Sang Gyu Park

Generation of reactive oxygen species in adipose-derived stem cells: friend or foe?

Introduction: Reactive oxygen species (ROS) participate in cellular apoptosis and are involved in pathophysiological etiology of degenerative diseases. However, recent studies suggest that ROS at low levels may play a pivotal role as second messengers and activate normal cellular processes. Intracellular ROS increase the proliferation, migration, and regenerative potential of adipose-derived stem cells (ASCs). In contrast, manipulations that diminish intracellular ROS levels interfere with normal ASC function. ROS generation therefore acts like a double-edged sword. Areas covered: This review discusses the following research questions: i) Do ROS stimulate or suppress ASCs? ii) How are ROS generated from ASCs? iii) Which function(s) is/are regulated by intracellular ROS generation? In addition, the antioxidant/antiapoptotic effect of ASCs is briefly introduced. Expert opinion: Whether ROS is harmful or beneficial is primarily a question of dosage. Low or moderate ROS generation increases the proliferation, migration and regenerative potential of ASCs. Therefore, it is beneficial to expose ASCs to moderate oxidative stress during manipulation. The addition of a ROS donor in culture can reduce the cost for the expansion of ASCs and a ROS preconditioning can enhance the regenerative potential of ASCs.

Functional Regulation of Adipose‐Derived Stem Cells by PDGF‐D

Platelet‐derived growth factor‐D (PDGF‐D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF‐D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF‐D in adipose‐derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF‐D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF‐D‐induced stimulation of ASCs. It is of interest that PDGF‐B is not expressed, but PDGF‐D and PDGF receptor‐β are expressed in ASCs. PDGF‐D showed the strongest mitogenic effect on ASCs, and PDGF‐D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF‐D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2‐related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF‐D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin‐binding EGF‐like growth factor. Therefore, the preconditioning of PDGF‐D enhanced the hair‐regenerative potential of ASCs. PDGF‐D‐induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen‐activated protein kinase pathway. In summary, PDGF‐D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF‐D also upregulates growth factor expression in ASCs. Therefore, PDGF‐D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation. Stem Cells 2015;33:542–556

Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK via PKCα-mediated MKP-1 induction

AIMS Hyperplasia of vascular smooth muscle cells (VSMCs) after blood vessel injury is one of the major pathophysiological mechanisms associated with neointima. Tauroursodeoxycholate (TUDCA) is a cytoprotective agent in a variety of cells including hepatocytes as well as an inducer of apoptosis in cancer cells. In this study, we investigated whether TUDCA could prevent neointimal hyperplasia by suppressing the growth and migration of VSMCs. METHODS AND RESULTS Transporters of TUDCA uptake in human VSMCs (hVSMCs) were analysed by RT-PCR and western blot. A knock-down experiment using specific si-RNA revealed that TUDCA was incorporated into hVSMCs via organic anion transporter 2 (OATP2). TUDCA reduced the viability of hVSMCs, which were mediated by inhibition of extracellular signal-regulated kinase (ERK) by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via protein kinase Cα (PKCα). The anti-proliferative effect of TUDCA was reversed by treatment with 7-hydroxystaurosporine, an inhibitor of PKC, and by the knock-down of MKP-1. In addition, TUDCA suppressed hVSMC migration, which was mediated by reduced matrix metalloproteinase-9 (MMP-9) expression by ERK inhibition, as well as reduced viability of hVSMCs. Rats with carotid artery balloon injury received oral administration of TUDCA; this reduced the increase in ERK and MMP-9 caused by balloon injury. TUDCA significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of the VSMCs. CONCLUSION TUDCA inhibits neointimal hyperplasia by reducing proliferation and inducing apoptosis of smooth muscle cells by suppression of ERK via PKCα-mediated MKP-1 induction.

Isolation of adipose-derived stem cells by using a subfractionation culturing method

Objective: Adipose-derived stem cells (ASCs) isolated from subcutaneous adipose tissue have been tested in clinical trials. However, ASCs isolated by enzyme digestion and centrifugation are heterogeneous and exhibit wide variation in regenerative potential and clinical outcomes. Therefore, we developed a new method for isolating clonal ASCs (cASCs) that does not use enzyme digestion or centrifugation steps. Research design and methods: In addition to cell surface markers and differentiation potential, we compared the mitogenic, paracrine and hair growth-promoting effects of ASCs isolated by the gradient centrifugation method (GCM) or by the new subfractionation culturing method (SCM). Results: We selected three cASCs isolated by SCM that showed high rates of proliferation. The cell surface markers expressed by ASCs isolated by GCM or SCM were very similar, and SCM-isolated ASCs could potentially differentiate into different cell lineages. However, cASC lines exhibited better mitogenic and paracrine effects than ASCs isolated by GCM. The expression of Diras3, Myb, Cdca7, Mki67, Rrm2, Cdk1 and Ccna2, which may play a key role in cASC proliferation, was upregulated in cASCs. In addition, cASCs exhibited enhanced hair growth-promoting effects in dermal papilla cells and animal experiments. Conclusions: SCM generates a highly homogeneous population of ASCs via a simple and effective procedure that can be used in therapeutic settings.

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

Fibroblast Growth Factor 12 is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate

Objective—Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. Approach and Results—Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. Conclusions—In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

Monoclonal and Polyclonal Antibodies Specific for Foot and Mouth Disease Virus Type A and Type O VP1

The foot and mouth disease virus (FMDV) is an RNA virus composed of single stranded positive sense RNA. FMDV has been known to infect cloven-hoofed animals, including pigs, cattle, and sheep. FMDV is rapidly spreading outward to neighboring regions, often leading to a high mortality rate. Thus, early diagnosis of FMDV is critical to suppress propagation of FMDV and minimize economic losses. In this study, we report the generation and characterization of polyclonal and six monoclonal antibodies against VP1 through immunoblotting and immunofluorescence microscopy analyses. These VP1 antibodies will be useful as tools to detect serotypes A and O of FMDVs for diagnostic usage.

The cKit Inhibitor, Masitinib, Prevents Diabetes-Induced Retinal Vascular Leakage

PURPOSE Stem cell factor (SCF) has recently demonstrated activity as a novel endothelial permeability factor that contributes to the development of diabetes-induced hyperpermeable retinal vasculature. This study investigated the therapeutic potential of masitinib, a pharmacologic inhibitor of the SCF receptor cKit, for prevention of diabetes-induced breakdown of blood retinal barrier (BRB). METHODS Permeability assays were performed with human retinal microvascular endothelial cells (HRMECs) and murine retinal vasculature. Localization of vascular endothelial (VE)-cadherin and activation of SCF signaling pathway was determined by immunofluorescence and Western blotting assays. Mice and rats with streptozotocin (STZ)-induced diabetes were used to investigate the role of cKit and masitinib in diabetes-induced retinal vascular hyperpermeability. RESULTS Masitinib substantially blocked SCF-induced phosphorylation of cKit in HRMECs. In vitro and in vivo vascular permeability assays showed that masitinib significantly inhibited SCF-induced endothelial hyperpermeability and junctional loss of VE-cadherin. Streptozotocin-induced diabetes was induced in cKit-mutant mice with low cKit expression in their endothelial cells. Although diabetic wild-type mice exhibited enhanced retinal vascular leakage, diabetic cKit-mutant mice showed no increase in retinal vascular leakage or alteration in the distribution of VE-cadherin; this indicates the crucial role of cKit in diabetes-induced breakdown of BRB. Moreover, in vivo prevention experiments showed that an intravitreal injection of masitinib substantially inhibited the development of hyperpermeable retinal vasculature. CONCLUSIONS These results provide the first demonstration that cKit inhibitors, such as masitinib, might be promising therapeutics for prevention of diabetes-induced breakdown of the BRB.

Antioxidant and Anti-Melanogenic Effect of the Novel Synthetic Hexapeptide (SFKLRY-NH2)

The novel synthetic hexapeptide, Angio-S (SFKLRY-NH2), induced angiogenesis in human endothelial cells and accelerated wound healing. Since the pathophysiology of a wound is similar to the skin-aging process, the antioxidant and anti-melanogenic effects of Angio-S were investigated in this study. The antioxidant effect was investigated in the dermal fibroblasts, and the skin-whitening effect was studied in melanoma B16 cells. Angio-S exhibited an antioxidant activity, which increased in a dose-dependent manner. A cell survival assay revealed that Angio-S aided dermal fibroblasts in the resistance of free radicals induced by tert-butyl hydroperoxide. In addition, activities of superoxide dismutase and glutathione peroxidase were enhanced after pre-treatment with Angio-S. Since antioxidants inhibit the chemical reactions leading to melanin formation, the anti-melanogenic effect of Angio-S was studied. Angio-S reduced the synthesis of melanin and inhibited the activity of tyrosinase in melanoma B16 cells. Although the underlying mechanism of inhibiting melanin synthesis was not fully studied, Angio-S may act as an anti-oxidant and directly inhibit tyrosinase during melanin biosynthesis. Collectively, these results indicate that Angio-S exhibits antioxidant and anti-melanogenic effects, and is a potential candidate for use as a skin rejuvenation agent considering the skin-rejuvenating effect at a relatively low concentration.