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At least 991 records · Page 55Linked to original sources
PubMed2002

A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor.

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.

Allantois↗
PubMed2005

Vascular leakage in chick embryos after expression of a secreted binding protein for fibroblast growth factors.

Fibroblast growth factors (FGFs) have been implicated in a variety of physiologic and pathologic processes from embryonic development to tumor growth and angiogenesis. FGFs are immobilized in the extracellular matrix of different tissues and require release from this storage site to trigger a response. Secreted FGF-binding proteins (FGF-BPs) can release immobilized FGFs, enhance the activity of locally stored FGFs and can thus serve as an angiogenic switch molecule in cancer. Here, we report on the effect of human FGF-BP transgene expression in chicken embryos. To establish the transgenic model, plasmid-based reporter vectors expressing luciferase, beta-galactosidase or green fluorescent protein were introduced through different routes into 4- to 5-day-old embryos grown outside their egg shell on top of the yolk sac. This allows for easy manipulation and continuous observation of phenotypic effects. Expression of human FGF-BP induced dose-dependent vascular permeability, hemorrhage and embryonic lethality. Light and electron microscopic studies indicate that this hemorrhage results from compromised microvascular structure. An FGF-1 expression vector with an added secretory signal mimicked this vascular leakiness phenotype whereas wild-type FGF-1 required coexpression of a threshold amount of FGF-BP. This model is a powerful tool for real-time monitoring of the effects of transient transgene expression during embryogenesis.

Animals↗
PubMed1995

Automatic vessel segmentation and quantification of the rat aortic ring assay of angiogenesis.

BACKGROUND: Pharmacologic control of angiogenesis is a promising new approach to the treatment of a variety of pathologic conditions including cancer. The recently developed in vitro rat aortic ring model provides a simple, reproducible assay for discovering angiogenic agonists and antagonists. However, quantification of results in this assay is time consuming, tedious, and subjective, because it involves visual inspection of images and manually counting the newly formed microvessels extending from the cultured aortic ring. This report describes an automated image analysis-based procedure for quantification of this assay that overcomes these difficulties. EXPERIMENTAL DESIGN: The designed image processing algorithm segments the vessels from gray scale images. A high-pass filter is used, and the results are separated into nonvascular and vessel compartments based on object size and shape. Quantification relies on identification of vessels intersecting a closed transect set a fixed distance from the aortic ring. The number and the total area of these vessels are determined. The entire operation has been automated and packaged in an application called Vessels. RESULTS: The correlation between computer-determined vessel area/vessel number and visual microvessel count is high (r2 = 0.91 and r2 = 0.86, respectively). CONCLUSION: Vessels offers high-speed, fully automatic batch processing including production of a hard copy for documentation. The application runs on the Apple family of computers. On a Quadra 800, the application can process approximately 30 images/hour, which is approximately 2.5 times faster than manual quantification of this assay.

Animals↗
PubMed2004

Embryonic cell lines with endothelial potential: an in vitro system for studying endothelial differentiation.

OBJECTIVE: Endothelial differentiation is a fundamental process in angiogenesis and vasculogenesis with implications in development, normal physiology, and pathology. To better understand this process, an in vitro cellular system that recapitulates endothelial differentiation and is amenable to experimental manipulations is required. METHODS AND RESULTS: Embryonic cell lines that differentiate exclusively into endothelial cells were derived from early mouse embryos using empirical but reproducible culture techniques without viral or chemical transformation. The cells were not pluripotent and expressed reduced levels of Oct 4 and Rex-1. They were non-tumorigenic with a population doubling time of approximately 15 hours. When plated on matrigel, they readily differentiated to form patent tubular structures with diameters of 30 to 150 microm. The differentiated cells endocytosed acetylated low-density lipoprotein (LDL) and began to express endothelial-specific markers such as CD34, CD31, Flk-1, TIE2, P-selectin, Sca-1, and thy-1. They also expressed genes essential for differentiation and maintenance of endothelial lineages, eg, Flk-1, vascular endothelial growth factor (VEGF), and angiopoietin-1. When transplanted into animal models, these cells incorporated into host vasculature. CONCLUSIONS: These cell lines can undergo in vitro and in vivo endothelial differentiation that recapitulated known endothelial differentiation pathways. Therefore, they are ideal for establishing an in vitro cellular system to study endothelial differentiation.

Animals↗
PubMed2003

Bone marrow-derived cells do not incorporate into the adult growing vasculature.

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

Animals↗
PubMed2002

The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis.

Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from the CD13/APN proximal promoter. Inhibition of these pathways and extracellular signal-regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APN transcription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor-induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.

Animals↗
PubMedDate not supplied

Aspects of human fetoplacental vasculogenesis and angiogenesis. III. Changes in complicated pregnancies.

Patterns of fetoplacental angiogenesis vary not only during the course of a normal pregnancy but also in certain pregnancy pathologies. Here, we review some of the molecular and morphological events which occur in complicated pregnancies. The pregnancy complications are chosen in an attempt to represent the possible different origins (preplacental, uteroplacental, postplacental) of fetal hypoxia. Molecular events focus on reported changes in hypoxia-inducible factors, angiopoietins and the vascular endothelial, basic fibroblast and placenta growth factors and their receptors. Morphological changes focus on patterns of angiogenesis (branching and non-branching) and a consistent set of morphometric descriptors (covering measures of total capillary growth, villous capillarization and capillary size and shape in transverse section). Apart from some uncertainties due to lack of information, or failure to resolve fully the effects of intrauterine growth restriction and pre-eclampsia, alterations in the angiogenic growth factors and morphologies of capillaries and villi in different complicated pregnancies seem to conform reasonably well to those predicted by the fetal hypoxia paradigm. However, it is clear that future studies on the effects of different origins of fetal hypoxia should exercise more care in the choice and interpretation of relevant descriptors and take more account of the parallel effects of possible confounders. In addition, rather than comparing uncomplicated and complicated pregnancies only at term, more information about molecular and morphological events that occur throughout gestation would be extremely valuable. This includes further studies on changes in growth factor receptors, the less-well-documented angiogenic factors (e.g. angiogenin, angiostatin, endostatin) and the associations between endothelial cells and pericytes. A more integrated approach involving also parallel analysis of the effects of erythropoietin and other potential vasoactive factors on the behaviour and morphology of fetal vessels would be beneficial.

Adult↗
PubMed1999

Potential roles of metalloprotease mediated ectodomain cleavage in signaling by the endothelial receptor tyrosine kinase Tie-1.

The orphan receptor tyrosine kinase Tie-1 is expressed predominantly in endothelial cells. Expression of this receptor is increased in physiologic angiogenesis and pathologic situations including tumor growth and arteriovenous malformations. Tie-1 is essential for vascular development where it acts in later stages of angiogenesis to suppress endothelial activation and stabilize the newly formed vessel. Stimulation of protein kinase C in endothelial cells results in endoproteolytic cleavage of Tie-1, releasing the extracellular ligand-binding domain of the receptor. We show that this is mediated by a metalloprotease. Immunoprecipitation and immunoblotting of lysates prepared from human placentas confirm that Tie-1 truncation occurs in vivo. We propose cleavage of this receptor may be a mechanism for inducing vessel destabilization by preventing ligand-activated signaling through Tie-1. Using an antibody that recognizes the carboxy terminus of the intracellular domain, we show that the Tie-1 endodomain formed on cleavage persists as a cell-associated fragment for several hours. Subcellular fractionation reveals this tyrosine kinase containing receptor fragment to be localized in the membrane fraction of the cell. Immunoprecipitation with antibodies recognizing phosphotyrosine demonstrates that cleavage of Tie-1 stimulates association of newly generated endodomain with cellular phosphoproteins. Furthermore, there was a marked induction of tyrosine phosphorylation of several proteins after PMA-induced endodomain generation. These data indicate that ectodomain cleavage may be a mechanism for down-regulating ligand-induced signaling through Tie-1 while activating an alternative ligand-independent signaling pathway in endothelial cells. Ectodomain cleavage occurs in some other receptor tyrosine kinases. We suggest that rather than solely being a means of down-regulating receptor activity, ectodomain cleavage may be a novel way for a receptor to switch between two alternative signaling pathways.

Cells, Cultured↗
PubMed2002

The role of the matrix metalloproteinases during in vitro vessel formation.

Matrix metalloproteinases (MMPs) constitute a large family of extracellular matrix degrading proteases implicated in a number of physiological and pathological processes, including angiogenesis. However, the relative importance of the individual MMPs in vessel formation is poorly understood. Using the three-dimensional rat aortic model, the role of the MMPs in angiogenesis in vitro was investigated both by the use of synthetic MMP inhibitors, and by a study of the expression of nine MMPs and three of their endogenous inhibitors (the TIMPs) during vessel formation. Inhibition of microvessel growth in this model by the MMP inhibitor Marimastat demonstrated the requirement of the MMPs for angiogenesis in both collagen and fibrin matrices (half-maximal inhibition at 5 and 80 nM, respectively). The profile of MMP expression was seen to be modified by both matrix composition and exogenous growth factors. For example, whilst the gelatinase MMP-2 and stromelysin MMP-3 were present at high levels in fibrin culture, the stromelysin MMP-11 and membrane-type-1-MMP were more highly expressed during vessel formation in collagen. The angiogenic basic fibroblast growth factor (bFGF) upregulated the expression of the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) and the interstitial collagenase MMP-13, whereas vascular endothelial growth factor (VEGF) led to a marked increase in expression of MMP-2 only. Together, the environment-dependent upregulation in expression of a number of MMPs during angiogenesis, and the total inhibition of vessel growth observed at nanomolar concentrations of synthetic MMP inhibitors, suggests a major collective role of these enzymes in angiogenesis, and provides a basis for further development of MMP inhibitors for anti-angiogenic therapy.

Animals↗
PubMed1997

Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine.

VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the endothelial cells lining nearby microvessels to proliferate, to migrate and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular endothelial cells hyperpermeable so that they spill plasma proteins into the extravascular space, leading to profound alterations in the extracellular matrix that favor angiogenesis. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma-derived matrix precede and accompany the onset of endothelial cell division and new blood vessel formation. It would seem, therefore, that tumors have made use of fundamental pathways that developed in multicellular organisms for purposes of tissue defense, renewal and repair. VPF/VEGF, therefore, has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important--perhaps essential--elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in sub-toxic concentrations that are more than sufficient to induce angiogenesis (Connolly et al., 1989a). Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating VPF/VEGF expression. In fact, there are already clear example of this. A number of putative angiogenic factors including small molecules (e.g. prostaglandins, adenosine) as well as many cytokines (e.g. TGF-alpha, bFGF, TGF-beta, TNF-alpha, KGF, PDGF) have all been shown to upregulate VPF/VEGF expression. Further studies that elucidate the crosstalk among various angiogenic factors are likely to contribute significantly to a better understanding of the mechanisms by which new blood vessels are formed in health and in disease.

Adult↗
PubMed2001

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Alkylation↗
PubMed2002

Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix.

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor alpha (bFGF/TNF-alpha) or vascular endothelial growth factor (VEGF)/TNF-alpha. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)-MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.

Adenoviridae↗
PubMed1998

[Radiotherapy of occult neovascularization in senile macular degeneration: initial results of a pilot study].

BACKGROUND: AMD is the leading cause of legal blindness in people aged of more than 50 in industrialized countries. Occult neovascularization accounts for more than 70% of all exsudatives forms of AMD. Radiotherapy has been proposed as a possible treatment for retrofoveal neovascularization. We present the first results of a pilot study actually ongoing at the AMD Center in Creteil. PATIENTS AND METHODS: 44 patients (46 eyes) presenting occult choroidal neovascularization involving the fovea, aged of > 50 years and with a minimal visual acuity of at least 0.1 were included. They received a total dose of 16 Gy in 4 sessions of 4 Gy. All the patients were checked after 3 months. RESULTS: Visual acuity was globally maintained (> 0.1 in 93% of the eyes). Only one eye suffered a significant loss of vision (> 6 lines) and 11% suffered a moderate loss of vision (> 3 lines). Moreover 67% of eyes still had a near vision of 0.5 or more with reading glasses. On fluorescein angiography 17.5% of the eyes experienced an increase of the size of the occult neovascularization of > 0.5 Disk Area (DA), 76% showed no modification and 6.5% demonstrated a decrease in size of > 0.5 DA. CONCLUSIONS: As it has been shown that the antimitotic action of radiotherapy is most effective after 3 months, the present results suggest better evolution than natural history. However, as occult choroidal neovascularization is a slow-evolving disease further follow-up is needed to confirm it.

Aged↗
PubMed1997

Choroidal neovascularization complicating epiretinal membrane removal.

PURPOSE: Surgical removal of epiretinal membranes generally leads to anatomic and functional improvement. Main complications include cataract, retinal breaks and detachment. We describe the onset of a juxtafoveal choroidal neovascularization 2 years after surgery of an epiretinal membrane. METHODS: The neovascular membrane was treated by argon laser photocoagulation. RESULTS: Complete obliteration of the neovascularization was obtained resulting in functional improvement. CONCLUSIONS: Although rare, choroidal neovascularization as a complication of epiretinal membrane surgery must be suspected in case of poor visual outcome or relapse of symptoms.

Adult↗