Interethnic Differences in the Distribution of Clinically Relevant Vascular Endothelial Growth Factor Genetic Polymorphisms

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein produced mostly in endothelial cells and its transcription is regulated by a variety of growth factors and cytokines. VEGF plays many relevant roles, and three functional polymorphisms in the promoter region of the VEGF gene (C-2578A, G-1154A, and G-634C) have been associated with disease conditions. Although some studies suggest that interethnic differences exist in the distribution of these variants, no previous study has examined this hypothesis in admixed populations. We examined the distribution of these three clinically relevant VEGF single-nucleotide polymorphisms in 175 white and 185 black subjects. We have also estimated the haplotype distribution and assessed associations between these variants. Although the A-2578 and A-1154 variants were more common in whites (39% and 29%, respectively) than in blacks (29% and 16%, respectively; both p < 0.05), no significant interethnic differences were found with regards to the G-634C polymorphism. While the haplotype including the C-2578, G-1154, and G-634 variants was the most common in both ethnic groups, it was more common in blacks than in whites (p < 0.05). The haplotype including the C-2578, A-1154, and G-634 alleles and the haplotype including the C-2578, A-1154, and C-634 alleles were more common in whites than in blacks (both p < 0.05). These results show marked interethnic differences in the distribution of genetic variants of VEGF that may explain, at least in part, interethnic disparities in the susceptibility to cardiovascular diseases.

Vascular Endothelial Growth Factor Genetic Polymorphisms and Haplotypes in Women with Migraine

Vascular endothelial growth factor (VEGF) production is regulated by growth factors and inflammatory cytokines, and VEGF plays a role in migraine. We examined for the first time whether three functional polymorphisms in the promoter region of VEGF gene (C(-2578)A, G(-1154A), and G(-634C)) and VEGF haplotypes are associated with migraine. We studied 114 healthy women without migraine and 175 women with migraine (129 without aura, and 46 with aura). We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAC haplotype was more frequent in controls than in migraine patients, and the AGC haplotype was more frequent in patients with migraine with aura than in controls (both p < 0.05). These findings suggest that VEGF haplotypes affect susceptibility to migraine.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction

In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in end-diastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Gene Therapy (2010) 17, 305-314; doi:10.1038/gt.2009.146; published online 10 December 2009

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage 1), developing (stage 11), developed (stage 111), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2 alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2a, and returned to pretreatment levels for the period 24-64 hr post-PGF2 alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of `B` and `C` splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the `B` and `C` spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Treatment With a Growth Factor-Protein Mixture Inhibits Formation of Mineralized Nodules in Osteogenic Cell Cultures Grown on Titanium

Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution (similar to 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti. (J Histochem Cytochem 57:265-276, 2009)

Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension

Pulmonary vascular remodeling is an important pathological feature of pulmonary hypertension, leading to increased pulmonary vascular resistance and reduced compliance. It involves thickening of all three layers of the blood vessel wall (due to hypertrophy and/or hyperplasia of the predominant cell type within each layer), as well as extracellular matrix deposition. Neomuscularisation of non-muscular arteries and formation of plexiform and neointimal lesions also occur. Stimuli responsible for remodeling involve transmural pressure, stretch, shear stress, hypoxia, various mediators [angiotensin II, endothelin (ET)-1, 5-hydroxytryptamine, growth factors, and inflammatory cytokines], increased serine elastase activity, and tenascin-C. In addition, there are reductions in the endothelium-derived antimitogenic substances, nitric oxide, and prostacyclin. Intracellular signalling mechanisms involved in pulmonary vascular remodeling include elevations in intracellular Ca2+ and activation of the phosphatidylinositol pathway, protein kinase C, and mitogen-activated protein kinase. In animal models of pulmonary hypertension, various drugs have been shown to attenuate pulmonary vascular remodeling. These include angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, ET receptor antagonists, ET-converting enzyme inhibitors, nitric oxide, phosphodiesterase 5 inhibitors, prostacyclin, Ca2+-channel antagonists, heparin, and serine elastase inhibitors. Inhibition of remodeling is generally accompanied by reductions in pulmonary artery pressure. The efficacy of some of the drugs varies, depending on the animal model of the disease. In view of the complexity of the remodeling process and the diverse aetiology of pulmonary hypertension in humans, it is to be anticipated that successful anti-remodeling therapy in the clinic will require a range of different drug options. (C) 2001 Elsevier Science Inc. All rights reserved.

Basic fibroblast growth factor and fibroblast growth factor receptors in adult olfactory epithelium

Basic fibroblast growth factor (FGF2) stimulates proliferation of the globose basal cells, the neuron:ll precursor in the olfactory epithelium. The present study investigates the expression of basic fibroblast growth factor and fibroblast growth factor receptors in the adult olfactory epithelium. FGF2 immunoreactivity was expressed widely in the olfactory epithelium, with the highest density of immunoreactivity in the supporting cells. In contrast, most cells in the epithelium expressed FGF2 mRNA. Fibroblast growth factor receptor-1 (FGFr1) immunoreactivity was densest in the basal cell and neuronal layers of the olfactory epithelium and on the apical surface of supporting cells. In the lamina propria FGF2 immunoreactivity and mRNA were densest in cells close to the olfactory nerve bundles. FGFr1 immunoreactivity was heaviest on the olfactory ensheathing cells. Using reverse transcriptase-polymerase chain reaction analysis, the olfactory epithelium was shown to express only three receptor splice variants, including one (FGFr1c) with which basic fibroblast growth factor has high affinity. Other receptor splice variants were present in the lamina propria. Taken together, these observations indicate endogenous sources of FGF? within the olfactory epithelium and lamina propria and suggest autocrine and paracrine pathways via which FGF2 might regulate olfactory neurogenesis. The observation of only three receptor splice variants in the olfactory epithelium limits the members of the fibroblast growth factor family which could act in the olfactory epithelium. The widespread distribution of receptors suggests that fibroblast growth factors may have roles other than proliferation of globose basal cells. (C) 2001 Published by Elsevier Science B.V.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 mum) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P

A novel bioassay for human somatogenic activity in serum samples supports the clinical reliability of immunoassays

OBJECTIVE Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states. DESIGN The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint. PATIENTS These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers. MEASUREMENTS In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay. RESULTS This bioassay was sensitive (5 mU/l or 2 mug/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state. CONCLUSIONS Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2(-/-) mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone-mediated downregulation of Ngn1.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.