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Showing papers on "Vascular endothelial growth factor A published in 2010"


Journal ArticleDOI
TL;DR: It is shown that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness, and a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis
Abstract: β-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a ‘miRNA sponge’ in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin. Metastases are responsible for more than 90% of cancer-related mortality. These secondary growths arise through a multistep process that begins when cancer cells within primary tumours break away from neighbouring cells and invade the basement membrane 1 . This local invasion may frequently be triggered by contextual signals that carcinoma cells receive from the nearby stroma, causing them to undergo an epithelial–mesenchymal transition (EMT) 2 . Subsequently, metastasizing cells enter the circulation either directly or through lymphatics. Size constraints in the microvasculature cause many of these cells to be arrested at distant sites, where they may extravasate and enter the foreign tissue parenchyma. There they may remain dormant or, with low efficiency, proliferate from occult micrometastases to form angiogenic, clinically detectable metastases. The absence of EMT-inducing signals in the foreign microenvironment may cause such disseminated cells to revert to an epithelial phenotype by means of a mesenchymal–epithelial transition. Critical regulators of the metastatic process include both proteins and miRNAs 3,4

1,238 citations


Journal ArticleDOI
09 Dec 2010-Nature
TL;DR: The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.
Abstract: Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133(+) fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133(+) stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133(+)/CD144(+) progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133(+) tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133(+) cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.

1,077 citations


Journal ArticleDOI
TL;DR: It was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months and provide new insights into their evolution and response to therapies.
Abstract: Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.

944 citations


Journal ArticleDOI
05 Aug 2010-Blood
TL;DR: It is shown that tissue macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction, and that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

927 citations


Journal ArticleDOI
TL;DR: Together with their spontaneous commitment to cardiac and angiogenic differentiation, transplanted CDCs serve as “role models,” recruiting endogenous regeneration and improving tissue resistance to ischemic stress.
Abstract: Rationale: Multiple biological mechanisms contribute to the efficacy of cardiac cell therapy. Most prominent among these are direct heart muscle and blood vessel regeneration from transplanted cells, as opposed to paracrine enhancement of tissue preservation and/or recruitment of endogenous repair. Objective: Human cardiac progenitor cells, cultured as cardiospheres (CSps) or as CSp-derived cells (CDCs), have been shown to be capable of direct cardiac regeneration in vivo. Here we characterized paracrine effects in CDC transplantation and investigated their relative importance versus direct differentiation of surviving transplanted cells. Methods and Results: In vitro, many growth factors were found in media conditioned by human adult CSps and CDCs; CDC-conditioned media exerted antiapoptotic effects on neonatal rat ventricular myocytes, and proangiogenic effects on human umbilical vein endothelial cells. In vivo, human CDCs secreted vascular endothelial growth factor, hepatocyte growth factor, and insuli...

628 citations


Journal ArticleDOI
TL;DR: It is found that hypoxia powerfully augmented macrophage-mediated T-cell suppression in vitro in a manner dependent on macrophages expression of HIF-1α, which links the innate immune hypoxic response to tumor progression through induction of T- cell suppression in the tumor microenvironment.
Abstract: T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their impact on tumor progression is poorly understood. Targeted deletion of the hypoxia responsive transcription factor HIF-1α in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although VEGF-A and vascularization was unchanged. Tumor associated macrophages can suppress tumor infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T cell suppression in the tumor microenvironment.

553 citations


Journal ArticleDOI
TL;DR: In this paper, a hypoxia-inducible factor-2alpha (HIF-2α) encoded by EPAS1 was identified as the most potent transactivator of COL10A1, which was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice.
Abstract: Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2alpha enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2alpha, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2alpha expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis development, and a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-kappaB (NF-kappaB) family member, as a potent inducer of HIF-2alpha expression. Hence, HIF-2alpha is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis.

470 citations


Journal ArticleDOI
08 Apr 2010-Nature
TL;DR: It is shown that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues, and may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.
Abstract: The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb(-/-) mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

428 citations


Journal ArticleDOI
TL;DR: A significant reduction in cell viability was revealed in CDF-treated cells compared with curcumin- treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity.
Abstract: Curcumin induces cancer cell growth arrest and apoptosis in vitro , but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E 2 , vascular endothelial growth factor, and NF-κB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue. Cancer Res; 70(9); 3606–17. ©2010 AACR.

418 citations


Journal ArticleDOI
TL;DR: A synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood, and the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system.
Abstract: Vascular endothelial growth factors (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro and the consequences of that increase are addressed here. Detailed analysis of the published literature has shown that in vivo and in vitro VEGF-mediated permeability differs in its time course, but has common involvement of many specific signalling pathways, in particular VEGF receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Pathways downstream of endothelial nitric oxide synthase appear to involve the guanylyl cyclase-mediated activation of the Rho–Rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organization of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.

410 citations


Journal ArticleDOI
22 Apr 2010-Nature
TL;DR: This work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
Abstract: Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

Journal ArticleDOI
11 Nov 2010-Blood
TL;DR: In this paper, a transcriptome analysis of retinal endothelial cells isolated from DLL4+/− and wild-type mice was performed to identify three clusters of tip cell-enriched genes encoding extracellular matrix degrading enzymes, basement membrane components and secreted molecules.

Journal ArticleDOI
21 Oct 2010-Nature
TL;DR: These findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.
Abstract: Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.

Journal ArticleDOI
TL;DR: A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement for maintenance, and to target antiangiogenic cancer therapies directed at VEGF-A or its receptors.
Abstract: The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement.

Journal ArticleDOI
TL;DR: Osteochondral angiogenesis in RA and OA is associated with growth factor expression by cells within subchondral spaces, vascular channels and by chondrocytes, and NGF expression and sensory nerve growth may link osteochondralAngiogenesis to pain in arthritis.
Abstract: Objectives. The osteochondral junction can be a source of pain in both RA and OA. Growth of blood vessels and nerves from the subchondral bone into articular cartilage may mediate the association between joint pathology and symptoms. We have investigated associations between angiogenesis, inflammation and neurovascular growth factor expression at the osteochondral junction in human arthritis. Methods. Osteochondral junctions from medial tibial plateaux of patients undergoing arthroplasty for RA (n = 10) or OA (n = 11), or from non-arthritic post-mortem controls (n = 11) were characterized by immunohistochemistry for CD34 and smooth muscle a-actin (blood vessels), CD68 (macrophages), CD3 (lymphocytes), proliferating cell nuclear antigen, vascular endothelial, platelet-derived and nerve growth factor (NGF). Results. Osteochondral angiogenesis was demonstrated as increased endothelial cell proliferation and vascular density in non-calcified articular cartilage, both in RA and OA. Osteochondral angiogenesis was associated with subchondral bone marrow replacement by fibrovascular tissue expressing VEGF, and with increased NGF expression within vascular channels. RA was characterized by greater lymphocyte infiltration and PDGF expression than OA, whereas chondrocyte expression of VEGF was a particular feature of OA. NGF was observed in vascular channels that contained calcitonin gene-related peptideimmunoreactive sensory nerve fibres.

Journal ArticleDOI
TL;DR: The effects of hypoxia on stem-like cells in glioblastoma (GBM) are examined, and it is believed HIF1alpha plays a causal role in these changes, as when oxygen-stable Hif1alpha is expressed in normoxic glioma cells CD133 is induced.
Abstract: Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1α (HIF1α) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1α plays a causal role in these changes, as when oxygen-stable HIF1α is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1α protein expression, HIF1α downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells, and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts, and suppressed expression of vascular endothelial growth factor.

Journal ArticleDOI
TL;DR: Matrix-bound VEGF elicits more distinct vascular effects than soluble V EGF, including prolonged VEGFR2 activation with altered patterns of tyrosine activation and downstream enhancement of the p38/MAPK pathway.
Abstract: VEGF can be secreted in multiple isoforms with variable affinity for extracellular proteins and different abilities to induce vascular morphogenesis, but the molecular mechanisms behind these effects remain unclear. Here, we show molecular distinctions between signaling initiated from soluble versus matrix-bound VEGF, which mediates a sustained level of VEGFR2 internalization and clustering. Exposure of endothelial cells to matrix-bound VEGF elicits prolonged activation of VEGFR2 with differential phosphorylation of Y1214, and extended activation kinetics of p38. These events require association of VEGFR2 with β1 integrins. Matrix-bound VEGF also promotes reciprocal responses on β1 integrin by inducing its association with focal adhesions; a response that is absent upon exposure to soluble VEGF. Inactivation of β1 integrin blocks the prolonged phosphorylation of Y1214 and consequent activation of p38. Combined, these results indicate that when in the context of extracellular matrix, activation of VEGFR2 is distinct from that of soluble VEGF in terms of recruitment of receptor partners, phosphorylation kinetics, and activation of downstream effectors.

Journal ArticleDOI
TL;DR: Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, it is shown that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall.
Abstract: Little is known about how metastatic cancer cells arrest in small capillaries and traverse the vascular wall during extravasation in vivo. Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, we show here that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall. Tumor cells do not damage or induce vascular leak at the site of extravasation, but rather induce local vessel remodeling characterized by clustering of endothelial cells and cell-cell junctions. Intravascular locomotion of tumor cells is independent of the direction of blood flow and requires β1-integrin-mediated adhesion to the blood-vessel wall. Interestingly, the expression of the pro-metastatic gene Twist in tumor cells increases their intravascular migration and extravasation through the vessel wall. However, in this case, Twist expression causes the tumor cells to switch to a β1-integrin-independent mode of extravasation that is associated with the formation of large dynamic rounded membrane protrusions. Our results demonstrate that extravasation of tumor cells is a highly dynamic process influenced by metastatic genes that target adhesion and intravascular migration of tumor cells, and induce endothelial remodeling.

Journal ArticleDOI
TL;DR: The approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application and shows that Id1 is required for increased proliferation and preservation of endothelial cell commitment.
Abstract: Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

Journal ArticleDOI
TL;DR: The results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.
Abstract: The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1–10 μM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5–100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.

Journal ArticleDOI
TL;DR: The results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.
Abstract: Stem cells hold great potential as cell-based therapies to promote vascularization and tissue regeneration. However, the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors and low cell viability after transplantation. Here, we have developed vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells for the purposes of promoting angiogenesis. Nonviral, biodegradable polymeric nanoparticles were developed to deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues. S.c. implantation of scaffolds seeded with VEGF-expressing stem cells (hMSCs and hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.

Journal ArticleDOI
TL;DR: By providing comprehensive real-time information, molecular imaging of VEGF pathway may accelerate the drug development process and will be of great help for patient stratification and therapeutic effect monitoring, which will promote effective personalized molecular cancer therapy.
Abstract: New blood vessel formation (angiogenesis) is fundamental to tumor growth, invasion, and metastatic dissemination. The vascular endothelial growth factor (VEGF) signaling pathway plays pivotal roles in regulating tumor angiogenesis. VEGF as a therapeutic target has been validated in various types of human cancers. Different agents including antibodies, aptamers, peptides, and small molecules have been extensively investigated to block VEGF and its pro-angiogenic functions. Some of these agents have been approved by FDA and some are currently in clinical trials. Combination therapies are also being pursued for better tumor control. By providing comprehensive real-time information, molecular imaging of VEGF pathway may accelerate the drug development process. Moreover, the imaging will be of great help for patient stratification and therapeutic effect monitoring, which will promote effective personalized molecular cancer therapy. This review summarizes the current status of tumor therapeutic agents targeting to VEGF and the applications of VEGF related molecular imaging.

Journal ArticleDOI
TL;DR: Evidence is provided that microRNA-93 (miR-93) regulates VEGF expression in experimental models of diabetes both in vitro and in vivo and indicates that high glucose decreases miR- 93 expression by down-regulating the promoter of the host MCM7 gene.

Journal ArticleDOI
TL;DR: Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, V EGF can be considered a promising tool to promote neuronal health and nerve repair.
Abstract: Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.

Journal ArticleDOI
TL;DR: In this article, the authors demonstrate that galectin-3 is a mediator of vascular endothelial growth factor and basic fibroblast growth factor (bFGF)-mediated angiogenic response.
Abstract: Recent studies have shown that a carbohydrate-binding protein, galectin-3, is a novel pro-angiogenic molecule. The mechanism by which galectin-3 promotes angiogenesis remains unknown. We demonstrate here that galectin-3 is a mediator of vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-mediated angiogenic response. Angiogenesis assays revealed that galectin-3 inhibitors, beta-lactose and dominant-negative galectin-3, reduce VEGF- and bFGF-mediated angiogenesis in vitro and that VEGF- and bFGF-mediated angiogenic response is reduced in galectin-3 knockdown cells and Gal3(-/-) animals. Integrin alphavbeta3 was identified as the major galectin-3-binding protein and anti-alphav, -beta3, and -alphavbeta3 integrin function-blocking antibodies significantly inhibited the galectin-3-induced angiogenesis. Furthermore, galectin-3 promoted the clustering of integrin alphavbeta3 and activated focal adhesion kinase. Knockdown of GnTV, an enzyme that synthesizes high-affinity glycan ligands for galectin-3, substantially reduced: (a) complex N-glycans on alphavbeta3 integrins and (b) VEGF- and bFGF-mediated angiogenesis. Collectively, these data suggest that galectin-3 modulates VEGF- and bFGF-mediated angiogenesis by binding via its carbohydrate recognition domain, to the GnTV synthesized N-glycans of integrin alphavbeta3, and subsequently activating the signaling pathways that promote the growth of new blood vessels. These findings have broad implications for developing novel, carbohydrate-based therapeutic agents for inhibition of angiogenesis.

Journal ArticleDOI
TL;DR: Data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour‐associated macrophages and their reverted polarization from M2 to M1 phenotype.
Abstract: It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB-2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour-free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour-associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin-10 was drastically down-regulated in favour of interferon-γ production. Peritoneal macrophages and tumour-associated macrophages of ZA-treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour-associated macrophages and their reverted polarization from M2 to M1 phenotype.

Journal ArticleDOI
08 Mar 2010-PLOS ONE
TL;DR: The results reveal that the IRE1α-XBP-1, PERK-ATF4, and ATF6α pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription.
Abstract: Background Angiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1α, PERK, and ATF6α, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis.

Journal ArticleDOI
TL;DR: In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma.
Abstract: BACKGROUND Corticosteroids are commonly used to treat infantile hemangioma, but the mechanism of action of this therapy is unknown. We investigated the effect of corticosteroids in a previously described in vivo model of infantile hemangioma and in cultured hemangioma-derived cells. METHODS We tested hemangioma-derived stem cells for vasculogenic activity in vivo after implantation into immune-deficient (nude) mice. We studied dexamethasone treatment of both the cells before implantation and the mice after implantation. We also tested hemangioma-derived stem cells for expression of vascular endothelial growth factor A (VEGF-A) in vitro and studied the inhibition of VEGF-A expression, using short hairpin RNA (shRNA) in vivo and in vitro. RESULTS Systemic treatment with dexamethasone led to dose-dependent inhibition of tumor vasculogenesis in the murine model. Pretreatment of hemangioma-derived stem cells in vitro before implantation also inhibited vasculogenesis. Dexamethasone suppressed VEGF-A production by hemangioma-derived stem cells in vitro but not by hemangioma-derived endothelial cells or human umbilical-vein endothelial cells. Silencing VEGF-A in hemangioma-derived stem cells reduced vasculogenesis in vivo. VEGF-A was detected in hemangioma specimens in the proliferating phase but not in the involuting phase and was shown by immunostaining to reside outside of vessels. Corticosteroid treatment suppressed other proangiogenic factors in hemangioma-derived stem cells, including urokinase plasminogen activator receptor, interleukin-6, monocyte chemoattractant protein 1, and matrix metalloproteinase 1. CONCLUSIONS In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma. The corticosteroid also inhibited the expression of VEGF-A by hemangioma-derived stem cells, and silencing of VEGF-A expression in these cells inhibited vasculogenesis in vivo.

Journal ArticleDOI
TL;DR: It is demonstrated that vascular endothelial growth factor‐A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs), and data indicating that VEGF‐driven angiogenesis is largely mediated through ROCK2 is provided, indicating that Rho/ROCK signaling is an important mediator in a number of angiogenic processes.
Abstract: The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate a number of cellular processes, including cell motility, proliferation, survival, and permeability. Pharmacological inhibitors of the Rho pathway reportedly block angiogenesis; however, the molecular details of this inhibition are largely unknown. We demonstrate that vascular endothelial growth factor-A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs). Moreover, the pharmacological inhibition of ROCK1/2 using 10 microM Y-27632 (the IC(50) for this compound in ECs) strongly disrupts vasculogenesis in pluripotent embryonic stem cell cultures, VEGF-mediated regenerative angiogenesis in ex vivo retinal explants, and VEGF-mediated in vitro EC tube formation. Furthermore, using small interfering RNA knockdown and mouse heterozygote knockouts of ROCK1 and ROCK2, we provide data indicating that VEGF-driven angiogenesis is largely mediated through ROCK2. These data demonstrate that Rho/ROCK signaling is an important mediator in a number of angiogenic processes, including EC migration, survival, and cell permeability, and suggest that Rho/ROCK inhibition may prove useful for the treatment of angiogenesis-related disorders.

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TL;DR: This review will focus on Vascular Endothelial (VE)-cadherin, an endothelial-specific cell-cell adhesion protein of the adherens junction complex that plays a key role in endothelial barrier function and angiogenesis.