Soon Chul Heo

Tumor Necrosis Factor-α-Activated Human Adipose Tissue–Derived Mesenchymal Stem Cells Accelerate Cutaneous Wound Healing through Paracrine Mechanisms

Human adipose tissue-derived mesenchymal stem cells (ASCs) stimulate regeneration of injured tissues by secretion of various cytokines and chemokines. Wound healing is mediated by multiple steps including inflammation, epithelialization, neoangiogenesis, and proliferation. To explore the paracrine functions of ASCs on regeneration of injured tissues, cells were treated with tumor necrosis factor-α (TNF-α), a key inflammatory cytokine, and the effects of TNF-α-conditioned medium (CM) on tissue regeneration were determined using a rat excisional wound model. We demonstrated that TNF-α CM accelerated wound closure, angiogenesis, proliferation, and infiltration of immune cells into the cutaneous wound in vivo. To assess the role of proinflammatory cytokines IL-6 and IL-8, which are included in TNF-α CM, IL-6 and IL-8 were depleted from TNF-α CM using immunoprecipitation. Depletion of IL-6 or IL-8 largely attenuated TNF-α CM-stimulated wound closure, angiogenesis, proliferation, and infiltration of immune cells. These results suggest that TNF-α-activated ASCs accelerate cutaneous wound healing through paracrine mechanisms involving IL-6 and IL-8.

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.

Ovarian cancer-derived lysophosphatidic acid stimulates secretion of VEGF and stromal cell-derived factor-1α from human mesenchymal stem cells

Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1α (SDF-1α) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA1 receptor abrogated the cancer CM-stimulated expression of α-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of α-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of α-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced α-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.

Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism.

Carcinoma-associated fibroblasts play a key role in tumorigenesis and metastasis by providing a tumor-supportive microenvironment. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to carcinoma-associated fibroblasts expressing α-smooth muscle actin, vascular endothelial growth factor, and stromal cell-derived factor-1. A549 conditioned medium-induced differentiation of hASCs to carcinoma-associated fibroblasts was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA(1)) expression in hASCs with small interfering RNA or lentiviral short hairpin RNA. Using a murine xenograft transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts in vivo. Knockdown of LPA(1) expression in hASCs abrogated hASCs-stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts. Moreover, A549 conditioned medium-treated hASCs stimulated tube formation of human umbilical vein endothelial cells by LPA(1)-dependent secretion of vascular endothelial growth factor. These results suggest that A549 cells induce in vivo differentiation of hASCs to carcinoma-associated fibroblasts, which play a key role in tumor angiogenesis within tumor microenvironment, through an LPA-LPA(1)-mediated paracrine mechanism.

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.

Lysophosphatidic acid-induced expression of periostin in stromal cells: prognoistic relevance of periostin expression in epithelial ovarian cancer

Lysophosphatidic acid (LPA) is a bioactive lipid crucial for the initiation and progression of ovarian cancer. Identification of LPA‐induced biomarkers is necessary for predicting prognosis of ovarian cancer patients. Here we report periostin, an extracellular matrix protein, as an LPA‐induced protein in stromal cells and as a prognostic marker in patients with epithelial ovarian cancer (EOC). In human EOC tissues, periostin was mainly expressed in cancer‐associated stromal fibroblasts, but not in cancer cells. The expression levels of periostin highly correlated with poor survival and tumor recurrence of ovarian cancer patients. Treatment of human adipose tissue‐derived stromal cells with LPA or conditioned media from human ovarian adenocarcinoma cell lines, such as SK‐OV‐3 and OVCAR‐3, induced expression of periostin. The periostin expression induced by cancer‐conditioned media was abrogated by silencing of the LPA receptor 1 expression using small hairpin RNA lentivirus. Recombinant periostin stimulated adhesion and invasion of SK‐OV‐3 human ovarian adenocarcinoma cells and induced expression of matrix metalloprotease‐2 in the cancer cells. These results suggest that LPA is associated with the expression of periostin in cancer‐associated fibroblasts of EOC.

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.

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.

Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells

Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca(2+), big-conductance Ca(2+)-activated K(+) (BKCa), and voltage-dependent K(+) (Kv) currents in TGF-β1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.