Vascular Tube Formation Research Articles

Increased expression of epidermal growth factor-like domain-containing protein 7 is predictive of poor prognosis in patients with hepatocellular carcinoma.

Epidermal growth factor-like domain-containing protein 7 (EGFL7) is an endothelial cell-derived secreted factor that regulates vascular tube formation. In human cancer, the specificity of expression is lost as EGFL7 has been detected in tumor cells, in addition to endothelial cells. This study evaluated the intricate relationship between hypoxia-inducible factor 1-alpha (HIF-1α) and EGFL7 under both hyperoxia and hypoxia states. In the present study, immunohistochemical staining and ELISA were applied to examine the relative level of EGFL7 in 182 cases of hepatocellular carcinoma (HCC) formalin-fixed and paraffin-embedded tissues and 110 cases of HCC serum samples. Quantitative polymerase chain reaction and Western blotting were applied to verify the correlation between serum EGFL7 level and anoxic microenvironment. Immunohistochemical staining was performed to determine the correlation between EGFL7 and HIF1-α. The correlations between EGFL7 expression and patients' age, tumor size, gender, N-stage, history of cirrhosis, M-stage, history of hepatitis C, and history of hepatitis B were statistically insignificant (P = 0.28, 0.34, 0.71, 0.15, 0.8, 0.2, 0.052, and 0.14, respectively). High level of EGFL7 was significantly correlated with overall survival as well as disease-free survival in 182 HCC patients (P = 0.0016 and P < 0.001, respectively). The correlations between serum EGFL7 and vascular invasion and extrahepatic metastasis were statistically significant (P < 0.0001). Among the 35 HIF1-α-positive HCC patients, 69% were medium positive and 31% were strong positive. EGFL7 protein expression level was oxygen dependent in HCC line (P < 0.05). EGFL7 was found to be a potential predictor for HCC survival and metastasis state; EGFL7 may be a promising biomarker and therapeutic target in human HCC.

PAC-1 and its derivative WF-210 Inhibit Angiogenesis by inhibiting VEGF/VEGFR pathway

Procaspase Activating Compound-1 (PAC-1) and its derivative WF-210 induce apoptosis in cancer cells by activating procaspase-3 to caspase-3. The aim of this study was to extend current knowledge about the mechanisms of PAC-1 and WF-210, particularly about their effects on tumor angiogenesis. PAC-1 and WF-210 restrained VEGF-induced human umbilical vascular endothelial cells (HUVECs) proliferation, invasion, and tube formation. PAC-1 and WF-210 abrogated VEGF-induced vessel sprouting from rat aortic rings and inhibited vascular formation in the Matrigel plug assay. PAC-1 and WF-210 suppressed phosphorylation of VEGFR2 and its downstream protein kinases c-Src, FAK, and AKT in both HUVECs and U-87 cells. When given to mice bearing subcutaneous or orthotopic xenograft, PAC-1 and WF-210 inhibited the tumor growth and tumor angiogenesis. Further tests showed that PAC-1 and WF-210 inhibited stemness and induced autophagy flux of U-87 cells. This study revealed mechanisms of PAC-1 and WF-210 other than inducing apoptosis, which provides additional support for their using in the clinic.

Inhibition of PI4K IIIα radiosensitizes in human tumor xenograft and immune-competent syngeneic murine tumor model.

Phosphatidylinositol (PI) 4-kinase (PI4K) has emerged as a potential target for anti-cancer treatment. We recently reported that simeprevir, an anti-hepatitis C viral (HCV) agent, radiosensitized diverse human cancer cells by inhibiting PI4K IIIα in vitro. In this study, we investigated the radiosensitizing effect of simeprevir in an in vivo tumor xenograft model and the mechanism of its interaction. The immune modulatory effect of PI4K IIIα was evaluated in an immune-competent syngeneic murine tumor model.In in vivo xenograft models using BT474 breast cancer and U251 brain tumor cells, inhibition of PI4K IIIα induced by simeprevir combined with radiation significantly delayed tumor growth compared to either treatment alone. PI4K IIIα inhibition led to eversion of the epithelial-mesenchymal transition as suggested by decreased invasion/migration and vascular tube formation. Simeprevir down-regulated PI3Kδ expression and PI3Kδ inhibition using RNA interference radiosensitized breast cancer cells. PI4K IIIα inhibition enhanced the radiosensitizing effect of anti-programmed death-ligand 1 (PD-L1) and decreased the expression of PI3Kδ, phosphorylated-Akt, and PD-L1 in breast cancer cells co-cultured with human T-lymphocytes. The immune modulatory effect in vivo was evaluated in immune-competent syngeneic 4T1 murine tumor models. Simeprevir showed significant radiosensitizing effect and immune modulatory function by affecting the CD4(+)/CD8(+) ratio of tumor infiltrating lymphocytes.These findings suggest that targeting PI4K IIIα with an anti-HCV agent is a viable drug repositioning approach for enhancing the therapeutic efficacy of radiation therapy. The immune regulatory function of PI4K IIIα via modulation of PI3Kδ suggests a strategy for enhancing the radiosensitizing effect of immune checkpoint blockades.

Parthenolide suppresses hypoxia-inducible factor-1α signaling and hypoxia induced epithelial-mesenchymal transition in colorectal cancer.

Activation of hypoxia-inducible factor 1α (HIF‑1α) is frequently observed in solid tumors and it has been associated with various pathophysiological processes, including epithelial‑mesenchymal transition (EMT). Previously, we reported that parthenolide (PT), an inhibitor of nuclear factor-κB (NF-κB), is a promising anticancer agent because it promotes apoptosis of human colorectal cancer (CRC). Here, we investigated a new molecular mechanism by which PT acts on HIF‑1α and hypoxia contributing to EMT by NF‑κB inhibition. Cell viability, DNA binding activity, vascular cell tube formation and cell motility were studied after treatment of PT in hypoxic or normoxic condition. Moreover, effects of PT on hypoxia signaling and hypoxia-induced EMT signaling were investigated. We also examined the inhibitory effect of PT on CRC progression in xenografts. We demonstrated that PT markedly inhibits hypoxia dependent HIF‑1α activity and angiogenesis by preventing NF-κB activation. We also report that PT decreases the level of proteins associated with glucose metabolism, angiogenesis, development and survival that are regulated by HIF‑1α. Furthermore, we verified that PT protects the morphological change from epithelial to mesenchymal state, inhibits matrix metalloproteinase (MMP) enzyme activity and decreases cell motility involved in the -regulation of the hypoxia-induced EMT markers. In addition, PT inhibits growth in CRC xenograft models and regulates NF‑κB, HIF‑1α and EMT specific marker in tissue specimens. Our data demonstrated that PT can inhibit HIF‑1α signaling and hypoxia-induced EMT, suggesting a novel molecular mechanism for HIF‑1α mediated cancer progression and metastasis.

SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR-153

Malignant glioma, the most common intracranial primary tumor, is characterized by increased angiogenesis. Accumulating evidence has shown that long non-coding RNAs (lncRNAs) play an important role in a variety of biological behaviors of tumors. However, the role of lncRNAs in the regulation of glioma vascular endothelial cell function remains to be investigated. To simulate the glioma microenvironment, we applied glioma conditioned medium (GCM) to human cerebral microvascular endothelial cells (hCMECs). In the present study, the lncRNA SNHG15 was found to be highly expressed in glioma vascular endothelial cells. Cell Counting Kit-8 (CCK-8), migration and tube formation assays demonstrated that knockdown of SNHG15 inhibited glioma vascular endothelial cell proliferation, migration and tube formation invitro. Furthermore, knockdown of SNHG15 downregulated the expression of VEGFA and Cdc42, which are known to promote angiogenesis. Bioinformatics software and dual-luciferase system analysis confirmed that SNHG15 affected endothelial cell function by targeting miR-153. Additionally, the present study showed that miR-153 targeted the 3'‑untranslated region of VEGFA and Cdc42 and downregulated their expression. In conclusion, knockdown of SNHG15 downregulated the expression of VEGFA and Cdc42 by targeting miR-153, consequently suppressing glioma vascular endothelial cell proliferation, migration and tube formation. Therefore, SNHG15 and miR-153 are new potential therapeutic targets for anti-angiogenesis treatment of glioma.

Reprogramming Bone Marrow Stem Cells to Functional Endothelial Cells in a Mini Pig Animal Model.

BackgroundThe aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model.Material/MethodsBone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 μg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs.ResultsReprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/106 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/106 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation.ConclusionsThe findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.

Identification of Nidogen 1 as a lung metastasis protein through secretome analysis.

Secreted proteins play crucial roles in mediating tumor-stroma interactions during metastasis of cancer to different target organs. To comprehensively profile secreted proteins involved in lung metastasis, we applied quantitative mass spectrometry-based proteomics and identified 392 breast cancer-derived and 302 melanoma-derived proteins secreted from highly lung metastatic cells. The cancer-specific lung metastasis secretome signatures (LMSSs) displayed significant prognostic value in multiple cancer clinical data sets. Moreover, we observed a significant overlap of enriched pathways between the LMSSs of breast cancer and melanoma despite an overall small overlap of specific proteins, suggesting that common biological processes are executed by different proteins to enable the two cancer types to metastasize to the lung. Among the novel candidate lung metastasis proteins, Nidogen 1 (NID1) was confirmed to promote lung metastasis of breast cancer and melanoma, and its expression is correlated with poor clinical outcomes. In vitro functional analysis further revealed multiple prometastatic functions of NID1, including enhancing cancer cell migration and invasion, promoting adhesion to the endothelium and disrupting its integrity, and improving vascular tube formation capacity. As a secreted prometastatic protein, NID1 may be developed as a new biomarker for disease progression and therapeutic target in breast cancer and melanoma.

Anti-angiogenic effect of arsenic trioxide in lung cancer via inhibition of endothelial cell migration, proliferation and tube formation.

Arsenic trioxide (As2O3) exhibits a remarkable effect on leukemia treatment; however, its effect on solid tumors remains poorly explored. The present study demonstrated the inhibitory effect of As2O3 on lung cancer and explored its possible mechanism. It was observed that As2O3 significantly inhibited the growth of lung cancer xenografts and tumor angiogenesis in vivo. The inhibitory effect of As2O3 on cell proliferation in vitro was more remarkable in vascular endothelial cells than in lung cancer cells. It was also observed that As2O3 inhibited the migration of vascular endothelial cells and disrupted vascular tube formation on Matrigel assays. In addition, a series of key signaling factors involved in multiple stages of angiogenesis, including matrix metalloproteinase (MMP)-2, MMP-9, platelet-derived growth factor (PDGF)-BB/PDGF receptor-β, vascular endothelial growth factor (VEGF)-A/VEGF receptor-2, basic fibroblast growth factor (FGF)/FGF receptor-1 and delta like canonical Notch ligand 4/Notch-1, were regulated by As2O3. These findings suggested that anti-angiogenesis may be an underlying mechanism of As2O3 anticancer activity in lung cancer.

MicroRNA-200c increases radiosensitivity of human cancer cells with activated EGFR-associated signaling.

MicroRNA-200c (miR-200c) recently was found to have tumor-suppressive properties by inhibiting the epithelial-mesenchymal transition (EMT) in several cancers. miR-200c also interacts with various cellular signaling molecules and regulates many important signaling pathways. In this study, we investigated the radiosensitizing effect of miR-200c and its mechanism in a panel of human cancer cell lines. Malignant glioma (U251, T98G), breast cancer (MDA-MB-468), and lung carcinoma (A549) cells were transfected with control pre-microRNA, pre-miR-200c, or anti-miR-200c. Then, RT-PCR, clonogenic assays, immunoblotting, and immunocytochemisty were performed. To predict the potential targets of miR-200c, microRNA databases were used for bioinformatics analysis. Ectopic overexpression of miR-200c downregulated p-EGFR and p-AKT and increased the radiosensitivity of U251, T98G, A549, and MDA-MB-468 cells. In contrast, miR-200c inhibition upregulated p-EGFR and p-AKT, and decreased radiation-induced cell killing. miR-200c led to persistent γH2AX focus formation and downregulated pDNA-PKc expression. Autophagy and apoptosis were major modes of cell death. Bioinformatics analysis predicted that miR-200c may be associated with EGFR, AKT2, MAPK1, VEGFA, and HIF1AN. We also confirmed that miR-200c downregulated the expression of VEGF, HIF-1α, and MMP2 in U251 and A549 cells. In these cells, overexpressing miR-200c inhibited invasion, migration, and vascular tube formation. These phenotypic changes were associated with E-cadherin and EphA2 downregulation and N-cadherin upregulation. miR-200c showed no observable cytotoxic effect on normal human fibroblasts and astrocytes. Taken together, our data suggest that miR-200c is an attractive target for improving the efficacy of radiotherapy via a unique modulation of the complex regulatory network controlling cancer pro-survival signaling and EMT.

Evaluation of the antitumor activity of NOV202, a novel microtubule targeting and vascular disrupting agent.

PurposeOverall, ~65% of patients diagnosed with advanced ovarian cancer (OC) will relapse after primary surgery and adjuvant first-line platinum- and taxane-based chemotherapy. Significant improvements in the treatment of OC are expected from the development of novel compounds having combined cytotoxic and antiangiogenic properties that make them effective on refractory tumors.MethodsPermeability of NOV202 was determined with Caco-2 monolayer assay. The compound’s pharmacokinetic profile and plasma:brain distribution were assessed in male C57Bl/6 mice. The compound’s impacts on tubulin, microtubules and cell cycle were investigated by using in vitro tubulin polymerization assay, cell-based immunofluorescence and live cell microscopy. The IC50 concentrations of NOV202 were assessed in a panel of eight cancer cell lines. Impact of the compound on vascular tube formation was determined using the StemKit and Chick chorioallantoic membrane assays. The in vivo efficacy of the compound was analyzed with an OC xenograft mouse model.ResultsNOV202 was found to suppress cancer cell proliferation at low nanomolar concentrations (IC50 2.3–12.0 nM) and showed equal efficacy between OC cell line A2780 (IC50 2.4 nM) and its multidrug-resistant subline A2780/Adr (IC50 2.3 nM). Mechanistically, NOV202 targeted tubulin polymerization in vitro in a dose-dependent manner and in cells induced an M phase arrest. In vivo, NOV202 caused a dose-dependent reduction of tumor mass in an A2780 xenograft model, which at the highest dose (40 mg/kg) was comparable to the effect of paclitaxel (24 mg/kg). Interestingly, NOV202 exhibited vascular disrupting properties that were similar to the effects of Combretastatin A4.ConclusionNOV202 is a novel tubulin and vascular targeting agent that shows strong anticancer efficacy in cells and OC xenograft models. The finding that the compound induced significantly more cell death in Pgp/MDR1 overexpressing OC cells compared to vincristine and paclitaxel warrants further development of the compound as a new therapy for OC patients with treatment refractory tumors and/or relapsing disease.

The Different Effects of VEGFA121 and VEGFA165 on Regulating Angiogenesis Depend on Phosphorylation Sites of VEGFR2.

The effects of VEGFA isoforms on the vascular permeability and structure are still unclear. In this study, we found that VEGFA121 and VEGFA165, 2 isoforms of VEGFA, exerted the opposing effects of antiangiogenesis and proangiogenesis on regulating vascular endothelia cells proliferation and tube formation. The 2 isoforms affected the protein expression of Ras-related protein 1-GTPase-activating protein 1 (Rap1GAP) and thrombospondin 1, 2 important signal molecules of Rap1GAP/thrombospondin 1 signal pathway in primary human umbilical vein endothelial cells by regulating 2 different phosphorylating sites of VEGFR2, Tyr(1175) and Tyr(1214). We also found that VEGFA121 and VEGFA165 regulating angiogenesis was related to their regulating VEGFR2 and Rap1GAP/thrombospondin 1 signal pathway with the technology of RNA intervening the gene expression of VEGFR2 and Rap1GAP. Meanwhile, 2 inhibitors of VEGFR2, cabozantinib malate and ZM 323881 HCl (ZM), were used to investigate the relationship among VEGFA(121 and 165), VEGFR2, and angiogenesis. It was demonstrated that cabozantinib malate blocked VEGFA121 and VEGFA165 binding to VEGFR2 and inhibited angiogenesis by specifically binding to VEGFR2 rather than changing VEGFR2 phosphorylation or regulating the expression of VEGFR2. However, ZM antagonized the effect of VEGFA on angiogenesis by specifically reversing the phosphorylation induced by VEGFA121 and VEGFA165. The experiments in vivo also demonstrated that obvious abnormality of VEGFA121 and VEGFA165 presented in the serum of ulcerative colitis (UC) rats compared with that of the normal rats. ZM could promote the repairation of the injuries of the vessels and tissues of colonic mucosa of UC rats and caused mild inflammation in colonic mucosa of normal rats. On the contrary, cabozantinib malate caused injury of vessels and inflammation in the colonic mucosa of normal rats and aggravated the injuries of the vessels and inflammation in the colonic mucosa of UC rats. Hence, our data indicated that the activation of different phosphorylation sites of VEGFR2 leaded to VEGFA121 and VEGFA165 exerting opposing effects on angiogenesis, and it might be an underlying pathogenesis of UC and a potential target for UC treatment.

MiR-193a-3p interaction with HMGB1 downregulates human endothelial cell proliferation and migration

Circulating endothelial colony forming cells (ECFCs) contribute to vascular repair where they are a target for therapy. Since ECFC proliferative potential is increased in cord versus peripheral blood and to define regulatory factors controlling this proliferation, we compared the miRNA profiles of cord blood and peripheral blood ECFC-derived cells. Of the top 25 differentially regulated miRNAs selected, 22 were more highly expressed in peripheral blood ECFC-derived cells. After validating candidate miRNAs by q-RT-PCR, we selected miR-193a-3p for further investigation. The miR-193a-3p mimic reduced cord blood ECFC-derived cell proliferation, migration and vascular tubule formation, while the miR-193a-3p inhibitor significantly enhanced these parameters in peripheral blood ECFC-derived cells. Using in silico miRNA target database analyses combined with proteome arrays and luciferase reporter assays of miR-193a-3p mimic treated cord blood ECFC-derived cells, we identified 2 novel miR-193a-3p targets, the high mobility group box-1 (HMGB1) and the hypoxia upregulated-1 (HYOU1) gene products. HMGB1 silencing in cord blood ECFC-derived cells confirmed its role in regulating vascular function. Thus, we show, for the first time, that miR-193a-3p negatively regulates human ECFC vasculo/angiogenesis and propose that antagonising miR-193a-3p in less proliferative and less angiogenic ECFC-derived cells will enhance their vasculo/angiogenic function.

Human and Swine Dental Pulp Stem Cells Form a Vascularlike Network after Angiogenic Differentiation in Comparison with Endothelial Cells: A Quantitative Analysis

IntroductionThe aim of this study was to quantify vascular network formation capacity after angiogenic induction of human and swine dental pulp stem cells (DPSCs) in comparison with endothelial cells. MethodsPrimary human DPSCs or swine DPSCs were induced in endothelial growth medium for 7 days. The expression of the endothelial marker von Willebrand factor was determined by immunostaining. Induced DPSCs (iDPSCs) and noninduced DPSCs (niDPSCs) were seeded at different cell numbers onto Matrigel (BD Biosciences, San Jose, CA) for vascular network formation assays and analyzed after 4, 8, 12, and 18 hours in comparison with human microvascular endothelial cells (hMECs). Quantitative analysis of vascular tubule formation was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The vascular network formation was also conducted by coculturing of niDPSCs and iDPSCs. ResultsVon Willebrand factor was detected by immunofluorescence in both niDPSCs and iDPSCs (human and swine). Time-lapse microscopic observation showed that the vascular network was formed by iDPSCs but not niDPSCs. After 4 hours, iDPSCs showed vascular network formation, whereas niDPSCs started to aggregate and formed clusters. Human iDPSCs displayed a similar capacity to form vascular networks in Matrigel compared with hMECs based on quantitative analysis. Swine iDPSCs had a higher capacity compared with human iDPSCs or hMECs (P < .05) in forming the network structures including segments, nodes, and mesh. A coculture experiment showed that human niDPSCs colocalized on the angiogenic tubules and vascular networks that were formed by human iDPSCs. ConclusionsOur findings indicate that iDPSCs in combination with their noninduced counterparts may be used as a future clinical strategy for enhancing angiogenesis during the process of pulp-dentin regeneration.

Elevated expression of placental growth factor is associated with airway-wall vascular remodelling and thickening in smokers with asthma

The increased expression of placental growth factor (PlGF) in chronic obstructive pulmonary disease and allergy-related asthma suggests its role in the pathogenesis of these diseases. In asthmatic smokers, airway remodelling is accompanied by an accelerated decline in lung function. However, whether PlGF contributes to the persistent airflow obstruction and vascular remodelling typically seen in asthmatic smokers is unknown. In this study we measured lung function, airway-wall thickening, and PlGF levels in serum and induced sputum in 74 asthmatic and 42 healthy smokers and never-smokers. Using human lung microvascular endothelial cells (HLMECs), we evaluated the in vitro effects of PlGF on each step of vascular remodelling, including proliferation, migration, stress-fibre expression, and tubule formation. Our data showed significantly higher serum and sputum PlGF levels in asthma patients, especially asthmatic smokers, than in healthy controls. Serum and sputum PlGF levels correlated negatively with post-bronchodilator forced expiratory volume in 1 s (FEV1) and the FEV1/forced vital capacity, but positively with airway-wall thickening. Stimulation of HLMECs with rhPlGF promoted all of the steps of airway-microvascular remodelling. These findings provide insights into the influence of cigarette smoking on the structural changes in the airways of asthmatics and the important pathogenic role played by PlGF.

\u03b12AP regulates vascular alteration by inhibiting VEGF signaling in systemic sclerosis: the roles of \u03b12AP in vascular dysfunction in systemic sclerosis

BackgroundSystemic sclerosis (SSc) is a connective tissues disease of unknown origin characterized by vascular damage and extensive fibrosis. Recently, we demonstrated that α2-antiplasmin (α2AP) is associated with the development of fibrosis in SSc. We herein investigate the roles of α2AP in vascular dysfunction in SSc.MethodsVascular damage in mice was determined by the levels of blood vessels and blood flow. Vascular functions in vascular endothelial cells (ECs) were determined by the levels of tube formation, cell proliferation, and endothelial junction-associated protein (VE-cadherin and PECAM1) production.ResultsThe administration of α2AP induced vascular damage in mice. Conversely, the α2AP neutralization improved vascular damage in a bleomycin-induced mouse model of SSc. Additionally, we showed that the SSc fibroblast-conditioned media induced the reduction of tube formation, cell proliferation, and endothelial junction-associated protein production in ECs, and that α2AP neutralization improved them. We also examined the mechanisms underlying the effects of α2AP on vascular alteration in SSc and found that α2AP attenuated vascular endothelial growth factor-induced tube formation, cell proliferation, and endothelial junction-associated protein production through the adipose triglyceride lipase/tyrosine phosphatase SHP2 axis in ECs.ConclusionOur findings demonstrate that α2AP is associated with vascular alteration, and that the blocking of α2AP improves vascular dysfunction in SSc.

Effect of THP-1 Cells on the Formation of Vascular Tubes by Endothelial EA.hy926 Cells in the Presence of Placenta Secretory Products.

We studied the effect of THP-1 cells on the formation of vessel-like structures by endothelial cells in the presence of placenta-conditioned media. Addition of THP-1 cells to endothelial cells cultured in the presence of media conditioned by first-trimester placentas led to an increase in the length of cell tubes and reduced their number in comparison with endothelial cell monoculture. In the presence of media conditioned by third-trimester placentas, THP-1 cells did not affect the length and number of cell tubes formed by endothelial cells. When evaluating the formation of vessel-like structures by endothelial cells in co-culture, marked decrease in the length of cell tubes in the presence of media conditioned by first-trimester placentas vs. third-trimester placentas was noted. No differences in the length and number of cell tubes formed by endothelial cells co-cultured with THP-1 cells in the presence of placental factors from women with preeclampsia and uncomplicated pregnancy were found. These findings can reflect the peculiarities of the influence of macrophages on the formation of blood vessels by endothelial cells in the placenta.

Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNF\u03b1-induced angiogenesis

Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell–cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.

P1.04-023 Thrombomodulin Inhibits the Growth and Angiogenesis of Human Lung Cancer via Blocking VEGFR2-Mediated JAK/STAT3 Signaling Pathway

Angiogenesis has been an attractive target for drug therapy because of its key role in the growth and metastatic spread of malignant tumor. Thrombomodulin has been shown to possess anti-inflammatory and vascular endothelial protection activities. However, its roles in tumor angiogenesis are unknown. The aim of this study was to investigate the roles of thrombomodulin in tumor angiogenesis and its anticancer activities. ex vivo aortic ring angiogenesis sprouting assay was used to detect neo-vascularization. Western blotting was performed to examine STAT3 signaling cascade. Thrombomodulin significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration and tube formation in vitro and blocked vascular endothelial growth factor (VEGF)-triggered neo-vascularization. VEGF receptor (VEGFR) 2 mediated-Janus Kinase 2/STAT3 signaling pathway was significantly inhibited by thrombomodulin in endothelial cells. In addition, the constitutively activated STAT3 protein, and the expression of STAT3-dependent target genes, including cyclin D1, c-Myc, Bcl-xL, and VEGF were also down-regulated in response for thrombomodulin in human lung cancer cells. Consistent with the above findings, thrombomodulin inhibited tumor cell cycle progression and induced cell apoptosis in vitro. Therefore, our provided the first evidence that thrombomodulin inhibited tumor angiogenesis and growth via inhibiting VEGFR2-mediated JAK/STAT3 signaling pathway with the potential of a drug candidate for cancer therapy.

Effects of interleukin-17 on human retinal vascular endothelial cell capillary tube formation invitro.

The present study aimed to investigate the effect of and mechanism underlying interleukin (IL)‑17 on human retinal vascular endothelial cell (HREC) capillary tube formation invitro. The expression of IL‑17 receptor (IL‑17R) in human HRECs was quantified using reverse transcriptase‑polymerase chain reaction (RT‑PCR) and western blot analyses. The roles of IL‑17 in HREC migration and capillary tube formation were detected using a wound scratching assay and three‑dimensional Matrigel assay, respectively, invitro. HREC proliferation was examined using a cell counting kit‑8 assay with administration of serial doses of IL‑17. The effects of IL‑17 on the expression of vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule (ICAM)‑1, IL‑6 and IL‑8 in HRECs were evaluated using RT‑PCR and western blot analyses. The results revealed that the HRECs expressed IL‑17R, and the number of intact capillary tubes formed by HRECs in the presence of IL‑17 was markedly higher, compared with that in the blank control group. The wound scratching assay showed that the numbers of migrated HRECs stimulated with IL‑17 at concentrations of 100 or 500ng/ml were significantly higher, compared with the number in the control group. The RT‑PCR and western blot analyses showed that IL‑17 significantly promoted the expression of VEGF, ICAM‑1, IL‑6 and IL‑8 by the HRECs. The proliferation of HRECs in the presence of IL‑17 was also significantly increased. Therefore, IL‑17 increased HREC capillary tube formation through promoting HREC migration, proliferation, and expression levels of VEGF, ICAM‑1, IL‑6 and IL-8.

JAK2V617F-Mutant Vascular Niche Promotes JAK2V617F Clonal Expansion in Myeloproliferative Neoplasms

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood, limiting the effectiveness of current treatments. Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs. In this work we test the hypothesis that the JAK2V617F-mutant vascular niche promotes JAK2V617F clonal expansion in MPNs.MethodsJAK2V617F Flip-Flop (FF1) mice (Radek Skoda, Switzerland) and Tie2-Cre mice (Mark Ginsberg, UC San Diego) were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and ECs (Tie2/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPrimary lung ECs (CD45-CD31+) were isolated from Tie2/FF1 mice (JAK2V617F ECs) and age-matched littermate control mice (JAK2WT EC). JAK2V617F ECs proliferated to a greater extent than JAK2WT ECs. A vascular tube formation assay was performed as a measure of in vitro angiogenesis and revealed that JAK2V617F ECs had significantly increased angiogenesis compared to JAK2WT EC. (Figure 1)To examine the role of JAK2WT and JAK2V617F ECs in MPN hematopoiesis in vitro, marrow Lin-cKit+ HSPCs were isolated from Tie2/FF1 and age-matched littermate controls and cultured on a feeder layer of JAK2WT or JAK2V617F ECs under serum-free conditions. While there was no difference between JAK2V617F and JAK2WT HSPC proliferation when co-cultured with JAK2WT EC, the JAK2V617F HSPC displayed a relative growth advantage over the JAK2WT HSPC (1.24-fold, p = 0.041) when co-cultured on JAK2V617F EC.Next, the effects of JAK2V617F-bearing vascular niche on MPN hematopoiesis were studied in vivo using marrow transplantation assays. First, wild-type CD45.1 marrow cells were transplanted into lethally irradiated (950cGy) 8-14 week old Tie2/FF1 mice or controls (CD45.2) (n=7 in each group). During a >8-month follow up, all wild-type recipients displayed full donor engraftment, while 4 of 7 Tie2/FF1 recipient mice displayed recovery of JAK2V617F-mutant hematopoiesis (mixed donor/recipient chimerism), suggesting that the JAK2V617F-mutant vascular niche may protect the JAK2V617F-mutant HSPCs from the lethal irradiation. Although marrow and splenic hematopoietic progenitors were significantly increased in the Tie2/FF1 recipients, there was no difference in peripheral blood (PB) cell count or PB hematopoietic progenitor cell numbers between the Tie2/FF1 mice and control mice. (Figure 2)Second, we performed a competitive marrow transplantation experiment in which 5 x 105 CD45.2 donor marrow cells from Tie2/FF1 mice were injected intravenously together with 5 x 105 competitor CD45.1 wild-type marrow cells intro lethally irradiated Tie2/FF1 mice or control mice (CD45.2) (n=5 in each group). During a 4-month follow up, Tie2/FF1 recipients displayed lower PB CD45.1 (i.e. wild-type donor) chimerism than the control mice (p = 0.025), suggesting that the mutant vascular niche in Tie2/FF1 promoted the JAK2V617F clonal maintenance/expansion and/or inhibited the wild-type hematopoiesis. By 18 weeks post-transplant, Tie2/FF1 recipients developed a MPN phenotype with elevated WBC count (22.7 vs. 9.85 x 109/L, p = 0.049), platelet count (5336 vs. 1029 x 109/L, p = 0.033), increased splenomegaly, increased total marrow and splenic hematopoietic progenitors, and increased CD150+CD48- E-SLAM cells (a highly enriched HSPC population) in both marrow (5-fold, p = 0.0004) and spleen (5.5-fold, p = 0.000004) compared to the control mice. To begin to understand the EC signals responsible for JAK2V617F clonal expansion and MPN pathogenesis, qRT-PCR of purified marrow ECs identified up-regulation of MPL and CXCL12 in both arterial (CD45-CD31+Sca1+) and sinusoidal (CD45-CD31+Sca1-) marrow ECs in Tie2/FF1 mice compared to controls.ConclusionsOur study establishes that JAK2V617F-bearing ECs form an important part of the MPN HSPC niche and contribute to mutant stem/progenitor cell expansion both in vitro and in vivo. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.