Search

Purpose: Elevated sFlt-1 and sEng is usually a clue for impending preeclampsia and intrauterine growth restriction. Likewise, uterine artery Doppler ultrasound is being investigated for prediction of similar conditions. In this study, we aimed to explore the possible relations of these two proteins in different body compartments with uterine artery Doppler indices (UtAD) in a healthy second trimester obstetric population., Methods: Levels of sFlt-1 and sEng were measured in serum and amniotic fluid samples of 43 patients. UtAD were measured on the days of sample collections. Findings were then analyzed for possible correlation., Results: There was a positive correlation between the levels of maternal serum sFlt-1 (MSsFlt-1) and sEng levels (MSsEng) (r= 0.516, p< 0.001). The negative correlation between MSsFlt-1 and UtAD was disappeared after elimination of poor obstetric outcome pregnancies (r= -0.371, p= 0.016). No correlation was found between UtAD and studied protein levels in amniotic fluid. Mean MSsFlt-1 level was 305.2 ± 220.1 pg/ml and mean AFsFlt-1 was 48.9 ± 11.8 ng/ml. Mean MSsEng level was 4.5 ± 1.3 ng/ml, mean AFsEng level was found 0.7 ± 0.3 ng/ml. Mean values for UtAD were 1.3 ± 0.4, 0.6 ± 0.1 and 3.5 ± 1.3 for PI, RI, and S/D, respectively., Conclusion: In normal second trimester pregnancies, there is a positive correlation between serum levels of sFlt-1 and sEng levels. Amniotic fluid levels of sEng and sFlt-1 are not correlated with UtAD in uncomplicated pregnancies.

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

Homocysteine (Hcy) is an intermediate non-diet amino acid connecting methionine and folate cycles. Elevated total Hcy level in blood, denoted as hyperhomocysteinemia, has emerged as a prevalent and strong risk factor for multiple diseases including atherosclerotic vascular disease in coronary, cerebral, and peripheral vessels. Its detrimental effect on vascular system implies the potential application as an inhibitor of angiogenesis. However, the detailed mechanism is unveiled. Inhibitory effect of Hcy was assessed on vascular endothelial growth factor (VEGF) induced cell proliferation and migration with endothelial cell (EC) culture system. Its effect on angiogenesis was further examined in vitro and in vivo After Hcy treatment, key angiogenic factors were measured by RT-qPCR. Cellular skeletal structure was also evaluated by actin stress fiber staining. VEGF-induced human umbilical vein EC (HUVEC) proliferation and migration were dramatically down-regulated by Hcy in a dose-responsive manner. Hcy treatment significantly inhibited the VEGF-induced angiogenesis in vitro by tube formation assay and chick chorioallantoic membrane (CAM) vessel formation in vivo Key angiogenic factors like VEGFR1/2 and angiopoietin (Ang)1/2 were substantially reduced by Hcy in HUVEC- and VEGF-induced actin stress fiber cytoskeletal structure was abolished. We demonstrated that Hcy could inhibit angiogenesis by targetting key angiogenic factor and disruption of actin cytoskeleton which is crucial for cell migration., (© 2017 The Author(s).)

Recent reports have revealed that hydrogen sulfide (H 2 S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate is one of the products formed during oxidative H 2 S metabolism, and thiosulfate has been used extensively and safely to treat calcific uremic arteriopathy in dialysis patients. Yet despite its significance, fundamental questions regarding how thiosulfate and H 2 S interact during redox signaling remain unanswered. In the present study, we examined the effect of exogenous thiosulfate on hypoxia-induced H 2 S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, we observed a decrease of GSH and GSSG levels in HUVECs at 0.5 and 4 h as well as decreased free H 2 S and acid-labile sulfide and increased bound sulfide at all time points. Treatment with exogenous thiosulfate significantly decreased the ratio of GSH/GSSG to total sulfide of HUVECs under 0.5 h of hypoxia but significantly increased this ratio in HUVECs under 4 h of hypoxia. These responses reveal that thiosulfate has different effects at low and high doses and under different O 2 tensions. In addition, treatment with thiosulfate also diminished VEGF-induced cystathionine-γ-lyase expression and reduced VEGF-induced HUVEC proliferation under both normoxic and hypoxic conditions. These results indicate that thiosulfate can modulate H 2 S metabolites and signaling under various culture conditions that impact angiogenic activity. Thus, thiosulfate may serve as a unique sulfide donor to modulate endothelial responses under pathophysiological conditions involving angiogenesis. NEW & NOTEWORTHY This report provides new evidence that different levels of exogenous thiosulfate dynamically change discrete sulfide biochemical metabolite bioavailability in endothelial cells under normoxia or hypoxia, acting in a slow manner to modulate sulfide metabolites. Moreover, our findings also reveal that thiosulfate surprisingly inhibits VEGF-dependent endothelial cell proliferation associated with a reduction in cystathionine-γ-lyase protein levels., (Copyright © 2017 the American Physiological Society.)

Diabetic retinopathy is a major complication of diabetes mellitus that can lead to retinal vascular abnormalities and visual impairment. While retinal endothelial pathology is well studied, retinal pigment epithelium (RPE) layer modifications and the patho-physiological regulations are not widely understood. The RPE is a highly specialized pigmented layer regulating not only physiological functions such as transport of nutrients, ions, absorption of light, phagocytosis of photoreceptor membranes, but also secretion of a number of cytokines, chemokines, angiogenic and anti-angiogenic factors. The RPE secretome, though crucial in health and disease, remains elusive in diabetic retinopathy. A knowledge of these secreted factors would help explain and correlate the clinical phase of the disease aiding in improved disease management. A comprehensive knowledge of the secreted factors of the RPE is a potential tool for understanding the differential treatment regime of early diabetic retinopathy, diabetic proliferative retinopathy and diabetic macular edema. In this review, we have delineated the importance of factors secreted by the retinal pigment epithelium and its regulation in the pathogenesis of diabetic retinopathy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Experimental studies have suggested that Wingless-related integration site 5A (WNT5A) is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Impaired angiogenesis in fat depots has been implicated in the development of adipose tissue capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction. We have recently demonstrated that impaired adipose tissue angiogenesis is associated with overexpression of antiangiogenic factor VEGF-A 165 b in human fat and the systemic circulation. In the present study, we postulated that upregulation of WNT5A is associated with angiogenic dysfunction and examined its role in regulating VEGF-A 165 b expression in human obesity. We biopsied subcutaneous and visceral adipose tissue from 38 obese individuals (body mass index: 44 ± 7 kg/m 2 , age: 37 ± 11 yr) during planned bariatric surgery and characterized depot-specific protein expression of VEGF-A 165 b and WNT5A using Western blot analysis. In both subcutaneous and visceral fat, VEGF-A 165 b expression correlated strongly with WNT5A protein ( r = 0.9, P < 0.001). In subcutaneous adipose tissue where angiogenic capacity is greater than in the visceral depot, exogenous human recombinant WNT5A increased VEGF-A 165 b expression in both whole adipose tissue and isolated vascular endothelial cell fractions ( P < 0.01 and P < 0.05, respectively). This was associated with markedly blunted angiogenic capillary sprout formation in human fat pad explants. Moreover, recombinant WNT5A increased secretion of soluble fms-like tyrosine kinase-1, a negative regulator of angiogenesis, in the sprout media ( P < 0.01). Both VEGF-A 165 b-neutralizing antibody and secreted frizzled-related protein 5, which acts as a decoy receptor for WNT5A, significantly improved capillary sprout formation and reduced soluble fms-like tyrosine kinase-1 production ( P < 0.05). We demonstrated a significant regulatory nexus between WNT5A and antiangiogenic VEGF-A 165 b in the adipose tissue of obese subjects that was linked to angiogenic dysfunction. Elevated WNT5A expression in obesity may function as a negative regulator of angiogenesis. NEW & NOTEWORTHY Wingless-related integration site 5a (WNT5A) negatively regulates adipose tissue angiogenesis via VEGF-A 165 b in human obesity., (Copyright © 2017 the American Physiological Society.)

Aims: Angiogenesis is a physiological process that has important impacts on the pathology and healing of various diseases, and its induction or inhibition by bioactive actinobacterial metabolites can help the treatment of some diseases. In this study, the effects of actinobacterial extract in the process of angiogenesis have been explored., Methods and Results: In this research, proangiogenic and antiangiogenic metabolites producing actinobacteria were isolated from soil samples and their fermentation broth were extracted and after evaluation of their toxicity by MTT assay, antiangiogenic and proangiogenic activities were screened against human umbilical vein endothelial cells (HUVECs) by in vitro tube formation and migration assay. Isolated strains were identified through molecular techniques. The results showed that Nocardiopsis arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts had a high potential of anti-angiogenic activity on HUVECs., Conclusions: For the first time proangiogenic potency of a rare actinobacterium, Kribbella sp. UTMC 522, was reported, and N. arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts inhibits the proliferation, migration and angiogenesis activity of HUVECs with reasonable potency., Significance and Impact of the Study: Metabolites of the introduced rare actinobacteria are potent proangiogenic and angiogenic inhibitors. Identification of angiogenic-antiangiogenic mechanisms and purification of the extracts would be useful in therapeutic angiogenesis., (© 2017 The Society for Applied Microbiology.)

Purpose of Review: Currently, severe retinopathy of prematurity (ROP) is diagnosed by clinical evaluation and not a laboratory test. Laser is still considered standard care. However, anti-vascular endothelial growth factor (VEGF) agents are being used and there are questions whether and/or if to use them, what dose or type of agent should be considered and what agent may be most beneficial in specific cases. Also unclear are the effects of laser or anti-VEGF on severe ROP, refractive outcomes or infant development. This article reviews recent studies related to these questions and other trials for severe ROP., Recent Findings: Imaging studies identify biomarkers of risk (plus disease, stage 3 ROP, and ROP in zone I). Intravitreal bevacizumab or ranibizumab are reported effective in treating aggressive posterior ROP in small series. Recurrences and effects on myopia vary among studies. Use of anti-VEGF agents affects cytokines in the infant blood and reduces systemic VEGF for up to 2 months, raising potential safety concerns. The effects of treatment vary based on infant size and are not comparable. Evidence for most studies is not high., Summary: Studies support experimental evidence that inhibiting VEGF reduces stage 3 ROP and peripheral avascular retina. Ongoing large-scale clinical trials may provide clarity for best treatments of severe ROP. Current guidelines hold for screening and treatment for type 1 ROP.

Vascular endothelial growth factor (VEGF) produced from tumor cells plays a crucial role in the pathogenesis and neovascularization of neuroblastoma. Inhibition of VEGF secretion by tumor cells, as well as VEGF-regulated signaling in endothelial cells, are important to reduce the angiogenesis and growth of neuroblastoma. Since melatonin has anti-angiogenic effects in tumor cell lines, the aim of the present study was to study melatonin modulation of the pro-angiogenic effects of VEGF in neuroblastoma cells (SH-SY5Y). We used co-cultures of SH-SY5Y and endothelial cells. VEGF expression and protein levels were analyzed by quantitative RT-PCR and ELISA, respectively. Endothelial cell migration was assessed by wound-healing assay and endothelial angiogenesis by a tube formation assay. Melatonin inhibited the pro-angiogenic effects of SH-SY5Y cells. The conditioned medium collected from the neuroblastoma cells was angiogenically active and stimulated proliferation, migration and tube formation in endothelial cells. This effect was significantly counteracted by the addition of either anti-VEGF or melatonin. Melatonin inhibited VEGF expression and secretion in SH-SY5Y cells, decreasing the levels of VEGF available for endothelial cells. Melatonin has anti-angiogenic effects at different steps of the angiogenic process in SH-SY5Y neuroblastoma cells, through the downregulation of VEGF.

Cyperenoic acid (1) is one of the major sesquiterpenes isolated from Croton crassifolius, which exhibited potent anti-angiogenic activity. Traditional structural modification of 1 is difficult to perform by chemical technology due to the remarkable stability of the patchoulane skeleton. In order to overcome chemical synthesis difficulties and obtain structurally diverse derivations, microbial transformation of 1 by using Cunninghamella elegans AS 3.2028 was studied for the first time. Five new hydroxylated products 2-6 were obtained. Furthermore, cytotoxicity and anti-angiogenic activities of all the biotransformation products were evaluated by MTT assay and ELISA in HepG2 and MCF-7 cells. These results indicated that hydroxylated modification products 2-4 significantly inhibited VEGF release, which suggest the potential use of hydroxylated modification products for cancer therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chronic chagasic cardiomyopathy (CCC) is arguably the most important form of the Chagas Disease, caused by the intracellular protozoan Trypanosoma cruzi; it is estimated that 10-30% of chronic patients develop this clinical manifestation. The most common and severe form of CCC can be related to ventricular abnormalities, such as heart failure, arrhythmias, heart blocks, thromboembolic events and sudden death. Therefore, in this study, we proposed to evaluate the anti-angiogenic activity of a recombinant protein from T. cruzi named P21 (rP21) and the potential impact of the native protein on CCC. Our data suggest that the anti-angiogenic activity of rP21 depends on the protein's direct interaction with the CXCR4 receptor. This capacity is likely related to the modulation of the expression of actin and angiogenesis-associated genes. Thus, our results indicate that T. cruzi P21 is an attractive target for the development of innovative therapeutic agents against CCC.

278: The Cytochrome P450 (CYP) system is involved in 90% of the human body's interactions with xenobiotics and due to this, it has become an area of avid research including the creation of transgenic mice. This paper proposes a three-compartment model which is used to explain the drug metabolism in the Hepatic Reductase Null (HRN) mouse developed by the University of Dundee (Henderson, C. J., Otto, D. M. E., Carrie, D., Magnuson, M. A., McLaren, A. W., Rosewell, I. and Wolf, C. R. (2003) Inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase. J. Biol. Chem. , 13480-13486). The model is compared with a two-compartment model using experimental data from studies using wild-type and HRN mice. This comparison allowed for metabolic differences between the two types of mice to be isolated. The three sets of drug data (Gefitinib, Midazolam and Thalidomide) showed that the transgenic mouse has a decreased rate of metabolism., (© The authors 2015. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

Melons have a good source of protease inhibitors. Its fruit and seeds have been used as a traditional medicine. However, its effects on angiogenesis and mechanism of its action remain elusive. Herein trypsin inhibitor from aqueous extract of C. melo seeds (TICMS) was purified. Its effects on different steps of angiogenesis were evaluated. Also, we examined its effects on migration and angiogenesis of endothelial cells. Three dimensional model of TICMS protein was accurately built in which TICMS docked to α V β 3 integrin and VEGFR1. Electrophoresis analysis of the purified protein revealed a single band with a molecular mass of about 3kDa. Treatment with TICMS at six doses resulted in a significant decrease of endothelial cell proliferation with an IC 50 value of about 20μg/ml. Tubulogenesis assay revealed that a dose dependent anti-angiogenic activity of TICMS (5-40μg/ml). Also, TICMS had inhibitory effects on VEGF, MMP-2 and MMP-9 secretion. Our docking result speculated that TICMS could bind to the cleft between the α V β 3 integrin and it able to decrease the activity of this receptor. The TICMS was also able to interact with VEGFR1 receptor, but with low probability. Based on our study, TICMS could be used as a specific angiogenesis inhibitor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.

Angiogenesis is modulated by a multitude of pro-angiogenic factors including VEGFR-2, Tie-2, and EphB4. Moreover, their crosstalk also had been well elaborated. We have identified several diarylurea-based VEGFR-2 inhibitors as potential anti-angiogenesis agents. As a continuation to our previous research, two series of diaryl malonamide and diaryl thiourea derivatives have been developed as multiplex VEGFR-2/Tie-2/EphB4 inhibitors. Interestingly, the biological evaluation indicated that several compounds bearing trifluoromethyl or trifluoromethoxyl exhibited promising multiplex inhibition against angiogenesis-related VEGFR-2, Tie-2, and EphB4. The representative compound (18a) displayed both potent multi-targeted RTK inhibition and considerable antiproliferative activities against human umbilical vein endothelial cells (EA.hy926). These results will contribute to the discovery of novel muti-targeted anti-angiogenesis agents., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Purpose: To determine the effects of bevacizumab, ranibizumab, and aflibercept on the permeability and the effects of anti-vascular endothelial growth factor (VEGF) on highly polarized retinal pigment epithelial cells (RPECs) in vitro., Methods: Highly polarized RPECs were cultured in the upper chamber of a Transwell system. Anti-VEGF antibodies were added to the upper chamber, and the concentrations of the drugs in the lower chambers were measured. The permeability rates of the three anti-VEGF drugs through the RPEC layer and the concentration of VEGF in each chamber were determined., Results: The permeability of aflibercept was significantly lower by about 40% than that of bevacizumab through the RPEC layer (P < 0.05). Ranibizumab was significantly more permeable through the RPECs than bevacizumab (180% of bevacizumab, P < 0.05). Although VEGF was almost absent in the upper chamber after exposure to the 3 antibodies, it was decreased more significantly with aflibercept than with bevacizumab in the lower chamber (2.8% vs. 65.8% of control; P < 0.01). Ranibizumab also decreased the VEGF level compared with bevacizumab (31.7% vs. 65.8% of control; P < 0.01)., Conclusion: The greater reduction of the amount of VEGF in the lower chamber by aflibercept and ranibizumab than bevacizumab may explain why aflibercept and ranibizumab are more effective than bevacizumab against type 1 choroidal neovascularization.

Preeclampsia (PE) is a disease unique to humans. PE complicates from 3 to 14% of pregnancies worldwide. Unknown cause yet, although part of poor placentation resulting placental ischemia release of proinfammatory cytokines and anti-angiogenic factors. Two PE types: early PE with maternal and fetal adverse events and late PE with low fetal involvement and more favorable maternal-fetal prognosis. As PE predictors being considered: increased uric acid of 3.15 mg/dl as a predictor of gestational hypertension in the first quarter and greater than 5.2 mg/dl for severe/eclampsia preeclampsia. The sFLT-1/PIGF (soluble FMS-like tyrosine kinase-1/angioge nic placental growth facto) ratio is above 85 is a marker of pregnancy complications and a good indicator that helps decision making. Hypertension in pregnancy can be the beginning of vascular and metabolic alterations future; indeed the PE/eclampsia is considered a specific risk factor in women in some guides cardiovascular risk. © 2017 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved., (Copyright © 2017 Sociedad Española de Hipertension-Liga Española para la Lucha de la Hipertensión Arterial (SEH-LELHA). Publicado por Elsevier España, S.L.U. All rights reserved.)

Hypoxia has been shown to be a key factor inhibiting the successful treatment of solid tumours. Existing strategies for reducing hypoxia, however, have shown limited efficacy and/or adverse side effects. The aim of this study was to investigate the potential for reducing tumour hypoxia using an orally delivered suspension of surfactant-stabilised oxygen nanobubbles. Experiments were carried out in a mouse xenograft tumour model for human pancreatic cancer (BxPc-3 cells in male SCID mice). A single dose of 100 μL of oxygen saturated water, oxygen nanobubbles or argon nanobubbles was administered via gavage. Animals were sacrificed 30 minutes post-treatment (3 per group) and expression of hypoxia-inducible-factor-1α (HIF1α) protein measured by real time quantitative polymerase chain reaction and Western blot analysis of the excised tumour tissue. Neither the oxygen saturated water nor argon nanobubbles produced a statistically significant change in HIF1α expression at the transcriptional level. In contrast, a reduction of 75% and 25% in the transcriptional and translational expression of HIF1α respectively (p<0.001) was found for the animals receiving the oxygen nanobubbles. This magnitude of reduction has been shown in previous studies to be commensurate with an improvement in outcome with both radiation and drug-based treatments. In addition, there was a significant reduction in the expression of vascular endothelial growth factor (VEGF) in this group and corresponding increase in the expression of arrest-defective protein 1 homolog A (ARD1A)., Competing Interests: Raymond Averre is a director of Avrox Technologies Ltd. Avrox Technologies Ltd. did not fund this work but did partially fund an earlier pilot study on oxygen nanobubbles. Joshua Owen, Raymond Averre and Eleanor Stride are co-inventors on a patent (Stride, E., Averre, R., Owen, J. (2015) Nanoencapsulated Oxygen. UK Patent Application No. 1512728.5), which has been filed in relation to the formulation described in the paper. Avrox Technologies Ltd. has licensed this patent. John Callan thanks Norbrook Laboratories Ltd. for an endowed chair. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Eriocalyxin B (EriB), a natural ent-kaurane diterpenoid isolated from the plant Isodon eriocalyx var. laxiflora, has emerged as a promising anticancer agent. The effects of EriB on angiogenesis were explored in the present study. Here we demonstrated that the subintestinal vein formation was significantly inhibited by EriB treatment (10, 15 μM) in zebrafish embryos, which was resulted from the alteration of various angiogenic genes as shown in transcriptome profiling. In human umbilical vein endothelial cells, EriB treatment (50, 100 nM) could significantly block vascular endothelial growth factors (VEGF)-induced cell proliferation, tube formation, cell migration and cell invasion. Furthermore, EriB also caused G1 phase cell cycle arrest which was correlated with the down-regulation of the cyclin D1 and CDK4 leading to the inhibition of phosphorylated retinoblastoma protein expression. Investigation of the signal transduction revealed that EriB inhibited VEGF-induced phosphorylation of VEGF receptor-2 via the interaction with the ATP-binding sites according to the molecular docking simulations. The suppression of VEGFR-2 downstream signal transduction cascades was also observed. EriB was showed to inhibit new blood vessel formation in Matrigel plug model and mouse 4T1 breast tumor model. EriB (5 mg/kg/day) treatment was able to decrease tumor vascularization and suppress tumor growth and angiogenesis. Taken together, our findings suggested that EriB is a novel inhibitor of angiogenesis through modulating VEGFR-2 signaling pathway, which could be developed as a promising anti-angiogenic agent for treatment of angiogenesis-related human diseases, such as cancer.

Background and Objectives: Hemangeol, approved for the treatment of proliferative infantile hemangiomas requiring systemic therapy, is metabolized by cytochrome P450 2D6 (CYP2D6), which is a highly polymorphic enzyme that metabolizes a large number of drugs. More than 100 CYP2D6 allelic variants have been reported so far, including 22 novel variants that discovered in our lab in the Chinese population. Our study aimed to probe the enzymatic activity of these variants toward hemangeol in vitro with recombinant microsomes that expressed in sf21 insect cells using a baculovirus-mediated expression system., Methods: The wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) were incubated with 1-200 μM hemangeol for 50 min at 37 °C. Then the products were extracted, and signal detection was performed by high-performance liquid chromatography with fluorescence detector., Results: All of the variants exhibited changed apparent Michaelis-Menten constant (Km) or maximum velocity of the reaction (V max ) values compared with that of wild-type protein. The intrinsic clearances (V max /Km) were significantly decreased by 0.37 to 42.74 %. However, CYP2D6.92 and CYP2D6.96 showed no or minimal enzymatic activity as no concentration of 4'-hydroxypropranolol was detected., Conclusions: The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards hemangeol in vivo.

Angiogenesis is the growth of new blood vessels from pre-existing microvessels. Peripheral arterial disease (PAD) is caused by atherosclerosis that results in ischemia mostly in the lower extremities. Clinical trials including VEGF-A administration for therapeutic angiogenesis have not been successful. The existence of anti-angiogenic isoform (VEGF 165b ) in PAD muscle tissues is a potential cause for the failure of therapeutic angiogenesis. Experimental measurements show that in PAD human muscle biopsies the VEGF 165b isoform is at least as abundant if not greater than the VEGF 165a isoform. We constructed three-compartment models describing VEGF isoforms and receptors, in human and mouse, to make predictions on the secretion rate of VEGF 165b and the distribution of various isoforms throughout the body based on the experimental data. The computational results are consistent with the data showing that in PAD calf muscles secrete mostly VEGF 165b over total VEGF. In the PAD calf compartment of human and mouse models, most VEGF 165a and VEGF 165b are bound to the extracellular matrix. VEGF receptors VEGFR1, VEGFR2 and Neuropilin-1 (NRP1) are mostly in 'Free State'. This study provides a computational model of VEGF 165b in PAD supported by experimental measurements of VEGF 165b in human and mouse, which gives insight of VEGF 165b in therapeutic angiogenesis and VEGF distribution in human and mouse PAD model.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation.

BACKGROUND This study investigated how miR-21 expression is reflected in acute myocardial infarction and explored the role of miR-21 and the PTEN/VEGF signaling pathway in cardiac microvascular endothelial cells. MATERIAL AND METHODS We used an in vivo LAD rat model to simulate acute myocardial infarction. MiR-21 mimics and miR-21 inhibitors were injected and transfected into model rats in order to alter miR-21 expression. Cardiac functions were evaluated using echocardiographic measurement, ELISA, and Masson staining. In addition, lenti-PTEN and VEGF siRNA were transfected into CMEC cells using standard procedures for assessing the effect of PTEN and VEGE on cell proliferation, apoptosis, and angiogenesis. MiR-21, PTEN, and VEGF expressions were examined by RT-PCR and Western blot. The relationship between miR-21 and PTEN was determined by the luciferase activity assay. RESULTS We demonstrated that miR-21 bonded with the 3'-UTR of PTEN and suppressed PTEN expressions. Established models significantly induced cardiac infarct volume and endothelial injury marker expressions as well as miR-21 and PTEN expressions (P<0.05). MiR-21 mimics exhibited significantly protective effects since they down-regulated both infarction size and injury marker expressions by increasing VEGF expression and inhibiting PTEN expression (P<0.05). In addition, results from in vitro research show that lenti-PTEN and VEGF siRNA can notably antagonize the effect of miR-21 on cell proliferation, apoptosis, and angiogenesis (P<0.05). CONCLUSIONS MiR-21 exerts protective effects on endothelial injury through the PTEN/VEGF pathway after acute myocardial infarction.

Introduction: Tyrosine kinase inhibitors (TKIs) are very attractive targeted drugs, although a large portion of patients remains unresponsive. PET imaging with EGFR targeting TKIs ([(11)C]erlotinib and [(18)F]afatinib) showed promise in identifying treatment sensitive tumors. The aim of this study was to synthesize two anti-angiogenic TKI tracers, [(11)C]axitinib and [(11)C]nintedanib, and to evaluate their potential for PET., Methods: Following successful tracer synthesis, biodistribution studies in VU-SCC-OE and FaDu xenograft bearing mice were performed. Furthermore, tracer stability studies in mice were performed employing (radio-)HPLC and LC-MS/MS techniques. For [(11)C]nintedanib an LC-MS/MS method was developed to detect the primary carboxylic acid metabolite, resulting from methylester cleavage, in plasma and tumors, because this metabolite is postulated to be important for nintedanib efficacy. LC-MS/MS was also explored to assess the metabolic fate of [(11)C]axitinib in vivo, since axitinib has an isomerizable double bond., Results: [(11)C]axitinib and [(11)C]nintedanib were successfully synthesized with 10.5±2.6% and 25.6±3.3% radiochemical yield (corrected for decay), respectively. Biodistribution studies only demonstrated tumor uptake of [(11)C]nintedanib in FaDu xenografts of 1.66±0.02% ID/g at 60min p.i. In vivo stability analysis of [(11)C]axitinib at 45min p.i. revealed the formation of predominantly non-polar metabolites (36.6±6.8% vs 47.1±8.4% of parent tracer and 16.3±2.1% of polar metabolites), while for [(11)C]nintedanib mostly polar metabolites were found (70.9±4.1 vs 26.7±3.9% of parent tracer and only 2.4±1.6 of a non-polar metabolites). No isomerization of [(11)C]axtinib was observed in vivo; however, a sulfoxide metabolite could be detected using LC-MS/MS. For [(11)C]nintedanib, LC-MS/MS revealed formation of the reported primary carboxylic acid metabolite when in vitro plasma incubations were performed, with large differences in plasmas from different species. In vivo metabolite analysis, however, did not demonstrate the presence of the carboxylic acid in plasma or tumor tissue., Conclusions: Reliable syntheses of [(11)C]axitinib and [(11)C]nintedanib were successfully developed. Tumor uptake was observed for [(11)C]nintedanib, albeit modest. The metabolic profiles of the tracers suggest that rapid metabolism is partly responsible for the modest tumor targeting observed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N‑BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06μM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The estrogen receptors have played important roles in breast cancer development and progression. Selective estrogen receptor modulators, such as Tamoxifen, have showed great benefits in the treatment and prevention of breast cancer. But the disadvantages of induction of endometrial cancer and drug resistance have limited their use. Multiple ligand which act at multiple biomolecular targets may exert favorable advantages of improved efficacy with lower incidence of side effects. In this work, we described the synthesis and evaluation of a series of 6-aryl-indenoisoquinolone derivatives as dual ERα and VEGFR-2 inhibitors. These compounds presented good ERα binding affinity and ERα antagonistic activity, as well as potent VEGFR-2 inhibitory potency. They also possessed excellent anti-proliferative activities against MCF-7, MDA-MB-231, Ishikawa and HUVEC cell lines. Further investigation of selective compound 21c showed that it was able to inhibit the activation of VEGFR-2 and the signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

Tea leaf (Camellia sinensis) is rich in catechins, which endow tea with various health benefits. There are more than ten catechin compounds in tea, among which epigallocatechingallate (EGCG) is the most abundant. Epidemiological studies on the association between tea consumption and the risk of breast cancer were summarized, and the inhibitory effects of tea catechins on breast cancer, with EGCG as a representative compound, were reviewed in the present paper. The controversial results regarding the role of tea in breast cancer and areas for further study were discussed.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

We examined the effect of plasma incubation from preeclampsia pregnant on the antiangiogenic miR-195-5p expression. Higher miR-195-5p expression was found in cultures incubated with preeclampsia plasma compared to those incubated with healthy pregnant plasma. Next, as VEGF is a target of miR-195-5p we have quantified its expression by real-time qPCR and ELISA. We found reduced VEGF levels in culture incubated with preeclampsia plasma. Therefore, we have concluded that the higher expression of miR-195-5p in endothelial cell cultures incubated with preeclampsia plasma may contribute to decreased expression of VEGFA (gene and protein) and increased antiangiogenic status in preeclampsia. Therefore, this miR may be an important target in preeclampsia., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The cytoprotective and anticancer action of dietary in-taken natural polyphenols has for long been attributed only to their direct radical scavenging activities. Currently it is well supported that those compounds display a broad spectrum of biological and pharmacological outcomes mediated by their complex metabolism, interaction with gut microbiota as well as direct interactions of their metabolites with key cellular signaling proteins. The beneficial effects of natural polyphenols and their synthetic derivatives are extensively studied in context of cancer prophylaxis and therapy. Herein we focus on cell signaling to explain the beneficial role of polyphenols at the three stages of cancer development: we review the recent proceedings about the impact of polyphenols on the cytoprotective antioxidant response and their proapoptotic action at the premalignant stage, and finally we present data showing how phenolic acids (e.g., caffeic, chlorogenic acids) and flavonols (e.g., quercetin) hamper the development of metastatic cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Flavone-8-acetic acid (FAA) has been proved to be a potent vascular-disrupting agent in mice. Unfortunately, FAA did not produce any anticancer activity in clinical trials. Previously, we had reported that FAA is metabolized by mouse microsomes into six metabolites, whereas it was poorly metabolized by human microsomes, with fewer metabolites formed in lesser amounts. Especially, 6-OH-FAA was not formed by human microsomes. In this work, two major available metabolites, 4'-OH-FAA and 6-OH-FAA, were tested and compared with the parent compound FAA for their potential antivascular activities in vitro. The ability of the products to induce morphological changes, disrupt preformed capillaries of EA.hy926 endothelial cells and inhibit tubulin polymerization in vitro was assessed. The action mechanism was determined using the RhoA and Rac1 inhibitors. At 25 µg/ml, 6-OH-FAA induced morphological changes and membrane blebbing, whereas 300 µg/ml of FAA and 4'-OH-FAA slightly changed the morphology without inducing membrane blebbing. At 300 µg/ml, 6-OH-FAA produced morphological changes that were 2.1-6.9-fold greater than that produced by FAA and 4'-OH-FAA, an effect that was consistent with its much greater inhibitory effect on tubulin polymerization compared with FAA and 4'-OH-FAA. 6-OH-FAA significantly disrupted the EA.hy926 cell capillaries. 6-OH-FAA activities were prevented in EA.hy926 cells pretreated with RhoA, but not Rac1, inhibitor. In this short communication we report for the first time that, in vitro, 6-OH-FAA, a mouse-specific FAA metabolite, exhibits significantly stronger antivascular activities compared with FAA and 4'-OH-FAA, which are mediated through the RhoA kinase pathway.

MicroRNAs (miRNAs) modulate complex physiological and pathological processes, including the regulation of angiogenesis. Our previous study reported that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into choroidal neovascularization lesions. miRNA-195 is highly expressed in MSCs, but its function remains unknown. In the present study, miR-195a-3p abundance was significantly decreased in hypoxia-treated murine MSCs; on the other hand, its overexpression reduced MSC proliferation and migration while increasing the activation of anti-angiogenic factor pigment epithelium-derived factor (PEDF). We further discovered that matrix metalloproteinase 2 (Mmp2) transcript is a target of miR-195a-3p, and that silencing Mmp2 phenocopied the reduced proliferation and migration of MSCs. The therapeutic potential of miR-195a-3p as an angiogenesis inhibitor was also demonstrated in a laser-induced choroidal neovascularization mouse model. These findings collectively indicate that miR-195a-3p is a negative modulator of angiogenesis, and could be used as an angiogenesis inhibitor. Mol. Reprod. Dev. 83: 413-423, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Sex hormone estrogen is one of the most active intrinsic angiogenesis regulators; its therapeutic use has been limited due to its carcinogenic potential. Plant-derived phytoestrogens are attractive alternatives, but reports on their angiogenic activities often lack in-depth analysis and sometimes are controversial. Herein, we report a data-mining study with the existing literature, using IPA system to classify and characterize phytoestrogens based on their angiogenic properties and pharmacological consequences. We found that pro-angiogenic phytoestrogens functioned predominantly as cardiovascular protectors whereas anti-angiogenic phytoestrogens played a role in cancer prevention and therapy. This bidirectional regulation were shown to be target-selective and, for the most part, estrogen-receptor-dependent. The transactivation properties of ERα and ERβ by phytoestrogens were examined in the context of angiogenesis-related gene transcription. ERα and ERβ were shown to signal in opposite ways when complexed with the phytoestrogen for bidirectional regulation of angiogenesis. With ERα, phytoestrogen activated or inhibited transcription of some angiogenesis-related genes, resulting in the promotion of angiogenesis, whereas, with ERβ, phytoestrogen regulated transcription of angiogenesis-related genes, resulting in inhibition of angiogenesis. Therefore, the selectivity of phytoestrogen to ERα and ERβ may be critical in the balance of pro- or anti-angiogenesis process., (Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions. Therefore we investigated the transport and intracellular localization of bevacizumab in immortalized bovine REC (iBREC) in detail, considering possible roles of vesicles and receptors mediating uptake and intracellular transport. By performing transcytosis assays with iBREC monolayers cultivated on porous membrane inserts, we demonstrated that bevacizumab was transported efficiently through a tight monolayer from the lower to the upper chamber or vice versa. When added to the lower chamber in excess, the internalized antibody was transported through the cells, but it was also recycled to be set free at the same side of the cell into a bevacizumab-free environment. The rates of both processes strongly depended on the concentration of fetal bovine serum (FBS) in the environment. This observation is important because in vivo REC might be exposed to varying amounts of serum, e.g. in patients with macular edema. FBS also affected the intracellular localization of bevacizumab as shown by analyses of subcellular fractions and direct immunofluorescence staining. When iBREC were cultivated in low-serum medium, most of the antibody was found in the fraction of cytoskeleton proteins and spots of high intensity of bevacizumab-specific staining close to the nuclei were observed. Cultivation in medium with FBS resulted in internalized bevacizumab predominately found in the membrane/organelle fraction in addition to its weaker association with proteins from the cytoskeleton and uniform staining of the cell. Bevacizumab-specific staining close to the cytoskeleton proteins α-tubulin or vimentin was also observed. Accumulation and association of the antibody with the cytoskeleton induced by serum reduction could be reversed by subsequent FBS addition. In uptake and transport of bevacizumab vesicles and binding to a receptor seems to be involved: Internalization was strongly temperature-dependent which ruled out paracellular passage and a fraction of the internalized bevacizumab was associated with early endosomes. Protein A inhibited transcytosis and affected intracellular localization suggesting a key role of the neonatal Fc receptor (FcRn). Interestingly, FcRn expression was decreased when iBREC were cultivated without FBS. Our results suggest this pathway of bevacizumab uptake and transition through iBREC: Independent of serum, bevacizumab is taken up through a nonspecific mechanism. The subsequent sorting into transport vesicles depends on the presence of serum as regulator of FcRn expression. Without sufficient amounts of the receptor being expressed, a likely obstructed exocytosis results in intracellular accumulation and an increased association with cytoskeleton proteins. Interaction of substantial amounts of bevacizumab with the cytoskeleton may be the reason for under these conditions suppressed migration of iBREC. If long-term therapies by intravitreal injection lead to accumulation of bevacizumab in REC in vivo and potentially harmful consequences, will have to be revealed by future investigations., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intact β2-glycoprotein I (iβ2GPI) is a glycoprotein that regulates coagulation and fibrinolysis. Nicked β2GPI (nβ2GPI) possesses an angiogenic property at a relatively low concentration, and an antiangiogenic property at a high concentration. Here we investigated the functions of βi 2GPI and nβ2GPI in vascular endothelial growth factor (VEGF)-A-induced endothelial cell proliferation and tube formation. We used noninvasive PET imaging to analyze the ｉｎ　ｖｉｖｏ distribution of intravenously injected β2GPI variants in tumor lesions in mice. iβ2GPI was incubated with plasmin to obtain nβ2GPI, and its N-terminal sequence was analyzed. nβ2GPI had at least one other cleavage site upstream of the β2GPI's domain V, whereas the former plasmin-cleavage site locates between K317 and T318. Both of intact and nicked β2GPI significantly inhibited the VEGF-A-induced cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs). PET imaging visualized considerably distributed intensities of all tested β2GPI variants in tumor lesions of pancreatic tumor cell-xenografts. These results indicate that β2GPI may be physiologically and pathophysiologically important in the regulation of not only coagulation and fibrinolysis, but also angiogenesis.

Lipocalin 2 (Lcn2) is a promising therapeutic target as well as a potential diagnostic biomarker for breast cancer. It has been previously shown to promote breast cancer progression by inducing the epithelial to mesenchymal transition in breast cancer cells as well as by enhancing angiogenesis. Lcn2 levels in urine and tissue samples of breast cancer patients has also been correlated with breast cancer status and poor patient prognosis. In this study, we have engineered a novel liposomal small interfering RNA (siRNA) delivery system to target triple negative breast cancer (TNBC) via a recently identified molecular target, intercellular adhesion molecule-1 (ICAM-1). This ICAM-1-targeted, Lcn2 siRNA- encapsulating liposome (ICAM-Lcn2-LP) binds human TNBC MDA-MB-231cells significantly stronger than non-neoplastic MCF-10A cells. Efficient Lcn2 knockdown by ICAM-Lcn2-LPs led to a significant reduction in the production of vascular endothelial growth factor (VEGF) from MDA-MB-231 cells, which, in turn, led to reduced angiogenesis both in vitro and in vivo. Angiogenesis (neovascularization) is a requirement for solid tumor growth and progression, and its inhibition is an important therapeutic strategy for human cancers. Our results indicate that a tumor-specific strategy such as the TNBC-targeted, anti-angiogenic therapeutic approach developed here, may be clinically useful in inhibiting TNBC progression.

N-Methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-1H-indazol-6-ylsulfanyl]-benzamide (axitinib) is an oral inhibitor of vascular endothelial growth factor receptors 1-3, which is approved for the treatment of advanced renal cell cancer. Human [(14)C]-labeled clinical studies indicate axitinib's primary route of clearance is metabolism. The aims of the in vitro experiments presented herein were to identify and characterize the enzymes involved in axitinib metabolic clearance. In vitro biotransformation studies of axitinib identified a number of metabolites including an axitinib sulfoxide, several less abundant oxidative metabolites, and glucuronide conjugates. The most abundant NADPH- and UDPGA-dependent metabolites, axitinib sulfoxide (M12) and axitinib N-glucuronide (M7) were selected for phenotyping and kinetic study. Phenotyping experiments with human liver microsomes (HLMs) using chemical inhibitors and recombinant human cytochrome P450s demonstrated axitinib was predominately metabolized by CYP3A4/5, with minor contributions from CYP2C19 and CYP1A2. The apparent substrate concentration at half-maximal velocity (Km) and Vmax values for the formation of axitinib sulfoxide by CYP3A4 or CYP3A5 were 4.0 or 1.9 µM and 9.6 or 1.4 pmol·min(-1)·pmol(-1), respectively. Using a CYP3A4-specific inhibitor (Cyp3cide) in liver microsomes expressing CYP3A5, 66% of the axitinib intrinsic clearance was attributable to CYP3A4 and 15% to CYP3A5. Axitinib N-glucuronidation was primarily catalyzed by UDP-glucuronosyltransferase (UGT) UGT1A1, which was verified by chemical inhibitors and UGT1A1 null expressers, with lesser contributions from UGTs 1A3, 1A9, and 1A4. The Km and Vmax values describing the formation of the N-glucuronide in HLM or rUGT1A1 were 2.7 µM or 0.75 µM and 8.9 or 8.3 pmol·min(-1)·mg(-1), respectively. In summary, CYP3A4 is the major enzyme involved in axitinib clearance with lesser contributions from CYP3A5, CYP2C19, CYP1A2, and UGT1A1., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Vascular endothelial growth factor 165 (VEGF165) is an important extracellular protein involved in pathological angiogenesis in diseases such as cancer, wet age-related macular degeneration (wet-AMD) and retinitis pigmentosa. VEGF165 exists in two different isoforms: the angiogenic VEGF165a, and the anti-angiogenic VEGF165b. In some angiogenic diseases the proportion of VEGF165b may be equal to or higher than that of VEGF165a. Therefore, developing therapeutics that inhibit VEGF165a and not VEGF165b may result in greater anti-angiogenic activity and therapeutic benefit. To this end, we report the selective binding properties of sulfated hyaluronic acid (s-HA). Selective biopolymers offer several advantages over antibodies or aptamers including cost effective and simple synthesis, and the ability to make nanoparticles or hydrogels for drug delivery applications or VEGF165a sequestration. Limiting sulfation to the C-6 hydroxyl (C-6 OH) in the N-acetyl-glucosamine repeat unit of hyaluronic acid (HA) resulted in a polymer with strong affinity for VEGF165a but not VEGF165b. Increased sulfation beyond the C-6 OH (i.e. greater than 1 sulfate group per HA repeat unit) resulted in s-HA polymers that bound both VEGF165a and VEGF165b. The C-6 OH sulfated HA (Mw 150 kDa) showed strong binding properties to VEGF165a with a fast association rate constant (Ka; 2.8 × 10(6) M(-1) s(-1)), slow dissociation rate constant (Kd; 2.8 × 10(-3) s(-1)) and strong equilibrium binding constant (KD; ∼1.0 nM)), which is comparable to the non-selective VEGF165 binding properties of the commercialized therapeutic anti-VEGF antibody (Avastin(®)). The C-6 OH sulfated HA also inhibited human umbilical vein endothelial cell (HUVEC) survival and proliferation and human dermal microvascular endothelial cell (HMVEC) tube formation. These results demonstrate that the semi-synthetic natural polymer, C-6 OH sulfated HA, may be a promising biomaterial for the treatment of angiogenesis-related disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a., (© 2015 American Heart Association, Inc.)

Introduction: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by microvascular damage, inflammation, and fibrosis. It has become increasingly evident that platelets, beyond regulating hemostasis, are important in inflammation and innate immunity. Platelets may be an important source of proinflammatory and profibrotic cytokines in the vascular microenvironment. In this study, we sought to assess the contribution of platelet-derived factors in patients with SSc to the angiogenesis of human dermal microvascular endothelial cells (DMVECs) in a tubule formation assay and to characterize the secretion of profibrotic and proinflammatory cytokines in these platelets., Methods: We analyzed platelets obtained from 30 patients with SSc and 12 healthy control subjects. Angiogenesis was evaluated in vitro with a DMVEC tubule formation assay on Matrigel and platelet-derived angiogenic factors such as vascular endothelial growth factor (VEGF), 165b isoform (VEGF165b), and cytokine secretion was evaluated. Platelet serotonin content was also determined., Results: When DMVECs were incubated with SSc platelet releasates, tubule formation was significantly inhibited (p < 0.01, t test), and higher expression of endothelin-1 in these cells was observed compared with control subjects (p < 0.05, Mann-Whitney U test). In SSc platelet releasates, VEGF165b was significantly higher (p < 0.05, t test), and the VEGF165b/VEGF ratio was increased compared with that of control subjects. Higher secretion of transforming growth factor β (p < 0.01, t test) and CD40L (p < 0.01, t test) was observed compared with control subjects. Also, intraplatelet serotonin levels were lower in platelets obtained from patients with diffuse SSc compared with patients with limited SSc and control subjects (p < 0.05, t test)., Conclusions: Our findings suggest that antiangiogenic factors such as VEGF165b, together with proinflammatory and profibrotic factors secreted by platelets, can contribute to the progression of peripheral microvascular damage, defective vascular repair, and fibrosis in patients with SSc.

Vascular endothelial growth factor (VEGF)-targeted antiangiogenic therapy significantly inhibits the growth of clear cell renal cell carcinoma (RCC). Eventually, therapy resistance develops in even the most responsive cases, but the mechanisms of resistance remain unclear. Herein, we developed two tumor models derived from an RCC cell line by conditioning the parental cells to two different stresses caused by VEGF-targeted therapy (sunitinib exposure and hypoxia) to investigate the mechanism of resistance to such therapy in RCC. Sunitinib-conditioned Caki-1 cells in vitro did not show resistance to sunitinib compared with parental cells, but when tested in vivo, these cells appeared to be highly resistant to sunitinib treatment. Hypoxia-conditioned Caki-1 cells are more resistant to hypoxia and have increased vascularity due to the upregulation of VEGF production; however, they did not develop sunitinib resistance either in vitro or in vivo. Human endothelial cells were more proliferative and showed increased tube formation in conditioned media from sunitinib-conditioned Caki-1 cells compared with parental cells. Gene expression profiling using RNA microarrays revealed that several genes related to tissue development and remodeling, including the development and migration of endothelial cells, were upregulated in sunitinib-conditioned Caki-1 cells compared with parental and hypoxia-conditioned cells. These findings suggest that evasive resistance to VEGF-targeted therapy is acquired by activation of VEGF-independent angiogenesis pathways induced through interactions with VEGF-targeted drugs, but not by hypoxia. These results emphasize that increased inhibition of tumor angiogenesis is required to delay the development of resistance to antiangiogenic therapy and maintain the therapeutic response in RCC., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The objectives of the present study were to develop functional targeting epirubicin liposomes for transferring drugs across the blood-brain barrier (BBB), treating glioblastoma, and disabling neovascularization. The studies were performed on glioblastoma cells in vitro and on glioblastoma-bearing mice. The results showed that the constructed liposomes had a high encapsulation efficiency for drugs (>95%), suitable particle size (109 nm), and less leakage in the blood component-containing system; were significantly able to be transported across the BBB; and exhibited efficacies in killing glioblastoma cells and in destroying glioblastoma neovasculature in vitro and in glioblastoma-bearing mice. The action mechanisms of functional targeting epirubicin liposomes correlated with the following features: the long circulation in the blood system, the ability to be transported across the BBB via glucose transporter-1, and the targeting effects on glioblastoma cells and on the endothelial cells of the glioblastoma neovasculature via the integrin β3 receptor. In conclusion, functional targeting epirubicin liposomes could be used as a potential therapy for treating brain glioblastoma and disabling neovascularization in brain glioblastomas.

Angiogenesis, the formation of new blood vessels from the pre-existing vasculature is essential for embryonic development and tissue homeostasis. It also plays critical roles in diseases such as cancer and retinopathy. A delicate balance between pro- and anti-angiogenic factors ensures normal physiological homeostasis. Endogenous angiogenesis inhibitors are proteins or protein fragments that are formed in the body and have the ability to limit angiogenesis. Many endogenous angiogenesis inhibitors have been discovered, and the list continues to grow. Endogenous protein/peptide inhibitors are relatively less toxic, better tolerated and have a lower risk of drug resistance, which makes them attractive as drug candidates. In this review, we highlight ten novel endogenous protein angiogenesis inhibitors discovered within the last five years, including ISM1, FKBPL, CHIP, ARHGAP18, MMRN2, SOCS3, TAp73, ZNF24, GPR56 and JWA. Although some of these proteins have been well characterized for other biological functions, we focus on their new and specific roles in angiogenesis inhibition and discuss their potential for therapeutic application.

Retinopathy of prematurity causes visual impairment due to destructive neoangiogenesis after degeneration of the retinal microvasculature. This study was aimed at analyzing whether local delivery of Semaphorin-3C (Sema3C) suppresses pathological retinal angiogenesis. Sema3C exerted potent inhibiting effects in cellular models of angiogenesis. In an endothelial cell xenotransplantation assay, Sema3C acted primarily on immature microvessels by inducing endothelial cell apoptosis. Intravitreal administration of recombinant Sema3C disrupted endothelial tip cell formation and cell-cell contacts, which led to decreased vascular bed expansion and vessel branching in the growing retinal vasculature of newborn mice, while not affecting mature vessels in the adult retina. Sema3C administration strongly inhibited the formation of pathological pre-retinal vascular tufts during oxygen-induced retinopathy. Mechanistically, Sema3C signaled through the receptors Neuropilin-1 and PlexinD1, which were strongly expressed on vascular tufts, induced VE-cadherin internalization, and abrogated vascular endothelial growth factor (VEGF)-induced activation of the kinases AKT, FAK, and p38MAPK. This disrupted endothelial cell junctions, focal adhesions, and cytoskeleton assembly resulted in decreased cell migration and survival. Thus, this study identified Sema3C as a potent and selective inhibitor of pathological retinal angiogenesis., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Lately, drug-coated balloons have been introduced in interventional cardiology as an approach to treat occluded blood vessel. They were developed for the rapid transfer of antiproliferative drugs during the angioplasty procedure in stenosed vessels with the intent to reduce the risk of restenosis. In this study five different paclitaxel (PTX) balloon coatings were tested in vitro in order to examine how solvents and additives influence coating stability and drug transfer rates. PTX-coated balloons were advanced through a guiding catheter and a simulated coronary artery pathway under perfusion and were then inflated in a hydrogel acceptor compartment. The fractions transferred to the gel, remaining on the balloon and the PTX lost in the simulated coronary pathway were then analysed. The results obtained suggest that the solvent used for the coating process strongly influences the surface structure and the stability of the coating. Ethanol/water and acetone based PTX coatings showed the lowest drug transfer rates to the simulated vessel wall (both <1%) due to their high drug losses during the prior passage through the coronary artery model (more than 95%). Balloons coated with PTX from ethyl acetate-solutions showed smaller drug loss (83%±9%), but most of the remaining PTX was not transferred (mean balloon residue approximately 15%). Beside the solvent, the use of additives seemed to have a great impact on transfer properties. The balloon pre-treatment with a crosslinked polyvinylpyrrolidone (PVP) film was able to increase the PTX transfer rate from less than 1% (without PVP) to approximately 6%. The best results in this study were obtained for balloon coatings with commercially available SeQuent© Please balloons containing the contrast agent iopromide. For this formulation drug transfer rates of approximately 17% were determined. Fluorescence microscopic imaging could visualize the particulate transfer of labelled PTX from the balloon surface during dilatation. The findings of this study underline the importance of drug adhesion and coating stability for the efficiency of PTX transfer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vascular endothelial growth factor (VEGF) is an angiogenic factor critically involved in tumor progression. Adenosine A2B receptor plays a pivotal role in promoting tumor growth. The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSCs) in the pro-angiogenic effects of A2B and to determine whether A2B blockade could enhance the effectiveness of anti-VEGF treatment. Mice treated with Bay60-6583, a selective A2B receptor agonist, showed enhanced tumor VEGF-A expression and vessel density. This effect was associated with accelerated tumor growth, which could be reversed with anti-VEGF treatment. Bay60-6583 increased the accumulation of tumor CD11b+Gr1+ cells. Depletion of MDSCs in mice significantly reduced A2B-induced VEGF production. However, A2B receptor stimulation did not directly regulate VEGF expression in isolated tumor myeloid cells. Mechanistically, Bay60-6583-treated melanoma tissues showed increased STAT3 activation. Inhibition of STAT3 significantly decreased the pro-tumor activity of Bay60-6583 and reduced tumor VEGF expression. Pharmacological blockade of A2B receptor with PSB1115 significantly reduced tumor growth by inhibiting tumor angiogenesis and increasing T cells numbers within the tumor microenvironment. These effects are, at least in part, dependent on impaired tumor accumulation of Gr1+ cells upon A2B receptor blockade. PSB1115 increased the effectiveness of anti-VEGF treatment.