Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells.

The generation of endothelial cells (ECs) from human pluripotent stem cells (PSCs) has been a promising approach for treating cardiovascular diseases for several years. Human PSCs, particularly induced pluripotent stem cells (iPSCs), are an attractive source of ECs for cell therapy. Although there is a diversity of methods for endothelial cell differentiation using biochemical factors, such as small molecules and cytokines, the efficiency of EC production varies depending on the type and dose of biochemical factors. Moreover, the protocols in which most EC differentiation studies have been performed were in very unphysiological conditions that do not reflect the microenvironment of native tissue. The microenvironment surrounding stem cells exerts variable biochemical and biomechanical stimuli that can affect stem cell differentiation and behavior. The stiffness and components of the extracellular microenvironment are critical inducers of stem cell behavior and fate specification by sensing the extracellular matrix (ECM) cues, adjusting the cytoskeleton tension, and delivering external signals to the nucleus. Differentiation of stem cells into ECs using a cocktail of biochemical factors has been performed for decades. However, the effects of mechanical stimuli on endothelial cell differentiation remain poorly understood. This review provides an overview of the methods used to differentiate ECs from stem cells by chemical and mechanical stimuli. We also propose the possibility of a novel EC differentiation strategy using a synthetic and natural extracellular matrix.

AGR2 is a target of canonical Wnt/ß-catenin signaling and is important for stemness maintenance in colorectal cancer stem cells.

Colorectal cancer is one of the leading causes of cancer-related deaths. Due to relapse after current therapy regimens, cancer stem cells (CSCs) are being studied to target this small tumor-initiating population. Anterior gradient 2 (AGR2), a disulfide isomerase protein, is a well-known pro-oncogenic/metastatic oncogene overexpressed in various tumor tissues, including colon cancer. We found that AGR2 was a novel stem cell marker that was regulated by the canonical Wnt/ß-catenin pathway in colon CSCs. AGR2 was highly co-expressed with surface stem cell markers in spheroidal culture. Silencing of AGR2 resulted in decreased sphere-forming ability and down-regulated expression of stem cell markers, whereas the opposite effects were seen with AGR2 overexpression. Moreover, patients with high ß-catenin and AGR2 expression showed lower overall survival than those with low expression. In conclusion, our study describes a novel role for AGR2 as a stem cell marker that is highly regulated by canonical Wnt/ß-catenin signaling in colorectal cancer.

Basic helix-loop-helix transcription factor Twist1 is a novel regulator of anterior gradient protein 2 homolog (AGR2) in breast cancer.

Anterior gradient protein 2 homolog (AGR2) belongs to the disulfide isomerase family of endoplasmic reticulum proteins. Itis overexpressed in several types of solid tumors, including tumors of the prostate, lung, and pancreas. However, the role of AGR2 in breast cancer and the regulatory mechanisms underlying AGR2 protein expressionare not fullyunderstood. We demonstrated that AGR2 levels are increased under hypoxic conditions and in breast cancer tumors. Mechanistically, Twist1 binds to, and activates the AGR2 promoter via an E-box sequence. Under hypoxic conditions, the increased expression of ARG2 is attenuated when Twist1 levels are reduced by shRNA. Conversely, Twist1 overexpression fully reverses decreased AGR2 levels upon HIF-1α knockdown. Notably, AGR2 is required for Twist1-induced proliferation, migration, and invasion of breast cancer cells. Collectively, these findings extend our understanding of AGR2 regulation in breast cancer and may contribute to development of Twist1-AGR2 targeting therapeutics for breast cancer.

Angiotensin II Attenuates the Bioactivities of Human Endothelial Progenitor Cells via Downregulation of ß2-Adrenergic Receptor.

Cross talks between the renin-angiotensin system (RAS), sympathetic nervous system, and vascular homeostasis are tightly coordinated in hypertension. Angiotensin II (Ang II), a key factor in RAS, when abnormally activated, affects the number and bioactivity of circulating human endothelial progenitor cells (hEPCs) in hypertensive patients. In this study, we investigated how the augmentation of Ang II regulates adrenergic receptor-mediated signaling and angiogenic bioactivities of hEPCs. Interestingly, the short-term treatment of hEPCs with Ang II drastically attenuated the expression of beta-2 adrenergic receptor (ADRB2), but did not alter the expression of beta-1 adrenergic receptor (ADRB1) and Ang II type 1 receptor (AT1R). EPC functional assay clearly demonstrated that the treatment with ADRB2 agonists significantly increased EPC bioactivities including cell proliferation, migration, and tube formation abilities. However, EPC bioactivities were decreased dramatically when treated with Ang II. Importantly, the attenuation of EPC bioactivities by Ang II was restored by treatment with an AT1R antagonist (telmisartan; TERT). We found that AT1R binds to ADRB2 in physiological conditions, but this binding is significantly decreased in the presence of Ang II. Furthermore, TERT, an Ang II-AT1R interaction blocker, restored the interaction between AT1R and ADRB2, suggesting that Ang II might induce the dysfunction of EPCs via downregulation of ADRB2, and an AT1R blocker could prevent Ang II-mediated ADRB2 depletion in EPCs. Taken together, our report provides novel insights into potential therapeutic approaches for hypertension-related cardiovascular diseases.

Cytoprotective Roles of a Novel Compound, MHY-1684, against Hyperglycemia-Induced Oxidative Stress and Mitochondrial Dysfunction in Human Cardiac Progenitor Cells.

Diabetic cardiomyopathy (DCM) is tightly linked to heart disorders and dysfunction or death of the cardiomyocytes including resident cardiac progenitor cells (CPCs) in diabetic patients. In order to restore loss of function of resident or transplanted CPCs, much research has focused on novel therapeutic strategies including the discovery of novel function-modulating factors such as reactive oxygen species (ROS) scavengers. Here, we developed and defined a novel antioxidant, MHY-1684, for enhancing the angiogenic potential of CPCs against ROS-related DCM. Short-term treatment with MHY-1684 restored ROS-induced CPC cell death. Importantly, MHY-1684 decreased hyperglycemia-induced mitochondrial ROS generation and attenuated hyperglycemia-induced mitochondrial fragmentation. We observed that the activation process of both Drp1 (phosphorylation at the site of Ser616) and Fis-1 is drastically attenuated when exposed to high concentrations of D-glucose with MHY-1684. Interestingly, phosphorylation of Drp1 at the site of Ser637, which is an inhibitory signal for mitochondrial fusion, is restored by MHY-1684 treatment, suggesting that this antioxidant may affect the activation and inhibition of mitochondrial dynamics-related signaling and mitochondrial function in response to ROS stress. In conclusion, our finding of the novel compound, MHY-1684, as an ROS scavenger, might provide an effective therapeutic strategy for CPC-based therapy against diabetic cardiomyopathy.

Long-Term Priming by Three Small Molecules Is a Promising Strategy for Enhancing Late Endothelial Progenitor Cell Bioactivities.

Endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs) play a pivotal role in vascular regeneration in ischemic tissues; however, their therapeutic application in clinical settings is limited due to the low quality and quantity of patient-derived circulating EPCs. To solve this problem, we evaluated whether three priming small molecules (tauroursodeoxycholic acid, fucoidan, and oleuropein) could enhance the angiogenic potential of EPCs. Such enhancement would promote the cellular bioactivities and help to develop functionally improved EPC therapeutics for ischemic diseases by accelerating the priming effect of the defined physiological molecules. We found that preconditioning of each of the three small molecules significantly induced the differentiation potential of CD34+ stem cells into EPC lineage cells. Notably, long-term priming of OECs with the three chemical cocktail (OEC-3C) increased the proliferation potential of EPCs via ERK activation. The migration, invasion, and tube-forming capacities were also significantly enhanced in OEC-3Cs compared with unprimed OECs. Further, the cell survival ratio was dramatically increased in OEC-3Cs against H2O2-induced oxidative stress via the augmented expression of Bcl-2, a prosurvival protein. In conclusion, we identified three small molecules for enhancing the bioactivities of ex vivo-expanded OECs for vascular repair. Long-term 3C priming might be a promising methodology for EPC-based therapy against ischemic diseases.

Oleuropein attenuates hydrogen peroxide-induced autophagic cell death in human adipose-derived stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with self-renewing properties; thus, transplanting functionally enhanced MSCs might be a promising strategy for cell therapy against ischemic diseases. However, extensive oxidative damage in ischemic tissue affects the cell fate of transplanted MSCs, eventually resulting in cell damage and autophagic cell death. Oleuropein (OLP) is a bioactive compound isolated from olives and olive oil that harbors antioxidant properties. This study aimed to investigate the potential cytoprotective effects of OLP against oxidative stress and autophagic cell death in MSCs. We found that short-term priming with OLP attenuated H2O2-induced apoptosis by regulating the pro-apoptotic marker Bax and the anti-apoptotic markers Bcl-2 and Mcl-1. Notably, OLP inhibits H2O2 -induced autophagic cell death by modulating autophagy-related death signals, including mTOR (mammalian target of rapamycin), ULK1 (unc-51 like autophagy activating kinase 1), Beclin-1, AMPK (AMP-activated protein kinase), and LC3 (microtubule-associated protein 1a/1b-light chain 3). Our data suggest that OLP might reduce H2O2-induced autophagy and cell apoptosis in MSCs by regulating both the AMPK-ULK axis and the Bcl-2-Mcl-1 axis. Consequently, short-term cell priming with OLP might enhance the therapeutic effect of MSCs against ischemic vascular diseases, which provides an important potential improvement for emerging therapeutic strategies.

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation.

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.

Adequate concentration of B cell leukemia/lymphoma 3 (Bcl3) is required for pluripotency and self-renewal of mouse embryonic stem cells via downregulation of Nanog transcription.

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the NF-κB family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity. [BMB Reports 2018; 51(2): 92-97].

Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells.

Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway.

High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker.

Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

Comparison of putative circulating cancer stem cell detection between the hepatic portal system and peripheral blood in colorectal cancer patients.

PURPOSE: The present pilot study was conducted to detect putative cancer stem cell (CSC) from the hepatic portal system and peripheral blood in the colorectal cancer patients and to compare them to healthy donor and diverticulitis patients. METHODS: Laboratory study was performed to identify the expression of cell surface markers, epithelial cell adhesion molecule (EpCAM), cytokeratin (CK) 18, CK20, CD44, and CD133, on several colon cancer cell lines. Clinical pilot study was conducted to detect putative circulating CSC as EpCAM(+)CD133(+) cell in colorectal cancer (n = 10), diverticulitis (n = 5), and four healthy donors, by using flow cytometry. Blood was drawn from the hepatic portal system and peripheral vein. RESULTS: On laboratory study, EpCAM was expressed in whole colon cancer cell lines, and CD44 and CD133 were simultaneously expressed in 50% of the cell lines with stemness phenotype, but CK18 and CK20 were not expressed in most of the cell lines. On clinical study, the mean EpCAM(+)CD133(+) cell counts of 11.6/10(5) in the hepatic portal system were somewhat lower than 15.4/10(5) in peripheral vein (P = 0.241). As for diverticulitis patients, EpCAM(+)CD133(+) cells were also detected to have steeper dropped to near zero, after the surgery. CONCLUSION: The numbers of putative CSC were not statistically different between the detection sites of the portal vein and peripheral vein in the colon cancer patients. Therefore, we may not have benefitted by getting the cells from the hepatic portal system. In addition, the CD133(+)EpCAM(+) cells in the colon cancer patients might contain normal stem cells from cancer inflammation similar to diverticulitis.

Cross talk with hematopoietic cells regulates the endothelial progenitor cell differentiation of CD34 positive cells.

INTRODUCTION: Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear. METHODS: In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34+ and CD34- cells, and determined the optimal concentrations of CD34+ cells and CD34- cells for spindle-shaped EPC differentiation. RESULTS: Functionally, the coculture of CD34+ and CD34- cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34+ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34+ and CD34- cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3+) markedly accelerated the early EPC status of CD34+ cells, while macrophages (CD11b+) or megakaryocytes (CD41+) specifically promoted large EPC colonies. CONCLUSION: Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34+ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.

Regular exercise training increases the number of endothelial progenitor cells and decreases homocysteine levels in healthy peripheral blood.

Endothelial progenitor cells (EPCs) are known to play an important role in the repair of damaged blood vessels. We used an endothelial progenitor cell colony-forming assay (EPC-CFA) to determine whether EPC numbers could be increased in healthy individuals through regular exercise training. The number of functional EPCs obtained from human peripheral blood-derived AC133 stem cells was measured after a 28-day regular exercise training program. The number of total endothelial progenitor cell colony-forming units (EPC-CFU) was significantly increased compared to that in the control group (p=0.02, n=5). In addition, we observed a significant decrease in homocysteine levels followed by an increase in the number of EPC-CFUs (p=0.04, n=5), indicating that the 28-day regular exercise training could increase the number of EPC colonies and decrease homocysteine levels. Moreover, an inverse correlation was observed between small-endothelial progenitor cell colony-forming units (small-EPC-CFUs) and plasma homocysteine levels in healthy men (r=-0.8125, p=0.047). We found that regular exercise training could increase the number of EPC-CFUs and decrease homocysteine levels, thus decreasing the cardiovascular disease risk in men.

Modulation of Human Cardiac Progenitors via Hypoxia-ERK Circuit Improves their Functional Bioactivities.

Recent accumulating studies have reported that hypoxic preconditioning during ex vivo expansion enhanced the self-renewal or differentiation of various stem cells and provide an important strategy for the adequate modulation of oxygen in culture conditions, which might increase the functional bioactivity of these cells for cardiac regeneration. In this study, we proposed a novel priming protocol to increase the functional bioactivity of cardiac progenitor cells (CPCs) for the treatment of cardiac regeneration. Firstly, patient-derived c-kit(+) CPCs isolated from the atrium of human hearts by enzymatic digestion and secondly, pivotal target molecules identifi ed their differentiation into specific cell lineages. We observed that hCPCs, in response to hypoxia, strongly activated ERK phosphorylation in ex vivo culture conditioning. Interestingly, pre-treatment with an ERK inhibitor, U0126, significantly enhanced cellular proliferation and tubular formation capacities of CPCs. Furthermore, we observed that hCPCs efficiently maintained the expression of the c-kit, a typical stem cell marker of CPCs, under both hypoxic conditioning and ERK inhibition. We also show that hCPCs, after preconditioning of both hypoxic and ERK inhibition, are capable of differentiating into smooth muscle cells (SMCs) and cardiomyocytes (CMs), but not endothelial cells (ECs), as demonstrated by the strong expression of α-SMA, Nkx2.5, and cTnT, respectively. From our results, we conclude that the functional bioactivity of patient-derived hCPCs and their ability to differentiate into SMCs and CMs can be effi ciently increased under specifically defined culture conditions such as shortterm hypoxic preconditioning and ERK inhibition.

Regulation of ROS-independent ERK signaling rescues replicative cellular senescence in ex vivo expanded human c-kit-positive cardiac progenitor cells.

BACKGROUNDS: Although the rescue of cellular senescence during ex vivo expansion of human-derived cardiac progenitor cells (hCPC) is critical for the application of autologous stem cell therapy in cardiovascular disease, the underlying molecular pathways during replicative senescence in hCPC have not been fully defined. Thus, we examined whether the regulation of mitogen-activated protein kinases activation could facilitate the recovery of human c-kit-positive hCPCs (hCPC(c-kit+)) and whether senescence is reactive oxygen species (ROS)-dependent or -independent. METHODS AND RESULTS: To investigate the molecular pathways of replicative cellular senescence, we first evaluated cellular senescence in ex vivo-expanded hCPC(c-kit+) by using senescence-associated ß-galactosidase (SA-ß-gal) activity with enlarged cytoplasm and observed increased expression of cell senescence-related pivotal molecules, including TP53, cleavage Mdm2 (cMdm2), and Mdm2. Unexpectedly, we found that the extracellular signal-regulated kinase (ERK) was markedly activated in aged hCPC(c-kit+), with reduced proliferative activity. SA-ß-gal activity and cytoplasm size in senescent hCPC(c-kit+) were significantly reduced, with reduced TP53 and cMdm2 expression after treatment with a specific ERK inhibitor (U0126). We examined whether the signaling in ERK inhibitory rescue of hCPC(c-kit+) senescence is ROS-dependent. Interestingly, the increased ROS level was not changed after treatment with a specific ERK inhibitor. Similarly, the increased expression levels of endogenous antioxidant enzymes, e.g., peroxiredoxin (Prdx)-1 and 2, in senescent hCPC(c-kit+) were not changed after treatment with a specific ERK inhibitor. CONCLUSIONS: From the above results, we conclude that the specific inhibition of ERK during cellular senescence might rescue bioactivities of senescent hCPC(c-kit+) in a ROS-independent manner.

Inhibitory effect of glyceollins on vasculogenesis through suppression of endothelial progenitor cell function.

SCOPE: Endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells, and have the ability to differentiate into mature endothelial cells and contribute to neovascularization. Glyceollins are a type of phytoalexin produced in soybeans under stress conditions. The aim of this study is to determine the effect of glyceollin treatment on EPCs during early tumor vasculogenesis. METHODS AND RESULTS: We found that glyceollin treatment significantly decreased the number of EPC colony-forming units in human cord blood-derived AC133⁺ cells and mouse bone-marrow-derived c-Kit⁺/Sca-1⁺/Lin⁻ cells. Glyceollin treatment diminished the number of lineage-committed EPC cells in a dose-dependent manner (1-20 µM). Glyceollin treatment inhibited EPC migration, tube formation and the mRNA expression of angiopoietin-1 (Ang-1), Tie-2, stromal-derived factor-1 (SDF-1), C-X-C-chemokine receptor-4 (CXCR4), and endothelial nitric oxide synthase (eNOS) in cultured EPCs. Glyceollin treatment suppressed activation of Akt, Erk, and eNOS induced by SDF-1α or vascular endothelial growth factor (VEGF). Treatment with 10 mg/kg glyceollins significantly reduced the number of tumor-induced circulating EPCs and the incorporation of EPCs into neovessels in bone marrow transplanted mice. CONCLUSION: These results suggest that glyceollins inhibit the function of EPCs in tumor neovascularization. Glyceollins from soybean elicitation could be beneficial in prevention of cancer development via vasculogenesis.

Identification of mouse colony-forming endothelial progenitor cells for postnatal neovascularization: a novel insight highlighted by new mouse colony-forming assay.

INTRODUCTION: Endothelial progenitor cells (EPCs) play a critical role in restoration of ischemic diseases. However, the actual status of EPC development and the mechanisms of EPC dysfunctions in patients with various ischemic diseases remain unknown. METHODS: To investigate the detailed function of EPCs in experimental murine models, we have established an EPC colony forming assay (EPC-CFA) in murine EPCs. The abilities of murine EPCs in differentiation, adhesive capacity, proliferative potency, and transplantation in vitro and in vivo were then examined. RESULTS: Peripheral blood mononuclear cells (PB-MNCs), bone marrow mononuclear cells (BM-MNCs) or bone marrow c-Kit+/Sca-1+ lineage negative (BM-KSL) cells differentiated into two types of EPC colony forming units (EPC-CFUs), large sized EPC (large-EPC)-CFUs and small sized EPC (small-EPC)-CFUs. Gene expression analysis demonstrated that both EPC-CFU-derived cells expressed eNOS, Flk-1 and VE-cadherin, markers of endothelial cells (ECs), although the small-EPCs derived from small-EPC-CFU were higher in number and showed more immature features (higher population of KSL cells). Functionally, the large-EPCs derived from large-EPC-CFU had higher adhesive capacity but lower proliferative potency than small-EPCs, showing improved tubular forming capacity and incorporation potency into primary EC-derived tube formation. Importantly, hindlimb ischemia increased the frequencies of large-EPC-CFUs differentiated from PB-MNCs and bone marrow. Actually, transplantation of large-EPCs into ischemic hindlimb enhanced neovascularization in hindlimb ischemia model, although small-EPCs or murine ECs did not, suggesting that large-EPC-CFUs might play an important role in restoration of ischemic diseases. CONCLUSIONS: We demonstrated, using a murine ischemia model, that the EPC-CFA could be a useful way to investigate the differentiation levels of murine EPCs, further providing a crucial clue that large-EPC-CFU status may be more functional or effective EPCs to promote neovascularization.

Amine-enriched surface modification facilitates expansion, attachment, and maintenance of human cardiac-derived c-kit positive progenitor cells.

BACKGROUND: Stem cells have a low expansion rate and are difficult to maintain in vitro. To overcome the problems of cardiovascular regeneration, we developed a novel method of stem cell cultivation in culture vessels with amine and carboxyl coatings. METHODS AND RESULTS: We isolated cardiac stem/progenitor cells from infant-derived heart tissue by using c-kit antibody (human cardiac-derived c-kit positive progenitor cells; hCPC(c-kit+)); the cells differentiated into endothelial cells, smooth muscle cells, and cardiomyocytes. To characterize the effect of surface modification on hCPC(c-kit+) expansion, cellular attachment, c-kit expression maintenance, and cardiomyocyte differentiation, we tested hCPC(c-kit+) cultured on non-coated (control), amine-coated (amine), and carboxyl-coated (carboxyl) vessels. Ex vivo proliferation, c-kit maintenance, and cellular attachment were significantly enhanced in the amine group. The amine coating also increased procollagen type I (pro-COL1) expression and increased phosphorylation signals, such as focal adhesion kinase (FAK) and cytosolic Src, as well as enhanced ERK/CDK2 signaling. In addition, there was significant downregulation of the stress signal transducer, JNK, in the amine group. However, cardiomyogenesis remained unchanged in the control, amine, and carboxyl groups. CONCLUSIONS: Although surface modifications had no effect on early induction cardiomyogenesis, amine-enriched surface modification may increase hCPC(c-kit+) expansion. The amine-enriched surface improved cellular proliferation and attachment during ex vivo hCPC(c-kit+) expansion, possibly by modulating intracellular signal transducers.