Yang Woo Kwon

Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.

WKYMVm‐Induced Activation of Formyl Peptide Receptor 2 Stimulates Ischemic Neovasculogenesis by Promoting Homing of Endothelial Colony‐Forming Cells

Endothelial colony‐forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G‐protein‐coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA‐mediated silencing of FPR2, but not FPR3, abrogated WKYMVm‐induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC‐mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm‐induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2‐dependent mechanism. Stem Cells 2014;32:779–790

TAZ Mediates Lysophosphatidic Acid-Induced Migration and Proliferation of Epithelial Ovarian Cancer Cells

Background: Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. Methods: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. Results and Conclusion: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells

Ovarian cancer has the highest mortality rate of all gynecological cancers with a high recurrence rate. It is important to understand the nature of recurring cancer cells to terminally eliminate ovarian cancer. The winged helix transcription factor Forkhead box P1 (FOXP1) has been reported to function as either oncogene or tumor-suppressor in various cancers. In the current study, we show that FOXP1 promotes cancer stem cell-like characteristics in ovarian cancer cells. Knockdown of FOXP1 expression in A2780 or SKOV3 ovarian cancer cells decreased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment, whereas overexpression of FOXP1 in A2780 or SKOV3 ovarian cancer cells increased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment. In addition, overexpression of FOXP1 increased promoter activity of ABCG2, OCT4, NANOG, and SOX2, among which the increases in ABCG2, OCT4, and SOX2 promoter activity were dependent on the presence of FOXP1-binding site. In xenotransplantation of A2780 ovarian cancer cells into nude mice, knockdown of FOXP1 expression significantly decreased tumor size. These results strongly suggest FOXP1 functions as an oncogene by promoting cancer stem cell-like characteristics in ovarian cancer cells. Targeting FOXP1 may provide a novel therapeutic opportunity for developing a relapse-free treatment for ovarian cancer patients.

Periostin mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in a xenograft lung adenocarcinoma model.

Mesenchymal stem cells stimulate tumor growth in vivo through a lysophosphatidic acid (LPA)-dependent mechanism. However, the molecular mechanism by which mesenchymal stem cells stimulate tumorigenesis is largely elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induces expression of periostin, an extracellular matrix protein, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated periostin expression was abrogated by pretreatment of hASCs with the LPA receptor 1 (LPA(1)) inhibitor Ki16425 or short hairpin RNA-mediated silencing of LPA(1), suggesting a key role of the LPA-LPA(1) signaling axis in A549 CM-stimulated periostin expression. Using a xenograft transplantation model of A549 cells, we demonstrated that co-injection of hASCs potentiated tumor growth of A549 cells in vivo and that co-transplanted hASCs expressed not only periostin but also α-smooth muscle actin (α-SMA), a marker of carcinoma-associated fibroblasts. Small interfering RNA- or short hairpin RNA-mediated silencing of periostin resulted in blockade of LPA-induced α-SMA expression in hASCs. In addition, silencing of periostin resulted in blockade of hASC-stimulated growth of A549 xenograft tumors and in vivo differentiation of transplanted hASCs to α-SMA-positive carcinoma-associated fibroblasts. Conditioned medium derived from LPA-treated hASCs (LPA CM) potentiated proliferation and adhesion of A549 cells and short interfering RNA-mediated silencing or immunodepletion of periostin from LPA CM abrogated proliferation and adhesion of A549 cells. These results suggest a pivotal role for hASC-secreted periostin in growth of A549 xenograft tumors within the tumor microenvironment.

Macrophages Regulate Smooth Muscle Differentiation of Mesenchymal Stem Cells via a Prostaglandin F2α−Mediated Paracrine Mechanism

Objective—Mesenchymal stem cells are useful for vascular regeneration of injured tissues. Macrophages are involved in acute or chronic inflammatory diseases, and interleukin-1&bgr; (IL-1&bgr;), a proinflammatory cytokine, plays a key role in the activation of macrophages within injured tissues. To explore the role of macrophages on mesenchymal stem cell–mediated vascular regeneration, we examined the effects of IL-1&bgr;–activated macrophages on differentiation of human adipose tissue–derived mesenchymal stem cells (hASCs) to smooth muscle cells (SMCs) and the vascular regenerative capacity of the differentiated SMCs in a hindlimb ischemia animal model. Methods and Results—We demonstrate that IL-1&bgr;–conditioned medium from RAW 264.7 macrophages induces differentiation of human adipose tissue–derived mesenchymal stem cells to &agr;-smooth muscle actin–positive SMCs, and the differentiated SMCs exhibited increased contractility in response to KCl and carbachol treatment. Transplantation of the differentiated SMCs attenuated severe hindlimb ischemia and promoted vascular regeneration. IL-1&bgr; treatment stimulated secretion of prostaglandin F2&agr; from RAW 264.7 cells. Small interfering RNA–mediated silencing of the prostaglandin F2&agr; receptor completely abrogated IL-1&bgr; conditioned medium–stimulated &agr;-smooth muscle actin expression. Moreover, prostaglandin F2&agr; treatment stimulated expression of &agr;-smooth muscle actin in human adipose tissue–derived mesenchymal stem cells. Conclusion—These results suggest that IL-1&bgr;–activated macrophages promote differentiation of human adipose tissue–derived mesenchymal stem cells to SMCs through a prostaglandin F2&agr;–mediated paracrine mechanism.

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid‐Mediated Autocrine Mechanism

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat‐stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non‐CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere‐forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC‐associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1‐specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA‐stimulated CSC properties. Autotaxin, an LPA‐producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA‐producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA‐dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin‐LPA‐LPA receptor 1‐AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs. Stem Cells 2016;34:551–564

Proteomic Identification of Betaig-h3 as a Lysophosphatidic Acid-Induced Secreted Protein of Human Mesenchymal Stem Cells: Paracrine Activation of A549 Lung Adenocarcinoma Cells by Betaig-h3

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor1/3 inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA1. LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA1– Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Oncostatin M promotes mesenchymal stem cell-stimulated tumor growth through a paracrine mechanism involving periostin and TGFBI

Oncostatin M, a member of the interleukin-6 family of cytokines, has been implicated in tumorigenesis of human prostate cancer. In the current study, we demonstrate that oncostatin M promotes human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in an in vivo xenograft transplantation model of the human prostate cancer cell line PC-3M-luc-C6, a PC3M cell line expressing the luciferase gene. Conditioned medium derived from oncostatin M-treated mesenchymal stem cells stimulated adhesion of PC-3M-luc-C6 cells. We identified TGFBI and periostin, extracellular matrix proteins implicated in tumorigenesis and metastasis, as oncostatin M-induced secreted proteins in mesenchymal stem cells. Treatment with oncostatin M stimulated secretion of periostin and TGFBI from mesenchymal stem cells in a time-dependent manner. Immunodepletion of TGFBI and periostin from conditioned medium derived from oncostatin M-treated mesenchymal stem cells resulted in abrogation of adhesion of PC-3M-luc-C6 cells stimulated by oncostatin M-conditioned medium. In addition, small interfering RNA-mediated silencing of TGFBI and periostin resulted in abrogation of cell adhesion stimulated by oncostatin M-conditioned medium. These results suggest that mesenchymal stem cell-derived TGFBI and periostin play a key role in tumorigenesis by stimulating adhesion of prostate cancer cells.

Therapeutic angiogenesis in a murine model of limb ischemia by recombinant periostin and its fasciclin I domain.

Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against β3 and β5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.