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Fine-tuning angiogenesis, the development of new blood vessels, is essential for maintaining a healthy circulatory and lymphatic system. The small glycoprotein vascular endothelial growth factors (VEGF) are the key mediators in this process, binding to their corresponding membrane-bound VEGF receptors (VEGFRs) to activate angiogenesis signaling pathways. These pathways are crucial throughout human life as they are involved in lymphatic and vascular endothelial cell permeability, migration, proliferation, and survival. Neovascularization, the formation of abnormal blood vessels, occurs when there is a dysregulation of angiogenesis and can result in debilitating disease. Hence, VEGFRs have been widely studied to understand their role in disease-causing angiogenesis. VEGFR1, also known as Fms-like tyrosine kinase-1 (FLT-1), is also found in a soluble form, soluble FLT-1 or sFLT-1, which is known to act as a VEGF neutralizer. It is incorporated into anti-VEGF therapy, designed to treat diseases caused by neovascularization. Here we review the journey of sFLT-1 discovery and delve into the alternative splicing mechanism that creates the soluble receptor, its prevalence in disease states, and its use in current and future potential therapies. [ABSTRACT FROM AUTHOR]

Background: The present study was aimed to explore the effects and the underlying mechanism of prophylactic low-molecular-weight heparin (LMWH) treatment on taurocholate-induced severe acute pancreatitis (SAP) in a rat model. Methods: Rat SAP model was induced by injection of 4% sodium taurocholate into the pancreatic duct. LMWH was applied half an hour before the induction of pancreatitis at the dose of 200 IU/kg subcutaneous injection. The rats were euthanized at 1 h, 6 h, and 12 h after taurocholate-induced SAP. The inflammatory and oxidative response markers were assessed. And the vascular endothelial growth factor (VEGF) and Fms-related tyrosine kinase 1 (Flt-1) expression were evaluated by immunohistochemistry (IHC) and western blot methods. Results: The expression of inflammatory and oxidative response markers increased after induction of SAP. IHC and western blot results showed the VEGF and Flt-1 expression were increased in SAP group. Prophylactic LMWH administration reduced the inflammatory and oxidative response markers expression and decreased the expression of VEGF and Flt-1. Conclusions: This study suggested that prophylactic LMWH treatment mitigated the severity of pancreatitis in rat SAP model by anti-inflammation and oxidative response. The underlying mechanism may result from downregulating VEGF/Flt-1 signaling of LMWH in SAP rat model. [ABSTRACT FROM AUTHOR]

We have previously characterized morphologic features of wounding-induced angiogenesis that occurs in response to acute and chronic gastric mucosal injury. As a means of investigating the molecular mechanisms underlying gastric angiogenesis, microvascular endothelial cells were isolated from stomachs of normal (non-injured) rats. The isolation procedure adapted and combined aspects of previous methods and employed positive selection using magnetic beads coated with monoclonal antibody specific for rat CD31 (PECAM-1), a cell surface marker restricted to platelets, monocytes, T lymphocytes and endothelial cells. The isolated microvascular endothelial cells expressed vascular endothelium-specific antigen and the endothelial-specific receptors, Tie2 and flt-1 (VEGFR1). When plated on growth factor-reduced matrigel, the isolated microvascular endothelial cells formed capillary-like structures reflecting in vitro angiogenesis. These cells were also responsive to vascular endothelial growth factor, VEGF, further verifying their endothelial nature. The rat microvascular endothelial cells isolated by this procedure should be useful in delineating molecular mechanisms and regulation of the angiogenesis that is essential for the healing of acute and chronic gastric injury.

Background/Aims: Patients with esophageal atresia (EA) and tracheoesophageal fistula (TEF) often suffer chronic respiratory tract disease. We previously reported that primary lung maldevelopment caused by deficient branching of embryonal airways in experimental EA-TEF was induced by Adriamycin. In this study, we investigated the Vascular endothelial growth factor (VEGF) pathway in the developing lung in an EA-TEF rat model. We further analyzed the effect of recombinant VEGF treatment in vitro on branching morphogenesis of embryo lungs in experimental EA-TEF. Methods: Pregnant rats received either Adriamycin or vehicle on E7, E8 and E9. Lungs were recovered at E15, E18 and E21. Expression of VEGF and receptors (Flk-1 and Flt-1) were assessed by quantitative PCR, immunohistochemistry and immunoblotting. E13 lungs were cultured for 72 hours with 50 ng/mL of recombinant rat VEGF in serum-free medium. The rates of increase in bud count and airway contour were evaluated. Results: Our results showed a significant downregulation of VEGF during pseudoglandular and canalicular stages. In contrast, there were significantly higher levels of the Flt-1 receptor in the canalicular stage, which may represent a compensatory response to decreased VEGF. However, both variables returned to normal levels at the saccular stage. Exogenous VEGF treatment enhanced hypoplastic lung growth, evidenced by the increase in bud count and airway contour. Conclusions: A VEGF signaling defect possibly plays an important role in defective embryonic airway branching. Additionally, VEGF treatment may accelerate lung growth in EA-TEF lungs.

Aerobic exercise benefits the body and brain. In the brain, benefits include neuroprotection and improved cognition. These exercise-induced changes are attributed in part to angiogenesis: the growth of new capillaries from preexisting vessels. One critical factor involved in the regulation of angiogenesis is VEGF and its receptors Flk-1 and Flt-1. Although exercise is generally found to be beneficial, there are wide variations in exercise regimens across experiments. This study standardized some of these variations. Rats were assigned to a voluntary or a forced wheel running exercise condition. Within each condition, animals ran for either a long (1000 m) or short distance (500 m) for up to 24 h. Additionally, one voluntary group had unrestricted access to the wheels for the full 24 h. Exercising animals were then compared to inactive controls, based on unbiased stereological quantification of Flk-1 and Flt-1 immunohistochemical labeling in the hippocampus and cerebellum. Findings indicated that voluntary exercise, but not forced exercise, could significantly increase Flk-1 and Flt-1 expression in the hippocampus. Interestingly, Flk-1 expression was elevated in astrocytes and Flt-1 in vessels. In the cerebellum long distance forced exercise resulted in the least Flk-1 expression compared to other conditions, and Flt-1 expression in exercising animals either did not change or was suppressed relative to inactive controls. [ABSTRACT FROM AUTHOR]

Background: Latest studies show that low-energy extracorporeal shock wave therapy (ESWT) can upregulate levels of vascular endothelial growth factor (VEGF). VEGF can ease nervous tissue harm after spinal cord injury (SCI). This study aims to explore whether low-energy ESWT can promote expression of VEGF, protect nervous tissue after SCI, and improve motor function. Methods: Ninety adult female rats were divided into the following groups: Group A (simple laminectomy), Group B (laminectomy and low-energy ESWT), Group C (spinal cord injury), and Group D (spinal cord injury and low-energy ESWT). Impinger was used to cause thoracic spinal cord injury. Low-energy ESWT was applied as treatment after injury three times a week, for 3 weeks. After SCI, the Basso, Beattie, and Bresnahan (BBB) scale was used to evaluate motor function over a period of 42 days at different time points. Hematoxylin and eosin (HE) staining was used to evaluate nerve tissue injury. Neuronal nuclear antigen (NeuN) staining was also used to evaluate loss of neurons. Polymerase chain reaction was used to detect mes- senger RNA (mRNA) expression of VEGF and its receptor fms-like tyrosine kinase 1 (Flt-1). Immunostaining was used to evaluate VEGF protein expression level in myeloid tissue. Results: BBB scores of Groups A and B showed no significant result related to dyskinesia. HE and NeuN staining indicated that only using low-energy ESWT could not cause damage of nervous tissue in Group B. Recovery of motor function at 7, 35, and 42 days after SCI in Group D was better than that in Group C (P<0.05). Compared with Group C, number of NeuN- positive cells at 42 days after SCI increased significantly (P<0.05). The mRNA levels of VEGF and Flt-1 and VEGF expression at 7 days after SCI in Group D were significantly higher than those in Group C (P<0.05). Conclusion: Low-energy ESWT promotes expression of VEGF, decreases secondary damage of nerve tissue, and improves recovery of motor function. It can be regarded as one mode of clinical routine adjunctive therapy for spinal injury. [ABSTRACT FROM AUTHOR]

Background: It is well known that platelet-rich plasma (PRP) preparations are not the same and that not all preparations include white blood cells, but the part that leukocytes play on the healing role of PRP is still unknown. Purpose: The primary aim of this study was to evaluate the influence of leukocytes in different PRP preparations with a special emphasis on growth factor concentrations. The secondary aim was to evaluate the influence of PRP on muscle healing. Study Design: Controlled laboratory study. Methods: Two PRP preparation procedures were evaluated. Blood fractions were stained with Rapid Panoptic, and growth factors (transforming growth factor beta 1 [TGF-β1], vascular endothelial growth factor [VEGF], insulin-like growth factor [IGF], epidermal growth factor [EGF], hepatocyte growth factor [HGF], and platelet-derived growth factor [PDGF]) were quantified by enzyme-linked immunosorbent assay. Western blotting analysis was performed for Fms-related tyrosine kinase 1 (Flt-1). A muscle contusion injury was created and treated with PRP at different time points. Results: Leukocytes were the main source of VEGF, and all other growth factors measured had a higher concentration in the preparations that included the buffy coat and consequently had a higher concentration of white blood cells. Flt-1 was also found in platelet-poor plasma (PPP). There were higher concentrations of PDGF and HGF in the preparations that encompassed the buffy coat. A PRP injection 7 days after the injury provided significantly increased exercise performance and decreased the fibrotic area when compared with other PRP-treated groups. Conclusion: VEGF is only present in PRP′s buffy coat, while Flt-1 is present in PPP. A PRP injection 7 days after an injury resulted in improved exercise performance. Clinical Relevance: The presence of Flt-1 in PRP provides yet another explanation for results described in the literature after a PRP injection. This information is relevant for selecting the best PRP for each type of injury. [ABSTRACT FROM AUTHOR]

Background/Aims: Patients with esophageal atresia (EA) and tracheoesophageal fistula (TEF) often suffer chronic respiratory tract disease. We previously reported that primary lung maldevelopment caused by deficient branching of embryonal airways in experimental EA-TEF was induced by Adriamycin. In this study, we investigated the Vascular endothelial growth factor (VEGF) pathway in the developing lung in an EA-TEF rat model. We further analyzed the effect of recombinant VEGF treatment in vitro on branching morphogenesis of embryo lungs in experimental EA-TEF. Methods: Pregnant rats received either Adriamycin or vehicle on E7, E8 and E9. Lungs were recovered at E15, E18 and E21. Expression of VEGF and receptors (Flk-1 and Flt-1) were assessed by quantitative PCR, immunohistochemistry and immunoblotting. E13 lungs were cultured for 72 hours with 50 ng/mL of recombinant rat VEGF in serum-free medium. The rates of increase in bud count and airway contour were evaluated. Results: Our results showed a significant downregulation of VEGF during pseudoglandular and canalicular stages. In contrast, there were significantly higher levels of the Flt-1 receptor in the canalicular stage, which may represent a compensatory response to decreased VEGF. However, both variables returned to normal levels at the saccular stage. Exogenous VEGF treatment enhanced hypoplastic lung growth, evidenced by the increase in bud count and airway contour. Conclusions: A VEGF signaling defect possibly plays an important role in defective embryonic airway branching. Additionally, VEGF treatment may accelerate lung growth in EA - TEF lungs. [ABSTRACT FROM AUTHOR]

Porcine embryo mortalities cause economic losses. Development of the placental vascular bed is required for successful gestation and postnatal survival. We studied the temporal and spatial distributions of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors, Flt-1 and Flk-1. We used crossbred swine placental tissues from 30, 60, 80, 90 and 114 (term) days of gestation. Both VEGF and PlGF were present during gestation. At early pregnancy and at term, VEGF probably acts through Flt-1; during intermediate periods, its function is mediated by Flk-1. By day 90, factors other than members of VEGF family appear to be involved. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is the most common hereditary muscular dystrophy caused by mutation in dystrophin, and there is no curative therapy. Dystrophin is a protein which forms the dystrophin-associated glycoprotein complex (DGC) at the sarcolemma linking the muscle cytoskeleton to the extracellular matrix. When dystrophin is absent, muscle fibers become vulnerable to mechanical stretch. In addition to this, accumulating evidence indicates DMD muscle having vascular abnormalities and that the muscles are under an ischemic condition. More recent studies demonstrate decreased vascular densities and impaired angiogenesis in the muscles of murine model of DMD. Therefore, generation of new vasculature can be considered a potentially effective strategy for DMD therapy. The pro-angiogenic approaches also seem to be pro-myogenic and could induce muscle regeneration capacity through expansion of the satellite cell juxtavascular niche in the mouse model. Here, we will focus on angiogenesis, reviewing the background, vascular endothelial growth factor (VEGF)/VEGF receptor-pathway, effect, and concerns of this strategy in DMD. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endotheliumdependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensinconverting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy. [ABSTRACT FROM AUTHOR]

Abstract: The vascular endothelial growth factor (VEGF) has beneficial effects on ovine oocytes during in vitro maturation and their subsequent early embryonic development, but the biochemical pathway underlying this effect has not been elucidated. Therefore, the focus of the present study was to investigate the activation of the mitogen-activated protein kinase (MAPK) pathway in response to the addition of VEGF to the maturation medium, and to study the subcellular localization of VEGF and its receptors during ovine oocyte maturation. We concluded that: (1) VEGF mainly localized in the cytoplasm, whereas its receptors, fms-tyrosine kinase-1 and kinase domain region (KDR), were localized on the plasma membrane of oocytes; (2) the addition of 5 ng/mL VEGF increased the percentage of oocytes with extruded first polar bodies (50.9 ± 2.2% vs. 34.6 ± 2.9%; treatment vs. control, respectively; P < 0.01) and the rate of oocytes competent to undergo nuclear maturation (70.6 ± 0.9% vs. 62.9 ± 1.9%, P < 0.01); and (3) as the expression of VEGF, fms-tyrosine kinase-1, and KDR increased after supplementation with 5 ng/mL, expression of VEGF, mitogen-activated protein kinase kinase (MEK), and MAPK mRNA, as well as MAPK phosphorylation, were stimulated in a time-dependent manner. We inferred that, in a paracrine manner, exogenous VEGF bound to KDR, its main receptor, and then activated the MAPK signaling pathway, which promoted maturation of ovine oocytes. However, the VEGF system also had an autocrine regulatory loop that contributed to creating an environment optimal for oocyte maturation. [Copyright &y& Elsevier]

Vascularization and angiogenicity of human nonvascularized nerve grafts in the second stage of facial reanimation were studied. Immunohistochemistry for endothelial markers (CD-31) and vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 was performed on distal end biopsies from 35 cross-facial nerve grafts. In grafted nonvascularized nerve, density of vascular structures (also clearly immunopositive for VEGF and both receptors) showed a mean of 166 vessels (range 78 to 267) per unit area, corresponding to control values. In addition, VEGF was expressed in axons and perineural structures. In control samples, VEGF expression was low and occurred in the myelin sheath. In nerve grafts, expression of Flt-1 and Flk-1 (less intense) was seen in axons and perineural structures. A higher density of vessels was associated with lower VEGF expression (not significant). In short, expression of VEGF and its receptors is described in human nerve grafts and compared with basic histology and p75 nerve growth factor receptor expression of the nerve graft and functional outcome of patients. [ABSTRACT FROM AUTHOR]

Vascularization and angiogenicity of human nonvascularized nerve grafts in the second stage of facial reanimation were studied. Immunohistochemistry for endothelial markers (CD-31) and vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 was performed on distal end biopsies from 35 cross-facial nerve grafts. In grafted nonvascularized nerve, density of vascular structures (also clearly immunopositive for VEGF and both receptors) showed a mean of 166 vessels (range 78 to 267) per unit area, corresponding to control values. In addition, VEGF was expressed in axons and perineural structures. In control samples, VEGF expression was low and occurred in the myelin sheath. In nerve grafts, expression of Flt-1 and Flk-1 (less intense) was seen in axons and perineural structures. A higher density of vessels was associated with lower VEGF expression (not significant). In short, expression of VEGF and its receptors is described in human nerve grafts and compared with basic histology and p75 nerve growth factor receptor expression of the nerve graft and functional outcome of patients. [ABSTRACT FROM AUTHOR]

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A. [ABSTRACT FROM AUTHOR]

Abstract: It is known that SPARC gates VEGF-A signal transduction towards KDR, the primary angiogenic VEGF receptor. We sought to determine whether inhibition of SPARC activity using anti-SPARC peptide could inhibit laser-induced CNV by promoting binding of VEGF-A to FLT-1. We created anti-SPARC l-peptide and retro-inverso anti-SPARC d-peptide. Anti-SPARC peptides or PBS were injected intravitreally 1day before or after laser induction. Intravitreal injection of anti-SPARC l-peptide 1day before laser induction promotes FLT-1 phosphorylation and inhibited laser-induced CNV and anti-SPARC d-peptide had no effect. Injection 1day after laser injury did not affect size of laser-induced CNV. Inhibition of SPARC activity could be complementary to existing anti-CNV therapy. [Copyright &y& Elsevier]

Abstract: Optimal vision is contingent upon transparency of the cornea. Corneal neovascularization, trauma and, surgical procedures such as photorefractive keratectomy and graft rejection after penetrating keratoplasty can lead to corneal opacification. In this article we identify the underlying basis of corneal transparency and factors that compromise the integrity of the cornea. With evidence from work on animal models and clinical studies, we explore the molecular mechanisms of both corneal avascularity and its dysfunction. We also seek to review therapeutic regimens that can safely salvage and restore corneal transparency. [Copyright &y& Elsevier]

Angiogenesis is the formation of new blood vessels from pre-existing vasculature. Pathologic angiogenesis in the eye can lead to severe visual impairment. In our review, we discuss the roles of both pro-angiogenic and anti-angiogenic molecular players in corneal angiogenesis, proliferative diabetic retinopathy, exudative macular degeneration and retinopathy of prematurity, highlighting novel targets that have emerged over the past decade. [ABSTRACT FROM AUTHOR]

Abstract: Aims: This study observed the relationship of angiogenesis and differential expression of growth factors and their receptors in micro- and macrovascular endothelial cells of diabetic and normal rats. Main methods: Myocardial microvascular endothelial cells (MMVEC) and aortic endothelial cells (AEC) were isolated from type 2 diabetic-Goto–Kakizaki (GK) rats and age-matched normal Wistar rats. In vitro and in vivo Angiogenesis assay were used to observe the difference between GK rats and Wistar rats. mRNA and protein expression were analyzed by Real-time RT-PCR and Western blotting. Key findings: MMVEC but not AEC of diabetic rats had reduced abilities of angiogenesis in vitro. Real-time RT-PCR showed increased mRNA levels of VEGF, fms-like tyrosine kinase (Flt-1) and kinase insert domain containing receptor (Flk-1) in GK-MMVEC, but not the hypoxia-inducible factor-1α (Hif-1α), basic fibroblast growth factor (bFGF), fibroblast growth factor receptor 1 (FGFR1), Angiopoietin-1(Ang-1), Angiopoietin-2(Ang-2), Tie-1 and Tie-2. In contrast, Western blotting showed decreased protein levels of VEGF and receptors, including the phosphorylation of receptors. No significant differences in the expression of theses genes were observed between AEC from diabetic and control rats. Anti-rat VEGF antibodies inhibited MMVEC angiogenic function including cell proliferation, adhesion, migration, scratch wound healing and capillary-like tube formation. The in vivo angiogenesis assay had similar results. Significance: These result indicated that decreased expression of VEGF and its receptors caused by post-transcription disorder in MMVEC may be responsible for diabetic impaired cardiac angiogenesis. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.Gene Therapy (2009) 16, 10–16; doi:10.1038/gt.2008.115; published online 17 July 2008 [ABSTRACT FROM AUTHOR]

Flt-1 is secreted by various cells and elevated concentrations are present in preeclampsia affecting vascular function. Microparticles from these cells may expose Flt-1. We evaluated whether Flt-1 is microparticle-associated in preeclampsia, and established the origin of Flt-1-exposing microparticles. Methods. The concentration of Flt-1 was measured in samples from preeclamptic patients, pregnant and nonpregnant women by enzyme-linked immunosorbent assay. Microparticles were analyzed by flow cytometry. Western blot determined the different forms of Flt-1. Results. Noncell bound Flt-1 was elevated in preeclampsia compared to controls. A fraction (5%) was associated with microparticles in preeclampsia. Flt-1-exposing microparticles were increased in preeclampsia compared to normotensive pregnancy (p 1/4 0.02). Full-length Flt-1, was identified in microparticles of platelet and placental origin. Conclusion. Full-length Flt-1 is associated with platelet and placenta-derived microparticles. Possibly, the presentation of Flt-1 on the membrane of a microparticle might alter its function, particularly if it acts in synergism with other exposed vasoactive molecules. [ABSTRACT FROM AUTHOR]

Abstract: Preeclampsia occurs in 3–14% of pregnancies and is defined by maternal hypertension with proteinurea, generally associated with edema, coagulation abnormalities, and disseminated intravascular coagulation. The conditions can lead to eclampsia, characterized by hyperreflexia and convulsions. Several organs are afflicted by the condition, most importantly the liver and kidneys. The direct cause of preeclampsia is unknown, but the initial events are linked to abnormalities of placentation. This implies abnormalities in trophoblast invasion and in physiological alterations of placental vessels required for adequate perfusion of the placenta, which leads to ischemia. The mechanisms that link the ischemic placenta to endothelial lesions and to stimulation of vasoconstrictors and inhibition of vasodilators are still subject of speculation. The only treatment of preeclampsia is delivery. Lowering of blood pressure and prevention of eclampsia with magnesium sulfate is indicated in severe preeclampsia. Despite numerous studies attempting to elucidate the exact etiopathogenesis of this complex multifactorial disease, prediction or prevention methods of preeclampsia are not available. [Copyright &y& Elsevier]

Abstract: Objective: There is experimental evidence that ionizing irradiation affects a proangiogenic response. However, the relevance of this effect on tumour growth in vivo is not in detail investigated yet. The present objectives were to examine the influence of ionizing radiation on the expression of the vascular endothelial growth factor (VEGF) and its receptors (flt-1 and flk-1), the microvessel density and the tumour proliferation, in head and neck squamous cell carcinoma (HNSCC). Methods: We used a HNSCC-cell line, derived from a hypopharyngeal tumour, for subcutaneous injection in 16 athymic nude mice. After reaching an average diameter of 12–14mm the xenografts were randomised and 8 out of the 16 animals (therapy group) were irradiated with a single fraction of 6 Gy while the control group remained without any intervention. The irradiated and the respective control tumours were prepared after 7 (T7) and 70 days (T70) for immunohistochemical analysis. The expression of VEGF, its receptors flk-1 and flt-1, the vessel density (CD31) and the proliferation rate (Ki67) were quantified. Results: At the point of time T7 we observed a reduction of the tumour growth rate, of the proliferative activity and of the VEGF- as well as of the VEGF-R-expression. At the point of time T70 we found increased values for proliferation, microvessel density, VEGF- and flk-1 expression in the therapy group compared to the therapy group at T7 as well as to the control group at T70. Conclusion: These changes might suggest a long-term proangiogenic effect of irradiation, which might result in growth promotion of the remaining tumour after the end of therapy. [Copyright &y& Elsevier]

Abstract: Introduction: Experimentally, laparotomy is associated with increased tumor growth. In humans, abdominal surgery is associated with immunosuppression and elevated plasma VEGF levels that might stimulate tumor growth early after surgery. Avoidance of these surgery-related changes and their consequences may be advantageous. Granulocyte-macrophage colony stimulating factor (GMCSF) is a non-specific immune system up-regulator that has also been associated, experimentally, with increased release of soluble VEGF Receptor 1 (sVEGFR1) which is an endogenous inhibitor of VEGF. This study''s purpose was to determine the impact of perioperatively administered recombinant human GMCSF (rhu-GMCSF) on both immune function and plasma sVEGFR1 levels in colorectal cancer patients. Methods: This randomized placebo-controlled study included 36 colorectal cancer patients who underwent minimally invasive resection (17 GMCSF, 19 Placebo). Patients received 7 subcutaneous injections of either rhu-GMCSF, 125μg/m2, or saline on preoperative days 3, 2 and 1 and on postoperative days (POD) 1, 2, 3 and 4. A number of immune parameters were followed and plasma levels of soluble VEGF Receptor 1 (sVEGFR1) and VEGF were determined. Results: The total WBC, neutrophil, eosinophil, and monocyte counts were significantly higher after surgery in the GMCSF group; no differences were noted for the other immune parameters. In the GMCSF group, median plasma sVEGFR1 levels were significantly elevated on POD 1 (188.1pg/ml), and on POD 5 (142.8pg/ml) when compared to pre-GMCSF levels (0pg/ml)(p-value<0.05 for all comparisons). In the placebo group, the POD5 median sVEGFR1 level (116.3pg/ml) was elevated and of borderline significance (p =0.05) vs the pre-treatment result (0pg/ml). Of note, both groups had significantly elevated median plasma VEGF levels on POD 5 (Control 435.7pg/ml; GMCSF 385.3pg/ml) when compared to their preoperative results (Control 183.3pg/ml, p =0.0013; GMCSF 171.5pg/ml, p =0.0055). Conclusions: Perioperative GMCSF was not associated with an immune function benefit in this study, however, such treatment leads to increased plasma sVEGFR1 levels. Colorectal resection, with or without GMCSF, was also associated with increased VEGF levels postoperatively. Increased plasma levels of sVEGFR1 after surgery might limit the pro-angiogenic tumor stimulatory effects of VEGF. Further study of GMCSF''s impact on angiogenesis appears warranted. [Copyright &y& Elsevier]

Abstract: To clarify the role of Fms-like tyrosine kinase-1 (Flt-1) signaling in bone dynamics, we examined C57BL/6J mice, aged 6, 9 and 16 weeks, with disruption of the flt1 tyrosine kinase domain gene (flt1 TK−/− ) and compared with age-matched wild-type (flt1 TK+/+ ) mice. Dynamic histomorphometric analysis confirmed a significant decrease in the values of mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the trabecular bone of the proximal tibiae of flt1 TK−/− mice compared with those in flt1 TK+/+ mice. The value of trabecular bone volume (BV/TV) was also significantly reduced in flt1 TK−/− mice compared with that in flt1 TK+/+ mice. The values of osteoclast surface (Oc.S/BS) and osteoclast number (Oc.N/BS) in flt1 TK−/− mice were somewhat lower than those in flt1 TK+/+ mice. The values of bending load of the femur significantly decreased in flt1 TK−/− mice. In addition, serum osteocalcin significantly decreased in flt1 TK−/− mice compared with those in flt1 TK+/+ mice. Furthermore, there was a significant decreased mineralization of bone marrow stromal cultures from flt1 TK−/− mice. These findings demonstrate that flt1 TK−/− mice show lower trabecular bone volume than flt1 TK+/+ mice, providing powerful evidence that vascular endothelial growth factor signal pathway through the Flt-1 tyrosine kinase domain could be implicated in osteoblast development. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) receptor activation regulates endothelial cell (EC) survival, migration and proliferation. Recently, it was suggested the cross-talk between the VEGF receptors-1 (FLT-1) and -2 (KDR) modulated several of these functions, but the detailed molecular basis for such interactions remained unexplained. Here we demonstrate for the first time that VEGF stimulation of EC monolayers induced a rapid FLT-1-mediated internalization of KDR to the nucleus, via microtubules and the endocytic pathway, internalization which required the activation of PI 3-kinase/AKT. KDR deletion mutants were generated in several tyrosine residues; in these, VEGF-induced KDR internalization was impaired, demonstrating this process required activation (phosphorylation) of the receptor. Furthermore, we demonstrate that in vitro wounding of EC monolayers leads to a rapid and transient internalization of VEGF+KDR to the nucleus, which is essential for monolayer recovery. Notably, FLT-1 blockade impedes VEGF and KDR activation and internalization, blocking endothelial monolayer recovery. Our data reveal a previously unrecognized mechanism induced by VEGF on EC, which regulates EC recovery following wounding, and as such indicate novel targets for therapeutic intervention. [Copyright &y& Elsevier]

Abstract: Background: Vascular endothelial growth factor (VEGF), a major angiogenic growth factor, is involved in the pathogenesis of cancer. Plasma VEGF is raised in breast cancer and falls after successful surgery. Less is known about angiopoietins 1 and 2 (Ang-1, Ang-2). All three growth factors act on cells via receptors; Flt-1 for VEGF and Tie-2 for the angiopoietins. Cancer is also marked by abnormalities in platelet activation (marked by soluble P selectin) and inflammation (interleukin-6 [IL6]). We hypothesised altered plasma Ang-1, Ang-2, Flt-1 and Tie-2 in breast cancer that would normalize after 3 and 12 months treatment (i.e., surgery plus chemo/radiotherapy). Methods: Baseline venous blood was obtained from 40 women with breast cancer and 30 age-matched women with benign breast disease (BBD) also requiring surgery. Samples were taken again 3 months and 1 year later. Soluble P selectin, IL6, VEGF, Ang-1, Ang-2, Flt-1 and Tie-2 were measured in citrated plasma by ELISA. Results: Women with breast cancer had raised VEGF (7-fold), Ang-1 (50% higher) and Tie-2 (2-fold), but lower Flt-1 (to 26%), compared to the BBD women that broadly correlated with markers of platelet activation and inflammation. A level of Tie-2 or VEGF >95th percentile of the BBD group correctly identified 68% and 52% of the women with breast cancer. After 3 months of treatment, VEGF and Ang-1 normalized (as did IL6 and soluble P selectin) but Tie-2 was significantly lower only after 1 year. There were no significant changes in the women with BBD. Conclusions: Treatment for breast cancer (surgery followed by chemotherapy and/or radiotherapy) is effective in reducing plasma VEGF, Tie-2 and Ang-1. These may be linked pathogenically with coagulation and inflammation. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote 3H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells. [Copyright &y& Elsevier]

Abstract: Fms-like tyrosine kinase 1 (Flt-1), a receptor for vascular endothelial growth factor (VEGF), have two isoforms: membrane-bound form (mFlt-1) and soluble form. In the present study, we quantitatively evaluated expression level of mFlt-1 mRNA and VEGF mRNA in non-small cell lung cancer, and demonstrated the clinical significance of the ratio of mFlt-1 mRNA to VEGF mRNA (mFlt-1/VEGF). High mFlt-1/VEGF tumor showed a significantly lower microvessel density (P=0.004), and patients with high mFlt-1/VEGF tumor had a significantly favorable survival (P=0.037). Thus, the ratio of mFlt-1 mRNA to VEGF mRNA was inversely correlated with tumor angiogenesis, and was a significant prognostic factor. [Copyright &y& Elsevier]

Abstract: Wear debris-induced vascularized granulomatous periprosthetic tissue may augment the progress of prosthetic loosening, a major clinical problem after total joint replacement. The purpose of this study is to investigate the association of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced inflammatory osteoclastogenesis and expression of RANK/RANKL and VEGF/VEGF receptors (Flt-1 and Flk-1) using a mouse osteolysis model. UHMWPE particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Mice were injected with either recombinant VEGF or VEGF inhibitor (VEGF R2/F c Chimera). Mice without drug treatment, as well as mice injected with saline alone were included. Each group contains 10 mice. Pouch tissues were harvested 2 weeks after bone implantation for histological and molecular analysis. UHMWPE stimulation significantly increased VEGF gene expression, and exerted a lower enhancement effect on the gene expression of Flt-1 and Flk-1. UHMWPE-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Immunofluorescent staining indicated that pouch tissue macrophages were the main source of both VEGF and Flt-1 production. A positive association was observed between tissue inflammation and the levels of VEGF and Flt-1 gene transcripts. Both RANK and RANKL gene transcripts were significantly increased by UHMWPE stimulation, which was subsequently reduced by VEGF inhibitor treatment (). VEGF treatment increased TRAP+ cells in pouches either with or without UHMWPE particle stimulation, and VEGF inhibitor treatment caused a significant reduction in the number of TRAP+ cells in UHMWPE-containing pouches. This study suggests that VEGF has a role in the regulation of RANK/RANKL-mediated osteoclastogenesis, and warrant future investigations to elucidate the role of VEGF signaling in the pathogenesis of prosthetic loosening. [Copyright &y& Elsevier]

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis. The inhibition effects of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an angiogenesis inhibitor, and gemcitabine, a chemotherapeutic agent, on expression of growth factors were investigated using human pulmonary adenocarcinoma cell line, A549. The A549 cells were divided into four groups: control group, 10−6 mg/ml gemcitabine treated group, 10−4 mg/ml TNP-470 treated group and gemcitabine+TNP-470 treated group. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and its receptors, FMS-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing receptor (KDR), in different groups were measured. The growth of A549 cell cultured with gemcitabine or TNP-470 was inhibited in an almost dose-dependent manner. Although gemcitabine (10−6 mg/ml) alone and TNP-470 (10−4 mg/ml) alone had no effect on the mRNA and protein expression of VEGF and its receptors (FLT-1, KDR) in A549 cells compared to the control ( P>0.05), 10−6 mg/ml gemcitabine in combination with 10−4 mg/ml TNP-470 had significant effect ( P<0.01). Moreover, combination of the two drugs significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone ( P<0.05). This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro. [ABSTRACT FROM AUTHOR]

It has been well established that hypoxia inducible factor-1α (HIF-1α) transcribes essential factors in cell preservation, including angiogenesis, during hypoxia. However, the transition of HIF-1α expression during liver regeneration remains unknown. In this study, a role of HIF-1α was experimentally elucidated in relation to sinusoidal endothelial reconstruction during liver regeneration. Methods: Expression of HIF-1α was evaluated in the regenerating liver following 70% hepatectomy in rats. Expressions of nuclear HIF-1α, vascular endothelial growth factor (VEGF) and fms-like tyrosine kinase-1 (flt-1) were measured by Western blot and liver blood flow by a laser Doppler. Sinusoidal endothelial cell area (SECA) and HIF-1α were localized by immunohistochemistry. HIF-1α mRNA was measured by reverse transcription-polymerase chain reaction. Result: Liver blood flow and SECA were lowest 36 and 72 h following hepatectomy, respectively. Nuclear HIF-1α peaked at 24 h and continuously increased 72–120 h following hepatectomy. This transition was fully supported by histological HIF-1α expression and HIF-1α mRNA up-regulation. VEGF and flt-1 peaked at 120 and 12 h, respectively. Conclusions: A significant evaluation in HIF-1α expression was revealed in regenerating rat livers. HIF-1α expression was preceded by VEGF and flt-1 expression and thus may be related to sinusoidal endothelial reconstruction. [ABSTRACT FROM AUTHOR]

Background: Our previous study found that silymarin (SM) and its major pure component silibinin (SB) have anti-angiogenic effects via decreased vascular endothelium growth factor (VEGF) secretion of LoVo cells (colon cancer). We designed this consecutive study to evaluate the anti-angiogenic effects of SM/SB in vivo, and on VEGF receptor (VEGFR) gene expression. Materials and methods: We used LoVo cells exposed to SM/SB in a modified chicken chorioallantoic membrane assay (CAM) to evaluate anti-angiogenic effects. We used EA.hy 926 cells (endothelial cells) exposed to SM/SB to evaluate the effect on VEGFR-1 (Flt-1) and VEGFR-2 (KDR), with 1-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: In CAM, SM/SB showed a dose-dependent decrease on the vascular density index (VDI) induced by LoVo cells, as did thalidomide in a concentration of 10 μg/ml. Adding escalating dosages of VEGF successfully reversed this inhibitory effect. RT-PCR revealed that SB up-regulated Flt-1 mRNA expression of EA.hy 926 cells. SM had a similar trend, although the effect was not statistically significant (P = 0.19). Neither drug effected KDR mRNA expression. Conclusion: We conclude that anti-angiogenic effects of SM/SB are associated with the up-regulation of VEGFR-1 (Flt-1) gene expression and that they are good candidates for combination therapy to treat colorectal cancer. [Copyright &y& Elsevier]

To study the relationship between the expression of VEGF, Flk-1 and Flt-1 proteins and clinical pathology in hepatocellular carcinoma. The expression of VEGF, Flk-1 and Flt-1 proteins in hepatocellular carcinomas from 60 patients was determined by immunohistochemistry (ABC method) and VEGF expression in relation to the clinicopathology evaluated. The positive rates of VEGF, Flk-1 and Flt-1 protein expression were 81.3%, 88.3%, 80.0% in tumor tissues, respectively, rates which were significantly higher than those in normal liver tissue ( P<0.05). The expression of VEGF protein was correlated with the histologic grade and metastases of the tumors. The results showed that, in hepatocellular carcinoma, a higher expression of VEGF protein was associated with a higher degree of malignancy and a greater tendency for metastases. VEGF, Flk-1 and Flt-1 play an important role in tumourgenesis. [ABSTRACT FROM AUTHOR]

The mechanisms responsible for age-associated hearing loss are still incompletely characterized. In this study, we used a murine model of age-dependent hearing loss and evaluated whether this condition is associated with vascular modifications of the structures of the inner ear. We used old C57BL/6J mice that are affected by rapid and severe age-related hearing loss, and analyzed the expression pattern of vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, and its receptors Flt-1 and Flk-1 in the inner ear. We report for the first time morphological and quantitative data about the expression of these crucial angiogenic molecules in the murine cochlea. We also show that in this animal model, cochlear VEGF expression is significantly reduced as a function of age. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, and presbycusis, suggesting that vascular abnormalities might play a role in aging-associated hearing loss, with potentially important fundamental and clinical implications. [Copyright &y& Elsevier]

A nontubulogenic endothelial cell line, NP31, can be transformed by the active form of the Flt-1 kinase (BCR-FLTm1) into Tb3 cells, which show a tubulogenic property only when cultured in Matrigel. By utilizing this strict dependence of NP31 on BCR-FLTm1 and Matrigel for experimental angiogenesis, we performed microarray analyses under several conditions and found 97 genes whose dynamically regulated profiles of gene expression are divided into nine groups, in two major clusters. In one major cluster, gene expression is interdependently regulated by BCR-FLTm1 or Matrigel. The second major cluster contains genes whose expression patterns under BCR-FLTm1 influence are reversed by Matrigel. Based on these gene expression patterns in NP31 driven by BCR-FLTm1 and/or Matrigel, we propose a model in which sequential and alternate stimulation by BCR-FLTm1 and Matrigel induces cooperative regulation of subsets of genes. Microarray analyses of Tb3 under 11 different conditions revealed 5 candidate genes whose gene expression regulation is most closely associated with tubulogenesis. [Copyright &y& Elsevier]

: BackgroundVEGF is best known for its angiogenic properties. We have found that VEGF expression is increased in neuroblastoma cells cocultured with hepatocytes. In addition, we have previously shown that neuroblastoma cells cultured with exogenous VEGF have an increase in the expression of VEGF receptors. Therefore, we hypothesized that the expression of VEGF receptors would be up-regulated in neuroblastoma cells grown in the coculture environment.: Materials and methodsTwo neuroblastoma cell lines (IMR-32 or SK-N-DZ) are used. These cells are cultured alone and in a coculture system with hepatocytes. Message for VEGF and the VEGF receptors KDR, flt-1, flt-4, neuropilin 1 (NRP-1), and neuropilin 2 (NRP-2) are measured with RT-PCR. Flt-4, NRP-1, and NRP-2 protein expression is measured with Western blot.: ResultsThe receptors KDR and flt-1 are not detected in either cell line in either control or coculture conditions. Message for VEGF and flt-4 is significantly increased in the cocultured IMR-32 cells, while that for NRP-1 and NRP-2 is unchanged in these cells. VEGF and its receptors are unchanged in cocultured SK-N-DZ cells.: ConclusionsNeuroblastoma cells express specific VEGF receptors that are differentially regulated in the different cell lines. These findings suggest that the heterogeneity of neuroblastomas may limit the utility of targeting VEGF and its receptors as sole treatments for the tumor, and that successful therapies will be dependent upon the specific biology of the tumor. [Copyright &y& Elsevier]

: ObjectiveUsing hyperstimulated rats, to elucidate the mechanisms of gonadotropin-releasing hormone agonist (GnRH-a) treatment to prevent early ovarian hyperstimulation syndrome (OHSS).: DesignDescriptive study of hyperstimulated rats as an early OHSS model with Western blot analysis, Northern blot hybridization, and vascular permeability assay.: SettingExperimental laboratory research.: Animal(s)Sprague-Dawley female rats were used for collecting ovarian samples.: Intervention(s)Hyperstimulated rats received consecutive GnRH-a treatment from the start of pregnant mare serum gonadotropin (PMSG) treatment through 2 days after hCG administration.: Main outcome measure(s)Expressions of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1: Flt-1), VEGF receptor-2 (VEGFR-2: KDR/Flk-1), and vascular permeability by Evans blue leakage.: Result(s)GnRH-a treatment significantly reduced expressions of VEGF, VEGFR-1, and VEGFR-2 both in mRNA and protein levels in the ovaries of hyperstimulated rats. GnRH-a treatment also reduced vascular permeability in the ovaries of hyperstimulated rats.: Conclusion(s)It is speculated that GnRH-a treatment may prevent early OHSS by reducing vascular permeability through the decrease in VEGF and its receptors. [Copyright &y& Elsevier]

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Angiogenesis, the formation of neovessels from the endothelium of preexisting vessels, is stimulated by soluble angiogenic factors. Insulin-like growth factor I (IGF-I) stimulates myogenesis and induces nerve regeneration after injury, and it has been shown to stimulate angiogenesis. However, the in vivo angiogenic effects of IGF-I in regenerating and diabetic muscle have yet to be described. Therefore, we studied the effects of human IGF-I (hIGF-I) delivered by a plasmid-mediated therapy on angiogenesis in mouse models of these two conditions. Plasmid hIGF-I was delivered to the injured tibialis muscle by direct intramuscular injection followed by electroporation. Initial experiments compared two muscle-specific hIGF-I-expressing constructs containing either a skeletal actin 3′UTR (pAV2001) or a human growth hormone (GH) 3′UTR (pAV2002). Skeletal actin 3′UTR mediates sequestration of hIGF-I in the muscle and was more active, while the GH 3′UTR mediated release of IGF-I into the circulation. Treatment of regenerating muscle with pAV2001 and sequestration of IGF-I in muscle led to increased expression of vascular endothelial growth factor (VEGF) and VEGF receptors fetal liver kinase-1 and FmS-like tyrosine kinase receptor-1, as well as platelet endothelial cell adhesion molecule-1, on endothelial cells. These results indicate that IGF-I can amplify angiogenic responses in regenerating muscle. In a mouse diabetic model, plasmid-mediated IGF-I therapy reversed diabetic microangiopathy, as shown by increased angiogenesis and arterial flow as analyzed by Doppler imaging. These studies show that plasmid IGF-I delivery and sequestration in muscle can augment angiogenesis in regenerating muscle and increase blood flow and angiogenesis in the diabetic limb. [Copyright &y& Elsevier]

Angiogenesis is gaining interest because of its importance in tumour growth and metastasis. Renal cell carcinoma (RCC) is known to be a well-vascularized tumour. The aim of this study was to evaluate the expression of VEGF mRNA and receptor flt-1 mRNA (VEGF R1) in a clinical material of RCCs compared with clinicopathological variables and serum VEGF levels. Total RNA was extracted from snap-frozen tumour tissue obtained from 61 patients. Expression of mRNA for VEGF121, VEGF165 and flt-1 were analysed using quantitative RT-PCR. Relative VEGF mRNA levels, corrected for corresponding cyclophilin value, were related to stage, grade, RCC type and survival time. Serum VEGF165 protein was analysed using a quantitative ELISA. Papillary RCC had significantly lower VEGF121 and flt-1 mRNA levels compared with conventional RCC (p=0.001). VEGF121 mRNA levels were significantly lower in locally advanced tumours in relation to tumours limited to the kidney and those with metastatic disease (p=0.047 and p=0.036). This statistical difference disappeared when only conventional RCCs were evaluated. No association was found between VEGF mRNA levels and nuclear grade. Patients with lower VEGF121 mRNA levels had significantly longer survival time compared with those with higher levels (when adjusted to stage, p=0.0097, log rank test). There was an inverse relation between VEGF165 mRNA and serum VEGF165 levels. The trend to lower VEGF121 mRNA levels in locally advanced RCC indicate that angiogenic activity and degradation might be up-regulated in tumours with a high ability to invade. The association with tumour progression shows that VEGF is a promising angiogenic factor especially important in conventional RCCs. VEGF expression might possibly be of help to identify RCCs susceptible for anti-angiogenic therapies. [ABSTRACT FROM AUTHOR]

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/ Kinase insert domain containing receptor (Flk-1/ KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors. [Copyright &y& Elsevier]

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5–E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF165, VEGF121, Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5–E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A. [Copyright &y& Elsevier]

The capillary network in the masseter muscle develops dramatically with the differentiation of muscle fibres after birth, especially around weaning. Here, developmental changes in mRNA expression for four splicing variants of vascular endothelial growth factor (VEGF) and for two distinct VEGF receptors (Fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1)) were studied in rat masseter. The relative abundance of VEGF120 mRNA was the highest, representing 35% of total VEGF mRNA on day 7 after birth and gradually decreased with age to become approximately 5% on day 37. In contrast, VEGF188 mRNA was very low in the newborn rat, but increased sharply before weaning and reached 40–50% of the total on day 50. Neither VEGF144 nor VEGF164 mRNA showed any significant change in abundance after birth. The expression of KDR/Flk-1 mRNA was transiently high in the early postnatal stage and gradually decreased with age, Flt-1 mRNA was stably expressed at a constant level after birth. These findings suggest that different combinations of VEGF isoforms and their receptors regulate angiogenesis in the development of the masseter muscle. [Copyright &y& Elsevier]

Phenotypically and functionally different types of microvascular endothelial cells (MVECs) derived from the developing corpus luteum were isolated and characterized by our group. We investigated whether these cytokeratin-positive (CK+) and cytokeratin-negative (CK−) MVECs differed in the expression of angiogenic factors and their regulation under hypoxia. Using quantitative RT-PCR, VEGF and its receptors, Flk-1 and Flt-1, were detected in CK− MVECs. The mRNA expression of Flk-1 mRNA was 100 times as high as that of Flt-1 mRNA. CK+ MVECs expressed VEGF and Flt-1 mRNA, but were devoid of Flk-1 transcripts. No Ang-1 mRNA was demonstrated in either cell type, and Ang-2 mRNA was found only in CK− MVECs. Tie-2 mRNA was detected in both MVEC types, but levels were 150 times as high in CK− MVECs as in CK+ MVECs. mRNA of hypoxia-inducible factors Hif-1α and Hif-1β was expressed in both MVEC types. After hypoxia, neither VEGF, nor Flk-1, nor Tie-2 mRNA expression was altered in either MVEC type. Flt-1 expression and Ang-2 mRNA expression were significantly increased at about 2.5-fold (P < 0.05) in CK− MVECs, but not in CK+ MVECs. Our study demonstrates the varying expression and regulation of angiogenesis-related factors and receptors in phenotypically different MVEC types. [Copyright &y& Elsevier]

The flt-1 gene encodes for both the full-length receptor Flt-1 (VEGFR-1) and a soluble form designated sFlt-1. sFlt-1 carries the VEGF-binding domain of Flt-1 as well as a 31-amino-acid stretch derived from an intron and tightly binds VEGF, suppressing its angiogenic activity. The flt-1 gene has so far been identified only in mammals and is highly expressed in placenta as well as in vascular endothelial cells. In placenta, sFlt-1 is abundant in the trophoblast layer during pregnancy, suggesting that it is a negative regulator to excess angiogenesis and vascular permeability at the feto–maternal border in mammals. However, we show here for the first time that the flt-1 gene exists and is highly conserved in chickens. Surprisingly, the chicken flt-1 gene also encodes for sFlt-1 in addition to the full-length receptor. Similar to the mammalian sFlt-1, chicken sFlt-1 carries the VEGF-binding domain and a 31-amino-acid carboxyl region derived from an intron, which was significantly homologous to that in mammals. Chicken sFlt-1 is expressed early in embryogenesis. These findings strongly suggest that the natural antiangiogenic molecule sFlt-1 is widely conserved in vertebrates and regulates the angiogenic process. [Copyright &y& Elsevier]

In denervated skeletal muscles, atrophy of muscle fibers and interstitial fibrosis are associated with alterations within the vascular bed. Our study has placed particular emphasis on changes occurring in resistance vessels and the microcirculatory bed of rat hindlimb skeletal muscles that had been denervated for 25 months. We found that the tunica media of the majority of long-term denervated resistance vessels undergoes deterioration. In small intramuscular arteries and arterioles, atrophic vascular smooth muscle cells (vSMCs) enclosed in a thick basal lamina are separated by expanded extracellular space. The remodeling and sclerotic changes in the arterial wall occasionally result in deformation of the lumen. It was also found that the microcirculatory bed undergoes significant alterations. In 25-month denervated extensor digitorum longus muscle, the capillary-to-fiber ratio is only 0.13 ± 0.01 and the mean number of capillaries per fascicle decreases almost ninefold compared to contralateral control muscle. Ultrastructural findings demonstrate that 24.67 ± 0.48% of capillaries examined in the chronically denervated fascicles show structural features typical for capillary regeneration. In addition, long cytoplasmic extensions of pericytes might develop a layer completely encircling the capillary endothelium. In pre- and postcapillary segments of the microcirculatory bed, some perivascular cells possess a phenotype that is intermediate between that of pericytes and atrophic vSMCs. RT-PCR and/or Western blot analyses showed that molecules participating in angiogenesis are detected in 25-month denervated skeletal muscle. We hypothesize that despite the fact that the microcirculatory bed of chronically denervated muscle undergoes significant reduction it still sustains the capacity for reparative capillary growth. [Copyright &y& Elsevier]