Search

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.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

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.

Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 (Fbn1) gene, has vascular manifestations including aortic aneurysm, dissection, and rupture. Its vascular pathogenesis is assumed to be attributed to increased transforming growth factor β (TGFβ) signaling and blockade of excessive TGFβ signaling has been thought to prevent dissection and aneurysm formation. Here, we investigated whether galunisertib, a potent small-molecule inhibitor of TGFβ receptor I (TβRI), attenuates aneurysmal disease in a murine model of MFS (Fbn1C1039G/+) and compared the impact of galuninsertib on the MFS-related vascular pathogenesis with that of losartan, a prophylactic agent routinely used for patients with MFS. Fbn1C1039G/+ mice were administered galunisertib or losartan for 8 weeks, and their ascending aortas were assessed for histopathological changes and phosphorylation of Smad2 and extracellular signal-regulated kinase 1/2 (Erk1/2). Mice treated with galunisertib or losartan barely exhibited phosphorylated Smad2, suggesting that both drugs effectively blocked overactivated canonical TGFβ signaling in Fbn1C1039G/+ mice. However, galunisertib treatment did not attenuate disrupted medial wall architecture and only partially decreased Erk1/2 phosphorylation, whereas losartan significantly inhibited MFS-associated aortopathy and markedly decreased Erk1/2 phosphorylation in Fbn1C1039G/+ mice. These data unexpectedly revealed that galunisertib, a TβRI inhibitor, showed no benefits in aneurysmal disease in MFS mice although it completely blocked Smad2 phosphorylation. The significant losartan-induced inhibition of both aortic vascular pathogenesis and Smad2 phosphorylation implied that canonical TGFβ signaling might not prominently drive aneurysmal diseases in MFS mice.

Peroxisome proliferator-activated receptor (PPAR)-α/γ dual agonists have been developed to treat metabolic diseases; however, most of them exhibit side effects such as body weight gain and oedema. Therefore, we developed a novel PPARα/γ dual agonist that modulates glucose and lipid metabolism without adverse effects. We synthesised novel compounds composed of coumarine and chalcone, determined their crystal structures, and then examined their binding affinity toward PPARα/γ. We investigated the expression of PPARα and PPARγ target genes by chemicals in HepG2, differentiated 3T3-L1, and C2C12 cells. We examined the effect of chemicals on glucose and lipid metabolism in db/db mice. Only MD001 functions as a PPARα/γ dual agonist in vitro. MD001 increased the transcriptional activity of PPARα and PPARγ, resulting in enhanced expression of genes related to β-oxidation and fatty acid and glucose uptake. MD001 significantly improved blood metabolic parameters, including triglycerides, free fatty acids, and glucose, in db/db mice. In addition, MD001 ameliorated hepatic steatosis by stimulating β-oxidation in vitro and in vivo. Our results demonstrated the beneficial effects of the novel compound MD001 on glucose and lipid metabolism as a PPARα/γ dual agonist. Consequently, MD001 may show potential as a novel drug candidate for the treatment of metabolic disorders.

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.

// Jin Gu Cho 1, 2 , Seho Park 3 , Chae Hyun Lim 4 , Hong Sook Kim 2 , Seung Yong Song 5 , Tae-Young Roh 4 , Jong-Hyuk Sung 6 , Wonhee Suh 7 , Seok-Jin Ham 4 , Key-Hwan Lim 2 , Sang Gyu Park 2 1 Department of Biomedical Science, CHA University, Sungnam-si, Gyunggi-do, Korea 2 Laboratory for Tracing of Gene Function, Department of Pharmacy, College of Pharmacy, Ajou University, Suwon, Gyunggi-do, Korea 3 Department of Surgery, Yonsei University College of Medicine, Seoul, Korea 4 Division of Integrative Biosciences & Biotechnology, Pohang University of Science & Technology (POSTECH), Pohang, Gyeongbuk, Korea 5 Department of Plastic and Reconstructive Surgery, Yonsei University College of Medicine, Seoul, Korea 6 Department of Pharmacy, College of Pharmacy, Yonsei University, Incheon, Korea 7 Department of Pharmacy, College of Pharmacy, Chung-Ang University, Seoul, Korea Correspondence to: Sang Gyu Park, e-mail: sgpark@ajou.ac.kr Keywords: ZNF224, miR-663a, p53, p21, apoptosis Received: December 22, 2015 Accepted: March 31, 2016 Published: April 20, 2016 ABSTRACT ZNF224 is a Kruppel-associated box-containing zinc-finger protein which represses gene transcription by interacting with various co-repressors. However, its consensus DNA sequences and target genes are not fully identified. In this study, we identified and characterized consensus DNA sequences containing 5‘-CAGC-3’ recognized by ZNF224 through ChIP-sequencing, which further confirmed by ELISA, SPR, qPCR, and luciferase activity assay. ZNF224 increased miR-663a transcription by binding to miR-663a promoter, which in turn binds to 3’ UTR of p53 and p21 to decrease their expression. miR-663a antagonist abolished ZNF224-mediated suppression of p21 and p53, resulting in the enhanced apoptosis by CPT. The analyses using human breast ductal carcinoma tissues exhibited that the expression of ZNF224 and miR-663a was increased in cancer compared to non-cancer region. Consequently, ZNF224 increases cell survival and decreases apoptosis by decreasing the expression of p53 and p21 via miR-663a as a transcriptional activator. Taken together, we identified and characterized DNA binding element of ZNF224, and its target genes, miR-663a, which provides a novel insight in the down-regulation of p21 and p53 via miR-663a by ZNF224 in breast cancer.

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.

Jung Eun Lee,1,* Koung Li Kim,2,* Danbi Kim,2 Yeongju Yeo,2 Hyounkoo Han,3 Myung Goo Kim,1,4 Sun Hwa Kim,4 Hyuncheol Kim,3,5 Ji Hoon Jeong,1,4 Wonhee Suh2 1School of Pharmacy, Sungkyunkwan University, Suwon, 2College of Pharmacy, Chung-Ang University, Seoul, 3Department of Chemical and Biomolecular Engineering, Sogang University, 4Center for Theragnosis Biomedical Research Institute, Korea Institute of Science and Technology (KIST), 5Department of Biomedical Engineering, Sogang University, Seoul, Korea *These authors contributed equally tothis work Abstract: Pathological angiogenesis is one of the major symptoms of severe ocular diseases, including corneal neovascularization. The blockade of vascular endothelial growth factor (VEGF) action has been recognized as an efficient strategy for treating corneal neovascularization. In this study, we aimed to investigate whether nanoparticle-based delivery of apatinib, a novel and selective inhibitor of VEGF receptor 2, inhibits VEGF-mediated angiogenesis and suppresses experimental corneal neovascularization. Water-insoluble apatinib was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro angiogenesis assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles potently inhibited VEGF-induced tube formation, scratch wounding migration, and proliferation of human endothelial cells. In a rat model of alkali burn injury-induced corneal neovascularization, a subconjunctival injection of Apa-HSA-PEG nanoparticles induced a significant decrease in neovascularization compared to that observed with an injection of free apatinib solution or phosphate-buffered saline. An in vivo distribution study using HSA-PEG nanoparticles loaded with fluorescent hydrophobic model drugs revealed the presence of a substantial number of nanoparticles in the corneal stroma within 24 h after injection. These in vitro and in vivo results demonstrate that apatinib-loaded nanoparticles may be promising for the prevention and treatment of corneal neovascularization-related ocular disorders. Keywords: apatinib, corneal neovascularization, nanoparticle, vascular endothelial growth factor

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.

Ji Hoon Jeong,1,* Hong Khanh Nguyen,2,* Jung Eun Lee,1 Wonhee Suh2 1School of Pharmacy, Sungkyunkwan University, Suwon, 2College of Pharmacy, Chung-Ang University, Seoul, Korea *These authors contributed equally tothis work Abstract: Apatinib, a novel and selective inhibitor of vascular endothelial growth factor (VEGF) receptor 2, has been demonstrated recently to exhibit anticancer efficacy by inhibiting the VEGF signaling pathway. Given the importance of VEGF in retinal vascular leakage, the present study was designed to investigate whether apatinib-loaded polymeric nanoparticles inhibit VEGF-mediated retinal vascular hyperpermeability and block diabetes-induced retinal vascular leakage. For the delivery of water-insoluble apatinib, the drug was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro paracellular permeability and transendothelial electric resistance assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles significantly inhibited VEGF-induced endothelial hyperpermeability in human retinal microvascular endothelial cells. In addition, they substantially reduced the VEGF-induced junctional loss and internalization of vascular endothelial-cadherin, a major component of endothelial junction complexes. In vivo intravitreal injection of Apa-HSA-PEG nanoparticles in mice blocked VEGF-induced retinal vascular leakage. These in vitro and in vivo data indicated that Apa-HSA-PEG nanoparticles efficiently blocked VEGF-induced breakdown of the blood–retinal barrier. In vivo experiments with streptozotocin-induced diabetic mice showed that an intravitreal injection of Apa-HSA-PEG nanoparticles substantially inhibited diabetes-induced retinal vascular leakage. These results demonstrated, for the first time, that apatinib-loaded nanoparticles may be a promising therapeutic agent for the prevention and treatment of diabetes-induced retinal vascular disorders. Keywords: permeability, retinal vascular endothelial cells, vascular endothelial growth factor

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.

BACKGROUND AND OBJECTIVES In our previous study, we found that the gene transfer of a potent derivative of cartilage oligomeric matrix protein Angiopoietin-1 (COMP-Ang-1) substantially prevented hypertension, microvascular rarefaction, and target organ damage in spontaneously hypertensive rats (SHRs). The purpose of the present study was to examine the role of nitric oxide (NO) in the therapeutic effects observed after COMP-Ang-1 gene transfer. MATERIALS AND METHODS To exclude the NO-mediated effects in COMP-Ang-1 gene therapy, the SHRs were treated with an NO synthase (NOS) inhibitor, N(w)-nitro-L-arginine methyl ester (L-NAME) before the electrophoretic gene transfer. RESULTS The pretreatment with L-NAME induced a severe and sustained increase in systolic blood pressure (BP) in a LacZ plasmid transferred control SHR. However, the electrophoretic transfer of a COMP-Ang-1 plasmid instead of LacZ plasmid in L-NAME-pretreated SHRs substantially blocked the development of hypertension without any significant difference in comparison with L-NAME-untreated COMP-Ang-1 plasmid transferred groups. In addition, the COMP-Ang-1 plasmid transfer substantially attenuated microvascular rarefaction and arteriole remodeling in the heart and kidney, which might account for the mild histological alterations observed in the COMP-Ang-1 plasmid transferred group, in contrast to the severe fibrosis and necrosis seen in the LacZ plasmid controls. CONCLUSION These therapeutic outcomes of COMP-Ang-1 gene transfer even in NOS inhibited SHRs suggested that the antihypertensive effect of COMP-Ang-1 was not merely secondary to NO-mediated vasorelaxation, but it may be associated with its ability to protect the vascular endothelium probably via an NO-independent mechanism which serves to attenuate microvascular rarefaction and target organ damage, and also to prevent hypertension by reducing peripheral vascular resistance.

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.

Aims The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. Methods and results To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. Conclusion The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.

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)

Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold.

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.

Human induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells into an embryonic stem cell-like state, such that vascular cells differentiated from hiPSCs might be a suitable autologous cell source for vascular regeneration. The goal of this study was to assess whether cotransplantation of endothelial cells (ECs) and smooth muscle cells (SMCs) differentiated from hiPSCs could promote neovascularization and tissue repair in a murine dermal wound model. hiPSCs were differentiated into ECs and SMCs; the differentiated cells displayed cell-specific surface markers. Compared to primary somatic cells, ECs and SMCs, which were differentiated from hiPSCs, strongly cooperated to enhance in vitro tubular network formation. In vivo gel assays in athymic nude mice showed that the coimplantation of differentiated ECs and SMCs significantly increased vascularization, unlike that observed in the case of implantation of differentiated ECs alone. In a murine full-thickness wound model, when compared with the transplantation of primary somatic cells or phosphate-buffered saline, cotransplantation of differentiated ECs and SMCs markedly enhanced neovascularization in injured tissues and accelerated wound healing. These results demonstrate that cotransplantation of hiPSC-derived ECs and SMCs may be feasible as a new autologous cell therapy for neovascularization and tissue repair.