Phenyl and Diaryl Ureas with Thiazolo[5,4‐d]pyrimidine Scaffold as Angiogenesis Inhibitors: Design,Synthesis and Biological Evaluation

Angiogenesis is crucial for tumor growth and inhibition of angiogenesis has been regarded as a promising approach for cancer therapy. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is an important factor in angiogenesis. In this work, a novel series of thiazolo[5,4‐d]pyrimidine derivatives inhibiting angiogenesis were rationally designed and synthesized. Their inhibitory activities against human umbilical vein endothelial cells (HUVEC) were investigated in vitro. 1‐(4‐Fluorophenyl)‐3‐{4‐[(5‐methyl‐2‐phenyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl)amino]phenyl}urea ( 19b ) and 1‐(3‐Fluorophenyl)‐3‐{4‐[(5‐methyl‐2‐phenyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl)amino]phenyl}urea ( 19g ) exhibited the most potent inhibitory effect on HUVEC proliferation (IC₅₀=12.8 and 5.3 μm , respectively). Compound 19g could inhibit the migration of human umbilical vein endothelial cells. These results support the further investigation of these compounds as potent anticancer agents.     

Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT9‐mediated uric acid uptake

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti‐inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA‐induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP‐1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor‐kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose‐dependent manner. In contrast, phloretin significantly attenuated pro‐inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor‐kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA‐induced endothelial injury via a synergic mechanism including direct anti‐inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia‐related cardiovascular diseases.     

Synthesis and Biological Evaluation of Novel Oxazolo[5,4‐d]pyrimidines as Potent VEGFR‐2 Inhibitors

Tumor angiogenesis is mediated by vascular endothelial growth factor receptor (VEGFR) and other protein kinases. Inhibition of these kinases presents an attractive approach for developing anticancer therapeutics. In this work, a series of 2,5,7‐trisubstituted oxazolo[5,4‐d]pyrimidines were synthesized, and their inhibitory activities were investigated against VEGFR‐2 and human umbilical vein endothelial cells (HUVEC) in vitro. Compound 9n exhibited the most potent inhibitory activity with IC₅₀ values of 0.33 and 0.29 μM for VEGFR‐2 kinase and HUVEC, respectively. A further kinase selectivity assay revealed that these compounds exhibit good VEGFR and moderate EGFR inhibitory activities. Docking analysis suggested a common mode of interaction at the ATP‐binding site of VEGFR‐2.     

Proteomic analysis of the secretome of human umbilical vein endothelial cells using a combination of free‐flow electrophoresis and nanoflow LC‐MS/MS

Human umbilical vein endothelial cells are the most widely used in vitro model for endothelial cells. Their secreted proteins, however, have not been comprehensively analysed so far. In this study, we accomplished to map the secretome of human umbilical vein endothelial cells by combining free‐flow electrophoresis with nanoflow LC‐MS/MS. This comprehensive analysis provides a basis for future comparative studies of protein secretion by endothelial cells in response to cardiovascular risk factors and is available on our website http://www.vascular‐proteomics.com .     

The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1

The signal transduction pathway involved in hepatocyte growth factor (HGF)-induced capillary morphogenesis of endothelial cells was investigated. HGF-induced capillary morphogenesis of the murine spleen endothelial cell line MSS31 was inhibited by a Src family kinase inhibitor, PP2. Stable expression of kinase-inactive Src in MSS31 cells inhibited HGF-induced activation of Src as well as capillary morphogenesis. The HGF-induced capillary morphogenesis of human umbilical vein endothelial cells was also inhibited by PP2 and was reduced by the downregulation of Src by small interfering RNA. These results suggest that HGF induces capillary morphogenesis of endothelial cells through Src.     

Diterpenoids and a Diarylheptanoid from Hedychium coronarium with Significant Anti‐Angiogenic and Cytotoxic Activities

Two new labdane diterpenoids, namely hedycoronals A and B ( 1 and 2 , resp.), were isolated from the rhizomes of Hedychium coronarium, together with eight known diterpenoids, 4 – 11 , and a known diarylheptanoid, 3 . The structures of 1 and 2 were established by detailed interpretation of their 1D‐ and 2D‐NMR spectra and HR‐ESI‐MS data. Inhibitory activities against human umbilical vein endothelial cells (HUMECs) proliferation and cytotoxic activities against four cancer cell lines were assessed for all the isolates. Most of these metabolites showed moderate or potent cytotoxic activities against four cancer cell lines. Moreover, compounds 3 and 8 exhibited promising inhibitory activities against HUMECs with the IC₅₀ values of 6.4 to 3.3 μM .     

A New 1,5‐Dihydroxy‐4‐methoxyisoquinoline from Scolopendra subspinipes mutilans

A new isoquinoline, 1,5‐dihydroxy‐4‐methoxyisoquinoline ( 1 ), was obtained from Scolopendra subspinipes mutilans. Compound 1 showed moderate cytotoxicity on tumour cells with IC₅₀ values ranging from 13 to 26 μm against five esophageal squamous cancer cells whereas low cytotoxicity against normal human esophageal epithelial cells. Isoquinoline ring oxidized at C(1), C(4), and C(5) can enhance its cytotoxicity. In addition, compound 1 showed potent inhibitory effect (inhibition rate > 50% at 13 μm ) on cell migration in human umbilical vein endothelial cells. This article mainly studies the structure and activity of 1 , and more modification of 1 as a potential anticancer agent.     

Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind ¹²⁵ I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.     

Acheron regulates vascular endothelial proliferation and angiogenesis together with Id1 during wound healing

RNA binding protein acheron has proved to be either the mediator of integrin‐extracellular matrix interactions or the regulatory factor that participates in vertebrate development, cell differentiation and cell death. We report the role of acheron in vascular endothelial proliferation, angiogenesis and wound healing post‐trauma. Co‐immunoprecipitation showed that Acheron forms a ternary complex with β1 integrin and Id1 in human umbilical vein endothelial cells following stimulation with serious trauma serum. Acheron, vascular endothelial growth factor (VEGF), and β1 integrin mRNA expression was apparently inhibited, and capillary density and wound healing rate also were reduced in Id1‐deficient mice trauma model. Acheron together with Id1 significantly induces VEGF, not CD105 level inhibition by serious trauma serum for 24 h. In conclusion, we have demonstrated that acheron may be an effective mediator of promoting endothelial proliferation, angiogenesis and wound healing probably by regulating VEGF together with Id1. Copyright © 2011 John Wiley & Sons, Ltd.     

Fibroblast growth factor-2-mediated capillary morphogenesis of endothelial cells requires signals via Flt-1/vascular endothelial growth factor receptor-1: possible involvement of c-Akt

Capillary morphogenesis is a crucial angiogenic response of endothelial cells. Although fibroblast growth factor-2 (FGF-2) potently induces capillary morphogenesis, the contribution of vascular endothelial growth factor-A (VEGF-A) in this response has not been clarified well. Here we examined the role of VEGF signaling in FGF-2-induced capillary morphogenesis by murine brain capillary endothelial cells (IBE cells) and human umbilical vein endothelial cells. FGF-2-treated IBE cells rapidly extended on Matrigel in association with actin reorganization. Chimeric protein, of which the extracellular domain of VEGF receptor-1 (VEGFR-1) fused to immunoglobulin Fc, inhibited FGF-2-induced cell extension, resulting in decreased capillary morphogenesis. Blocking antibody against VEGFR-1 inhibited FGF-2-induced capillary formation. Also, anti-VEGF-A antibody inhibited FGF-2-induced capillary morphogenesis, which was restored by the addition of placental growth factor-1. Similar results were obtained by the experiments with human umbilical vein endothelial cells. Expression of kinase-inactive c-Akt in IBE cells showed impaired capillary morphogenesis promoted by FGF-2. Conversely, stable cell lines expressing activated c-Akt demonstrated ligand-independent capillaries, which were resistant to the treatment with anti-VEGFR-1 blocking antibody. Upstream of c-Akt, calmodulin-dependent signals seemed to be involved. Taken together, signals via VEGFR-1 were required for FGF-2-induced capillary morphogenesis by endothelial cells, and c-Akt activity seemed to be involved in this process.     

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.     

Synthesis,structure–activity relationships and biological evaluation of barbigerone analogues as anti-proliferative and anti-angiogenesis agents

A series of barbigerone analogues (7a–7w, 13a–13x) were designed, synthesized and biologically evaluated for their anti-proliferative and anti-angiogenic activities. Among these compounds, compound 13a exhibited the most potent inhibitory effect on the proliferation of HUVECs, HepG2, A375, U251, B16, and HCT116 cells (IC₅₀ = 3.80, 0.28, 1.58, 3.50, 1.09 and 0.68 μM, respectively). Compound 13a inhibited the angiogenesis in zebrafish embryo assay in a concentration-dependent manner. Furthermore, 13a also effectively inhibited the migration and capillary like tube formation of human umbilical vein endothelial cell in vitro. These results support the further investigation of this class of compounds as potential anti-proliferative and anti-angiogenesis agents.     

Novel leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry

The over-expression of aminopeptidase N on diverse malignant cells is associated with the tumor angiogenesis and metastasis. In this report, one new series of leucine ureido derivatives containing the triazole moiety was designed, synthesized and evaluated as APN inhibitors. Among them, compound 13v showed the best APN inhibition with an IC₅₀ value of 0.089?±?0.007?μM, which was two orders of magnitude lower than that of bestatin (IC₅₀?=?9.4?±?0.5?μM). Compound 13v also showed dose-dependent anti-angiogenesis activities. Even at the lower concentration (10?μM), compound 13v presented similar anti-angiogenesis activity compared with bestatin at 100?μM in both the human umbilical vein endothelial cells (HUVECs) capillary tube formation assay and the rat thoracic aorta rings test. Moreover, compared with bestatin, 13v exhibited comparable, if not better in vivo anti-metastasis activity in a mouse H22 pulmonary metastasis model.     

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.     

Cytoprotective effect of caffeic acid phenethyl ester (CAPE) and catechol ring-fluorinated CAPE derivatives against menadione-induced oxidative stress in human endothelial cells

Caffeic acid phenethyl ester (CAPE), a natural polyphenolic compound with many biological activities, has been shown to be protective against ischemia-reperfusion injury. We have synthesized six new catechol ring-fluorinated CAPE derivatives and evaluated their cytotoxic and cytoprotective effects against menadione-induced cytotoxicity in human umbilical vein endothelial cells. These results provide some insights into the structural basis of CAPE cytoprotection in this assay, which does not appear to be based solely on direct antioxidant properties.     

Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling

abstract

The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.     

Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

Synthesis and promotion angiogenesis effect of chrysin derivatives coupled to NO donors

Two types of new chrysin derivatives were prepared by coupling NO donors of alkyl nitrate and furazan derivatives and were fully characterized by ¹H NMR and other techniques. These compounds were tested in human umbilical vein endothelial cells (HUVECs-12) and all the compounds exhibited cell proliferation. Notable effects of promoting angiogenesis were observed for all the modified compounds using chick chorioallantoic membrane (CAM) assay.     

Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.     

瑞舒伐他汀对OX-LDL诱导的人脐静脉内皮细胞PPAR-gamma表达的影响

目的：探讨经氧化低密度脂蛋白（OX-LDL）刺激后,人脐静脉内皮细胞（PPAR-gamma）表达的变化,以及瑞舒伐他汀对动脉粥样硬 化的影响。方法：将实验标本随机分为2 组,分为（OX-LDL）刺激组、瑞舒伐他汀干预组。应用RT-PCR及Western blot 技术,观察 OX-LDL 诱导的人脐静脉内皮细胞PPAR-gamma表达情况及瑞舒伐他汀对人脐静脉内皮细胞PPAR-gamma及浓度依赖的方式降低了人脐静脉内皮细胞PPAR-gamma的表达;2）瑞舒伐他汀可以逆转OX-LDL 对人脐静脉内细胞的影响 并可能与甲羟戊酸有关。结论：OX-LDL可降低人脐静脉内皮细胞PPAR-gamma的表达。瑞舒伐他汀可以抑制OX-LDL诱导人脐静脉 内皮细胞PPAR-gamma表达的增强,从而可能抑制了OX-LDL信号通路介导的与炎症有关的血管损伤,延缓动脉粥样硬化的进程。