Capillary-like Tube Formation Research Articles

Diarylureas and Diarylamides with Oxazolo[5,4‐d]pyrimidine Scaffold as Angiogenesis Inhibitors

A series of oxazolopyrimidine-based ureas and amides were designed, synthesized, and biologically evaluated for their antiproliferative and antiangiogenic activities. These compounds were identified to exhibit inhibitory activities against human umbilical vein endothelial cells (HUVEC) in vitro. Among these compounds, compound 22 effectively inhibited the migration and capillary-like tube formation of human umbilical vein endothelial cells. It also exhibited a concentration-dependent inhibition on capillary sprouting from the rat aorta rings. Preliminary mechanistic studies revealed that compound 22 suppressed protein kinases activation, by decreasing PI3K and ERK 1/2 phosphorylation. These results support the further investigation of this class of compounds as potential anticancer agents.

Abstract 392: Decreased ARNT Mediates Cardiovascular Endothelial Dysfunction in Diabetic Cardiomyopathy

Background: Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy. Methods and Results: Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT -/- ) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT -/- mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway. Conclusion: Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.

Abstract 3390: CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors

Abstract CTHRC1 (collagen triple helix repeat-containing 1), a protein secreted during the tissue repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 plays an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here, we demonstrate a novel role of CTHRC1 as a potent endothelial activator, promoting angiogenesis by recruiting bone marrow-derived cells into the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, consistent with observed increases in neovascularization in vivo. Moreover, rCTHRC1 induced upregulation of angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody abrogated Ang-2 expression in ECs in vitro. Moreover, CTHRC1-neutralizing antibody treatment in a xenograft mouse model reduced tumor burden and infiltration of TEMs in tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the conclusion that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers. Citation Format: Seongyea Jo, Jaemin Lee, jinhoi song. CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3390.

Artemisinic acid exhibits antitumor activity in MCF-7 breast cancer cells through the inhibition of angiogenesis, VEGF, m-TOR and AKT signalling pathways

<p>The aim of the present study was to evaluate the antitumor and anti-angiogenic effects of artemisinic acid in MCF-7 human breast cancer cells. Various cell signalling pathways (VEGF, m-TOR and AKT signalling pathways) and MTT assay were used. The in vivo antitumor activity of artemisinic acid was evaluated by means of tumor xenograft mouse model. Transwell cell migration assay was used to examine the chemotactic motility of the human umbilical vascular endothelial cells (HUVECs), while as endothelial cell capillary-like tube formation assay was used to evaluate the effect of artemisinic acid on the tube formation in HUVECs. We found that artemisinic acid considerably reduced both the volume and weight of concrete tumors and reduced angiogenesis in a xenograft mouse tumor model in vivo. Further, artemisinic acid suppressed the VEGF-induced cell migration and capillary-like tube formation of HUVECs in a dose-dependent manner. Artemisinic acid was found to suppress the VEGF-induced phosphorylation of VEGFR2 and also the activity of AKT and m-TOR.</p><p><strong>Video Clip:</strong></p><p><a href="https://youtube.com/v/w7jZqcDvMTc">Western blot assay:</a> 4 min 33 sec </p>

MiRNA involvement in angiogenesis in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Late-stage AMD is characterized by choroidal neovascularization (CNV). miR-93 appears to play a role in regulating vascular endothelial growth factor-A (VEGF-A), a known factor involved in neovascularization. Understanding its biological significance might enable development of therapeutic interventions for diseases like AMD. We aimed to determine the role of miR-93 in AMD using a laser-induced CNV mouse model. CNV was induced by laser photocoagulation in C57BL/6 mice. The CNV mice were transfected with scrambled miR or miR-93 mimic. The treatment effect was assessed by fundus photography and fluorescein angiography and confirmed by choroidal flatmount. The expression of miR-93 and VEGF-A in ocular tissues was analysed by quantitative polymerase chain reaction (qPCR) and Western blot. The overexpression effects of miR-93 were also proved on human microvascular endothelial cells (HMECs). Significantly decreased expression of miR-93 was observed by qPCR analysis in CNV mice compared to untreated mice (p < 0.05). VEGF-A messenger RNA (mRNA) and protein expression were upregulated with CNV; these changes were ameliorated by restoration of miR-93 (p < 0.05). CNV was reduced after miR-93 transfection. Transfection of miR-93 reduced the proliferation of HMECs (p < 0.01), but no significant changes were observed in 2D capillary-like tube formation (p > 0.05) and migration (p > 0.05) compared with that in the untreated cells. miR-93 has been shown to be a negative modulator of angiogenesis in the eye. All together, these results highlight the therapeutic potential of miR-93 and suggest that it may contribute as a putative therapeutic target for AMD in humans.

The anti-angiogenic effect and novel mechanisms of action of Combretastatin A-4.

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.

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88–92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88–92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

Design, Synthesis, and Evaluation of in Vitro and in Vivo Anticancer Activity of 4-Substituted Coumarins: A Novel Class of Potent Tubulin Polymerization Inhibitors.

In this paper, a series of novel 4-substituted coumarin derivatives were synthesized. Among these compounds 34, 39, 40, 43, 62, 65, and 67 exhibited significant antiproliferative activity toward a panel of tumor cell lines at subnanomolar IC50 values. Compound 65 showed potent antiproliferative ability (IC50 values of 7-47 nM) and retained full activity in multidrug resistant cancer cells. Compound 65 caused G2/M phase arrest and interacted with the colchicine-binding site in tubulin, as confirmed by immune-fluorescence staining, microtubule dynamics assays, and competition assays with N,N'-ethylene-bis(iodoacetamide). Compound 65 reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, compound 65 significantly and dose-dependently reduced tumor growth in four xenografts models including paclitaxel sensitive and resistant ovarian tumors (A2780s and A2780/T), adrmicycin sensitive and resistant breast tumors (MCF-7 and MCF-7/ADR), suggesting that compound 65 is a promising novel antimitotic compound for the potential treatment of cancer.

Regulation of retinal angiogenesis by phospholipase C-β3 signaling pathway.

Angiogenesis has an essential role in many pathophysiologies. Here, we show that phospholipase C-β3 (PLC-β3) isoform regulates endothelial cell function and retinal angiogenesis. Silencing of PLC-β3 in human umbilical vein endothelial cells (HUVECs) significantly delayed proliferation, migration and capillary-like tube formation. In addition, mice lacking PLC-β3 showed impaired retinal angiogenesis with delayed endothelial proliferation, reduced endothelial cell activation, abnormal vessel formation and hemorrhage. Finally, tumor formation was significantly reduced in mice lacking PLC-β3 and showed irregular size and shape of blood vessels. These results suggest that regulation of endothelial function by PLC-β3 may contribute to angiogenesis.

The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50>800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

Re-Endothelialization Study on Endovascular Stents Seeded by Endothelial Cells through Up- or Downregulation of VEGF.

We studied the effects of gene transfection of endothelial cells with vascular endothelial growth factor (VEGF) on re-endothelialization and inhibition of in-stent restenosis. Transfected endothelial cells (ECs) exposed to different VEGF levels were seeded on a stent surface for evaluation in vitro. VEGF121(++) ECs and VEGF121(--) ECs were established using lentiviral-mediated HUVECs transfection. VEGF RNA transcription level and VEGF protein expression were detected by qPCR, Western blot, and ELISA. Methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, and in vitro HUVEC tube formation assay showed that VEGF overexpression promoted cell proliferation, migration, and endothelial capillary-like tube formation. Downregulation of VEGF expression inhibited these activities. Using a rotational culturing system, cells tightly adhered on the stent surface. Stents seeded with transfected ECs at different VEGF levels were implanted in abdominal aortas of New Zealand white rabbits to study re-endothelialization and inhibition of in-stent restenosis. Stents with cells exposed to excess VEGF expression were almost completely covered with cells after stent implantation for 1 week (w). In the VEGF interference group this process was delayed over 4 w due to RNAi-mediated silencing of VEGF. Cryosectioning after 12 w showed that stents seeded with HUVECs exposed to excess VEGF expression significantly reduced the neointima area and stenosis when compared with bare metal stents and stents from the VEGF interference group. Transgenic HUVECs were not found in tissues of experimental animals. Furthermore, cells from these tissues were similar to those from normal tissue. In conclusion, VEGF-mediated endothelialization was found. Furthermore, ECs exposed to VEGF overexpression reduced neointimal hyperplasia, promoted endothelialization, and reduced in-stent restenosis.

Tetraspanin CD151 and integrin α6β1 mediate platelet‐enhanced endothelial colony forming cell angiogenesis

ESSENTIALS: Platelet releasates (PRs) enhance endothelial colony forming cell (ECFC) angiogenesis. The impact of platelet membrane components on ECFC angiogenesis was studied by a tube formation assay. Platelets enhanced ECFC angiogenesis more potently than PR, via tetraspanin CD151 and integrin α6β1. Optimal enhancement of ECFC angiogenesis by platelets requires both membrane proteins and PR. Platelets promote angiogenesis of endothelial colony forming cells (ECFCs), with the underlying mechanisms not being fully understood. To investigate if platelets regulate the angiogenic property of ECFCs via mechanisms beyond platelet-released angiogenic regulators. Endothelial colony forming cells were generated by ECFC-directed cell culture of peripheral blood mononuclear cells. Capillary-like tube formation of ECFCs was assessed using a Matrigel assay. Platelets promoted ECFC tube formation in both basic and complete ECFC medium. Importantly, the ECFC angiogenic responses induced by platelets were stronger than those induced by platelet releasates. Thus, the branching points of ECFC tube formation (30.5 ± 9.0/field, ECFC alone) were increased by platelet releasates (58.2 ± 8.3/field) and even more profoundly by platelets (95.5 ± 17.6/field), indicating that platelet membrane components also promoted ECFC tube formation. The latter was further supported by evidence that fixed platelets did enhance ECFC tube formation. Subsequent experiments revealed that the promotion was dependent on platelet-surface glycoproteins, as removal of sialic acid from platelet glycoproteins by neuraminidase abolished the enhancement. Furthermore, platelet-expressed, but not ECFC-expressed, CD151 was important for the enhancement, as pretreatment of platelets, but not ECFCs, with a CD151-blocking antibody attenuated the effect. Integrin α6β1 on both ECFCs and platelets also participated in platelet-promoted tube formation, as integrin α6 or β1 blockade of either cell type markedly or totally inhibited the phenomenon. Moreover, platelets exerted the enhancement via the Src-PI3K signaling pathway of ECFCs. Platelet-enhanced ECFC angiogenesis requires platelet tetraspanin CD151 and α6β1 integrin, as well as ECFC α6β1 integrin and Src-PI3K signaling.

Enhanced in vitro angiogenic behaviour of human umbilical vein endothelial cells on thermally oxidized TiO2 nanofibrous surfaces.

One of the major challenges in bone grafting is the lack of sufficient bone vascularization. A rapid and stable bone vascularization at an early stage of implantation is essential for optimal functioning of the bone graft. To address this, the ability of in situ TiO2 nanofibrous surfaces fabricated via thermal oxidation method to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) was investigated. The cellular responses of HUVECs on TiO2 nanofibrous surfaces were studied through cell adhesion, cell proliferation, capillary-like tube formation, growth factors secretion (VEGF and BFGF), and angiogenic-endogenic-associated gene (VEGF, VEGFR2, BFGF, PGF, HGF, Ang-1, VWF, PECAM-1 and ENOS) expression analysis after 2 weeks of cell seeding. Our results show that TiO2 nanofibrous surfaces significantly enhanced adhesion, proliferation, formation of capillary-like tube networks and growth factors secretion of HUVECs, as well as leading to higher expression level of all angiogenic-endogenic-associated genes, in comparison to unmodified control surfaces. These beneficial effects suggest the potential use of such surface nanostructures to be utilized as an advantageous interface for bone grafts as they can promote angiogenesis, which improves bone vascularization.

A Heterodimeric Cytokine, Consisting of IL-17A and IL-17F, Promotes Migration and Capillary-Like Tube Formation of Human Vascular Endothelial Cells.

The interleukin (IL)-17 family, consisting of six homodimeric cytokines IL-17A, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, and IL-17F, mediates a variety of biological activities including regulation of chemokine secretion and angiogenesis. Among the IL-17 family members, IL-17A and IL-17E/IL-25 are angiogenesis stimulators, while IL-17B and IL-17F are angiogenesis inhibitors. Recently, IL-17A/F heterodimer, comprised of the IL-17A and IL-17F subunits, was found as another member of the IL-17 cytokine family. However, to date, it has been unknown whether IL-17A/F has biological actions to affect the angiogenesis-related vascular endothelial functions. Therefore, in this study, we investigated the biological effects of IL-17A/F on the growth, migration and capillary-like tube formation of vascular endothelial cells. Recombinant IL-17A/F protein had no direct effects on the growth of human dermal microvascular endothelial cells (HMVECs), whereas, after 4-hour incubation in a modified Boyden Chemotaxicell chamber, IL-17A/F significantly induced migration of HMVECs over a wide range of doses via the phosphatidylinositol-3 kinase (PI3K) signaling pathway. We further investigated the biological effect of IL-17A/F on capillary-like tube formation using a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), which mimicked the in vivo microenvironment. In this co-culture system, IL-17A/F significantly promoted capillary-like endothelial tube formation in a dose-dependent fashion via the PI3K and extracellular signal-regulated kinase (ERK) signaling pathways. Additionally, IL-17A/F up-regulated secretion of angiogenic growth factors such as IL-8 and growth-related oncogene (GRO)-α by HDFs. These findings identify a novel biological function for IL-17A/F as an indirect angiogenic agent.

Enhanced vascularization of VEGF synergizing with 2-N,6-O-sulfated chitosan

Event Abstract Back to Event Enhanced vascularization of VEGF synergizing with 2-N,6-O-sulfated chitosan Yuanman Yu1, 2, Rui Chen1, 2, Xiang Peng2, Jing Wang1, 2, 3 and Changsheng Liu1, 2, 3 1 East China University of Science and Technology, The State Key Laboratory of Bioreactor Engineering, China 2 East China University of Science and Technology, Engineering Research Center for Biomedical Materials of Ministry of Education, China 3 East China University of Science and Technology, Key Laboratory for Ultrafine Materials of Ministry of Education, China Introduction: Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications[1],[2]. While vascular endothelial growth factor (VEGF) which regards as an angiogenic regulator holds greatest potential to promote angiogenesis[3]. However, soluble VEGF will be quickly degraded by enzymes in vivo and clinical applications with high dose of VEGF are often associated with severe side effects[4],[5]. Thus, reducing the dosage of VEGF and enhancing its activity becomes particularly important. 2-N,6-O-sulfated chitosan (26SCS) as a synthetic sulfated polysaccharide has been demonstrated that it could efficiently associate with VEGF to promote angiogenesis in vitro during our previous work[6]. Nevertheless, the mechanism of the interaction between 26SCS and VEGF is not clear. This study aimed at the synergy of 26SCS with VEGF as well as its pro-angiogenesis in vitro and in vivo. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) which purchased from the American Type Culture Collection was used as a cell model. A series concentrations of 26SCS was used to characterize the interaction with VEGF (2 ng/mL) on cell viability. Subsequently, two experimental groups which named as 40 SCS/VEGF (26SCS, 40 ng/mL) and 160 SCS/VEGF (26SCS, 160 ng/mL) respectively were chosen to investigate the cell migration, sprouting and qRT-PCR analysis in vitro. Furthermore, we employed the corresponding concentration ratio of 26SCS and VEGF to further investigate the pro-angiogenesis activity in vivo. Subcutaneous implantation of mice (VEGF, 100 ng) and chicken chorioallantoic membrane (CAM) assay (VEGF, 20 ng) were used as experimental models, respectively. Results and Discussion: The pro-angiogenesis of 26SCS synergizing with VEGF was found and mainly studied. As shown in Figure 1, 26SCS synergizing with VEGF could directly improve HUVECs cell proliferation through dose-dependently regulating the expression of relevant angiogenic genes, and the group of 40 SCS/VEGF exhibited optimal results. Moreover, the repaired area of 40 SCS/VEGF was almost 1.5 folds compared to negative control in scratch wound healing assay, and 40 SCS/VEGF generated more branch points and forming tubes in capillary-like tube formation assay. In addition, using gelatin sponge loaded with VEGF and different content of 26SCS as samples implanted in subcutaneous model of mice, we investigated the influence of 26SCS and VEGF facilitate vascularization in vivo at day 7 (Figure 2A). The results of histological analysis showed that 26SCS and VEGF could significantly promote the repair rate of vascular network at the defect site (Figure 2B). Through the CAM assay by implanting filter paper containing different ratios of 26SCS and VEGF compounds into fertilized eggs (Figure 2C), the results further revealed that 26SCS synergizing VEGF could signally promote angiogenesis. Conclusions: In this study, 26SCS was demonstrated that synergizing with VEGF could significantly promote angiogenesis in vitro and in vivo. This findings evidence that 26SCS have the potential to improve the VEGF activity and will be promising prospects as a synergistic factor of VEGF for vascularization in clinical applications. The National Basic Research Program of China (973 Program, No. 2012CB933600); The National Natural Science Foundation of China (Grant Nos. 31271011, 31330028, and 31470923); The New Century Excellent Talents in University (Grant No. NCET-12-0856); The National Science and Technology Support Program (Grant No. 2012BAI17B02)

MiR-126 Contributes to Human Umbilical Cord Blood Cell-Induced Neurorestorative Effects After Stroke in Type-2 Diabetic Mice.

Diabetes mellitus (DM) is a high risk factor for stroke and leads to more severe vascular and white-matter injury than stroke in non-DM. We tested the neurorestorative effects of delayed human umbilical cord blood cell (HUCBC) treatment of stroke in type-2 diabetes (T2DM). db/db-T2DM and db/+-non-DM mice were subjected to distal middle cerebral artery occlusion (dMCAo) and were treated 3 days after dMCAo with: (a) non-DM + Phosphate buffered saline (PBS); (b) T2DM + PBS; (c) T2DM + naïve-HUCBC; (d) T2DM + miR-126(-/-) HUCBC. Functional evaluation, vascular and white-matter changes, neuroinflammation, and miR-126 effects were measured in vivo and in vitro. T2DM mice exhibited significantly decreased serum and brain tissue miR-126 expression compared with non-DM mice. T2DM + HUCBC mice exhibited increased miR-126 expression, increased tight junction protein expression, axon/myelin, vascular density, and M2-macrophage polarization. However, decreased blood-brain barrier leakage, brain hemorrhage, and miR-126 targeted gene vascular cell adhesion molecule-1 and monocyte chemotactic protein 1 expression in the ischemic brain as well as improved functional outcome were present in HUCBC-treated T2DM mice compared with control T2DM mice. MiR-126(-/-) HUCBC-treatment abolished the benefits of naïve-HUCBC-treatment in T2DM stroke mice. In vitro, knock-in of miR-126 in primary cultured brain endothelial cells (BECs) or treatment of BECs with naïve-HUCBCs significantly increased capillary-like tube formation, and increased axonal outgrowth in primary cultured cortical neurons; whereas treatment of BECs or cortical neurons with miR-126(-/-) HUCBC attenuated HUCBC-treatment-induced capillary tube formation and axonal outgrowth. Our data suggest delayed HUCBC-treatment of stroke increases vascular/white-matter remodeling and anti-inflammatory effects; MiR-126 may contribute to HUCBC-induced neurorestorative effects in T2DM mice.

Baicalein Promotes Angiogenesis and Odontoblastic Differentiation via the BMP and Wnt Pathways in Human Dental Pulp Cells.

Baicalein is an active flavonoid extracted from the root of Scutellaria baicalensis that has anticancer and anti-inflammatory properties; its effects on osteoblastic and angiogenic potential are controversial. The aim of this study was to investigate the effects of baicalein on odontoblastic differentiation and angiogenesis and the underlying mechanism in human dental pulp cells (HDPCs). Baicalein (1-10[Formula: see text][Formula: see text]M) had no cytotoxic effects and promoted alkaline phosphatase (ALP) activity, mineralization assayed by Alizarin Red-S staining, and the mRNA expression of marker genes, in a concentration-dependent manner. In addition, baicalein upregulated angiogenic factors and increased in vitro capillary-like tube formation. Moreover, baicalein upregulated bone morphogenetic protein (BMP)-2 mRNA and phosphorylation of Smad 1/5/8 and Wnt ligand mRNA, glycogen synthase kinase-3, and nuclear [Formula: see text]-catenin. The odontogenic and angiogenic effects of baicalein were abolished by the BMP antagonist noggin and the Wnt/[Formula: see text]-catenin receptor antagonist DKK-1. These results demonstrate that baicalein promoted odontoblastic differentiation and angiogenesis of HDPCs by activating the BMP and Wnt/[Formula: see text]-catenin signal pathways. Our findings suggest that baicalein may contribute to dental pulp repair and regenerative endodontics.

Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells.

Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50–200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

Nucleoside Transport Inhibition by Dipyridamole Prevents Angiogenesis Impairment by Homocysteine and Adenosine.

Adenosine plays an important role in the pathogenesis of homocysteine-associated vascular complications. This study examined the effects of dipyridamole, an inhibitor for nucleoside transport, on impaired angiogenic processes caused by homocysteine and adenosine in human cardiovascular endothelial cell line (EAhy926). The results showed that dipyridamole restored the extracellular adenosine and intracellular S-adenosylhomocysteine concentrations disrupted by the combination of homocysteine and adenosine. Dipyridamole also ameliorated the impaired proliferation, migration and formation of capillary-like tubes of EAhy926 cells caused by the combination of homocysteine and adenosine. Mechanism analysis revealed that dipyridamole induced the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinases (ERK) and its effect on cell growth was attenuated by the MEK inhibitor, U0126. Dipyridamole protected against impaired angiogenesis caused by homocysteine and adenosine, at least in part, by activating the MEK/ERK signalling pathway, and this could be associated with its effects in suppressing intracellular S-adenosylhomocysteine accumulation. This is the first paper showing that nucleoside transport inhibition by dipyridamole reduced impaired angiogenic process caused by homocysteine and adenosine.