To express human vascular endothelial growth factor121 (VEGF121) in insect cells.

A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.

Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.

Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

Hydrogels have been extensively studied as a carrier of various hydrophilic molecular compounds and cells for local delivery and subsequent controlled release. One of key design parameters in the hydrogel assembly is an ability to control spatiotemporal gel degradation, in order to tailor release rates of multiple drugs and also regulate phenotypic activities of co-cultured cells. To achieve this goal, this study presents a simple but innovative implantable, microfabricated hydrogel patch that undergoes micropatterned surface erosion at controlled rates and subsequently discharges two molecular compounds of interests at desired rates. This device was prepared by first fabricating a non-degradable poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel patch containing micro-pockets of controlled spacing and subsequently filling micro-pockets with a hydrogel of poly(ethylene imine) (PEI) and PEG diacrylate (PEGDA) that was tailored to degrade at controlled rates. Separate incorporation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)<em>121</em> and VEGF165, known to orchestrate <em>vascular</em> development, into the PEI-PEGDA gel and PEGDMA hydrogel resulted in enhanced neo<em>vascular</em>ization at the implantation sites due to bimodal, sequential release of two VEGF isoforms. We believe that the hydrogel patch fabricated in this study will be highly useful to better understand a broad array of complex biological processes and also improve the efficacy of molecular cargos in varied applications.

BACKGROUND

A living, self-supporting pulp tissue replacement in vitro and for transplantation is an attractive yet unmet bioengineering challenge. Our aim is to create 3-dimensional alginate-based microenvironments that replicate the shape of gutta-percha and comprise key elements for the proliferation of progenitor cells and the release of growth factors.

METHODS

An RGD-bearing alginate framework was used to encapsulate dental pulp stem cells and human umbilical vein <em>endothelial</em> cells in a ratio of 1:1. The alginate hydrogel also retained and delivered 2 key <em>growth</em> <em>factors</em>, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> and fibroblast <em>growth</em> <em>factor</em>, in a sufficient amount to induce proliferation. A method was then devised to replicate the shape of gutta-percha using RGD alginate within a custom-made mold of thermoresponsive N-isopropylacrylamide. Plugs of alginate containing different permutations of <em>growth</em> <em>factor</em>-based encapsulates were tested and evaluated for viability, proliferation, and release kinetics between 1 and 14 days.

RESULTS

According to scanning electron microscopic and confocal microscopic observations, the encapsulated human endothelial cells and dental pulp stem cell distribution were frequent and extensive throughout the length of the construct. There were also high levels of viability in all test environments. Furthermore, cell proliferation was higher in the growth factor-based groups. Growth factor release kinetics also showed significant differences between them. Interestingly, the combination of vascular endothelial growth factor and fibroblast growth factor synergize to significantly up-regulate cell proliferation.

CONCLUSIONS

RGD-alginate scaffolds can be fabricated into shapes to fill the pulp space by simple templating. The addition of dual growth factors to cocultures of stem cells within RGD-alginate scaffolds led to the creation of microenvironments that significantly enhance the proliferation of dental pulp stem cell/human umbilical vein endothelial cell combinations.

BACKGROUND

Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials.

METHODS

We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin-inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues.

RESULTS

Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P(SV40); murine or human cytomegalovirus immediate-early promoter, P(mCMV), P(hCMV)) or regulated (streptogramin-inducible) promoters (P(PIR)ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap-dependent manner and the two subsequent ones by internal ribosome entry site (IRES)-mediated translation initiation. (ii) The triple-transcript expression configuration, in which a combination of well-established (P(SV40), P(hCMV), P(mCMV)) and novel synthetic constitutive promoters (P(GTX)) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high-level expression of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin-inducible tricistronic SAMY-VEGF(<em>121</em>)-SEAP expression provided excellent regulation performance-high-level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near-undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility-in all cell types, in particular in NRCs and NRC-derived myocardial microtissues.

CONCLUSIONS

Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor <em>vascular</em>ization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory <em>factor</em> would be equally effective in arresting tumor <em>growth</em>. To this goal, we investigated the effectiveness of ABRaA-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em> (VEGF<em>121</em>), an anti<em>vascular</em> drug constructed by us. It is a fusion protein composed of VEGF<em>121</em>, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor <em>growth</em>. ABRaA-VEGF<em>121</em> is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF<em>121</em> anti<em>vascular</em> agent with immunostimulatory cytokine IL-12 indeed inhibits tumor <em>growth</em> more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4(+) and CD8(+) as compared to control mice. It seems that bringing together anti<em>vascular</em> strategy and the action of immunostimulating agents indeed inhibits <em>growth</em> of tumors.

OBJECTIVE

To evaluate the effect of 2 <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoforms (<em>121</em> and 165) and 2 anti-VEGF compounds (ranibizumab and pegaptanib sodium) on the permeability of human retinal pigment epithelium (RPE) cells in vitro.

METHODS

The RPE permeability was assessed on ARPE19 cells grown onto inserts of polytetrafluoroethylene previously treated with ammonia gas plasma. Paracellular permeability to ions was measured by mean of transepithelial electrical resistance (TEER). Permeability to non-ionic molecules was gathered by the amount of fluorescein dextran (FD) passing across the monolayer within 2 hours.

RESULTS

Only VEGF165 applied at the apical side of the monolayer induced a statistically significant decrease of TEER (p<0.001). No changes in TEER were observed when pegaptanib sodium or ranibizumab were apically administered together with VEGF165. Both VEGF isoforms significantly increased permeability to 4 kDa dextran (p<0.01). Apical administration of ranibizumab or pegaptanib sodium as well as coadministration of pegaptanib sodium with VEGF<em>121</em> or VEGF165 induced a statistically significant increase of permeability to 4 kDa FD.

CONCLUSIONS

Both VEGF isoforms and anti-VEGF compounds exert an effect on human RPE permeability in vitro.

OBJECTIVE

To investigate the distribution of vascular endothelial growth factor (VEGF) isoforms and soluble form of VEGF receptor 1 (sFlt-1) in the follicular fluid (FF) of in vitro fertilization (IVF) patients in relationship to age, body mass index (BMI), diagnosis of polycystic ovary syndrome (PCOS), and their correlation with IVF outcomes.

METHODS

Prospective study.

METHODS

VEGF( 121) and VEGF(165) isoforms were detected using Western blotting and pixel density analysis. The concentration of sFlt-1 was determined by enzyme-linked immunosorbent assay (ELISA). In vitro fertilization outcomes measured included number of oocytes retrieved, fertilization rate, and clinical pregnancy. Statistical analysis used the Kruskal-Wallis and Mann-Whitney U test where appropriate.

RESULTS

There was a statistically significant association between higher VEGF(165) levels and the diagnosis of PCOS, BMI ≥ 30, and age ≥40 years. In vitro fertilization cycles resulting in pregnancy were linked to statistically lower VEGF(165) levels in the FF. No statistically significant trend was identified in levels of VEGF(121) or sFlt-1 relative to patient characteristics or IVF outcomes.

CONCLUSIONS

Our results suggest that elevated VEGF(165) levels are associated with less favorable patient characteristics and clinical IVF outcomes.

Currently, at least five different mRNA species encoding <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) have been characterized. These variants result from alternative splicing of the VEGF-A transcript and encode human isoforms of VEGF protein of <em>121</em>, 145, 165, 189, and 206 amino acids. In the rat, a similar profile of VEGF-A splice variants has been described, each encoding one fewer amino acid than the human species. Studies of mammalian tissues have shown that these mRNA isoforms vary in abundance. Whereas VEGF 188/189, 164/165, and 120/<em>121</em> (rat/human, respectively) are the predominant forms expressed in most tissues and cells examined, VEGF 144/145 and 205/206 are rare variants. Previously, VEGF 144/145 had been detected only in placental and uterine tissues and endometrial carcinoma cell lines, whereas VEGF 205/206 was detected only in fetal liver and placenta. Using an RT-PCR technique, cDNA cloning, and sequencing, we have detected and confirmed the presence of mRNA encoding VEGF 144/145 and 205/206 in both adult rat lung and penis. Therefore, these low-abundance VEGF splice variants have a more diverse expression pattern than originally predicted.

BACKGROUND

Choroidal neovascularisation is often associated with pathological myopia. Bisphosphonates (BP), the preferred drug for treatment of osteoporosis, are known to have anti-angiogenic effects.

OBJECTIVE

To compare the therapeutic effects of oral BP with anti-vascular endothelial growth factor therapy (anti-VEGF) and photodynamic therapy (PDT) for myopic choroidal neovascularisation (mCNV) over 2 years of follow-up.

METHODS

One hundred eyes of 96 consecutive patients with mCNV who underwent oral BP treatment (alendronate 5 mg/day or 35 mg/week), anti-VEGF therapy, PDT or observation only were followed up for 2 years. The best-corrected visual acuity (BCVA) and the central retinal thickness (CRT) in optical coherence tomography were compared among the treatment groups.

RESULTS

The mean BCVA of the patients was maintained for up to 2 years in the BP and PDT groups. In the anti-VEGF group, the mean BCVA was significantly improved but was significantly worse in the no-treatment group. The visual outcomes were significantly better in the BP, PDT and anti-VEGF groups than the no-treatment group over 2 years of follow-up (-0.28, -0.26 and -0.39 logMAR units, p=0.032, 0.021 and 0.0004, respectively). The mean CRT was significantly decreased in all treatment groups (-84, -121 and -122 mm, p=0.0025, 0.017 and 0.000025, respectively).

CONCLUSIONS

Oral BP should be investigated further as possible therapeutic and preventive drugs for mCNV.

OBJECTIVE

Loss of regulation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) production and action disturbs <em>vascular</em> homeostasis leading to pathology. Primary varicose veins (VVs) demonstrate aberrant production/release of VEGF. Our aim was to examine transcription of genes for VEGF (VEGF(<em>121</em>)/VEGF(165)) and its receptors (KDR, flt-1, s.flt-1) in VVs, in relation to underlying venous incompetence.

METHODS

Samples of varicose (n=83, 18 patients) or normal (n=14, five subjects) great saphenous vein were divided into segments, determined by anatomical position from the sapheno-femoral junction (SFJ). SFJ and segmental incompetence were determined from duplex scans. Gene transcripts were amplified by RT-PCR, analysed by scanning densitometry, and the levels of transcription determined by ratio to control gene GADPH-3 (GAP-3).

RESULTS

VEGF(<em>121</em>)/(165), KDR and flt-1 transcription was elevated in VVs overall (p<0.001), and in VVs with an incompetent SFJ (p<0.001), but not when the SFJ was functional; s.flt-1 was unaltered. Notably, gene transcription was unaffected by segmental position, or incompetence. Position below the SFJ correlated with increased transcription of s.flt-1 when the SFJ was incompetent (p<0.04), and s.flt-1 and VEGF(<em>121</em>) when the segment was incompetent (p<0.03).

CONCLUSIONS

SFJ incompetence is associated with altered transcription of VEGF and its receptors reflecting an aetiological mechanism or later stage of disease development. Altered VEGF(<em>121</em>) and s.flt-1 transcription may be an early event in varicogenesis.

BACKGROUND

Cardiovascular disease (CVD) is the leading cause of death in developed countries. Previous studies have shown that hormone replacement therapy reduces the risk of CVD in postmenopausal women, however, the mechanism remains unclear. This study was designed to evaluate the effect of estrogen on serum vascular endothelial growth factor (VEGF) concentration in normotensive and hypertensive ovariectomized rats.

METHODS

Forty-eight female rats were ovariectomized and randomly divided into 6 groups. Hypertension was induced by DOCA-Salt method. DOCA was injected 30 mg/kg of body weight subcutaneously, twice a week with NaCl 1% instead of tap water for drinking throughout the experiment. Estradiol valerate (Es) was injected 2 mg/week i.m. The groups were as follows: (i) DOCA (4 weeks) and DOCA+Es (6 weeks); (ii) DOCA (10 weeks); (iii) Normal saline (N/S) (4 weeks) and Es (6 weeks); (iv) N/S 10 weeks; (v) DOCA (4 weeks), and (vi) N/S (4 weeks). Serum VEGF concentration was measured in groups 1 to 4.

RESULTS

Results showed that in normotensive animals that received estrogen treatment, serum VEGF concentration was significantly higher than those not receiving estrogen (269+/-41 vs. 106+/-36 pg/ml) (P<0.05). In hypertensive group, serum VEGF level was also increased after estrogen therapy compare to those not receiving estrogen (326+/-55 vs. 121+/-28 pg/ml (P<0.05).

CONCLUSIONS

It is possible that the increase in serum VEGF concentration after estrogen therapy may contribute to the cardiovascular effects of estrogen in normotensive and hypertensive conditions.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF-A) is a key angiogenic <em>growth</em> <em>factor</em> which regulates vertebrate embryonic vascularization, adult physiology such as wound healing and reproduction as well as many human diseases. To understand the evolution and regulation of this gene in vertebrates, we have isolated and characterized the zebrafish vegf-A gene and compared it with VEGF-A genes of human, mouse as well as an in silico isolated VEGF-A homologue from pufferfish. Our results indicate that the zebrafish vegf-A gene is organized similarly to mammalian and Fugu VEGF-A genes, with eight exons interrupted by seven introns. However, zebrafish vegf-A introns are generally larger than mammalian introns while Fugu VEGF-A introns are much smaller. Furthermore, zebrafish exon 6 (z6) has a unique sequence while Fugu's exon 6 is highly homologous to the mammalian counterparts. Alternative splicing generates multiple vegf-A mRNA isoforms in zebrafish with Vegf(<em>121</em>) as the dominant isoform in adult and Vegf(165) as the dominant isoform in early embryos. The exon z6 containing isoform Vegf(12345z678) is only detected in heart, muscle, and early embryos while another isoform Vegf-A(1234577)(a)(8) is only detected in heart. Furthermore, no conserved 5' flanking sequences between zebrafish and Fugu were observed while numerous conserved regions exist between human and mouse in this area. These results suggest both conserved and diverged functions of VEGF-A from fish to mammals since the separation of these two groups from their common ancestor about 450 million years ago and a diverged regulation of this gene since the separation of zebrafish from Fugu. These data will be valuable for future studies of VEGF-A gene regulation and function in different vertebrates.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent angiogenic <em>factor</em> that has a strong association with <em>growth</em> and metastasis of various cancers. We analyzed the expression of VEGF mRNA levels in human breast-tumor derived GI-101A cells and in human promyelocytic leukemia derived HL-60 cells using RT-PCR technique. During our RT-PCR analysis we detected the expression of three splice variants of VEGF mRNA at 400, 520 and, 650 bp lengths, which were amplified by a single set of VEGF specific forward and reverse primers. The three RT-PCR products detected by us in these cells correspond to the mRNA splice variants coding for the three isoforms of VEGF respectively, VEGF(<em>121</em>), VEGF(165), and VEGF(189). Treatment of GI-101A and HL-60 cells with phorbol 12, 13-dibutyrate (PDB) or diethylstilbestrol (DES) resulted in a significant increase of VEGF mRNA levels in a dose dependent manner. Both treatments increased the levels of all three splice variants of VEGF mRNA and a maximum increase was detected with 10 microM concentrations of PDB or DES treatments after 2 h. Interestingly, both PDB and DES mediated stimulation of VEGF mRNA expression was completely blocked by the PKC inhibitor chelerythrine. Quantitation of VEGF levels by ELISA technique confirmed that changes seen in mRNA levels following different treatments altered the release of VEGF. Our results suggest that PDB and DES mediated effects on VEGF expression in GI-101A and HL-60 cells occur at the gene transcription level.

Differences in placental mass and <em>vascular</em>ity exist between cows gestating single vs. multiple fetuses. Therefore, the association between fetal number and placental development or function was assessed by comparing concentrations of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), pregnancy-associated glycoproteins (PAG), IGF-I, and progesterone in the maternal blood of cattle selected for twin births and gestating 1 (n = 23) vs. 2 (n = 17) fetuses. Samples of jugular venous blood were collected serially at a mean of 57, <em>121</em>, 192, and 234 d (range within groups was 20 d) after AI. Plasma concentrations of VEGF, IGF-I, and progesterone were measured by double-antibody RIA, and of PAG by an indirect sandwich ELISA. Concentrations of VEGF and progesterone were greater (P < 0.05) in dams with twin vs. single fetuses. Maternal VEGF concentrations did not differ among collection times, but progesterone concentrations increased (P < 0.01) between d 192 and 234. Conversely, PAG concentrations were low at d 57 and <em>121</em> and did not differ between dams carrying singles or twins. However, the subsequent increase (P < 0.01) in PAG was greater in dams with twins, resulting in greater (P < 0.01) PAG concentrations for dams with twins at d 192 and 234 (type of birth x time; P < 0.01). Maternal IGF-I concentrations were unaffected by fetal number. Because corpora lutea persisted for the duration of the evaluation period, maternal progesterone concentrations were likely related to the number of corpora lutea rather than the number of fetuses. It is postulated that the greater PAG and VEGF concentrations in the blood of dams gestating twins are the result of a larger uteroplacental mass, including increased numbers of binucleate cells and increased angiogenesis and vasculogenesis associated with a twin pregnancy. Although PAG and VEGF were elevated in dams gestating twins, variability within and among birth groups limits the use of PAG or VEGF measurements for the diagnosis of twins.

Following the discovery of RNA interference (RNAi) and related phenomena, novel regulatory processes, attributable to small non-protein-coding RNAs, continue to emerge. Capitalizing on the ability of artificial short interfering RNAs (siRNAs) to trigger degradation of specific target transcripts, and thereby silence desired gene expression, we designed and characterized a generic transcription-translation network in which it is possible to fine-tune heterologous protein production by coordinated transcription and translation interventions using macrolide and tetracycline antibiotics. Integration of siRNA-specific target sequences (TAGs) into the 5' or 3' untranslated regions (5'UTR, 3'UTR) of a desired constitutive transcription unit rendered transgene-encoded protein (erythropoietin, EPO; human placental alkaline phosphatase, SEAP; human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em>, VEGF(<em>121</em>)) production in mammalian cells responsive to siRNA levels that can be fine-tuned by macrolide-adjustable RNA polymerase II- or III-dependent promoters. Coupling of such macrolide-responsive siRNA-triggered translation control with tetracycline-responsive transcription of tagged transgene mRNAs created an antibiotic-adjustable two-input transcription-translation network characterized by elimination of detectable leaky expression with no reduction in maximum protein production levels. This transcription-translation network revealed transgene mRNA depletion to be dependent on siRNA and mRNA levels and that translation control was able to eliminate basal expression inherent to current transcription control modalities. Coupled transcription-translation circuitries have the potential to lead the way towards composite artificial regulatory networks, to enable complex therapeutic interventions in future biopharmaceutical manufacturing, gene therapy and tissue engineering initiatives.

BACKGROUND

Hypoxia caused by sleep apnea might be associated with an increased risk of cardiovascular events in subjects with metabolic syndrome. The aim of this study was to examine the effect of hypoxia on the left ventricular (LV) myocardium and evaluate the cardioprotective effect of an angiotensin-II receptor blocker (ARB) in diabetic rats.

RESULTS

Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 30 weeks of age (n=30) were divided into 2 groups that were treated with vehicle or candesartan 0.2 mg x kg(-1) x day (-1). The animals were housed in a hypoxic gas chamber (oxygen, 10.0+/-0.5%, mean +/- standard deviation) for 2 weeks. Hypoxia increased right ventricular (RV) systolic pressure (hypoxia; 78+/-14 mmHg vs control; 22+/-5, p<0.05), but did not increase LV systolic pressure (131+/-23 mmHg vs <em>121</em>+/-10). Hypoxia exacerbated the degeneration of cardiomyocytes, and accelerated the expression of hypoxia inducible <em>factor</em>-1alpha (HIF-1 alpha) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the myocardium. Treatment with ARB decreased RV and LV pressures (46+/-7 and 100+/-18 mmHg, respectively), suppressed the expression of HIF-1alpha and VEGF, and preserved the fine structure of the LV myocardium.

CONCLUSIONS

ARB exhibited cardioprotection under hypoxia, in part through the reduction of blood pressure and cytokine expression, in OLETF rats. Thus, ARB might be a potent agent for the treatment of diabetic patients with the complication of sleep apnea.

The blocking of angiogenesis provides a novel therapeutic target to inhibit tumour spreading. In this study, we investigated the effect of linomide on angiogenesis induced in vivo by highly angiogenic breast carcinoma cells. The rabbit cornea was used to assess neo<em>vascular</em> <em>growth</em> in the absence of a tumour mass. MCF-7 cells stably transfected with the cDNA encoding for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>) (V12 clone) were used to elicit a potent VEGF-dependent corneal angiogenesis. After tumour cell implant, albino rabbits received 100 mg kg(-1) day(-1) linomide for 5 consecutive days. Daily observation of neo<em>vascular</em> progression indicated that linomide blocked angiogenesis. The antiangiogenic effect of linomide was apparent within 48 h from the beginning of the treatment and was both angiosuppressive and angiostatic. The block of neo<em>vascular</em> <em>growth</em> lasted over 10 days from treatment suspension, and preformed vessels, which had regressed, remained dormant, suggesting the persistence of unfavourable conditions for capillary progression. Linomide (50-200 microg ml[-1]) was not cytotoxic in vitro on resting capillary <em>endothelial</em> cells but blocked <em>endothelial</em> cell replication induced by VEGF. Our data indicate that linomide can efficiently and persistently block VEGF-dependent angiogenesis in vivo in the absence of a <em>growing</em> tumour mass. These data suggest that linomide could be a chemopreventive drug in breast cancer patients and a valuable tool in clinical settings in which metastatic spreading occurs in the absence of a detectable tumour mass.

BACKGROUND

Intravitreal anti-VEGF (vascular endothelial growth factor) therapy with ranibizumab has been shown to be an effective therapeutic option for foveal diabetic macular edema (DME). This prospective study evaluated the functional and morphological retinal changes after intravitreal ranibizumab treatment.

METHODS

A consecutive prospective series of DME patients treated with intravitreal ranibizumab were examined before and after 3 and 6 months of intravitreal ranibizumab therapy. Best-corrected visual acuity (BCVA) according to the ETDRS protocol, retinal thickness in the macular area and central retinal thickness (CRT) measured with spectral-domain optical coherence tomography (SD-OCT) was determined. In addition, microperimetric functional macular mapping was determined before therapy and 4 weeks after the third injection.

RESULTS

A total of 41 eyes from 33 patients were evaluated. During the 6-month observational period patients received a mean number of 5.2 injections. The mean BCVA increased significantly from 26 ± 14 to 33 ± 13 letters 4 weeks after the third injection and to 34 ± 14 letters 6 months after starting the treatment. The mean CRT decreased significantly from 509 ± 147 µm to 385 ± 121 µm after the third injection and to 383 ± 110 µm after 6 months. After 3 injections, the thickness of the most prominent central retinal area was less than 445 µm in 68.3% of patients and after a further 3 months of treatment in 78.0%.

CONCLUSIONS

The presented data demonstrate that intravitreal ranibizumab is effective for DME in everyday clinical practice and results are comparable to those of registration trials. After three initial injections significant structural and functional improvements were observed in a considerable number of patients.

OBJECTIVE

To compare the mRNA expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> and 165 isoforms and thrombospondin-1 (TSP-1) after raloxifene, 17beta-E(2), and P administration in cultured Ishikawa cells.

METHODS

Prospective basic research study.

METHODS

Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia.

METHODS

None.

METHODS

Ishikawa cells were cultured in vitro. Raloxifene, 17beta-E(2), and P at concentrations of 0.01, 0.1, and 1.0 microM were added to confluent cells.

METHODS

The VEGF <em>121</em> and 165 isoforms and TSP-1 mRNA expression from treated Ishikawa cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

17beta-Estradiol increased both VEGF <em>121</em> and 165 mRNA compared to control. Raloxifene did not increase either VEGF <em>121</em> or 165 mRNA above control values. Progesterone at all concentrations did not increase VEGF <em>121</em> isoform mRNA, whereas VEGF 165 isoform was minimally increased by P at the lowest concentration. Progesterone and raloxifene increased TSP-1 mRNA expression. 17beta-Estradiol did not stimulate TSP-1 mRNA expression at any concentration.

CONCLUSIONS

Raloxifene did not stimulate VEGF <em>121</em> and 165, whereas it increased TSP-1 mRNA synthesis in Ishikawa cells. Our hypothesis is that raloxifene's lack of endometrial stimulation may be partly mediated by an antiangiogenic effect.

OBJECTIVE

Recent reports of animal models have shown that growth factors have stimulating effect on brain perfusion via the development of blood vessels. However, studies on the effect of growth factors on brain perfusion in humans are lacking. The aim of our study was to prospectively investigate in humans the relation between growth factors and brain perfusion.

METHODS

We analyzed circulating levels of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating growth factor (GM-CSF), tumor necrosis factor alpha (TNFalpha) and basic fibroblast growth factor (bFGF) in 121 consecutive patients (99 men and 22 women, age 58+/-10 years) who were enrolled in a prospective cohort study of patients with symptomatic atherosclerotic disease. In all patients regional cerebral blood flow (rCBF; in mL/min/100g) measurements were performed with arterial spin labeling magnetic resonance imaging. Cerebrovascular risk factors were assessed by means of a questionnaire and physical, ultrasonographic and laboratory examination.

RESULTS

Increasing levels of TNFalpha were significantly associated with a higher rCBF (beta=7.0; 95% confidence interval 0.7; 13.9), independent of the presence of cerebrovascular risk factors. No significant association was found for VEGF, GM-CSF and bFGF.

CONCLUSIONS

Increasing levels of TNFalpha are associated with increased rCBF, independent of the presence of cerebrovascular risk factors.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) Flays a central role in solid tumour angiogenesis, although its expression has never been previously documented in circulating lymphoma or leukaemia cells. Here we have quantified mRNA encoding VEGF isoforms <em>121</em> and 165 in peripheral lymphocytes of 6/6 CLL patients using Northern blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques. No VEGF mRNA could be detected in control lymphocytes. VEGF expression in primary lymphoma cells was lower than in normoxic human colorectal carcinoma cells (LS174T), implying that VEGF expression within tumour cells must be regulated by a range of malignancy-associated <em>factors</em>.

Overexpression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is related to tumour progression and xenotransplant-ability in various human solid tumours, but the specific impact of the VEGF-subtypes is still under discussion. The aim of this study was to analyse a possible association of the major VEGF-isoforms and the <em>growth</em> characteristics of xenotransplanted human head and neck squamous cell carcinomas in nude mice. Seven SCC cell lines were analysed by quantitative RT-PCR using the TaqMan-System. We investigated the expression of VEGF-total-mRNA and of the major subtypes VEGF-<em>121</em>, -165, and -189 by using subtype specific primers. The cell lines were xenotransplanted in three mice each, and the data of tumour <em>growth</em> and progression were correlated to the expression of VEGF-isoforms. Six out of the seven cell lines analysed expressed all isoforms of VEGF in different quantities. One cell line expressed generally low levels of VEGF and no VEGF-189 at all. In this cell line xenotransplantation failed in one mice out of three. In a second cell line transplantation also failed in one out of seven mice. Success rates for the other five cell lines were 100%. The cell lines with higher transplantation success were expressed higher VEGF-<em>121</em>/165-189 ratios compared to those without success. In contrast, linearity of tumour <em>growth</em> and lack of necrosis were associated with a lower VEGF-<em>121</em>/165-189 ratio. The findings demonstrate a predominant expression of VEGF-165 and VEGF-189, compared to VEGF-<em>121</em>. We discuss an association of '<em>growth</em> response rate' measured by tumour <em>growth</em> per detected VEGF-level. In highly proliferating tumours this rate appeared to be about 10 times higher than in low proliferating tumours. We conclude that the ratio between the VEGF-subtypes during tumour implantation and <em>growth</em> is a prerequisite for progression and hypothesise an individual and different response of each tumour cell line to VEGF.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> is a multipotent angiogenic <em>factor</em> implicated in cell survival and proliferation. The objective was to determine effects of exogenous recombinant human VEGFA (or VEGFA165) in culture media on porcine oocyte maturation and parthenote development. Adding 5 ng/mL VEGFA to the culture medium improved the maturation rate of denuded oocytes (P < 0.05), although 5, 50, or 500 ng/mL did not significantly affect nuclear maturation of oocytes. Parthenotes from oocytes cultured either in in vitro maturation or in vitro culture medium supplemented with 5 or 50 ng/mL VEGFA had an improved blastocyst rate and increased total numbers of cells (P < 0.05). Moreover, those treated with 5 ng/mL of VEGFA had a higher hatched blastocyst rate (average of <em>121</em> cells per blastocyst). All VEGFA-treated oocytes had reduced apoptotic indices (P < 0.05), except for those with a higher dose (500 ng/mL) of VEGFA which had more apoptotic cells (P < 0.05). Adding 5 ng/mL VEGFA to oocytes during the last 22 h of in vitro maturation improved (P < 0.05) blastocyst rates and total numbers of cells, with reduced apoptosis indices similar to that of long-term (44 h) culture. Furthermore, Axitinib (VEGFR inhibitor) reversed the effects of VEGFA on parthenote development (P < 0.05). Follicular fluids from medium (2-6 mm) to large (>6 mm) follicles contained 5.3 and 7.0 ng/mL <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> protein, respectively, higher (P < 0.05) than concentrations in small (<2 mm) follicles (0.4 ng/mL). Also, VEGFA and its receptor (VEGFR-2) were detected (immunohistochemistry) in <em>growing</em> follicles and developing blastocysts. In addition, VEGFA inhibited caspase-3 activation in matured oocytes (P < 0.05). In conclusion, this is apparently the first report that VEGFA has proliferative and cytoprotective roles in maturing porcine oocytes and parthenotes. Furthermore, an optimal VEGFA concentration promoted porcine oocyte maturation and subsequent development.

Background: Imatinib, a tyrosine kinase inhibitor, causes <em>growth</em> failure in children with chronic myeloid leukemia probably by targeting the <em>growth</em> hormone (GH)/insulin like <em>growth</em> <em>factor</em>-1 (IGF-1) axis. We aim to explore the imatinib targets expression in pituitary adenomas and study the effect of imatinib on GH secretion in somatotropinoma cells and GH3 cell line. Materials and Methods: The expression pattern of imatinib's targets (c-kit, VEGF, and PDGFR-α/β) was studied using immunohistochemistry and immunoblotting 157 giant (≥4 cm) pituitary adenomas (<em>121</em> non-functioning pituitary adenomas, 32 somatotropinomas, and four prolactinomas) and compared to normal pituitary (n = 4) obtained at autopsy. The effect imatinib on GH secretion, cell viability, immunohistochemistry, electron microscopy, and apoptosis was studied in primary culture of human somatotropinomas (n = 20) and in rat somato-mammotroph GH3 cell-line. A receptor tyrosine kinase array was applied to human samples to identify altered pathways. Results: Somatotropinomas showed significantly higher immunopositivity for c-kit and platelet-derived <em>growth</em> <em>factor</em> receptor-β (PDGFR-β; P < 0.009 and P < 0.001, respectively), while staining for platelet-derived <em>growth</em> <em>factor</em> receptor-α (PDGFR-α) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) revealed a weaker expression (P < 0.001) compared to normal pituitary. Imatinib inhibited GH secretion from both primary culture (P < 0.01) and GH3 cells (P < 0.001), while it did not affect cell viability and apoptosis. The receptor tyrosine kinase array showed that imatinib inhibits GH signaling via PDGFR-β pathway. Conclusion: Imatinib inhibits GH secretion in somatotropinoma cells without affecting cell viability and may be used as an adjunct therapy for treating GH secreting pituitary adenomas.