Predicting and understanding synergistic pairwise compound combinations of Shexian Baoxin Pill (SBP) using network biology

To investigate inhibition effects and the mechanism of EPA on the proliferation of human umbilical vascular endothelial cells (HUVEC). Different concentrations of EPA were added to the cultured HUVEC in vitro. The time cause and does response for the inhibition the cells proliferation in all groups were measured by the MTT assay. Light absorption values and cytostasis ratios in all groups were compared. One-way ANOVA in the SPSS 13.0 version statistical software was used. The effect of EPA on cell cycle, proliferative index (PI) and apoptosis of HUVEC in vitro were observed by Flow Cytometry. chi2-test of R x C contingency table was used as a method for statistical analysis. When the concentration of EPA was equal to or more than 0.15 g/L, MTT assay showed a significant difference of light absorption value in the cultured cell after EPA exposure compared with control, the suppressing effects enhanced as the treatment time increased. The peak time of the inhibition of the cell proliferation induced by EPA was at 60 hours and the effect was last until 72 hours. The proliferative index in the treatment group was 23.9%, which was lower than that in the control group (26.9%). No apoptosis was found in the cell in each group. EPA plays an important role of inhibition of proliferation of cultured HUVEC in vitro. No apoptosis was induced by the exposure HUVEC to EPA, therefore, it suggests a potential application for clinical trial.

Antiproliferative and antiangiogenic effects of 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, in human umbilical vascular endothelial cells

Local application of platelets represents a promising tool to enhance bone regeneration. New bone formation strictly requires blood vessel formation, a sequential process involving matrix degradation, migration, proliferation, and tube formation of endothelial cells. Here we investigated the impact of secreted granula products from activated platelets on endothelial cells, and determined the involvement of extracellular signal-regulated kinase (ERK) signaling. The effects of platelet-released supernatant on endothelial cells were investigated using in vitro models. Matrix metalloproteinase-2 (MMP-2) release, migration, proliferation, and tube formation of human umbilical vascular endothelial cells (HUVEC) were determined in response to platelet-released supernatant by gelatine zymography, Boyden chamber assay, 3[H]thymidine incorporation, and basement membrane assay, respectively. All experiments were performed in the presence of the ERK signaling inhibitor PD98059. ERK phosphorylation was detected by Western blot analysis. Incubation with platelet-released supernatant increased the production of MMP-2, migration, proliferation, and tube formation of HUVEC. Platelet-released supernatant also stimulated ERK phosphorylation in HUVEC. Inhibition of ERK signaling decreased platelet-released supernatant-stimulated endothelial cell proliferation, but not MMP-2 activity, migration, and the formation of capillary tubes. Our data suggest that secreted granula products from platelets can enhance different stages of blood vessel formation, and that ERK signaling is required to mediate the mitogenic effects of the supernatant. These findings support the hypothesis of a potential link between platelet activation and blood vessel formation during bone regeneration.

The β 2 -glycoprotein I (β 2 GPI) is an endothelial cell ligand, accessible for systemic, autocrine and paracrine signaling.In vivo, β 2 GPI is immobilized by binding, mainly to the endothelial cell membrane heparan sulfate but also to anionic phospholipids, and functional receptors.The β 2 GPI was attributed antiangiogenic properties both in vitro and in vivo.This work was designed to evaluate the antiangiogenic effects of native, monomeric, and dimeric β 2 GPI.Monomeric as well as dimeric forms were purified from human plasma, and the native protein was obtained as a balanced mixture of both components present in human plasma.The proliferation, migration, and differentiation of Human Umbilical Vascular Endothelial Cells (HUVEC) were considered in an in vitro angiogenesis model based on tridimensional cultures and quantitative digital image processing techniques.The early events of the in vitro HUVEC growth and differentiation in the tridimensional cultures microenvironment were addressed by the morphological analysis.The morphological aspects were correlated to the cell growth, oxidative balance outcome and mitochondrial toxicity assays, leading to the evidence that non-confluent HUVEC cultures temporarily stop growing in the presence of the native protein, but remain competent to proliferate.The β 2 GPI monomer allowed the in vitro differentiation of the HUVECs into typical trabeculae and incomplete capillary-like tubes, along with lowering the available proliferation fraction.In opposition, the dimer rich purification fraction exposure halted cell elongation and migration, and prevented the organization of the tubular structures in tridimensional cultures, maintaining cell growth.The morphological approach was useful to attribute to β 2 GPI dimerization the cell migration inhibition modulation, which potentially leads to overcome the diminished sprouting antiangiogenic effect of the monomer fraction of the native protein.

IntroductionAtherosclerosis is a continuously worsening chronic condition that starts in the arteries and may then affect other blood vessels. There is increasing evidence linking microRNAs to the development of arteriosclerosis obliterans (ASO). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied in this study to detect miR-210-5p (miR-210) expression, and significant upregulation was observed in human arterial walls where ASO was prevalent.Material and methodsThe proliferation of human umbilical vascular endothelial cells (HUVECs) with various levels of miR-210 expression was assessed via MTT and colony-formation assays. Cell proliferation was significantly promoted in HUVECs with upregulated miR-210 levels and reduced in HUVECs with downregulated miR-210 levels.ResultsFlow cytometric analysis of cells stained with annexin V-FITC and propidium iodide data demonstrated that miR-210 inhibited apoptosis, while miR-210 inhibition promoted apoptosis by mediating pro-apoptotic protein expression levels. These results were verified using a dual-luciferase reporter gene assay system, which showed that Janus kinase 1 (JAK1) was directly targeted by miR-210, while an miR-210 mimic significantly decreased downstream JAK1 and signal transducer and activator of transcription 3 (STAT3) activation at a post-transcriptional level in HUVECs, as detected by western blotting and qRT-PCR. Further inhibition of either JAK1 or STAT3 counteracted the effect of miR-210 on HUVEC proliferation and apoptosis. These findings suggest that miR-210 promotes HUVEC proliferation, at least in part, by targeting the JAK1-STAT3 signaling axis.ConclusionsThis study provides insights into the contribution of the miR-210-JAK1-STAT3 axis and its underlying mechanisms to ASO pathology.

Endothelial cell apoptosis driven by inflammation (TNF-α) plays a critical role in the pathogenesis of atherosclerosis, but the exact molecular mechanisms are not clearly elucidated. MicroRNA (miR)-29 families (a/b/c) take important roles in pathophysiological processes of atherosclerosis, also the underlying mechanisms have not been fully clarified. The aims are to explore whether or not miR-29 families mediate the apoptotic effects of TNF-α on endothelial cells and uncover the underlying molecular mechanisms. In this study, MTT assay and flow cytometer analysis were employed respectively to determine the proliferation and apoptosis of human umbilical vascular endothelial cells (HUVECs) under TNF-α exposure. Real-time quantitative PCR and western blot were performed to detect the levels of target RNAs and proteins/their phosphorylation in HUVECs.TNF-α could inhibit HUVEC proliferation and induce HUVEC apoptosis in a positive dose- and time-dependent manner, with a similar way of miR-29a upregulation, but no effects on miR-29b/c. Upregulation of miR-29a with its mimics enhanced the apoptotic effect of TNF-α on HUVECs, but downregulation of miR-29a using anti-miR-29a blocked up its apoptotic effect. MiR-29a inhibited the expression of PI3Kp85α and Bcl-2 and blocked up the signal transduction of PI3K/AKT/Bcl-2 axis to mediate the apoptotic effect of TNF-α on HUVECs. Mediating the inflammation-driven endothelial cell apoptosis is an important biology mechanism by which miR-29a promotes atherosclerosis and its complications. MiR-29a will be a potential diagnostic and therapeutic target for atherosclerotic cardiovascular diseases; it is worthwhile to further study.

In an attempt to find a strategy to modulate the proliferation of vascular endothelial cells, we examined whether constitutive activation of proto-oncogen protein p21 (Ras) induced the reentry of confluent human umbilical vascular endothelial cells (HUVECs) into the S phase. When an adenovirus construct expressing a constitutively active Ras mutant (Ad/RasG12V) was infected into HUVECs, their morphology changed strikingly and they appeared to be transformed. However, Ad/RasG12V-infected HUVECs did not enter the S phase, as determined by assessing 3H-thymidine incorporation. In accordance with the above results, the expression of cyclin A both at the transcript and protein levels did not increase in Ad/RasG12V-infected HUVECs relative to that in control cells, although the expression of cyclin D1 was induced in Ad/RasG12V-infected cells. Interestingly, the expression of the cyclin-dependent kinase (CDK) inhibitor p21cip1 was remarkably increased while that of p27kip1 did not decrease in Ad/RasG12V-infected HUVECs. Furthermore, CDK2 activity was not induced in Ad/RasG12V-infected HUVECs. These results suggested that the constitutive activation of Ras promoted the reentry of confluent HUVECs in the G0 phase into the G1 phase, but not into the S phase. The results also indicated that the constitutive activation of Ras might have induced the persistent expression of p21cip1 and p27kip1, and that this induction of p21cip1 and p27kip1 expression possibly caused the cell cycle arrest at the G1 phase.

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) have been strongly implicated in the growth and metastasis of gastric cancer. The purpose of this study was to examine the effects of ZD6474, an inhibitor of inhibitor of VEGF receptor (VEGFR) tyrosine kinase with additional activity against EGF receptor (EGFR), on tumor growth and angiogenesis in an orthotopic model of gastric cancer. In vitro, ZD6474 inhibited human umbilical vascular endothelial cell and TMK-1 human gastric tumor cell proliferation in a dose-dependent fashion. EGF-mediated activation of EGFR and Erk-1/2 was decreased in tumor cells after ZD6474 treatment. In addition, VEGF-mediated activation of VEGFR2 and Erk-1/2 was decreased in human umbilical vascular endothelial cells. TMK-1 human gastric adenocarcinoma cells were injected into the gastric wall of nude mice. ZD6474 therapy was initiated on day 10. Mice (n = 14 per group) were treated p.o. with (a) 1% Tween 80 (control), (b) 50 mg/kg/d ZD6474, or (c) 100 mg/kg/d ZD6474. Mice were sacrificed on day 33. Tumors from each group were stained for markers of blood vessels, pericytes, proliferation, and apoptosis. ZD6474 at both 50 and 100 mg/kg/d led to marked inhibition of tumor growth (P < 0.05). ZD6474 reduced tumor cell proliferation by 48% in the 50 mg/kg/d group and 65% in the 100 mg/kg/d group (P < 0.03) and increased tumor cell apoptosis (P < 0.001) in vivo. ZD6474 led to a 69% decrease in microvessel density in the 50 mg/kg/d group (P < 0.001) and a 62% decrease in the 100 mg/kg/d group (P < 0.001). Although microvessel density was decreased by ZD6474, the remaining vessels showed a relatively higher percentage of pericyte coverage (3-fold increase; P < 0.001), perhaps reflecting selective loss of uncovered vessels in the ZD6474 group. In conclusion, therapies such as ZD6474 that target two distinct aspects of tumor growth, angiogenesis and tumor cell proliferation, warrant further investigation.

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0-120 microM) dose-dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 microM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB-induced inhibition of cell growth rate is reversible. [(3)H]thymidine incorporation revealed that TB reduced the [(3)H]thymidine incorporation into HUVEC during the S-phase of the cell-cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB-induced cell-cycle arrest in HUVEC occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB-induced inhibition of [(3)H]thymidine incorporation. Taken together, these results suggest an involvement of the p21-associated signaling pathway in the TB-induced antiproliferation in HUVEC. Capillary-like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis.

Human cytomegalovirus (HCMV) infection has been linked to the pathogenesis of vasculopathy by inducing dysfunction of vascular cells such as endothelial cells. Hcmv-miR-UL112 is the most well-characterized HCMV-encoded microRNA occurring in the plasma of patients with cardiovascular diseases such as hypertension, while the specific underlying pathophysiological mechanisms are yet to be defined. The current study investigated the effect of hcmv-miR-UL112 on the growth and proliferation of human umbilical vascular endothelial cells (HUVECs); it might also be associated with signaling pathways. An adenovirus vector was designed and synthesized to stably express hcmv-miR-UL112 in HUVECs. Cell Counting Kit-8 results showed that ectopically expressed hcmv-miR-UL112 can significantly increase the proliferation of HUVECs (p<0.05). Flow cytometry revealed that the S-phase fraction in the cell cycle analysis was raised significantly after overexpression of hcmv-miR-UL112 (p<0.05). Gene expression profile analysis, using the microarray technology, revealed 303 up-regulated and 62 down-regulated genes in HUVECs by comparing the AD-hcmv-miR-UL112-infected and control groups (p<0.05 and>2 fold change). Kyoto Encyclopedia of Genes and Genomes and Reactome Pathway, chosen as the functional annotation categories, were affected by hcmv-miR-UL112 adenovirus vector. The significantly altered pathways mainly include the mitogen-activated protein kinase signaling pathway, cell adhesion molecules, chemokine signaling pathway, cytokine-cytokine receptor interaction, circadian rhythm-mammal, mineral absorption, protein processing in the endoplasmic reticulum, proximal tubule bicarbonate reclamation, vasopressin-regulated water reabsorption, and arachidonic acid metabolism. In conclusion, hcmv-miR-UL112 could serve as a potential biomarker, and the miRNA-mediated regulation of signaling pathways might play significant roles in the physiological effects of hcmv-associated diseases.

Background. Potent inhibitors of histone binding modules have the potential to suppress, selectively, transcription of specific genes, including MYC family of transcription factors. JQ1 binds competitively to acetyl-lysine recognition motifs, or bromodomains. JQ1 inhibits BET bromodomain, and down-regulates MYC transcription. Ewing sarcoma (EWS) and alveolar rhabdomyosarcoma (ARMS) are associated with high expression of c-MYC and MYCN, respectively (Delmore JE, Cell 2011; Filippakopoulos P, Nature 2010). Consequently, we have evaluated sensitivity of EWS and ARMS cell lines and xenograft models to JQ1. Methods. EWS and ARMS cells were incubated with JQ1, or JQ1R (non-active enantiomer) for 96 hrs. Cell viability was determined by Alamar Blue staining. C-MYC and MYCN were determined by immunoblotting. Xenograft tumor lines were grown in CB17 female SCID mice. JQ1 was administered P.O. at 30 mg/kg (BID) or 50 mg/kg (SID) for up to 3 weeks. Assessment of JQ1 activity was as described (Houghton P, Ped Blood &amp; Cancer, 2007). The effect of JQ1 on proliferation and tube formation of human umbilical vascular endothelial cells (HUVECs) was as described (Bid H, Mol Cancer Ther, 2012). Results. For all cell lines, the IC50 concentration for JQ1R (inactive enantiomer) was &amp;gt; 10 microM. EWS cell lines (n=9) segregated into two distinct groups with IC50 &amp;lt; 1 microM, (ES-1, ES-2, ES-3, ES-4, ES-7, ES-8) and cell lines with significantly higher IC50s (IC50 &amp;gt; 7 microM ES-6, EW8, CHLA-258). Seven of 9 ARMS cell lines demonstrated sensitivity to JQ1 (IC50 &amp;lt; 1 microM) whereas 2 lines were insensitive (IC50 &amp;gt; 10 microM Rh18, Rh30). JQ1 (500 nM, 24 hr) markedly down-regulated c-MYC in Rh41, and to a lesser extent in Rh36. MYCN expression in Rh5 ARMS cell line was also suppressed by JQ1. Of interest, JQ1 did not suppress c-MYC in any EWS cell line. In vivo, BID and SID dosing was well tolerated (mortality 1.2%) and gave similar antitumor activity. JQ1 significantly retarded growth of all 4 sarcoma models (Rh10, Rh28 ARMS and EW5, EW8 EWS lines. T/C at day 21 range 0.30 – 0.43). Of note, while EW8 cells in vitro were resistant to JQ1 (IC50 &amp;gt; 10 microM), the xenografts were equally sensitive as other lines to JQ1 treatment, suggesting JQ1 exerted effects on stroma/angiogenesis. In vitro, JQ1 (500 nM) suppressed tube formation of HUVECs without decreasing viability. Conclusions. JQ1 potently inhibited proliferation of EWS and ARMS cells in vitro, with distinct groups demonstrating either sensitivity or resistance. Sensitivity was associated with decreased levels of MYCN or c-MYC in some cell lines. In vivo, JQ1 demonstrated significant antitumor activity in 4 xenograft lines, including EW8 derived from cells resistant to JQ1 in vitro. The potential for JQ1 to exert anti-angiogenic effects is suggested by its potent inhibition of HUVECs differentiation. Supported through a grant from SWOG Citation Format: Linlin Xiao, Doris A. Phelps, Laurence A. Baker, Jun Qi, Hemant K. Bid, Peter J. Houghton. Targeting bromodomain and extra-terminal (BET) family member BRD4 for treatment of pediatric sarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2013-2753

Combretastatin A-4 (CA4) is the lead compound of a relatively new class of vascular disrupting agents that target existing tumor blood vessels. Recent studies showed the CA4 might inhibit angiogenesis. However, the underlying molecular mechanisms by which CA4 exerts its anti-angiogenic effects are not fully understood. In this study, we revealed that CA4 inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs). In in vivo assay, CA4 suppressed neovascularization in chicken chorioallantoic membrane (CAM) model and decreased the microvessel density in tumor tissues of a breast cancer MCF-7 xenograft mouse model. In addition, CA4 decreased the expression level and secretion of VEGF both in MCF-7 cells and HUVECs under hypoxia, as well as the activation of VEGFR-2 and its downstream signaling mediators following VEGF stimulation in HUVECs. Moreover, VEGF and VEGFR-2 expression in tumor tissues of the mouse xenograft model were down-regulated following CA4 treatment. Taken together, results from the current work provide clear evidence that CA4 functions in endothelial cell system to inhibit angiogenesis, at least in part, by attenuating VEGF/VEGFR-2 signaling pathway.

Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is β-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration. In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated. Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI. The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future.

Angiogenesis provides necessary nutrients and oxygen for tumor growth and metastasis; thus, every stage of angiogenesis process is the potential target for cancer therapies. Ursolic acid (UA) is reported to decrease tumor burden through anti-angiogenesis pathway, but its poor water solubility greatly limits its efficiency and clinical application. Here, a simple method for preparing UA-loaded chitosan nanoparticles (CH-UA-NPs) with anti-angiogenesis and anti-tumor activity was demonstrated. In vitro, CH-UA-NPs could significantly inhibit the proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs). After uptake by HUVECs, CH-UA-NPs were mainly localized in lysosomes and mitochondria, but not nuclei. CH-UA-NPs induced the destruction of lysosome membrane integrity, collapse of mitochondrial membrane potential, and reorganization of cell cytoskeleton. All these changes led to the apoptosis or necrosis in HUVECs. In vivo, CH-UA-NPs could inhibit the angiogenesis in chicken chorioallantoic membrane (CAM) model and H22 xenograft model. Notably, comparing with free UA, such synthesized CH-UA-NPs could save about tenfold of UA doses, implying that this could significantly decrease the side effects induced by high doses of UA in biological organism. Our data showed that CH-UA-NPs and this nanoparticle-based drug delivery system could be as a potential drug candidate foranti-angiogenesis treatment.