Capillary-like Tube Formation Research Articles

Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway

Hyaluronan (HA), a component of the extracellular matrix, modulates cellular behavior including angiogenesis. However, little is known about the effect of HA on lymphangiogenesis in fibrosis model. In this study, we investigated the roles of HA in lymphangiogenesis of unilateral ureteral obstruction (UUO). We found that HA cooperated synergistically with vascular endothelial cell growth factor-C to stimulate capillary-like tube formation and increase migration of cells in a haptotaxis assay. Accumulation of HA in the cortical interstitial space was positively correlated with the number of lymphatic vessels after UUO. Depletion of macrophages with clodronate decreased UUO-induced HA accumulation and lymphangiogenesis. Additionally, hyaluronan synthase (HAS) mRNA expression and HA production were increased in bone marrow-derived macrophages upon stimulation with TGF-β1. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive macrophages to SCID mice resulted in a partial decrease in UUO-induced lymphangiogenesis. HA increased expression of vascular endothelial cell growth factor-C in macrophages. Vascular endothelial cell growth factor-C expression and LYVE-1-positive lymphatic area was significantly lower in the UUO-kidney from TLR4 null mice than that from TLR4 wild-type mice. Collectively, these results suggest that HA increases lymphangiogenesis in renal fibrosis model and also stimulates vascular endothelial cell growth factor-C production from macrophages through Toll-like receptor 4-dependent signal pathway.

Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.

Hypoxia-induced fibroblast growth factor 11 stimulates capillary-like endothelial tube formation.

Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis.

Imidazole-based alkaloid derivative LCB54-0009 suppresses ocular angiogenesis and lymphangiogenesis in models of experimental retinopathy and corneal neovascularization.

Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these diseases. In vitro and in vivo angiogenesis and/or lymphangiogenesis were assessed in VEGF- or hypoxia-stimulated endothelial and retinal cells and in animal models of oxygen-induced retinopathy (OIR), streptozotocin-induced diabetic retinopathy (SIDR), suture-induced inflammatory corneal neovascularization (SICNV) and silver nitrate-induced corneal neovascularization. HUVECs and retinal cells were cultured under hypoxic conditions or incubated with VEGF to identify the molecular mechanisms involved. The imidazole-based alkaloid derivative LCB54-0009 inhibited capillary-like tube formation in VEGF-induced HUVECs without inducing cytotoxic effects. Intravitreal injection of LCB54-0009 into retinas suppressed the formation of the pathological neovascular tufts and increased vascular permeability in both OIR of mice and SIDR of rats. Furthermore, subconjunctival injection of LCB54-0009 into the cornea suppressed corneal inflammation and inflammation-associated angiogenesis and lymphangiogenesis in SICNV of mice and silver nitrate cauterization of rats. These pharmacological activities were associated with effects on HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity through its antioxidant activities. LCB54-0009 also inhibited the hypoxia-induced expression of angiopoietin-2, and VEGF-induced VEGFR-2 activation and downstream signalling, resulting in the down-regulation of the expression of pro-angiogenic factors and pro-inflammatory mediators and an up-regulation of the expression of anti-angiogenic factors. LCB54-0009 is a potential candidate molecule for blocking pathological angiogenesis and lymphangiogenesis mediated by HIF-1α- angiopoietin-2 expression and VEGFR-2 activation.

CPU-12, a novel synthesized oxazolo[5,4-d]pyrimidine derivative, showed superior anti-angiogenic activity

Angiogenesis is a crucial requirement for malignant tumor growth, progression and metastasis. Tumor-derived factors stimulate formation of new blood vessels which actively support tumor growth and spread. Various of drugs have been applied to inhibit tumor angiogenesis. CPU-12, 4-chloro-N-(4-((2-(4-methoxyphenyl)-5-methyloxazolo[5,4-d] pyrimidin-7-yl)amino)phenyl)benzamide, is a novel oxazolo[5,4-d]pyrimidine derivative that showed potent activity in inhibiting VEGF-induced angiogenesis in vitro and ex-vivo. In cell toxicity experiments, CPU-12 significantly inhibited the human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner with a low IC50 value at 9.30 ± 1.24 μM. In vitro, CPU-12 remarkably inhibited HUVEC's migration, chemotactic invasion and capillary-like tube formation in a dose-dependent manner. In ex-vivo, CPU-12 effectively inhibited new microvessels sprouting from the rat aortic ring. In addition, the downstream signalings of vascular endothelial growth factor receptor-2 (VEGFR-2), including the phosphorylation of PI3K, ERK1/2 and p38 MAPK, were effectively down-regulated by CPU-12. These evidences suggested that angiogenic response via the induction of VEGFR through distinct signal transduction pathways regulating proliferation, migration and tube formation of endothelial cells was significantly inhibited by the novel small molecule compound CPU-12 in vitro and ex-vivo. In conclusion, CPU-12 showed superior anti-angiogenic activity in vitro.

Synthesis and antitumor evaluation of novel hybrids of phenylsulfonylfuroxan and epiandrosterone/dehydroepiandrosterone derivatives

Thirteen novel furoxan-based nitric oxide (NO) releasing hybrids (14a–e, 15a–e, 17b–d) of 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated against the MDA-MB-231, HCC1806, SKOV-3, DU145, and HUVEC cell lines for their in vitro anti-proliferative activity. Most of the compounds displayed potent anti-proliferative effects. Among them, 17c exhibited the best activity with IC50 values of 20–1.4nM against four cell lines (MDA-MB-231, SKOV-3, DU145, and HUVEC), and 1.03μM against a tamoxifen resistant breast cancer cell line (HCC1806). Furthermore, five compounds (14a, 15a, 17b–d) were selected to screen for VEGF inhibitory activity. Compounds 15a, 17b,c showed obviously better activity than 2-Methoxyestradiol (2-ME) on reducing levels of VEGF secreted by MDA-MB-231 cell line. In a Capillary-like Tube Formation Assay, compounds 17b,c exhibited a significant suppression of the tubule formation in the concentration of 1.75nM and 58nM, respectively. The preliminary SAR showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 17c merited to be further investigated as a promising anti-cancer candidate.

Biphasic effect of PTK7 on KDR activity in endothelial cells and angiogenesis

Protein tyrosine kinase 7 (PTK7) is a member of the defective receptor protein tyrosine kinase family which lacks catalytic activity. Expression of PTK7 is increased in various cancers but its role in carcinogenesis is not well understood. We previously showed that disruption of PTK7 function suppresses VEGF-induced angiogenic phenotypes in HUVECs and mice. Here, we investigated molecular mechanisms for modulating VEGF-induced physiological effects by PTK7. Treatment with a high concentration of extracellular domain of PTK7 (soluble PTK7; sPTK7) or knockdown of PTK7 inhibited VEGF-induced phosphorylation of kinase insert domain receptor (KDR) but did not inhibit phosphorylation of fms-related tyrosine kinase 1 (FLT-1) in HUVECs. PTK7, more specifically sPTK7, interacted with KDR but not with FLT-1 in HUVECs and HEK293 cells. In vitro binding assay showed that sPTK7 formed oligomers with the extracellular domain of KDR (sKDR) up to an approximately 1:3 molar ratio, and vice versa. sPTK7 at lower molar ratios than sKDR enhanced the binding of VEGF to sKDR. At the same or higher molar ratios, it reduced the binding of VEGF to sKDR. Increasing concentrations of sPTK7 or increasing levels of PTK7 expression first increased and then decreased VEGF-induced KDR phosphorylation, migration, and capillary-like tube formation of HUVECs, as well as in vivo angiogenesis. Taken together, our data demonstrates that PTK7 regulates the activity of KDR biphasically by inducing oligomerization of KDR molecules at lower concentrations and by surrounding KDR molecules at higher concentrations.

IPP51, a chalcone acting as a microtubule inhibitor with in vivo antitumor activity against bladder carcinoma.

We previously identified 1-(2,4-dimethoxyphenyl)-3-(1-methylindolyl) propenone (IPP51), a new chalcone derivative that is capable of inducing prometaphase arrest and subsequent apoptosis of bladder cancer cells. Here, we demonstrate that IPP51 selectively inhibits proliferation of tumor-derived cells versus normal non-tumor cells. IPP51 interfered with spindle formation and mitotic chromosome alignment. Accumulation of cyclin B1 and mitotic checkpoint proteins Bub1 and BubR1 on chromosomes in IPP51 treated cells indicated the activation of spindle-assembly checkpoint, which is consistent with the mitotic arrest. The antimitotic actions of other chalcones are often associated with microtubule disruption. Indeed, IPP51 inhibited tubulin polymerization in an in vitro assay with purified tubulin. In cells, IPP51 induced an increase in soluble tubulin. Furthermore, IPP51 inhibited in vitro capillary-like tube formation by endothelial cells, indicating that it has anti-angiogenic activity. Molecular docking showed that the indol group of IPP51 can be accommodated in the colchicine binding site of tubulin. This characteristic was confirmed by an in vitro competition assay demonstrating that IPP51 can compete for colchicine binding to soluble tubulin. Finally, in a human bladder xenograft mouse model, IPP51 inhibited tumor growth without signs of toxicity. Altogether, these findings suggest that IPP51 is an attractive new microtubule-targeting agent with potential chemotherapeutic value.

587. MiRNA-92a Is Involved in the Regulation of Adipose-Derived Stromal Cell (ADSC) Angiogenic Properties

Reparative and regenerative processes after tissue damage are strongly dependent on successful restoration of vascular network. Mesenchymal stem/stromal cells (MSC) including ADSC located within the vessel wall and closely interacted with endothelial cells appear to be key regulators of angiogenesis and regeneration in adult tissues. Mechanisms of MSC ability to stimulate blood vessel growth and remodeling are extensively studying during last decades. microRNAs (miRs), small noncoding RNAs that regulate gene expression by binding to target mRNAs reducing their stability and/or inhibiting translation, play an important role in MSC regulation. It was demonstrated that miR-92a expressed in endothelial cells was induced in ischemia and involved in the regulation of angiogenesis and vessel patterning. We studied miR-92a impact in ADSC angiogenic properties regulation.ADSC were isolated from subcutaneous fat tissue of healthy young donors (n=4) and cultured up to 2-3 passages. ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45-/CD31- for all samples and these cells were capable of adipogenic and osteogenic differentiation. We analyzed miR profile in ADSC using Illumina microarrays and found that miR-92a was one of the highly expressed miRs in all ADSC samples. ADSC transfection by pre-miR-92a or anti-miR-92a led to the coordinated changes of miR-92a well-known target genes expression (integrin alpha 5, ITGA5, and mitogen activated protein kinase, MEK4). ADSC with over-expression of miR-92a completely lost the ability to stimulate capillary-like tube formation by endothelial cells (HUVEC) in vitro compared to ADSC transfected by the control gene. However, transfection of ADSC by anti-miR-92a did not significantly increase their stimulating effect in tube formation assay. As paracrine mechanisms are considered to be the most important for realizing of MSC angiogenic potential, we analyzed gene expression (qRT-PCR) and secretion (Bio-Plex assay and ELISA) of some key angiogenic factors in ADSC. We found that mRNA level of vascular endothelial growth factor (VEGF), angiogenin and leptin was slightly increased in ADSC transfected by anti-miR-92a and this tendency was also observed for VEGF secretion. When miR-92a was over-expressed in ADSC secretion of hepatocyte growth factor (HGF) and angiopoetin-1 by these cells significantly decreased compared to the control cells.We conclude that miR-92a might play an important role in the regulation of ADSC ability to stimulate angiogenesis and key angiogenic factors such as VEGF, HGF and angiopoetin-1 are involved in this process. The underlying mechanisms of miR-92a-mediated direct or indirect effects in ADSC need to be further investigated. Anyway, miR-92a could be considered as a promising target for the modulation of ADSC angiogenic potential.

Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2 and JAK/STAT3 Signalling.

Vascular endothelial growth factor receptor-2 (VEGFR-2) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signalling are important for tumor angiogenesis and metastasis. In this study, we identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB03-0110) as a potent angiogenesis inhibitor. LCB03-0110 inhibited VEGFR-2 and JAK/STAT3 signalling in primary cultured human endothelial cells and cancer cells. An in vitro kinase assay and molecular modelling revealed that LCB03-0110 inhibited VEGFR-2, c-SRC and TIE-2 kinase activity via preferential binding at the ATP-binding site of their kinases. LCB03-0110 successfully occupied the hydrophobic pocket of VEGFR-2, c-SRC and TIE-2. LCB03-0110 also inhibited hypoxia-induced HIF/STAT3 and EGF- or angiopoietin-induced signalling cascades. In addition, LCB03-0110 inhibited VEGF-induced proliferation, viability, migration and capillary-like tube formation. LCB03-0110 also suppressed the sprouting of endothelial cells in the rat aorta and the formation of new blood vessels in the mouse Matrigel plug assay, but also suppressed pulmonary metastasis and tumor xenograft in mice. Our results suggest that LCB03-0110 is a potential candidate small molecule for blocking angiogenesis mediated by aberrant activation of VEGFR-2 and JAK/STAT3 signalling.

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.

Inhibition of human vascular endothelial cell migration and capillary-like tube formation by the microtubule-stabilizing agent peloruside A.

Peloruside A is a microtubule-stabilizing agent that is currently under investigation as a potential anticancer agent. Peloruside A binds to a site on β-tubulin that is distinct to that of the taxanes (paclitaxel and docetaxel) and the epothilones. An attractive clinical quality of microtubule-stabilizing agents is their ability to target multiple mechanisms of tumour growth. In addition to inducing tumour cell apoptosis by arresting cells in mitosis, microtubule-stabilizing agents also inhibit angiogenesis, a process needed by tumor cells for growth and metastasis. In this study, the effects of peloruside A on endothelial cell processes important for angiogenesis were assessed in comparison to docetaxel. Both peloruside A and docetaxel potently inhibited the proliferation of human umbilical vein endothelial cells, with IC50 values of 1.4 and 1.7 nM, respectively. Peloruside also potently blocked endothelial cell migration during wound closure and the three-dimensional organization of the endothelial cells into capillary-like tubes. In the wound scratch assay, peloruside A inhibited wound recovery with an IC50 of 6.3 nM after 18 h. Docetaxel was approximately 3-fold more potent than peloruside A. The number of capillary-like tubes that formed after 16 h culture in Matrigel™ was also inhibited in a dose-dependent manner with an IC50 of 4.5 nM. Docetaxel was about 2-fold more potent than peloruside A in preventing tube formation. This inhibition of endothelial cell function occurred at relatively non-cytotoxic concentrations over the 16-18 h incubations for both stabilizing agents, suggesting that anti-angiogenic effects are likely to occur before therapeutically relevant doses begin to inhibit tumor growth or adverse side effects develop.

Paclitaxel impairs adipose stem cell proliferation and differentiation

BackgroundCancer patients with chemotherapy-induced immunosuppression have poor surgical site wound healing. Prior literature supports the use of human adipose–derived stem cell (hASC) lipoinjection to improve wound healing. It has been established that multipotent hASCs facilitate neovascularization, accelerate epithelialization, and quicken wound closure in animal models. Although hASC wound therapy may benefit surgical cancer patients, the chemotherapeutic effects on hASCs are unknown. We hypothesized that paclitaxel, a chemotherapeutic agent, impairs hASC growth, multipotency, and induces apoptosis. MethodshASCs were isolated and harvested from consented, chemotherapy and radiation naive patients. Growth curves, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide), and EdU (5-ethynyl-2-deoxyguridine) assays measured cytotoxicity and proliferation. Oil Red O stain, Alizarin Red stain, matrigel tube formation assay, and quantitative polymerase chain reaction analyzed hASC differentiation. Annexin V assay measured apoptosis. Immunostaining and Western blot determined tumor necrosis factor α (TNF-α) expression. ResultshASCs were selectively more sensitive to paclitaxel (0.01–30 μM) than fibroblasts (P < 0.05). After 12 d, paclitaxel caused hASC growth arrest, whereas control hASCs proliferated (P = 0.006). Paclitaxel caused an 80.6% reduction in new DNA synthesis (P < 0.001). Paclitaxel severely inhibited endothelial differentiation and capillary-like tube formation. Differentiation markers, lipoprotein lipase (adipogenic), alkaline phosphatase (osteogenic), CD31, and van Willebrand factor (endothelial), were significantly decreased (all P < 0.05) confirming paclitaxel impaired differentiation. Paclitaxel was also found to induce apoptosis and TNF-α was upregulated in paclitaxel-treated hASCs (P < 0.001). ConclusionsPaclitaxel is more cytotoxic to hASCs than fibroblasts. Paclitaxel inhibits hASC proliferation, differentiation, and induces apoptosis, possibly through the TNF-α pathway. Paclitaxel's severe inhibition of endothelial differentiation indicates neovascularization disruption, possibly causing poor wound healing in cancer patients receiving chemotherapy.

An evaluation of the anti-angiogenic effect of the Korean medicinal formula "Sa-mi-yeon-geon-tang" in vitro and in ovo.

BackgroundAngiogenesis is a general hallmark of cancer; therefore, the inhibition of tumor-derived angiogenesis is considered to be an attractive target in the development of anti-cancer agents. Sa-mi-yeon-geon-tang (SMYGT), a decoction that consists of four natural medicinal products, has been traditionally prescribed in Oriental medicine to treat diverse diseases, including cancer. In the present study, we investigated the anti-angiogenic potential of SMYGT in vitro and in ovo.MethodsThe anti-angiogenic potential of SMYGT was evaluated using conventional in vitro assays with human umbilical vein endothelial cells (HUVECs) and chorioallantoic membrane (CAM) assays with fertilized eggs. The expression changes of pro-angiogenic proteins and intracellular signaling in HUVECs following SMYGT treatment were determined by quantitative polymerase chain reaction, gelatinase zymography, and western blot analysis.ResultsSMYGT efficiently inhibited three-dimensional capillary-like tube formation by HUVECs on extracellular matrix supports, as well as new vessel formation on CAMs. SMYGT inhibited cell adhesion to the extracellular matrix and HUVEC cell invasion through Matrigel without affecting cell proliferation, viability, and motility. These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity.ConclusionsSMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions. We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0573-z) contains supplementary material, which is available to authorized users.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor-reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P1 and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.

In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis.

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.

Sildenafil ameliorates long term peripheral neuropathy in type II diabetic mice.

Diabetic peripheral neuropathy is a common complication of long-standing diabetes mellitus. To mimic clinical trials in which patients with diabetes enrolled have advanced peripheral neuropathy, we investigated the effect of sildenafil, a specific inhibitor of phosphodiesterase type 5 enzyme, on long term peripheral neuropathy in middle aged male mice with type II diabetes. Treatment of diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 36 weeks with sildenafil significantly increased functional blood vessels and regional blood flow in the sciatic nerve, concurrently with augmentation of intra-epidermal nerve fiber density in the skin and myelinated axons in the sciatic nerve. Functional analysis showed that the sildenafil treatment considerably improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal stimulus sensitivity compared with the saline treatment. In vitro studies showed that mouse dermal endothelial cells (MDE) cultured under high glucose levels exhibited significant down regulation of angiopoietin 1 (Ang1) expression and reduction of capillary-like tube formation, which were completely reversed by sildenafil. In addition, incubation of dorsal root ganglia (DRG) neurons with conditioned medium harvested from MDE under high glucose levels suppressed neurite outgrowth, where as conditional medium harvested from MDE treated with sildenafil under high glucose levels did not inhibit neurite outgrowth of DRG neurons. Moreover, blockage of the Ang1 receptor, Tie2, with a neutralized antibody against Tie2 abolished the beneficial effect of sildenafil on tube formation and neurite outgrowth. Collectively, our data indicate that sildenafil has a therapeutic effect on long term peripheral neuropathy of middle aged diabetic mice and that improvement of neurovascular dysfunction by sildenafil likely contributes to the amelioration of nerve function. The Ang1/Tie2 signaling pathway may play an important role in these restorative processes.

Stimulation of angiogenesis and survival of endothelial cells by human monoclonal Tie2 receptor antibody

Angiopoietin-1 (Ang1) and its endothelium-specific receptor, tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2), play critical roles in vascular development. Although the Ang1/Tie2 system has been considered a promising target for therapeutic neovascularization, several imitations of large-scale production have hampered the development of recombinant Ang1 for therapeutics. In this study, we produced a fully human agonistic antibody against Tie2, designated 1–4h, and tested the applicability of 1–4h as an alternative to native Ang1 in therapeutic angiogenesis. 1–4h significantly enhanced the phosphorylation of Tie2 in a dose- and time-dependent manner in human Tie2-expressing HEK293 cells and human umbilical vein endothelial cells. Moreover, 1–4h induced the activation of Tie2-mediated intracellular signaling such as AKT, eNOS, MAPK, and Focal Adhesion Kinase p125FAK. In addition, 1–4h increased the chemotactic motility and capillary-like tube formation of endothelial cells in vitro and enhanced the survival of serum-deprived endothelial cells. Taken together, our data clearly suggest that a human Tie2 agonistic antibody is a potentially useful therapeutic approach for the treatment of several ischemic diseases including delayed-wound healing and ischemic heart and limb diseases.

Enhanced angiogenesis promoted by human umbilical mesenchymal stem cell transplantation in stroked mouse is Notch1 signaling associated

Cellular therapy has provided hope for restoring neurological function post stroke through promoting endogenous neurogenesis, angiogenesis and synaptogenesis. The current study was based on the observation that transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) promoted the neurological function improvement in stroked mice and meanwhile enhanced angiogenesis in the stroked hemisphere. Grafted hUCMSCs secreted human vascular endothelial growth factor A (VEGF-A). Notch1 signaling was activated after stroke and also in the grafted hUCMSCs. To address the potential mechanism that might mediate such pro-angiogenic effect, we established a hUCMSC-neuron co-culture system. Neurons were subjected to oxygen glucose deprivation (OGD) injury before co-culturing to mimic the in vivo cell transplantation. Consistent with the in vivo data, co-culture medium claimed from hUCMSC-OGD neuron co-culture system significantly promoted the capillary-like tube formation of brain-derived endothelial cells. Moreover, coincident with our in vivo data, Notch 1 signaling activation was detected in hUCMSCs after co-cultured with OGD neurons as demonstrated by the up-regulation of key Notch1 signaling components Notch1 and Notch1 intercellular domain (NICD). In addition, OGD-neuron co-culture also increased the VEGF-A production by hUCMSCs. To verify whether Notch1 activation was involved in the pro-angiogenic effect, γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was added into the co-culture medium before co-culture. It turned out that DAPT significantly prevented the Notch1 activation in hUCMSCs after co-culture with OGD neurons. More importantly, the pro-angiogenic effect of hUCMSCs was remarkably abolished by DAPT addition as demonstrated by inhibited capillary-like tube formation and less VEGF-A production. Regarding how Notch1 signaling was linked with VEGF-A secretion, we provided some clue that Notch1 effector Hes1 mRNA expression was significantly up-regulated by OGD-neuron co-culturing and down-regulated after additional treatment of DAPT. In summary, our data provided evidence that the VEGF-A secretion from hUCMSCs after being triggered by OGD neurons is Notch1 signaling associated. This might be a possible mechanism that contributes to the angiogenic effect of hUCMSC transplantation in stroked brain.