study the different cardioprotective effects of heterogeneous expression of brown adipocytes. We found that antioxidant and antiapoptotic factors in H9c2 cardiomyocytes were upregulated by high levels of secreted FGF21 in HBAC conditioned medium (HBAC-CM), whereas FGF21 in HBAC-CM did not affect antioxidative or antiapoptotic cell death in H9c2 cardiomyocytes with Nrf2 knockdown. These results show that NRF2 mediates antioxidative and antiapoptotic effects through the HBAC-secreted factor FGF21. Consistent with this finding, the expression of antioxidant and antiapoptotic genes was upregulated by highly secreted FGF21 after HBAC-CM treatment compared to LBAC-CM treatment in H9c2 cardiomyocytes via NRF2 activation. Furthermore, HBAC-CM significantly attenuated ischemic rat heart tissue injury via NRF2 activation. Based on these findings, we propose that HBAC-CM exerts beneficial effects in rat cardiac ischemia/reperfusion injury by modulating NRF2 and has potential as a promising therapeutic agent for myocardial infarction.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

ADSC-Based Cell Therapies for Musculoskeletal Disorders: A Review of Recent Clinical Trials.

Recently published clinical trials involving the use of adipose-derived stem cells (ADSCs) indicated that approximately one-third of the studies were conducted on musculoskeletal disorders (MSD). MSD refers to a wide range of degenerative conditions of joints, bones, and muscles, and these conditions are the most common causes of chronic disability worldwide, being a major burden to the society. Conventional treatment modalities for MSD are not sufficient to correct the underlying structural abnormalities. Hence, ADSC-based cell therapies are being tested as a form of alternative, yet more effective, therapies in the management of MSDs. Therefore, in this review, MSDs subjected to the ADSC-based therapy were further categorized as arthritis, craniomaxillofacial defects, tendon/ligament related disorders, and spine disorders, and their brief characterization as well as the corresponding conventional therapeutic approaches with possible mechanisms with which ADSCs produce regenerative effects in disease-specific microenvironments were discussed to provide an overview of under which circumstances and on what bases the ADSC-based cell therapy was implemented. Providing an overview of the current status of ADSC-based cell therapy on MSDs can help to develop better and optimized strategies of ADSC-based therapeutics for MSDs as well as help to find novel clinical applications of ADSCs in the near future.

MicroRNA-26a/b-5p promotes myocardial infarction-induced cell death by downregulating cytochrome c oxidase 5a.

Myocardial infarction (MI) damage induces various types of cell death, and persistent ischemia causes cardiac contractile decline. An effective therapeutic strategy is needed to reduce myocardial cell death and induce cardiac recovery. Therefore, studies on molecular and genetic biomarkers of MI, such as microRNAs (miRs), have recently been increasing and attracting attention due to the ideal characteristics of miRs. The aim of the present study was to discover novel causative factors of MI using multiomics-based functional experiments. Through proteomic, MALDI-TOF-MS, RNA sequencing, and network analyses of myocardial infarcted rat hearts and in vitro functional analyses of myocardial cells, we found that cytochrome c oxidase subunit 5a (Cox5a) expression is noticeably decreased in myocardial infarcted rat hearts and myocardial cells under hypoxic conditions, regulates other identified proteins and is closely related to hypoxia-induced cell death. Moreover, using in silico and in vitro analyses, we found that miR-26a-5p and miR-26b-5p (miR-26a/b-5p) may directly modulate Cox5a, which regulates hypoxia-related cell death. The results of this study elucidate the direct molecular mechanisms linking miR-26a/b-5p and Cox5a in cell death induced by oxygen tension, which may contribute to the identification of new therapeutic targets to modulate cardiac function under physiological and pathological conditions.

Suppressing Pyroptosis Augments Post-Transplant Survival of Stem Cells and Cardiac Function Following Ischemic Injury.

The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1ß production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.

Neutralization of hexokinase 2-targeting miRNA attenuates the oxidative stress-induced cardiomyocyte apoptosis.

Hexokinase 2 (HK2) is a metabolic sensor that couples glycolysis and oxidative phosphorylation of mitochondria by binding to the outer mitochondrial membrane (OMM), and it also has been implicated in induction of apoptotic process by regulating the integrity of OMM. When HK2 detaches from the mitochondria, it triggers permeability increase of the OMM and subsequently facilitates the cytosolic release of cytochrome c, a major apoptosis-inducing factor. According to previous studies, a harsh microenvironment created by ischemic heart disease such as low tissue oxygen and nutrients, and increased reactive oxygen species (ROS) can cause cardiomyocyte apoptosis. Under these conditions, the expression of HK2 in heart significantly decrease and such down-regulation of HK2 was correlated to the increased apoptosis of cardiomyocytes. Therefore, prevention of HK2 down-regulation may salvage cardiomyocytes from apoptosis. MicroRNAs are short, non-coding RNAs that either inhibit transcription of target mRNAs or degrade the targeted mRNAs via complementary binding to the 3'UTR (untranslated region) of the targeted mRNAs. Since miRNAs are known to be involved in virtually every biological processes, it is reasonable to assume that the expression of HK2 is also regulated by miRNAs. Currently, to my best knowledge, there is no previous study examined the miRNA-mediated regulation of HK2 in cardiomyocytes. Thus, in the present study, miRNA-mediated modulation of HK2 during ROS (H2O2)-induced cardiomyocyte apoptosis was investigated. First, the expression of HK2 in cardiomyocytes exposed to H2O2 was evaluated. H2O2 (500 µM) induced cardiomyocyte apoptosis and it also decreased the mitochondrial expression of HK2. Based on miRNA-target prediction databases and empirical data, miR-181a was identified as a HK2-targeting miRNA. To further examine the effect of negative regulation of the selected HK2-targeting miRNA on cardiomyocyte apoptosis, anti-miR-181a, which neutralizes endogenous miR-181a, was utilized. Delivery of anti-miR-181a significantly abrogated the H2O2-induced suppression of HK2 expression and subsequent disruption of mitochondrial membrane potential, improving the survival of cardiomyocytes exposed to H2O2. These findings suggest that miR-181a-mediated down-regulation of HK2 contributes to the apoptosis of cardiomyocytes exposed to ROS. Neutralizing miR-181a can be a viable and effective means to prevent cardiomyocyte from apoptosis in ischemic heart disease.

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition.

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.

Novel Therapeutic Effects of Pterosin B on Ang II-Induced Cardiomyocyte Hypertrophy.

Pathological cardiac hypertrophy is characterized by an abnormal increase in cardiac muscle mass in the left ventricle, resulting in cardiac dysfunction. Although various therapeutic approaches are being continuously developed for heart failure, several studies have suggested natural compounds as novel potential strategies. Considering relevant compounds, we investigated a new role for Pterosin B for which the potential life-affecting biological and therapeutic effects on cardiomyocyte hypertrophy are not fully known. Thus, we investigated whether Pterosin B can regulate cardiomyocyte hypertrophy induced by angiotensin II (Ang II) using H9c2 cells. The antihypertrophic effect of Pterosin B was evaluated, and the results showed that it reduced hypertrophy-related gene expression, cell size, and protein synthesis. In addition, upon Ang II stimulation, Pterosin B attenuated the activation and expression of major receptors, Ang II type 1 receptor and a receptor for advanced glycation end products, by inhibiting the phosphorylation of PKC-ERK-NF-κB pathway signaling molecules. In addition, Pterosin B showed the ability to reduce excessive intracellular reactive oxygen species, critical mediators for cardiac hypertrophy upon Ang II exposure, by regulating the expression levels of NAD(P)H oxidase 2/4. Our results demonstrate the protective role of Pterosin B in cardiomyocyte hypertrophy, suggesting it is a potential therapeutic candidate.

Proteome Analysis of Human Natural Killer Cell Derived Extracellular Vesicles for Identification of Anticancer Effectors.

Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, ß-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.

Isoliquiritigenin Derivatives Inhibit RANKL-Induced Osteoclastogenesis by Regulating p38 and NF-κB Activation in RAW 264.7 Cells.

Bone diseases may not be imminently life-threatening or a leading cause of death such as heart diseases or cancers. However, as aging population grows in almost every part of the world, they surely impose significant socioeconomic burden on the society, not to mention the patients and their families. Osteoporosis is the most common type of bone disease, which frequently develops in seniors, especially in postmenopausal women. Although currently several anti-osteoclastic drugs designed to suppress excessive osteoclast activation, a major cause of osteoporosis, are commercially available, accompanying adverse effects ranging from mild to severe have been reported as well. Natural products have become increasingly popular because of their effectiveness with fewer side effects. Isoliquiritigenin (ILG), a natural flavonoid from licorice, has been reported to suppress osteoclast differentiation and activation. In the present study, newly synthesized ILG derivatives were screened for their anti-osteoporotic activity as more potent substitute candidates to ILG. Out of the 12 ILG derivatives tested, two compounds demonstrated significantly improved bone loss in vitro by inhibiting both osteoclastogenesis and osteoclast activity. The results of the present study indicate that these compounds may serve as a potential drug for osteoporosis and warrant further studies to evaluate their in vivo efficacy.

A Combinational Therapy of Articular Cartilage Defects: Rapid and Effective Regeneration by Using Low-Intensity Focused Ultrasound After Adipose Tissue-Derived Stem Cell Transplantation.

BACKGROUND: Although low-intensity pulsed ultrasound has been reported to be potential cartilage regeneration, there still unresolved treatment due to cartilage fibrosis and degeneration by a lack of rapid and high-efficiency treatment. The purpose of this study was to investigate the effect of a combination therapy of focused acoustic force and stem cells at site for fast and efficient healing on cartilage regeneration. METHODS: Using a rat articular cartilage defects model, one million adipose tissue-derived stem cells (ASCs) were injected into the defect site, and low-intensity focused ultrasound (LOFUS) in the range of 100-600 mV was used for 20 min/day for 2 weeks. All experimental groups were sacrificed after 4 weeks in total. The gross appearance score and hematoxylin and eosin (H&E), Alcian blue, and Safranin O staining were used for measuring the chondrogenic potential. The cartilage characteristics were observed, and type II collagen, Sox 9, aggrecan, and type X collagen were stained with immunofluorescence. The results of the comprehensive analysis were calculated using the Mankin scoring method. RESULTS: The gross appearance scores of regenerated cartilage and chondrocyte-like cells in H&E images were higher in LOFUS-treated groups compared to those in negative control or ASC-treated groups. Safranin O and Alcian blue staining demonstrated that the 100 and 300 mV LOFUS groups showed greater synthesis of glycosaminoglycan and proteoglycan. The ASC + LOFUS 300 mV group showed positive regulation of type II collagen, Sox 9 and aggrecan and negative regulation of type X collagen, which indicated the occurrence of cartilage regeneration based on the Mankin score result. CONCLUSION: The combination therapy, which involved treatment with ASC and 300 mV LOFUS, quickly and effectively reduced articular cartilage defects.

Differentiation of adipose-derived stem cells into functional chondrocytes by a small molecule that induces Sox9.

Osteoarthritis (OA) is a common joint disease that results from the disintegration of joint cartilage and the underlying bone. Because cartilage and chondrocytes lack the ability to self-regenerate, efforts have been made to utilize stem cells to treat OA. Although various methods have been used to differentiate stem cells into functional chondrocytes, the currently available methods cannot induce stem cells to undergo differentiation into chondrocyte-like cells without inducing characteristics of hypertrophic chondrocytes, which finally lead to cartilage disintegration and calcification. Therefore, an optimized method to differentiate stem cells into chondrocytes that do not display undesired phenotypes is needed. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a small molecule that regulated the expression of Sox9 as a key factor in cartilage development and then explored its ability to treat OA. We selected ellipticine (ELPC), which induces chondrocyte differentiation of ASCs, using a GFP-Sox9 promoter vector screening system. An in vivo study was performed to confirm the recovery rate of cartilage regeneration with ASC differentiation into chondrocytes by ELPC in a collagenase-induced animal model of OA. Taken together, these data indicate that ellipticine induces ASCs to differentiate into mature chondrocytes without hypertrophic chondrocytes in vitro and in vivo, thus overcoming a problem encountered in previous studies. These results indicate that ELPC is a novel chondrocyte differentiation-inducing drug that shows potential as a cell therapy for OA.

Small G protein signaling modulator 3 (SGSM3) knockdown attenuates apoptosis and cardiogenic differentiation in rat mesenchymal stem cells exposed to hypoxia.

Connexin 43 (Cx43) may be important in cell death and survival due to cell-to-cell communication-independent mechanisms. In our previous study, we found that small G protein signaling modulator 3 (SGSM3), a partner of Cx43, contributes to myocardial infarction (MI) in rat hearts. Based on these previous results, we hypothesized that SGSM3 could also play a role in bone marrow-derived rat mesenchymal stem cells (MSCs), which differentiate into cardiomyocytes and/or cells with comparable phenotypes under low oxygen conditions. Cx43 and Cx43-related factor expression profiles were compared between normoxic and hypoxic conditions according to exposure time, and Sgsm3 gene knockdown (KD) using siRNA transfection was performed to validate the interaction between SGSM3 and Cx43 and to determine the roles of SGSM3 in rat MSCs. We identified that SGSM3 interacts with Cx43 in MSCs under different oxygen conditions and that Sgsm3 knockdown inhibits apoptosis and cardiomyocyte differentiation under hypoxic stress. SGSM3/Sgsm3 probably has an effect on MSC survival and thus therapeutic potential in diseased hearts, but SGSM3 may worsen the development of MSC-based therapeutic approaches in regenerative medicine. This study was performed to help us better understand the mechanisms involved in the therapeutic efficacy of MSCs, as well as provide data that could be used pharmacologically.

Substrate-enzyme affinity-based surface modification strategy for endothelial cell-specific binding under shear stress.

Establishing an endothelial cell (EC) monolayer on top of the blood contacting surface of grafts is considered to be a promising approach for creating a hemocompatible surface. Here we utilized the high affinity interactions between the EC plasma membrane expressed enzyme called endothelin converting enzyme-1 (ECE-1) and its corresponding substrate big Endothelin-1 (bigET-1) to engineer an EC-specific binding surface. Since enzymatic cleavage of substrates require physical interaction between the enzyme and its corresponding substrate, it was hypothesized that a surface with chemically immobilized synthetic bigET-1 will preferentially attract ECs over other types of cells found in vascular system such as vascular smooth muscle cells (VSMCs). First, the expression of ECE-1 was significantly higher in ECs, and ECs processed synthetic bigET-1 to produce ET-1 in a cell number-dependent manner. Such interaction between ECs and synthetic bigET-1 was also detectible in blood. Next, vinyl-terminated self-assembled monolayers (SAMs) were established, oxidized and activated on a glass substrate as a model to immobilize synthetic bigET-1 via amide bonds. The ECs cultured on the synthetic bigET-1-immobilized surface processed larger amount of synthetic bigET-1 to produce ET-1 compared to VSMCs (102.9±5.13 vs. 9.75±0.74 pg/ml). The number of ECs bound to the synthetic bigET-1-immobilized surface during 1 h of shearing (5dyne/cm2) was approximately 3-fold higher than that of VSMCs (46.25±12.61 vs. 15.25±3.69 cells/100×HPF). EC-specific binding of synthetic bigET-1-immobilized surface over a surface modified with collagen, a common substance for cell adhesion, was also observed. The present study demonstrated that using the substrate-enzyme affinity (SEA) of cell type-specific enzyme and its corresponding substrate can be an effective method to engineer a surface preferentially binds specific type of cells. This novel strategy might open a new route toward rapid endothelialization under dynamic conditions supporting the long-term patency of cardiovascular implants.

Cytotoxic effects of ex vivo-expanded natural killer cell-enriched lymphocytes (MYJ1633) against liver cancer.

BACKGROUND: Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a targeted method of controlling the immune system against cancer. Despite their significant therapeutic potential, efficient methods to generate adequate numbers of NK cells are lacking and ex vivo-expansion and activation of NK cells is currently under intensive investigation. The primary purpose of this study was to develop an effective method for expansion and activation of the effector cells with high proportion of NK cells and increasing cytotoxicity against liver cancer in a short time period. METHODS: Expanded NK cell-enriched lymphocytes (NKL) designated as "MYJ1633" were prepared by using autologous human plasma, cytokines (IL-2, IL-12 and IL-18) and agonistic antibodies (CD16, CD56 and NKp46) without an NK cell-sorting step. The characteristics of NKL were compared to those of freshly isolated PBMCs. In addition, the cytotoxic effect of the NKL on liver cancer cell was examined in vitro and in vivo. RESULTS: The total cell number after ex vivo-expansion increased about 140-fold compared to that of freshly isolated PBMC within 2 weeks. Approximately 78% of the expanded and activated NKL using the house-developed protocol was NK cell and NKT cells even without a NK cell-sorting step. In addition, the expanded and activated NKL demonstrated potent cytotoxicity against liver cancer in vitro and in vivo. CONCLUSION: The house-developed method can be a new and effective strategy to prepare clinically applicable NKL for autologous NK cell-based anti-tumor immunotherapy.