Search

The fibroblast growth factor (FGF) family has various biological functions, including cell growth, tissue regeneration, embryonic development, metabolism, and angiogenesis. In the case of hair growth, several members of the FGF family, such as FGF1 and FGF2, are involved in hair growth, while FGF5 has the opposite effect. In this study, the regulation of the hair growth cycle by FGF12 was investigated. To observe its effect, the expression of FGF12 was downregulated in mice and outer root sheath (ORS) by siRNA transfection, while FGF12 overexpression was carried out using FGF12 adenovirus. For the results, FGF12 was primarily expressed in ORS cells with a high expression during the anagen phase of hair follicles. Knockdown of FGF12 delayed telogen-to-anagen transition in mice and decreased the hair length in vibrissae hair follicles. It also inhibited the proliferation and migration of ORS cells. On the contrary, FGF12 overexpression increased the migration of ORS cells. FGF12-overexpressed ORS cells induced the telogen-to-anagen transition in the animal model. In addition, FGF12 overexpression regulated the expression of PDGF-CC, MDK, and HB-EGF, and treatment of these factors exhibited hair growth promotion. Altogether, FGF12 promoted hair growth by inducing the anagen phase of hair follicles, suggesting the potential for hair loss therapy.

Diabetic retinopathy is the leading cause of blindness which is associated with excessive angiogenesis. Using the structure of wondonin marine natural products, we previously created a scaffold to develop a novel type of antiangiogenesis agent that possesses minimized cytotoxicity. To overcome its poor pharmaceutical properties, we further modified the structure. A new scaffold was derived in which the stereogenic carbon was changed to nitrogen and the 1,2,3-triazole ring was replaced by an alkyl chain. By comparing the bioactivity versus cytotoxicity, compound 31 was selected, which has improved aqueous solubility and an enhanced selectivity index. Mechanistically, 31 suppressed angiopoietin-2 (ANGPT2) expression induced by high glucose in retinal cells and exhibited in vivo antiangiogenic activity in choroidal neovascularization and oxygen-induced retinopathy mouse models. These results suggest the potential of 31 as a lead to develop antiangiogenic small-molecule drugs to treat diabetic retinopathy and as a chemical tool to elucidate new mechanisms of angiogenesis.

Loss of BMP (bone morphogenic protein) signaling induces a phenotype switch of pulmonary arterial smooth muscle cells (PASMCs), which is the pathological basis of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Here, we identified FGF12 (fibroblast growth factor 12) as a novel regulator of the BMP-induced phenotype change in PASMCs and elucidated its role in pulmonary vascular remodeling during PAH development. Using murine models of PAH and lung specimens of patients with PAH, we observed that FGF12 expression was significantly reduced in PASMCs. In human PASMCs, FGF12 expression was increased by canonical BMP signaling. FGF12 knockdown blocked the antiproliferative and prodifferentiation effect of BMP on human PASMCs, suggesting that FGF12 is required for the BMP-mediated acquisition of the quiescent and differentiated PASMC phenotype. Mechanistically, FGF12 regulated the BMP-induced phenotype change by inducing MEF2a (myocyte enhancer factor 2a) phosphorylation via p38MAPK signaling, thereby modulating the expression of MEF2a target genes involved in cell proliferation and differentiation. Furthermore, we observed that TG (transgenic) mice with smooth muscle cell–specific FGF12 overexpression were protected from chronic hypoxia–induced PAH development, pulmonary vascular remodeling, and right ventricular hypertrophy. Consistent with the in vitro data using human PASMCs, FGF12 TG mice showed increased MEF2a phosphorylation and a substantial change in MEF2a target gene expression, compared with the WT (wild type) controls. Overall, our findings demonstrate a novel BMP/FGF12/MEF2a pathway regulating the PASMC phenotype switch and suggest FGF12 as a potential target for the development of therapeutics for ameliorating pulmonary vascular remodeling in PAH.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are used for the treatment of type 2 diabetes mellitus (DM). Recent studies have shown that beyond their effect in lowing glucose, DPP-4 inhibitors mitigate DM-related microvascular complications, such as diabetic retinopathy. However, the mechanism by which pathological retinal neovascularization, a major clinical manifestation of diabetic retinopathy, is inhibited is unclear. This study sought to examine the effects of evogliptin, a potent DPP-4 inhibitor, on pathological retinal neovascularization in mice and elucidate the mechanism by which evogliptin inhibits angiogenesis mediated by vascular endothelial growth factor (VEGF), a key factor in the vascular pathogenesis of proliferative diabetic retinopathy (PDR). In a murine model of PDR, an intravitreal injection of evogliptin significantly suppressed aberrant retinal neovascularization. In human endothelial cells, evogliptin reduced VEGF-induced angiogenesis. Western blot analysis showed that evogliptin inhibited the phosphorylation of signaling molecules associated with VEGF-induced cell adhesion and migration. Moreover, evogliptin substantially inhibited the VEGF-induced activation of adenosine 5′-diphosphate ribosylation factor 6 (Arf6), a small guanosine 5′-triphosphatase (GTPase) that regulates VEGF receptor 2 signal transduction. Direct activation of Arf6 using a chemical inhibitor of Arf-directed GTPase-activating protein completely abrogated the inhibitory effect of evogliptin on VEGF-induced activation of the angiogenic signaling pathway, which suggests that evogliptin suppresses VEGF-induced angiogenesis by blocking Arf6 activation. Our results provide insights into the molecular mechanism of the direct inhibitory effect of the DPP-4 inhibitor evogliptin on pathological retinal neovascularization. In addition to its glucose-lowering effect, the antiangiogenic effect of evogliptin could also render it beneficial for individuals with PDR., Retinal disease: How anti-diabetic drug can help eyes Pathological retinal angiogenesis, the damaging formation of new blood vessels in the retina, which is associated with various diseases including diabetes, could be reduced using the anti-diabetic drug evogliptin to inhibit the effects of a vascular growth factor. Researchers in South Korea led by Wonhee Suh and Koung Li Kim at Chung-Ang University in Seoul investigated the molecular mechanism underlying evogliptin’s effects. In studies using mice and cultured human cells they found that evogliptin inhibited the activation of signaling molecules that mediate the effects of vascular endothelial growth factor. They also identified an enzyme in the signaling pathway that is directly inhibited by evogliptin. The results offer molecular level insights into the additional benefit gained from using evogliptin to treat diabetes, distinct from the drug’s established effects in lowering blood glucose.

Objective: Aberrant neovascularization is a leading cause of blindness in several eye diseases, including age-related macular degeneration and proliferative diabetic retinopathy. The identification of key regulators of pathological ocular neovascularization has been a subject of extensive research and great therapeutic interest. Here, we explored the previously unrecognized role of cKIT and its ligand, SCF (stem cell factor), in the pathological ocular neovascularization process. Approach and Results: Compared with normoxia, hypoxia, a crucial driver of neovascularization, caused cKIT to be highly upregulated in endothelial cells, which significantly enhanced the angiogenic response of endothelial cells to SCF. In murine models of pathological ocular neovascularization, such as oxygen-induced retinopathy and laser-induced choroidal neovascularization models, cKIT and SCF expression was significantly increased in ocular tissues, and blockade of cKIT and SCF using cKit mutant mice and anti-SCF neutralizing IgG substantially suppressed pathological ocular neovascularization. Mechanistically, SCF/cKIT signaling induced neovascularization through phosphorylation of glycogen synthase kinase-3β and enhancement of the nuclear translocation of β-catenin and the transcription of β-catenin target genes related to angiogenesis. Inhibition of β-catenin-mediated transcription using chemical inhibitors blocked SCF-induced in vitro angiogenesis in hypoxia, and injection of a β-catenin agonist into cKit mutant mice with oxygen-induced retinopathy significantly enhanced pathological neovascularization in the retina. Conclusions; Our data reveal that SCF and cKIT are promising novel therapeutic targets for treating vision-threatening ocular neovascular diseases.

In a previous study, we have demonstrated that S-methylmethionine sulfonium (SMMS) confers wound-healing and photoprotective effects on the skin, suggesting that SMMS can be used as a cosmetic raw material. However, it has an unpleasant odor. Therefore, in the present study, we synthesized odor-free SMMS derivatives by eliminating dimethyl sulfide, which is the cause of the unpleasant odor and identified two derivatives that exhibited skin-protective effects: one derivative comprised (2S,4S)- and (2R,4S)-2-phenylthiazolidine-4-carboxylic acid and the other comprised (2S,4R)-, (2S,4S)-, (2R,4R)-, and (2R,4S)-2-phenyl-1,3-thiazinane-4-carboxylic acid. We performed in vitro proliferation assays using human dermal fibroblasts (hDFs) and an immortalized human keratinocyte cell line (HaCaT). The two SMMS derivatives were shown to increase hDF and HaCaT cell proliferation as well as improve their survival by protecting against ultraviolet exposure. Moreover, the derivatives regulated the expression of collagen type I and MMP mRNAs against ultraviolet exposure in hDFs, suggesting that these derivatives can be developed as cosmetic raw materials.

The wet type of age-related macular degeneration (AMD) accompanies the subfoveal choroidal neovascularization (CNV) caused by the abnormal extension or remodeling of blood vessels to the macula and retinal pigment epithelium (RPE). Vascular endothelial growth factor (VEGF) is known to play a crucial role in the pathogenesis of the disease. In this study, we tried to repurpose an investigational anticancer drug, rivoceranib, which is a selective inhibitor of VEGF receptor-2 (VEGFR2), and evaluate the therapeutic potential of the drug for the treatment of wet-type AMD in a laser-induced CNV mouse model using microsphere-based sustained drug release formulations. The PLGA-based rivoceranib microsphere can carry out a sustained delivery of rivoceranib for 50 days. When administered intravitreally, the sustained microsphere formulation of rivoceranib effectively inhibited the formation of subfoveal neovascular lesions in mice.

The Src kinase family (SKF) includes non-receptor tyrosine kinases that interact with many cellular cytosolic, nuclear and membrane proteins, and is involved in the progression of cellular transformation and oncogenic activity. However, there is little to no evidence on the effect of SKF or its inhibitors on melanogenesis. Therefore, the present study investigated whether C-terminal Src kinase inhibition can induce melanogenesis and examined the associated signaling pathways and mRNA expression of melanogenic proteins. First, whether stimulators of melanogenesis, such as ultraviolet B and α-melanocyte-stimulating hormone, can dephosphorylate Src protein was evaluated, and the results revealed that SU6656 and PP2 inhibited the phosphorylation of Src in G361 cells. Src inhibition by these chemical inhibitors induced melanogenesis in G361 cells and upregulated the mRNA expression levels of melanogenesis-associated genes encoding microphthalmia-associated transcription factor, tyrosinase-related protein 1 (TRP1), TRP2, and tyrosinase. In addition, Src inhibition by small interfering RNA induced melanogenesis and upregulated the mRNA expression levels of melanogenesis-associated genes. As the p38 mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element binding (CREB) pathways serve key roles in melanogenesis, the present study further examined whether Src mediates melanogenesis via these pathways. As expected, Src inhibition via SU6656 or PP2 administration induced the phosphorylation of p38 or CREB, as determined by western blotting analysis, and increased the levels of phosphorylated p38 or CREB, as determined by immunofluorescence staining. In addition, the induced pigmentation and melanin content of G361 cells by Src inhibitors was significantly inhibited by p38 or CREB inhibitors. Taken together, these data indicate that Src is associated with melanogenesis, and Src inhibition induces melanogenesis via the MAPK and CREB pathways in G361 cells.

It has previously been demonstrated that hypoxia has diverse stimulatory effects on adipose‑derived stem cells (ASCs), however, metabolic responses under hypoxia remain to be elucidated. Thus, the present study aimed to investigate the glucose uptake and metabolism of ASCs under hypoxic conditions, and to identify the underlying molecular mechanisms. ASCs were cultured in 1% oxygen, and experiments were conducted in vitro. As determined by proteomic analysis and western blotting, GAPDH and enolase 1 (ENO1) expression were upregulated under hypoxia. In addition, lactate production was significantly increased, and mRNA levels of glycolytic enzymes, including GAPDH, ENO1, hexokinase 2 (HK2), and lactate dehydrogenase α (LDHα) were upregulated. Hypoxia‑inducible factor 1‑α (HIF‑1α) expression was increased as demonstrated by western blotting, and a pharmacological inhibitor of HIF‑1α significantly attenuated hypoxia‑induced lactate production and expression of glycolytic enzymes. It was also observed that hypoxia significantly increased glucose uptake in ASCs, and glucose transporter (GLUT)1 and GLUT3 expression were upregulated under hypoxia. Pharmacological inhibition of the HIF‑1α signaling pathways also attenuated hypoxia‑induced GLUT1 and GLUT3 expression. These results collectively indicate that hypoxia increases glucose uptake via GLUT1 and GLUT3 upregulation, and induces lactate production of ASCs via GAPDH, ENO1, HK2, and LDHα. Furthermore, HIF‑1α is involved in glucose uptake and metabolism of ASCs.

Successful cellular delivery of synthetic siRNA depends mainly on the capability of a carrier to form a stable complex with siRNA, which can provide efficient protection of the siRNA from enzyme-mediated degradation and improved cellular uptake. However, due to its short length and rigid structure, cellular delivery of siRNA is often not as efficient as that of plasmid DNA using conventional cationic polymer- and lipid-based carriers. Herein, we synthesized a dendritic gold nanoparticle (Au@MC)-based siRNA delivery system, which provides efficient protection of siRNA and improved cellular uptake. The Au@MC can be readily synthesized from a block copolymer micelle template with a dendritic structure. Au@MC can efficiently form a stable complex with the short and rigid siRNA by localizing it in the space between the branches of the Au@MC. The stability and cellular uptake efficiency were significantly influenced by the structural features of Au@MC, such as size, surface charge, and gap width between the branches. A selected Au@MC/siRNA formulation could successfully achieve highly efficient siRNA transfection in the absence and presence of serum proteins without significant cell toxicity, suggesting the formulation as a potential candidate for siRNA-based clinical gene therapy.

Duplex RNA harboring the 50-terminal triphosphate RNA is hypothesized to not only execute selective gene silencing via RNA interference, but also induce type I interferon (IFN) through activation of the ret- inoic acid inducible gene I (RIG-I). We evaluated gene silencing efficacy of the shRNA containing 5 0 -tri- phosphate (3p-shRNA) targeting the hepatitis C virus (HCV) RNA genome in hepatic cells. Gene silencing efficacy of the 3p-shRNA was diminished due to the presence of the 5 0 -triphosphate moiety in shRNA, whereas the shRNA counterpart without 5 0-triphosphate (HO-shRNA) showed a strong antiviral activity without significant induction of type I IFN in the cells. 3p-shRNA was observed to be a better acti- vator of the RIG-I signaling than the HO-shRNA with an elevated induction of type I IFN in cells that express RIG-I. Taken together, we suggest that competition for the duplex RNA bearing 5 0 -triphosphate between RIG-I and RNA interference factors may compromise efficacy of selective gene silencing.

Purpose Vascular endothelial growth factor (VEGF) signaling via VEGF receptor 2 (VEGFR2) plays a crucial role in pathologic ocular neovascularization. In this study, we investigated the antiangiogenic effect of apatinib, a pharmacologic inhibitor of VEGFR2 tyrosine kinase, against oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) in mice. Methods Western blotting and in vitro angiogenesis assays were performed using human retinal microvascular endothelial cells (HRMECs). OIR was induced in neonatal mice by exposure to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17. Experimental CNV was induced in mice using laser photocoagulation. Apatinib was intravitreally and orally administered to mice. Neovascularization and phosphorylation of VEGFR2 were evaluated by immunofluorescence staining. Results Apatinib inhibited VEGF-mediated activation of VEGFR2 signaling and substantially reduced VEGF-induced proliferation, migration, and cord formation in HRMECs. A single intravitreal injection of apatinib significantly attenuated retinal or choroidal neovascularization in mice with OIR or laser injury-induced CNV, respectively. Retinal or choroidal tissues of the eyes treated with apatinib exhibited substantially lower phosphorylation of VEGFR2 than those of controls injected with vehicle. Intravitreal injection of apatinib did not cause noticeable ocular toxicity. Moreover, oral administration of apatinib significantly reduced laser-induced CNV in mice. Conclusions Our study demonstrates that apatinib inhibits pathologic ocular neovascularization in mice with OIR or laser-induced CNV. Apatinib may, therefore, be a promising drug for the prevention and treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.

Purpose Vascular endothelial growth factor (VEGF) is a principal mediator of pathological ocular neovascularization, which is the leading cause of blindness in various ocular diseases. As Src, a non-receptor tyrosine kinase, has been implicated as one of the major signaling molecules in VEGF-mediated neovascularization, the present study aimed to investigate whether dasatinib, a potent Src kinase inhibitor, could suppress pathological ocular neovascularization in murine models of oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). Methods Tube formation, scratch wounding migration, and cell proliferation assays were performed to measure the inhibitory effect of dasatinib on VEGF-induced angiogenesis in human retinal microvascular endothelial cells. Murine models of OIR and laser-induced CNV were used to assess the preventive effect of an intravitreal injection of dasatinib on pathological neovascularization in the retina and choroid. Neovascularization and Src phosphorylation were evaluated with immunofluorescence staining. Results Dasatinib efficiently inhibited VEGF-induced endothelial proliferation, wounding migration, and tube formation. In mice with OIR and laser injury-induced CNV, eyes treated with a single intravitreal injection of dasatinib exhibited significant decreases in pathological neovascularization compared with that of controls injected with vehicle. The dasatinib-treated OIR mice also showed a decrease in Src phosphorylation in the periretinal tufts. The intravitreal injection of dasatinib did not cause ocular toxicity at the treatment dose administered. Conclusions These results demonstrated that dasatinib suppressed pathological neovascularization in the mouse retina and choroid. Therefore, dasatinib may be indicated for the treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.

Objective— Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. Approach and Results— We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase–mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF–neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. Conclusions— Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

In this study, in situ forming gelatin hydrogels via horseradish peroxidase (HRP)-catalyzed cross-linking were developed to serve as bioactive wound dressings with suitable tissue adhesive properties to deliver dermal fibroblasts (DFBs). The DFB-encapsulated gelatin hydrogels with different stiffnesses, GH-soft (1.1 kPa) and GH-hard (6.2 kPa), were prepared by controlling the hydrogen peroxide (H2O2) concentrations. The GH-soft hydrogel was capable of facilitating the proliferation of DFBs and the synthesis of extracellular components, as compared to GH-hard hydrogels. In addition, the subcutaneously injected GH-soft hydrogel with bioluminescent reporter cells provided enhanced cell survival and local retention over 14 days. In vivo transplantation of DFB-encapsulated GH-soft hydrogels accelerated wound contraction, and promoted collagen deposition and neovascularization within the incisions performed on mice skin. Therefore, we expect that HRP-catalyzed in situ forming gelatin hydrogels can be useful for local delivery of cells with high viability in wounds, which holds great promise for advancing wound healing technologies and other tissue engineering applications.

Tcea3 is present in high concentrations in mouse embryonic stem cells (mESCs) and functions to activate Lefty1, a negative regulator of Nodal signaling. The Nodal pathway has numerous biological activities, including mesoderm induction and patterning in early embryogenesis. Here, we demonstrate that the suppression of Tcea3 in mESCs shifts the cells from pluripotency into enhanced mesoderm development. Vascular endothelial growth factor A (VEGFA) and VEGFC, major transcription factors that regulate vasculogenesis, are activated in Tcea3 knocked down (Tcea3 KD) mESCs. Moreover, differentiating Tcea3 KD mESCs have perturbed gene expression profiles with suppressed ectoderm and activated mesoderm lineage markers. Most early differentiating Tcea3 KD cells expressed Brachyury-T, a mesoderm marker, whereas control cells did not express the gene. Finally, development of chimeric embryos that included Tcea3 KD mESCs was perturbed.

Identification of differentially expressed genes in angioblasts derived from human embryonic stem cells (hESCs) is of great interest for elucidating the molecular mechanisms underlying human vasculogenesis. The aim of this study was to define hESC-derived angioblasts at the clonal level and to perform comparative transcriptional analysis to characterize their distinct gene expression profiles. In a clonal analysis performed in cell-specific differentiation media, hESC-derived CD34(+)CD31(+) cells were identified as angioblasts in that they exhibited a significantly higher ability to form endothelial cell (EC) and smooth muscle cell (SMC) colonies than CD34(+)CD31(-) and CD34(-) cell populations did. Microarray analysis showed that many genes involved in vascular development and signaling transduction were overexpressed in hESC-derived CD34(+)CD31(+) cells, whereas those related to mitosis, the DNA damage response, and translation were substantially downregulated. In addition, comparative gene expression profiling of hESC-derived CD34(+)CD31(+) cells and human somatic primary vascular cells demonstrated that hESC-derived CD34(+)CD31(+) cells expressed key genes involved in the EC and SMC differentiation processes, which supports the result that hESC-derived CD34(+)CD31(+) cells are bipotent angioblasts. Our results may provide insights into the identity and function of hESC-derived angioblasts and may also facilitate further investigation of the molecular mechanisms regulating human embryonic vasculogenesis.

Background Our goal was to investigate the correlation between the dysregulation of transforming growth factor-β1 (TGF-β1) and cystic medial degeneration in the aortic aneurysmal tissues of in Marfan syndrome (MFS) patients. Although aortic aneurysm in animal models of MFS is related to the dysregulation of TGF-β, it has yet to be determined whether TGF-β dysregulation correlates with pathogenic aneurysmal characteristics in MFS patients. Methods and results Compared with aortic tissue from normal individuals, the medial layers of aortic tissue from MFS patients exhibited profound cystic medial degeneration and cellular apoptosis. These histopathologic changes positively correlated with the extent of TGF-β1 signaling activation (Smad2 phosphorylation) in aneurysmal aortic tissue. In addition, the level of TGF-β1 expression in peripheral blood and aneurysmal aortic tissues was significantly elevated in MFS patients. A significant positive correlation was observed between the plasma level of active TGF-β1 in MFS patients and the severity of cystic medial degeneration and Smad2 phosphorylation in aneurysmal aortic medial layers. Conclusions We found a strong association between the dysregulation of TGF-β1 and aortic pathogenesis in human MFS patients. This suggests that the plasma concentration of TGF-β1 in MFS patients might be a useful biomarker of the progression of aortic aneurysms.

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.

Src kinase signaling is important in the regulation of microvascular barrier function and endothelial hyperpermeability. This study was designed to evaluate the protective effect of dasatinib, a potent Src inhibitor used clinically for the treatment of cancer, against the breakdown of the blood-retinal barrier (BRB) and the retinal vascular leakage caused by vascular endothelial growth factor (VEGF) and diabetes. We examined the effects of dasatinib on VEGF-induced endothelial hyperpermeability and the loss of vascular endothelial (VE)-cadherin, an endothelial junctional protein. Dasatinib inhibited VEGF-induced phosphorylation of Src in human retinal microvascular endothelial cells (HRMECs). In vitro and in vivo vascular permeability assays showed that dasatinib blocked the VEGF-enhanced hyperpermeability of HRMECs and decreased VEGF-mediated retinal vascular leakage in mice. Immunofluorescent staining of VE-cadherin showed that dasatinib abolished the junctional disappearance of VE-cadherin in VEGF-treated HRMECs and murine retinal vasculature. In addition, we examined the protective effect of dasatinib against diabetes-induced retinal vascular leakage in streptozotocin-induced diabetic rats. An intravitreal injection of dasatinib substantially inhibited the development of hyperpermeable retinal vasculature. Our results indicate that dasatinib is a promising agent for the prevention and treatment of diabetes-induced retinal vascular leakage.

In 2006, Shinya Yamanaka first reported that in vitro reprogramming of somatic cells toward pluripotency was achieved by simple induction of specific transcription factors. Induced pluripotent stem cell (iPSC) technology has since revolutionized the ways in which we explore the mechanisms of human diseases and develop therapeutics. Here, I describe the recent advances in human iPSC-based disease modeling and drug discovery and discuss the current challenges. Additionally, I outline potential future applications of human iPSCs in classifying patients based on their response to drugs in clinical trials and elucidating optimal patient-specific therapeutic strategies, which will contribute to reduced attrition rates and the development of precision medicine.

Cardiac stem/progenitor cells can be differentiated into cardiomyocytes in vitro using several differentiation methodologies. However, the methodology of cardiomyogenic induction in human c-kit positive progenitor cells (hCPCsc-kit+) was not fully demonstrated. Thus, the purpose of our study was to directly evaluate each cardiomyocyte induction system using hCPCsc-kit+. In this study, cardiomyocyte induction methodologies were divided into the following three groups; treatment with dexamethasone, 5-azacytidine, and co-treatment with 5-azacytidine and Transforming Growth Factor Beta 1 (TGF-β1), using different serum concentrations [2% or 10% fetal bovine serum (FBS)]. GATA4 and Nkx2-5, cardiac-specific transcription factors, were expressed in our hCPCsckit+. However, the GATA4 and Nkx2-5 expressions were significantly decreased in 10% FBS/cardiomyogenic induction system (p 0.05). GATA4 and Nkx2-5 is crucial roles in cardiac development, thus we considered the low serum conditions more affected in our cardiomyogenic induction system. In addition, c-kit expression decreased significantly during cardiomyogenic differentiation. Importantly, we demonstrated that co-treated with 5-azacytidine and TGF-β1 led to an earlier expression pattern of alpha-sarcomeric actin (α-SA), implying that this cardiomyocyte induction system facilitates early cardiomyocyte differentiation of hCPCsc-kit+. Thus, the present study provides a pivotal cardiomyogenic differentiation methodology using hCPCsc-kit+for basic or clinical research.

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Vasculogenic progenitor cells (VPCs) circulate in the blood and have the ability to differentiate into endothelial cells that make up the lining of blood vessels. Therefore, VPC transplantation is a new strategy for the treatment of ischemic diseases. Because priming/preconditioning of VPCs before transplantation enhances their regenerative potential, the present study investigated whether ent-16α,17-dihydroxy-kauran-19-oic acid (DHK) isolated from Siegesbeckia pubescens could stimulate/activate VPCs in vitro. Therefore, the effect of DHK (1-100 µM concentration) on the proliferation, migration, and tube forming of VPCs was examined in various systems, and related signaling pathways were identified. DHK treatment significantly increased the proliferation, migration, and tube formation of VPCs in a dose-dependent manner. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and Akt was significantly increased by DHK, but chemical inhibitors against ERK1/2 (U0126) and Akt (LY294002) significantly attenuated DHK-enhanced proliferation, migration, and tube formation of VPCs. Collectively, these results indicated that DHK shows promise as a novel VPC primer/activator.

For clinical application of human embryonic stem cells (hESCs), it is critical to develop hESC culture techniques that completely exclude the use of animal feeder cells, mitotic inhibition, and enzyme treatments used in conventional hESC culture systems. Toward this goal, we attempted to maintain hESCs and induced pluripotent stem (iPS) cells on porous membranes (PMs) with proliferative human adipose-derived stromal cells (ASCs) seeded on the bottom surface of inverted PMs. This culture condition will ensure that the two cell types are separate from each other, yet retain the ability to interact through the pores of the membrane. We found that hESCs and iPS cells can be maintained stably and mechanically transferred without the need for enzyme treatment. In addition, the pluripotency of hESCs and iPS cells was stably maintained, as evidenced by immunostaining of Oct4, SSEA3/4 and TRA-1-60 as well as RT-PCR analyses of Nanog, Oct4 and Sox2 expression. Furthermore, hESCs cultured on PMs showed a normal karyotype and in vivo teratoma formation containing all three germ layers.

Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.

Background Recently the potential of myocardial repair by transplantation of autologous bone marrow stem cells has been suggested. Whether the additional intracoronary transplantation of autologous peripheral blood stem cells (PBSC), which were mobilized by granulocyte-colony-stimulating factor (G-CSF), could safely improve myocardial function in patients with acute myocardial infarction (AMI) was investigated. Methods and Results Seventy-three patients with AMI who had successfully undergone percutaneous coronary intervention (PCI) were enrolled in the present prospective nonrandomized open-labeled study. Ten patients with elective PCI received G-CSF for 4 days followed by intracoronary PBSC transplantation. Thirty-two patients with primary PCI and 31 patients with recent AMI and elective PCI served as controls. The left ventricular (LV) function was evaluated using echocardiography and magnetic resonance imaging. G-CSF and intracoronary transplantation of PBSC did not incur any periprocedural myocardial damage. After 6 months, the LV ejection fraction was significantly improved in the cell therapy group. For 2 years of the follow-up period, there was no adverse clinical events, except one asymptomatic in-stent restenosis. However, comparable improvement of the LV ejection fraction was also identified in the primary PCI and elective PCI control groups. Conclusions In the present study, additional intracoronary infusion of PBSC was safe and feasible for the patients with AMI who had undergone PCI, but did not lead to a significant improvement in LV function compared to standard reperfusion treatment. (Circ J 2007; 71: 486 - 494)

Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrin1a messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development. We also show that netrins accelerate neovascularization in an in vivo model of ischemia and that they reverse neuropathy and vasculopathy in a diabetic murine model. We propose that the attractive vascular and neural guidance functions of netrins offer a unique therapeutic potential.

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microg/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

Targeting is one of the primary considerations in designing a specific and efficient gene delivery system. Here, an angiogenic endothelial cell-targeted polymeric gene delivery carrier was developed by conjugating an alpha(v)beta3/alpha(v)beta5 integrin-binding RGD peptide, ACDCRGDCFC, into the cationic polymer polyethyleneimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer. The incorporation of PEG into PEI improved the poor physicochemical properties of PEI-DNA complexes. At a neutral charge ratio, DNA complexes with PEI were polydisperse and substantially aggregated, whereas DNA complexes with PEI-g-1PEG-RGD were homogeneous with 100-200 nm effective diameter. Their surface charge was also significantly reduced due to the charge shielding effect of PEG. However, the extensive grafting of PEI with PEG was shown to inhibit the DNA condensation process, significantly decreasing transfection efficiency. In in vitro transfection experiments with angiogenic endothelial cells, PEI-g-1PEG-RGD showed an approximately fivefold increase in transfection efficiency over PEI, due to an integrin-mediated internalization pathway. PEI-g-1PEG-RGD also exhibited high specificity to angiogenic endothelial cells compared with normal endothelial cells, which was confirmed by in vitro transfection experiments with non-targeting PEI-g-1PEG-RAE in angiostatic endothelial cells.

Marfan syndrome (MFS) is a dominantly inherited connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1) and is characterized by aortic dilatation and dissection, which is the primary cause of death in untreated MFS patients. However, disease progression-associated changes in gene expression in the aortic lesions of MFS patients remained unknown. Using a mouse model of MFS, FBN1 hypomorphic mouse (mgR/mgR), we characterized the aortic gene expression profiles during the progression of the MFS. Homozygous mgR mice exhibited MFS-like phenotypic features, such as fragmentation of elastic fibers throughout the vessel wall and were graded into mgR1-4 based on the pathological severity in aortic walls. Comparative gene expression profiling of WT and four mgR mice using microarrays revealed that the changes in the transcriptome were a direct reflection of the severity of aortic pathological features. Gene ontology analysis showed that genes related to oxidation/reduction, myofibril assembly, cytoskeleton organization, and cell adhesion were differentially expressed in the mgR mice. Further analysis of differentially expressed genes identified several candidate genes whose known roles were suggestive of their involvement in the progressive destruction of aorta during MFS. This study is the first genome-wide analysis of the aortic gene expression profiles associated with the progression of MFS. Our findings provide valuable information regarding the molecular pathogenesis during MFS progression and contribute to the development of new biomarkers as well as improved therapeutic strategies.

We have designed the gene delivery carrier targeted to Molt 4 cells, human leukemia T cells, using monoclonal antibody against leukemia-specific JL1 antigen, anti-JL1 antibody, as a targeting moiety. Anti-JL1 antibody has been proven to bind to JL1 antigen and subsequently be internalized into Molt 4 cells, demonstrating that anti-JL1 antibody has the potential as a targeting ligand for leukemia-specific gene transfer. Anti-JL1 antibody was modified with the heterobifunctional crosslinker, PDPH, at carbohydrate sites and conjugated to thiolated poly- l -lysine (PLL) via disulfide bridges. The composition and antigen binding affinity of antibody–PLL conjugates were analyzed by the amino acid analysis and the flow cytometry, respectively. Antibody–PLL conjugates neutralized pSV-β-galactosidase plasmid DNA at 5:1 weight ratio and condensed into about 200–300-nm complexes. DNA/antibody–PLL complexes were effectively internalized into Molt 4 cells after 4 h incubation at 37°C and showed significantly higher in vitro transfection efficiency than DNA/PLL complexes and DNA/Lipofectin™ formulation due to the targeting effect of receptor-mediated endocytosis induced by anti-JL1 antibody.

This study aimed to gain mechanistic insights into the nature of the pore pathway of fully hydrated human stratum corneum from lag time data obtained using a model polar permeant, urea. Lag times were deduced from transport experiments with human epidermal membranes and with human epidermal membranes after ethanol or chloroform–methanol treatment. A tortuous pore pathway transport model and a `bottleneck' transport model were employed for data analysis, and their appropriateness for the observed data was examined. Important outcomes from the present study with intact and with delipidized stratum corneum were as follows. Long lag times (around 60–800 min) for the transport of urea in human epidermal membranes were generally observed. These results were consistent with an extremely tortuous pore pathway as would be expected if it is associated with the polar/aqueous region of the stratum corneum intercellular lipids (i.e. the bilayers in the intercellular region). The permeability of the stratum corneum increased after ethanol treatment, and, at the same time, the tortuosity decreased but remained relatively high. Chloroform–methanol treatment further increased the permeability and further decreased the tortuosity. Since delipidization by ethanol and chloroform–methanol treatments decreased the tortuosity of the pore pathway, these results suggest that the effectively highly tortuous pathway for polar permeants in stratum corneum may be associated with the polar regions of the intercellular lipids. Untreated skin samples that had high electrical resistance were observed to have longer lag times than those with low resistance; this is consistent with the hypothesis that skin samples of high resistance have less appendage routes or less damage and transport polar permeants predominantly via the tortuous pathways involving the intercellular lipid regions of the stratum corneum. Neither the tortuous pathway transport model alone nor the `bottleneck' transport model alone seems to perfectly represent the experimental data, and a modified model (a hybrid of the two models) has been proposed to be more consistent with the lag time data and the morphology of fully hydrated stratum corneum. The present study has demonstrated the usefulness of lag times obtained with a polar permeant in better understanding the transport mechanisms involved with the pore pathway.

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.

Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCsc-kit+). The two protocols employed enzymatically digested hCPCsc-kit+ (ED-hCPCs) with tissue-expanded hCPCc-kit+ (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.

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.

Aims Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. Methods and results In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. Conclusion In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

Transplantation of human cord blood-derived endothelial progenitor cells (EPCs) is reported to contribute to neovascularization in various ischemic diseases. However, the possible beneficial role and underlying mechanisms in diabetes-impaired wound healing have been less well characterized. In this study, EPC transplantation stimulated keratinocyte and fibroblast proliferation substantially as early as 3 days after injury, leading to significantly accelerated wound closure in streptozotocin-induced diabetic nude mice, compared to PBS control. RT-PCR analysis showed that EPCs secreted various wound healing-related growth factors. Among them, keratinocyte growth factor and platelet-derived growth factor were highly expressed in the EPCs and were present at substantial levels in the EPC-injected dermal tissue. Using EPC-conditioned medium (CM), we found that paracrine factors from EPCs directly exerted mitogenic and chemotactic effects on keratinocytes and fibroblasts. Moreover, injection of EPC-CM alone into the same diabetic wound mice promoted wound healing and increased neovascularization to a similar extent as achieved with EPC transplantation. These results indicate that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their direct paracrine action on keratinocytes, fibroblasts, and endothelial cells, rather than through their physical engraftment into host tissues (vasculogenesis). In addition, EPC-CM was shown to be therapeutically equivalent to EPCs, at least for the treatment of diabetic dermal wounds, suggesting that conditioned medium may serve as a novel therapeutic option that is free from allograft-associated immune rejection concern.

The use of cellular therapy in the treatment of dermal wounds is currently an active area of investigation. Multipotent adult stem cells are an attractive choice for cell therapy because they have a large proliferative potential, the ability to differentiate into different cell types and produce a variety cytokines and growth factors important to wound healing. This review focused on the roles of adult stem cells such as endothelial progenitor cells, bone marrow and adipose-derived mesenchymal stem cells, during dermal wound healing process and their therapeutic potentials for the treatment of chronic wounds, which remain a major clinical problem, especially in diabetic patients.

Rationale : Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized. Objective : To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs). Methods and Results : Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell–derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing. Conclusions : Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell–derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy.

Objective: To evaluate the efficacy of the curcumin-coating stent (CCS) on the inhibition of restenosis in a rabbit iliac artery stent model. Results: Curcumin, pigment naturally acquired from the rhizome of the plant curcuma longa, is known to have antiproliferative, antimigratory, and anti-inflammatory effects. However, it is still unclear that curcumin can inhibit neointimal proliferation of the injured vessel. Methods: Dose-dependent inhibition of cell growth was observed over a dose range from 10 nM to 10 μM. CCS was prepared by a dip-coating method (high-dose: HD, low-dose: LD). The release profile of the HD CCS showed that drug release persisted until day 21. Scanning electron microscopy of the CCS showed an intact surface of the stent even after expansion. To test the efficacy of CCS in vivo, LD CCS, HD CCS, and bare metal stents (BMS) were implanted in random order in one iliac artery (N = 30 arteries) of male New Zealand White rabbits (N = 15). Results: After 28 days, the LD and HD CCS groups had a 43% and 55% reduction in the neointimal area, compared with the BMS group (BMS 3.3 ± 1.0 mm2, LD 1.9 ± 0.8 mm2, and HD 0.9 ± 0.5 mm2, P < 0.05). There appeared to be no cytotoxicity related to curcumin at the indicated doses. Conclusions: Curcumin, a natural compound in the human diet, seems to be a safe and effective candidate drug for use in a drug-eluting stent for the prevention of stent restenosis following angioplasty. © 2009 Wiley-Liss, Inc.

ObjectivesThe goal of this study was to investigate the expression of early growth response-1 (Egr-1), a vascular pathogenic transcription factor, and its potential relationship with tissue factor (TF), a key player during the thrombus formation in the abdominal aortic aneurysm (AAA) wall.BackgroundAlthough intraluminal thrombus is a common finding in human AAA, the molecular mechanism of the thrombus formation has not been studied.MethodsDuring the elective AAA repair, specimens were taken from the thrombus-covered and thrombus-free portions of the aneurysmal wall in each of 16 patients with AAA and analyzed to assess the differential expression of Egr-1 and TF. The proinflammatory and prothrombogenic activities of Egr-1 in vasculature were evaluated in vitro and in vivo by overexpressing it using adenovirus.ResultsThe expression of both Egr-1 and TF was significantly increased in the thrombus-covered wall compared with the thrombus-free wall, in which their up-regulation in the thrombus-covered wall was strongly correlated with each other (p < 0.005, r = 0.717). Adenoviral overexpression of Egr-1 in human vascular smooth muscle and endothelial cells was found to up-regulate the expression of TF and inflammation-related genes. Moreover, Egr-1 overexpression in endothelial cells increased their adhesiveness to monocytes and also substantially promoted the intravascular thrombus formation in vivo, as shown in the inferior vena cava ligation experiment of the rat.ConclusionsThe present study demonstrates the differential up-regulation of Egr-1 in the thrombus-covered wall of human AAA and also suggests its possible contribution to the thrombogenic and inflammatory pathogenesis in human AAA.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures1. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system2,3; however, their role in the mammalian vasculature remains ill defined4–8. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165–induced migration, tube formation and permeability in vitro and VEGF-165–stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.

Objective: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. Methods and results: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. Conclusions: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pattern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each pathway revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.