CONGENITAL ANOMALIES, Issue 2 2001
Yoshiko Yasuda
ABSTRACT, Vascular endothelial growth factor (VEGF) is induced by hypoxic environment and contributes to vascular formation in both developing embryos and adults. Exogenous retinoic acid (RA) induces avascular yolk sacs with anemic stunted embryos of day 9 and 10 of gestation when RA is given to pregnant mice on day 6, 6.5 or 7 of pregnancy (Yasuda et al., 1996). We undertook the present studies to find out whether VEGF is activated and plays any role in those RA-exposed embryos. Embryos were obtained from dams given 60 mg/kg of RA on day 6 or 7 of pregnancy and sacrificed three days later. Most RA-exposed embryos showed edematous swelling without prominent vascular nets, but had beating heart tubes on day 9 and day 10 of gestation. Microscopic examination of developing tissue components showed various degrees of degeneration, and distension of the dorsal aorta when the body cavity was dosed. Northern blot analysis revealed expression of VEGF mRNA in the RA-exposed and control embryos. The highest expression of VEGF mRNA was seen in the embryos of day 10 exposed to RA on day 7, and these embryos had a significantly lower ATP content than did the controls (p < 0.01). Immunoreactive VEGF was detectable in both experimental and control embryos; in the former it was especially visible in the distended neuroepithelium, endothelium and membranes. These VEGF-immunoreactive regions also expressed another permeability factor, bradykinin. These findings suggest that VEGF upregulated by hypoxic conditions in edematous embryos induced by RA exposure in utero acts as hyperpermeability. [source]

Adventures in multivalency, the Harry S. Fischer memorial lecture CMR 2005; Evian, France

CONTRAST MEDIA & MOLECULAR IMAGING, Issue 1 2006
Michael F. Tweedle
Abstract This review discusses multivalency in the context of drug discovery, specifically the discovery of new diagnostic imaging and related agents. The aim is to draw attention to the powerful role that multivalency plays throughout research involving molecular biology, in general, and much of biochemically targeted contrast agent research, in particular. Two examples from the author's laboratory are described. We created small (,5,kDa) peptide ,dimers' composed of two different, chemically linked peptides. The monomer peptides both bound to the same target protein with Kd,,,100,s,nM, while the heterodimers had sub-nM Kd values. Biological activity was evident in the heterodimers where none or very little existed in homodimers, monomers or monomer mixtures. Two different tyrosine kinases (KDR and C-Met) and four peptide families produced consistent results: multivalent heterodimers were uniquely different. The second example begins with making two micron ultrasound bubbles coated with the peptide, TKPPR (a Tuftsin antagonist) as a negative control for bubbles targeted with angiogenesis target-binding peptides. Unexpected binding of a ,negative' control, (TKPPR)-targeted bubble to endothelial cells expressing angiogenesis targets, led to the surprising result that TKPPR, only when multimerized, binds avidly, specifically and actively to neuropilin-1, a VEGF co-receptor. VEGF is the primary stimulator of angiogenesis. Tuftsin is a small peptide (TKPR) derived from IgG that binds to macrophages during inflammation, and has been studied for over 30 years. The receptor has never been cloned. The results led to new conclusions about Tuftsin, neuropilin-1 and the purpose, up to now unknown, of exon 8 in VEGF. Multivalency can be used rationally to solve practical problems in drug discovery. When targeting larger structures, multivalency is frequently unavoidable, and can lead to unpredictable and useful biochemical information, as well as to new drug candidates. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Time course of changes in angiogenesis-related factors in denervated muscle

ACTA PHYSIOLOGICA, Issue 4 2006
A. Wagatsuma
Abstract Aim:, Denervation leads to capillary regression in skeletal muscle. To gain insight into the regulation of this process, we investigated the time course of changes in capillary supply and gene expression of angiogenesis-related factors during muscle denervation. Method:, Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were isolated at 12 h, 1, 3, 5, 10, 20, or 30 days after surgery. The capillary supply was assessed by immunohistochemistry using anti-PECAM-1/CD31 antibody. The mRNA levels for angiogenesis-related factors were analysed using a real-time polymerase chain reaction. Results:, We found that the capillary-to-fibre ratio began to decrease 10 days after muscle denervation and decreased by 52% after 30 days. The levels of mRNA for vascular endothelial growth factor (VEGF), its receptors [fms-like tyrosine kinase (Flt-1) and a kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1)], angiopoietin-1 and angiopoietin-2 of denervated muscle were immediately down-regulated after 12 h and remained lower than control muscle until 30 days after muscle denervation. The levels of mRNA for the VEGF receptor, neuropilin-1, angiopoietin receptor and Tie-2 decreased within 12,24 h, but returned to near those of control muscle after 10,20 days, and again decreased after 30 days. Conclusions:, These findings suggest that denervation-induced capillary regression may be associated with down-regulation of VEGF and angiopoietin signalling. [source]

Role of VEGF and tissue hypoxia in patterning of neural and vascular cells recruited to the embryonic heart

DEVELOPMENTAL DYNAMICS, Issue 11 2009
Hongbin Liu
Abstract We hypothesized that oxygen gradients and hypoxia-responsive signaling may play a role in the patterning of neural or vascular cells recruited to the developing heart. Endothelial progenitor and neural cells are recruited to and form branched structures adjacent to the relatively hypoxic outflow tract (OFT) myocardium from stages 27,32 (ED6.5,7.5) of chick development. As determined by whole mount confocal microscopy, the neural and vascular structures were not anatomically associated. Adenoviral delivery of a VEGF trap dramatically affected the remodeling of the vascular plexus into a coronary tree while neuronal branching was normal. Both neuronal and vascular branching was diminished in the hearts of embryos incubated under hyperoxic conditions. Quantitative analysis of the vascular defects using our recently developed VESGEN program demonstrated reduced small vessel branching and increased vessel diameters. We propose that vascular and neural patterning in the developing heart share dependence on tissue oxygen gradients but are not interdependent. Developmental Dynamics 238:2760,2769, 2009. © 2009 Wiley-Liss, Inc. [source]

VEGF-mediated fusion in the generation of uniluminal vascular spheroids

DEVELOPMENTAL DYNAMICS, Issue 10 2008
Carmine Gentile
Abstract Embryonic mouse allantoic tissue (E8.5) was cultured in hanging drops to generate a three-dimensional vascular micro-tissue. The resulting tissue spheroids had an inner network of small diameter vessels expressing platelet endothelial cell adhesion molecule-1 (PECAM-1) and an outer layer of cells expressing SM,A, SM22-,, and SM-MHC. In a subsequent phase of culture, the fusion-promoting activity of vascular endothelial growth factor (VEGF) was used to transform the inner network of small diameter endothelial tubes into a contiguous layer of cells expressing PECAM-1, CD34, and VE-cadherin that circumscribed a central lumen-like cavity. The blood vessel-like character of the VEGF-treated spheroids was further demonstrated by their physiologically relevant vasodilatory and contractile responses, including contraction induced by KCl and relaxation stimulated by high-density lipoproteins and acetylcholine-induced nitric oxide production. Developmental Dynamics 237:2918,2925, 2008. © 2008 Wiley-Liss, Inc. [source]

In vivo and in vitro analysis of the vasculogenic potential of avian proepicardial and epicardial cells,

DEVELOPMENTAL DYNAMICS, Issue 4 2006
Juan A. Guadix
Abstract Coronary vessel formation is a special case in the context of embryonic vascular development. A major part of the coronary cellular precursors (endothelial, smooth muscle, and fibroblastic cells) derive from the proepicardium and the epicardium in what can be regarded as a late event of angioblastic and smooth muscle cell differentiation. Thus, coronary morphogenesis is dependent on the epithelial,mesenchymal transformation of the proepicardium and the epicardium. In this study, we present several novel observations about the process of coronary vasculogenesis in avian embryos, namely: (1) The proepicardium displays a high vasculogenic potential, both in vivo (as shown by heterotopic transplants) and in vitro, which is modulated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor signals; (2) Proepicardial and epicardial cells co-express receptors for platelet-derived growth factor-BB and VEGF; (3) Coronary angioblasts (found all through the epicardial, subepicardial, and compact myocardial layers) express the Wilms' tumor associated transcription factor and the retinoic acid-synthesizing enzyme retinaldehyde-dehydrogenase-2, two markers of the coelomic epithelium involved in coronary endothelium development. All these results contribute to the development of our knowledge on the vascular potential of proepicardial/epicardial cells, the existent interrelationships between the differentiating coronary cell lineages, and the molecular mechanisms involved in the regulation of coronary morphogenesis. Developmental Dynamics 235:1014,1026, 2006. © 2006 Wiley-Liss, Inc. [source]

VEGF signaling is required for the assembly but not the maintenance of embryonic blood vessels

DEVELOPMENTAL DYNAMICS, Issue 3 2002
W. Scott Argraves
Abstract Here we investigated the importance of vascular endothelial growth factor (VEGF) signaling to the de novo formation of embryonic blood vessels, vasculogenesis, as opposed to the maintenance of blood vessels. We found that antagonizing the activity of the VEGF signaling pathway by using soluble VEGF receptor 1 (sFlt1) or VEGF antibodies inhibited vasculogenesis that occurs in embryos and in cultures of 7.5 days postcoitus prevascular mesoderm. Antagonist treatment resulted in the formation of clusters of endothelial cells not normally observed during vasculogenesis. In contrast, when embryos with established vasculatures or cultures of vascularized mesoderm were treated with sFlt1 or VEGF antibodies, no discernible alterations to the preexisting blood vessels were observed. These observations indicate that, although VEGF signaling is required to promote the mesenchymal to epithelial transition by which angioblasts assemble into nascent endothelial tubes, it is not required by endothelial cells to maintain their organization as an endothelium. © 2002 Wiley-Liss, Inc. [source]

Vascular regression is required for mesenchymal condensation and chondrogenesis in the developing limb

DEVELOPMENTAL DYNAMICS, Issue 3 2001
Melinda Yin
Abstract Vascular regression occurs during limb mesenchymal cell condensation and chondrogenesis, but it is unclear whether it is required for these processes or is a secondary phenomenon without major regulatory roles. To address this issue, beads presoaked with the potent angiogenic factor vascular endothelial growth factor (VEGF) were implanted in the vicinity of the prospective digit 2 in early chick embryo wing buds and the effects on angiogenesis and digit development were determined over time. We found that VEGF treatment caused a marked local increase in blood vessel number and density. Strikingly, this was accompanied by inhibition of digit 2 development as revealed by lack of expression of chondrogenic transcription factor Sox9 and absence of Alcian blue staining. Vascular distribution and skeletal development in adjacent areas remained largely unaffected. Inhibition of digit formation and excess vascularization were both reversible upon further embryonic growth and dissipation of VEGF activity. When supernumerary digits were induced at the anterior limb margin by retinoic acid treatment, their development was also preceded by vascular regression; interestingly, cotreatment with VEGF inhibited supernumerary digit development as well. Direct exposure of limb mesenchymal cells in micromass cultures to VEGF caused no obvious effects on condensation and chondrogenesis, indicating that VEGF effects are not due to direct action on skeletal cells. Our results are the first to provide evidence that vascular regression is required for mesenchymal condensation and chondrogenesis. A model of how patterning mechanisms and vascular regression may intersect and orchestrate limb skeletogenesis is proposed. © 2001 Wiley-Liss, Inc. [source]

Vascular endothelial growth factor prevents G93A-SOD1-induced motor neuron degeneration

DEVELOPMENTAL NEUROBIOLOGY, Issue 13 2009
J. Simon Lunn
Abstract Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu2+/Zn2+ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A-SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase-3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1-induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A-SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A-SOD1 toxicity and neuroprotection involves phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source]

Signalling pathways involved in retinal endothelial cell proliferation induced by advanced glycation end products: inhibitory effect of gliclazide

DIABETES OBESITY & METABOLISM, Issue 2 2004
J.-C. Mamputu
Aim:, We have previously demonstrated that advanced glycation end products (AGEs) stimulate bovine retinal endothelial cell (BREC) proliferation through induction of vascular endothelial growth factor (VEGF) production by these cells. We have also shown that gliclazide, a sulfonylurea which decreases oxidative stress, inhibits this effect. The aim of the present study was to characterize the signalling pathways involved in AGE-induced BREC proliferation and VEGF production and mediating the inhibitory effect of gliclazide on these biological events. Methods:, BRECs were treated or not treated with AGEs in the presence or absence of gliclazide, antioxidants, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) or nuclear factor-,B (NF-,B) inhibitors. BREC proliferation was assessed by measuring [3H]-thymidine incorporation into DNA. Activation of PKC, MAPK and NF-,B signal transduction pathways and determination of VEGF expression were assessed by Western blot analysis using specific antibodies. MAPK activity was also determined by an in vitro kinase assay. Results:, Treatment of BRECs with AGEs significantly increased cell proliferation and VEGF expression. AGEs induced PKC-, translocation, extracellular signal-regulated protein kinase 1/2 and NF-,B activation in these cells. Pharmacological inhibition of these signalling pathways abolished AGE effects on cell proliferation and VEGF expression. Exposure of BRECs to gliclazide or antioxidants such as vitamin E or N -acetyl- l -cysteine resulted in a significant decrease in AGE-induced activation of PKC-, MAPK- and NF-,B-signalling pathways. Conclusions:, Our results demonstrate the involvement of PKC, MAPK and NF-,B in AGE-induced BREC proliferation and VEGF expression. Gliclazide inhibits BREC proliferation by interfering with these intracellular signal transduction pathways. [source]

Protective role of pigment epithelium-derived factor (PEDF) in early phase of experimental diabetic retinopathy

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 7 2009
Yumiko Yoshida
Abstract Background Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that PEDF may protect against proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. We investigated here whether and how PEDF could prevent the development of diabetic retinopathy. Methods Streptozotocin-induced diabetic rats were treated with or without intravenous injection of PEDF for 4 weeks. Early neuronal derangements were evaluated by electroretinogram (ERG) and immunofluorescent staining of glial fibrillary acidic protein (GFAP). Expression of PEDF and 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative stress, was localized by immunofluorescence. Vascular endothelial growth factor (VEGF) and p22phox expression were evaluated with western blots. Breakdown of blood retinal barrier (BRB) was quantified with fluorescein isothiocynate (FITC)-conjugated dextran. NADPH oxidase activity was measured with lucigenin luminescence. Results Retinal PEDF levels were reduced, and amplitudes of a- and b-wave in the ERG were decreased in diabetic rats, which were in parallel with GFAP overexpression in the Müller cells. Further, retinal 8-OHdG, p22phox and VEGF levels and NADPH oxidase activity were increased, and BRB was broken in diabetic rats. Administration of PEDF ameliorated all of the characteristic changes in early diabetic retinopathy. Conclusions Results suggest that PEDF could prevent neuronal derangements and vascular hyperpermeability in early diabetic retinopathy via inhibition of NADPH oxidase-driven oxidative stress generation. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2003
Ruth B. Caldwell
Abstract Retinal neovascularization and macular edema are central features of diabetic retinopathy, the major cause of blindness in the developed world. Current treatments are limited in their efficacy and are associated with significant adverse effects. Characterization of the molecular and cellular processes involved in vascular growth and permeability has led to the recognition that the angiogenic growth factor and vascular permeability factor vascular endothelial growth factor (VEGF) plays a pivotal role in the retinal microvascular complications of diabetes. Therefore, VEGF represents an exciting target for therapeutic intervention in diabetic retinopathy. This review highlights the current understanding of the mechanisms that regulate VEGF gene expression and mediate its biological effects and how these processes may become altered during diabetes. The cellular and molecular alterations that characterize experimental models of diabetes are considered in relation to the influence of high glucose-mediated oxidative stress on VEGF expression and on the mechanisms of VEGF's actions under hyperglycemic induction. Finally, potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological effects in the diabetic retina are considered. Copyright © 2003 John Wiley & Sons, Ltd. [source]

An adipocentric view of signaling and intracellular trafficking

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2002
Silvia Mora
Abstract Adipocytes have traditionally been considered to be the primary site for whole body energy storage mainly in the form of triglycerides and fatty acids. This occurs through the ability of insulin to markedly stimulate both glucose uptake and lipogenesis. Conventional wisdom held that defects in fuel partitioning into adipocytes either because of increased adipose tissue mass and/or increased lipolysis and circulating free fatty acids resulted in dyslipidemia, obesity, insulin resistance and perhaps diabetes. However, it has become increasingly apparent that loss of adipose tissue (lipodystrophies) in both animal models and humans also leads to metabolic disorders that result in severe states of insulin resistance and potential diabetes. These apparently opposite functions can be resolved by the establishment of adipocytes not only as a fuel storage depot but also as a critical endocrine organ that secretes a variety of signaling molecules into the circulation. Although the molecular function of these adipocyte-derived signals are poorly understood, they play a central role in the maintenance of energy homeostasis by regulating insulin secretion, insulin action, glucose and lipid metabolism, energy balance, host defense and reproduction. The diversity of these secretory factors include enzymes (lipoprotein lipase (LPL) and adipsin), growth factors [vascular endothelial growth factor (VEGF)], cytokines (tumor necrosis factor-,, interleukin 6) and several other hormones involved in fatty acid and glucose metabolism (leptin, Acrp30, resistin and acylation stimulation protein). Despite the large number of molecules secreted by adipocytes, our understanding of the pathways and mechanisms controlling intracellular trafficking and exocytosis in adipocytes is poorly understood. In this article, we will review the current knowledge of the trafficking and secretion processes that take place in adipocytes, focusing our attention on two of the best characterized adipokine molecules (leptin and adiponectin) and on one of the most intensively studied regulated membrane proteins, the GLUT4 glucose transporter. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Increased levels of monokine induced by interferon-, (Mig) in the vitreous of patients with diabetic retinopathy

DIABETIC MEDICINE, Issue 7 2008
Y. Wakabayashi
Abstract Aim To determine the intravitreous concentration of monokine induced by interferon-, (Mig) in patients with diabetic retinopathy (DR) and the relation between Mig and vascular endothelial growth factor (VEGF). Research design and methods Vitreous samples were obtained at the time of vitrectomy from 41 eyes of 38 DR patients (30 with active DR and 11 with inactive DR) and from 15 eyes of 15 non-diabetic patients who had macular disease (control subjects). Human Mig and VEGF were quantified using a FACS Caliber® flow cytometer. Results The vitreous concentration of Mig was increased significantly in patients with both active and inactive DR [148.0 (31.6,997.2; median range) and 82.3 (25.7,347.7) pg/ml, respectively] compared with control subjects [21.0 (5.2,74.3) pg/ml; P < 0.0001 and P < 0.001, respectively]. In DR patients, a significant (P < 0.01) correlation was observed between vitreous concentrations of Mig and VEGF. Conclusion Our results suggest that Mig may play an important role in the pathogenesis of DR and works in consort with VEGF in the progression of pathological angiogenesis in DR. [source]

Protein alterations in ESCC and clinical implications: a review

DISEASES OF THE ESOPHAGUS, Issue 1 2009
D.-C. Lin
SUMMARY Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer in Asia, characterized by high incidence and mortality rate. Although significant progress has been made in surgery and adjuvant chemoradiotherapy, the prognosis of the patients with this cancer still remains poor. Investigation into protein alterations that occurred in tumors can provide clues to discover new biomarkers for improving diagnosis and guiding targeted therapy. Hundreds of papers have appeared over the past several decades concerning protein alterations in ESCC. This review summarizes all the dysregulated proteins investigated in the disease from 187 published papers and analyzes their contributions to tumor development and progression. We document protein alterations associated with tumor metastasis and the transition from normal esophageal epithelia to dysplasia in order to reveal the most useful markers for prediction of clinical outcome, early detection, and identification of high-risk patients for targeted therapies. In particluar, we discuss the largest and most rigorous studies on prognostic implications of proteins in ESCC, in which cyclin D1, p53, E-cadherin and VEGF appeared to have the strongest evidence as independent predictors of patient outcome. [source]

Vascular endothelial growth factor gene polymorphisms are associated with the risk of developing adenomyosis

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 5 2009
Shan Kang
Abstract Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis and vascular permeability, may play a key role in the development of adenomyosis. The aim of this study was to investigate whether these four VEGF polymorphisms (,2578C/A, ,1154G/A, ,460C/T, and +936C/T) were associated with the risk of adenomyosis development. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in 174 adenomyosis patients and 199 frequency-matched control women. There were significant differences between patients and control group in allele frequencies and genotype distributions of the ,2578C/A polymorphisms (P = 0.010 and 0.044, respectively). Compared with the C/C genotype, the A/A + C/A genotype could significantly modify the risk of developing adenomyosis [odds ratio (OR) = 0.64, 95% confidence interval (CI) = 0.42,0.97]. For the ,1154G/A polymorphism, the allele frequencies and genotype distributions in patient group were significant different from those of the controls (P = 0.001 and 0.007, respectively). Compared with the G/G genotype, the A/A + G/A genotype could significantly decrease the risk of developing adenomyosis (OR = 0.51, 95% CI = 0.33,0.80). However, the genotype distributions and allele frequencies of the ,460C/T and +936C/T polymorphisms did not significantly differ between controls and patients (all P value > 0.05). The haplotype analysis suggested that the TGA (VEGF ,460/,1154/,2578) and CGA haplotypes exhibited a significant decrease in the risk of developing adenomyosis compared with the haplotype of TGC (OR = 0.64, 95% CI = 0.41,1.00; OR = 0.44, 95% CI = 0.21,0.93, respectively). The study indicated that the ,2578A or ,1154A allele of VEGF gene could significantly decrease the risk of adenomyosis and might be potentially protective factors for adenomyosis development. Environ. Mol. Mutagen., 2009. © 2009 Wiley-Liss, Inc. [source]

Parathyroid hormone stimulates the endothelial expression of vascular endothelial growth factor

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2008
G. Rashid
ABSTRACT Background, We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro-atherosclerotic and pro-inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra-cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions. Materials and methods, Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10,12,10,10 mol L,1 PTH. The VEGF-165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra-cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated. Results, PTH (10,10 mol L,1) significantly increased VEGF-165 mRNA expression (P < 0·05). The addition of 50 nmol L,1 protein kinase C (PKC) and 10 µmol L,1 protein kinase A (PKA) inhibitors significantly reduced the VEGF-165 mRNA expression (P = 0·01). We also examined whether nitric oxide (NO) may be involved in the PTH-induced stimulation of VEGF-165 expression. Pre-treatment of the cells with 200 µmol L-nitro arginine methyl ester (L-NAME, NO synthase inhibitor) was found to inhibit VEGF-165 mRNA expression (P = 0·006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression. Conclusion, The stimulatory effect of PTH on endothelial VEGF-165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent. [source]

Expression of angiogenic factors in chronic myeloid leukaemia: role of the bcr/abl oncogene, biochemical mechanisms, and potential clinical implications

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2004
C. Sillaber
Abstract Chronic myeloid leukaemia (CML) is a stem cell disease characterized by an increased production and accumulation of clonal BCR/ABL,positive cells in haematopoietic tissues. The chronic phase of CML is inevitably followed by an accelerated phase of the disease, with consecutive blast crisis. However, depending on genetic stability, epigenetic events, and several other factors, the clinical course and survival appear to vary among patients. Recent data suggest that angiogenic cytokines such as vascular endothelial growth factor (VEGF), are up-regulated in CML, and play a role in the pathogenesis of the disease. These factors appear to be produced and released in leukaemic cells in patients with CML. In line with this notion, increased serum-levels of angiogenic growth factors are measurable in CML patients. In this study we provide an overview of angiogenic growth factors expressed in CML cells, discuss the possible pathogenetic role of these cytokines, the biochemical basis of their production in leukaemic cells, and their potential clinical implications. [source]

Angiopoietin/tie-2 as mediators of angiogenesis: a role in congestive heart failure?

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 1 2004
A. Y. Chong
Abstract Angiogenic factors, in particular vascular endothelial growth factor (VEGF) and the angiopoietins, Ang-1 and -2, have recently generated significant interest, especially in oncology. The process of angiogenesis is also thought to occur in response to ischaemic conditions, which lie at the core of cardiovascular disease states such as coronary artery disease and congestive heart failure. However, current data do not conclusively show evidence of angiogenesis per se in these conditions, despite (for example) the presence of high levels of VEGF and Ang-2. High levels of these angiogenic factors in heart disease also have not translated into clinically significant new vessel formation, as in accelerated cancer growth or proliferative retinopathy. Indeed, we would hypothesize that these angiogenic markers , especially the angiopoietins , do not necessarily translate into new vessel formation in congestive heart failure (CHF), but may well reflect disturbances of endothelial integrity in CHF. [source]

Prognostic value of interleukin-6, plasma viscosity, fibrinogen, von Willebrand factor, tissue factor and vascular endothelial growth factor levels in congestive heart failure

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2003
B. S. P. Chin
Abstract Background, Congestive heart failure (CHF) carries a poor prognosis with a high mortality rate, frequent hospitalizations and increased risk of thrombotic complications such as stroke. Cytokines may contribute to the progression and prothrombotic state of CHF, including the pro-inflammatory interleukin-6 (IL-6) and the pro-angiogenic vascular endothelial growth factor (VEGF), both of which are raised in CHF. The procoagulant properties of both cytokines may be mediated via tissue factor (TF), a potent clotting activator. We hypothesized that plasma levels of these markers, as well as levels of plasma viscosity, fibrinogen, soluble P-selectin and von Willebrand factor (markers of abnormal rheology, clotting, platelet activation, and endothelial damage, respectively) will be useful in predicting morbidity and mortality in chronic stable CHF. Methods and results, One hundred and twenty consecutive out-patients with chronic stable CHF (92 males; mean [SD] age 64 [11] years, mean [SD] left ventricular ejection fraction of 29 [6]%) were recruited and followed for 2 years during which 42 patients reached a clinical end-point of all-cause mortality and cardiovascular hospitalizations, including stroke and myocardial infarction. Plasma IL-6 (P = 0·003) and TF (P = 0·013) levels, but not other research indices, were higher in those who suffered events compared with those without events. Predictors of end-points were high (, median) TF (P = 0·011), and IL-6 (P = 0·023) levels, as well as the lowest quartile of a left ventricular ejection fraction (P = 0·007). A strong correlation was present between TF and IL-6 levels (r = 0·59; P < 0·0001) and with VEGF levels (r = 0·43; P < 0·0001). Conclusion, IL-6 and TF are predictors of poor prognosis in chronic CHF, raising the hypothesis that IL-6 may contribute to the progression and thrombotic complications of CHF via its actions on TF expression. Although VEGF did not independently predict outcome in chronic CHF, the possibility arises that it may act with IL-6 to induce TF expression. [source]

Basic fibrobrast growth factor induces the secretion of vascular endothelial growth factor by human aortic smooth muscle cells but not by endothelial cells

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003
F. Belgore
Abstract Background, Endothelial cell dysfunction and smooth muscle cell (SMC) proliferation are major events in atherogenesis. Both cells are a source of growth factors that mediate cellular proliferation and chemotaxis. Inappropriate production of, and/or response to, these growth factors (such as vascular endothelial growth factor, VEGF, and basic fibroblast growth factor (bFGF)) may contribute to atherogenesis and therefore to disease progression. Methods, Production of VEGF and its soluble receptor (sFlt-1) by human SMCs and human umbilical endothelial cells (HUVECs) after stimulation with bFGF were examined by ELISA of cell culture media and by Western blotting. Results, Smooth muscle cells produced significantly more VEGF than HUVECs (P < 0·05) after 24 h of culture with bFGF levels , 0·001 µg mL,1. bFGF induced dose-dependent production of VEGF by SMCs, where maximum production was present in 1 µg mL,1 of bFGF. Conversely, the SMCs produced less sFlt-1 than HUVECs (P < 0·05). However, bFGF induced dose-dependent phosphorylation of Flt1 and another VEGF receptor, KDR, in HUVECs but not SMCs. There was no VEGF or sFLT-1 after 6 h of culture in any dose of bFGF in either type of cell. Conclusions, Differences in the production of VEGF and sFlt-1 by SMCs and HUVECs are consistent with the role of these cells in angiogenesis. Induction of VEGF production and expression by bFGF in these cells indicates that this growth factor may participate in angiogenesis indirectly by the induction of VEGF. The production of sFlt-1 by both cell types is in agreement with the notion that sFlt-1 may be involved in the regulation of VEGF activity. Additionally, the ability of bFGF to induce dose-dependent phosphorylation of KDR in HUVECs highlights the important role of bFGF in VEGF-mediated angiogenic processes. [source]

Plasma angiopoietin-1, angiopoietin-2 and Tie-2 in breast and prostate cancer: a comparison with VEGF and Flt-1

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003
G. J. Caine
Abstract Background, Angiogenesis is essential for tumour growth and metastasis, and is coordinated by several classes of growth factors mediating their effect through receptors linked, in turn, to tyrosine kinase. These growth factors include angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF), which act through receptors Flt-1 and Tie-2. Materials and methods, In order to further determine abnormalities in levels of Ang-1, Ang-2, Tie-2, sFlt-1 and VEGF in human cancer (and their interrelationships), these molecules were measured in plasma from 30 patients with breast cancer, 30 patients with prostate cancer and 12 healthy controls per cancer group. Results, In breast cancer, levels of Ang-1 (P = 0·0005), Ang-2 (P = 0·0173), Tie-2 (P = 0·0001), and VEGF (P = 0·0001) were all significantly raised, and plasma levels of sFlt-1 (P = 0·045) were significantly reduced compared with controls. However, in prostate cancer, only levels of VEGF and Tie-2 were significantly higher (both P= 0·001). There were no significant differences between levels of any molecule between the two groups of cancer. The only difference between the healthy control groups was lower Ang-1 in the women compared with men. Significant correlations were found between levels of Ang-1 and Tie-2 both in breast (r = 0·498, P= 0·005) and prostate cancer (r = 0·643, P= < 0·001). Angiopoietin-1 was also positively correlated with Ang-2 in both breast (r = 0·422, P= 0·02) and prostate cancer (r = 0·543, P= 0·002). Conclusions, Abnormal levels of Ang-1, Ang-2 and their receptor, Tie-2, are present in breast and prostate cancer, and their interrelationships may be important in the pathophysiology of these conditions. [source]

Measurement of the soluble angiopoietin receptor tie-2 in patients with coronary artery disease: development and application of an immunoassay

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2003
N. A. Y. Chung
Abstract Background The angiopoietin family has emerged as a group of crucial growth factors to normal angiogenesis. They are essential to the development of the mature vessel wall and interact with the endothelium via endothelial cell-specific tyrosine kinase receptors, tie-1 and tie-2. The role of the tie-2 receptor has been extensively examined in neovascularization associated with malignancy, but little is known about the role it may play in atherosclerosis, a condition whose pathophysiology also involves angiogenesis. Soluble tie-2 has been detected in the plasma of healthy controls, but this has yet to be applied to patients in the clinical setting. Materials and methods We developed an ELISA to detect plasma tie-2 levels and applied these to a clinical setting. The intra- and interassay coefficients of variation for the assay were 4·7% and 9·6%, respectively. We then measured levels of tie-2, vascular endothelial growth factor (VEGF), another factor associated with angiogenesis, and the soluble VEGF receptor Flt-1 (sFlt-1) in 75 patients with coronary artery disease [25 with acute myocardial infarction (AMI), 25 with acute coronary syndromes (ACS) and 25 with stable angina] and 25 healthy controls. Results Median [IQR, interquartile range] levels of tie-2 were significantly higher in the coronary artery disease patients (AMI 12 [10,17] ng mL,1, ACS 10 [9,14] ng mL,1, stable angina 9 [3,11] ng mL,1) when compared with the controls (7·5 [7,9] ng mL,1P = 0·004). As expected, levels of VEGF and sFlt were significantly different from those in the healthy controls (P = 0·011 and P < 0·001, respectively). Significant correlations were found between levels of tie-2 and VEGF (Spearman r = 0·59, P < 0·001), tie-2 and sFlt-1 (r = 0·45, P < 0·001) and VEGF and sFlt-1 (r = 0·56, P < 0·001) in the whole study group. Conclusion We suggest that tie-2 may be potentially used as a marker of angiogenesis in atherosclerosis and may help elucidate the role of the angiopoietin/tie-2 system in atherogenesis. [source]

Increased expression of VEGF following exercise training in patients with heart failure

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2001
T. Gustafsson
Background and aims During the last decades several angiogenic factors have been characterized but so far it is unknown whether local muscle exercise training increases the expression of these factors in patients with moderate heart failure. Expression of the major putative angiogenic factor vascular endothelial growth factor (VEGF) at the level of messneger RNA (mRNA) and/or protein was therefore studied before and after 8 weeks of training in patient with chronic heart failure. Methods VEGF mRNA and protein concentrations were determined in skeletal muscle biopsies before and after 8 weeks of one-legged knee extension training in patients with chronic heart failure (New York Heart Association II,III). Results Exercise training increased the citrate synthase activity and peripheral exercise capacity by 46% and 36%, respectively, in parallel with a two-fold increase in VEGF at both the mRNA (P = 0·03) and protein (P = 0·02) levels Conclusion The increase in VEGF gene expression in response to exercise training indicates VEGF to be one possible mediator in exercise-induced angiogenesis and may therefore regulate an important and early step in adaptation to increased muscle activity in patient with chronic heart failure. [source]

Serum vascular endothelial growth factor in adult haematological patients with neutropenic fever: a comparison with C-reactive protein

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2009
Sari Hämäläinen
Abstract Objectives:, Vascular endothelial growth factor (VEGF) is considered to be of importance in patients with sepsis. No data are available on VEGF kinetics in haematological patients with neutropenic fever. Methods:, Forty-two haematological patients were included into this prospective study. Median age was 57 yr (range 18,70). Fifteen patients received therapy for acute myeloid leukaemia and 27 patients received autologous stem cell transplantation for haematological malignancy. Laboratory samples for the determination of C-reactive protein (CRP) and VEGF were collected at the start of fever (d0) and then daily. Results:, The median serum VEGF concentrations were low in all study patients. In patients with severe sepsis (n = 5) the median VEGF on d0 was higher than in septic patients without signs of hypoperfusion or hypotension (n = 37) (77 pg/mL vs. 52 pg/mL, P = 0.061). Also on d1 the median VEGF concentration was higher in patients with severe sepsis (82 pg/mL vs. 56 pg/mL, P = 0.048). There were no statistically significant differences in CRP values on any day during the study period between patients with severe sepsis and those without. Time from d0 to the peak VEGF concentration (mean 1.02, SE 0.18 d) was shorter than that to the peak CRP concentration (mean 1.93, SE 0.15 d) (P = 0.002). Conclusion:, Compared to CRP, serum VEGF was a more rapid indicator for sepsis in our haematological patients with neutropenic fever. Those with severe sepsis had higher VEGF concentrations than those without on d0 and d1 after the onset of fever. Further studies on VEGF are warranted in haematological patients. [source]

Placenta growth factor stimulates the growth of Philadelphia chromosome positive acute lymphoblastic leukemia cells by both autocrine and paracrine pathways

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2005
Toshiko Ikai
Abstract:, Vascular endothelial growth factor (VEGF) and its associated molecule, placenta growth factor (PlGF) are now known to support normal hematopoiesis, and leukemia cell growth. In this study, expression of VEGF and PlGF in acute lymphoblastic leukemia (ALL) cells was examined by real time reverse transcription-polymerase chain reaction in 20 patient samples. Expression of PlGF was more intense in Philadelphia chromosome positive (Ph+) ALL than in Ph, ALL cases. On the other hand, expression level of VEGF was not different between Ph+ and Ph, cases. Then, PlGF was added to the two ALL cell lines, CRL1929 (Ph+), and Nalm6 (Ph,). The PlGF stimulated the growth of CRL1929 in time- and dose-dependent manners, although the growth of Nalm6 was not affected by PlGF. The growth stimulation of CRL1929 by PlGF was confirmed by the increase of S phase cells. And the growth promoting effect of PlGF on CRL1929 was cancelled by simultaneous addition of VEGFR1/Fc (which binds to PlGF and abrogates its function), but was not cancelled by VEGFR2/Fc (which does not bind to PlGF). Then, addition of VEGFR1/Fc to the simple culture of CRL1929 demonstrated growth inhibitory effect. These observations demonstrated that PlGF stimulates the growth of Ph+ ALL cells by both autocrine and paracrine pathways. Finally, PlGF-VEGFR1 loop might be a therapeutic target to improve the prognosis of Ph+ ALL. [source]

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2003
Øystein Bruserud
Abstract: Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1,, IL6, IL8, tumor necrosis factor ,] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects. [source]

Involvement of hypoxia-inducible factor-1 HiF(1,) in IgE-mediated primary human basophil responses

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009
Vadim V. Sumbayev
Abstract Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the ,-subunit of hypoxia-inducible factor 1, (HIF-1,), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1,, although the latter crucially affected degranulation. Abrogating HIF-1, expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1, protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4. [source]

Serum VEGF levels in acute ischaemic strokes are correlated with long-term prognosis

EUROPEAN JOURNAL OF NEUROLOGY, Issue 1 2010
S.-C. Lee
Background and purpose:, We investigated whether serum vascular endothelial growth factor (VEGF) levels in acute-stage ischaemic stroke patients with small vessel disease (SVD) or large vessel disease (LVD) are correlated with long-term prognoses, based on the difference in NIH Stroke Scale (NIHSS) scores between acute and chronic stages. Methods:, From March 2007 to May 2008, we evaluated patients who experienced an ischaemic stroke for the first time, defined as SVD (n = 89) or LVD (n = 91) using the TOAST classification. Serum samples were taken immediately after admission (within 24 h of stroke onset) to evaluate VEGF levels. After 3 months, follow-up NIHSS scores were collected for all patients. Results:, Serum VEGF levels in the acute stage (within 24 h of stroke onset) were higher in the LVD group than in the SVD group and were correlated with infarction volume. The increase in serum VEGF levels in the acute stage was proportional to an improved NIHSS score after 3 months. After adjustment for covariates, serum VEGF levels in the acute stage were still significantly correlated with the long-term prognosis of ischaemic stroke. Conclusion:, Serum VEGF levels are correlated with long-term prognoses in acute ischaemic stroke patients. [source]

Erythropoietin protects the in vitro blood,brain barrier against VEGF-induced permeability

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2003
Ofelia María Martínez-Estrada
Abstract The blood,brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions. [source]