Increased Vascular Endothelial Growth Factor mRNA Research Articles

Pro-Angiogenetic Effects of Purified Extracts from Helix aspersa during Zebrafish Development

Helix aspersa is a species of land snail belonging to the Helicidae family, widespread in the Mediterranean and continental area up to Northern Europe. In some areas it is appreciated as a food, but is mostly considered a parasite of gardens and cultivated fields. The mucus of Helix aspersa has found multiple applications in the cosmetic and health fields. In the present study, we investigated for the first time the angiogenetic properties of purified extracts from Helix aspersa using a transgenic zebrafish line Tg (kdrl:EGFP). The angiogenesis induced by purified snail extracts was demonstrated by their capability to increase the three well-established parameters of angiogenesis: generation of intersegmental vessels, modeling of caudal venous plexus, and formation of sub-intestinal venous plexus. The effects appeared to be mediated by the vascular endothelial growth factor (VEGF) pathway, being prevented by pretreatment of embryos with the selective VEGF receptor antagonist SU5416, and supported by the increased VEGF mRNA levels found in snail-extract-treated embryos. Insufficient vascular supply is underlined by low VEGF signaling, primarily because of its indispensable role in preventing capillary loss. Our findings might have a pharmacological impact by counteracting VEGF hypofunction and promoting angiogenesis to maintain adequate microvascular and vascular density in normal and suffering tissues and organs.

α1-Adrenergic receptor mediates adipose-derived stem cell sheet-induced protection against chronic heart failure after myocardial infarction in rats

Cell-based therapy using adipose-derived stem cells (ADSCs) has emerged as a novel therapeutic approach to treat heart failure after myocardial infarction (MI). The purpose of this study was to determine whether inhibition of α1-adrenergic receptors (α1-ARs) in ADSCs attenuates ADSC sheet-induced improvements in cardiac functions and inhibition of remodeling after MI. ADSCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ADSCs, we determined the mRNA levels of vascular endothelial growth factor (VEGF)-A and α1-AR under normoxia or hypoxia and the effects of norepinephrine and an α1-blocker, doxazosin, on the mRNA levels of angiogenic factors. Hypoxia increased α1-AR and VEGF mRNA levels in ADSCs. Norepinephrine further increased VEGF mRNA expression under hypoxia; this effect was abolished by doxazosin. Tube formation of human umbilical vein endothelial cells was promoted by conditioned media of ADSCs treated with the α1 stimulant phenylephrine under hypoxia but not by those of ADSCs pretreated with phenylephrine plus doxazosin. In in vivo studies using rats with MI, transplanted ADSC sheets improved cardiac functions, facilitated neovascularization, and suppressed fibrosis after MI. These effects were abolished by doxazosin treatment. Pathway analysis from RNA sequencing data predicted significant upregulation of α1-AR mRNA expression in transplanted ADSC sheets and the involvement of α1-ARs in angiogenesis through VEGF. In conclusion, doxazosin abolished the beneficial effects of ADSC sheets on rat MI hearts as well as the enhancing effect of norepinephrine on VEGF expression in ADSCs, indicating that ADSC sheets promote angiogenesis and prevent cardiac dysfunction and remodeling after MI via their α1-ARs.

Antibacterial Biopolymer Gel Coating on Meshes Used for Abdominal Hernia Repair Promotes Effective Wound Repair in the Presence of Infection.

Prosthetic mesh infection is a devastating complication of abdominal hernia repair which impairs natural healing in the implant area, leading to increased rates of patient morbidity, mortality, and prolonged hospitalization. This preclinical study was designed to assess the effects on abdominal wall tissue repair of coating meshes with a chlorhexidine or rifampicin-carboxymethylcellulose biopolymer gel in a Staphylococcus aureus (S. aureus) infection model. Partial abdominal wall defects were created in New Zealand white rabbits (n = 20). Four study groups were established according to whether the meshes were coated or not with each of the antibacterial gels. Three groups were inoculated with S. aureus and finally repaired with lightweight polypropylene mesh. Fourteen days after surgery, implanted meshes were recovered for analysis of the gene and protein expression of collagens, macrophage phenotypes, and mRNA expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Compared to uncoated meshes, those coated with either biopolymer gel showed higher collagen 1/3 messenger RNA and collagen I protein expression, relatively increased VEGF mRNA expression, a significantly reduced macrophage response, and lower relative amounts of MMPs mRNAs. Our findings suggest that following mesh implant these coatings may help improving abdominal wall tissue repair in the presence of infection.

Absence of TRPC1 Ca2+-permeable Channel Causes Placental and Fetal Growth Restriction in Mice

ObjectivesPlacental tissue intracellular calcium (Ca2+) regulates placental development and growth (e.g., blastocyst development through branching morphogenesis). Maternal high-fat (HF) diet results in placental lipid accumulation, increase in inflammation, reduction in nutrient transport expression and intra uterine growth restriction (IUGR). Currently, whether maternal HF diet affects placental and fetal growth and development differentially under reduction in Ca2 + influx is not known. Thus, we hypothesized that maternal HF diet feeding decreases placental growth and development resulting in IUGR. We further hypothesized that reduction of Ca2 + influx in placenta worsens the maternal HF-induced placental dysfunction. MethodsTwo-month old female B6129SF2/J wild type (WT) and transient receptor potential canonical 1 (TRPC1) protein deficient (KO) mice were fed normal fat (NF, 16% fat) and high fat (HF, 45%) diets for 12 weeks. Fetuses and placentae were examined at mid- (D12) and late- (D19) gestation. ResultsPlacental length, width, and weight as well as fetal weight were decreased in the TRPC1KO mice at D12 and D19 compared to that of WT mice. Expression of placental growth factor (PLGF) mRNA was decreased at D12 in TRPC1 KO mice while vascular endothelial growth factor (VEGF) mRNA levels were increased at D19 compared to WT mice. ConclusionsThese findings suggest that genotypic differences rather than maternal HF diet alter placental size and weight as well as fetal weight. Decreased in PLGF mRNA may be responsible for the placental and fetal growth restriction while increase in VEGF mRNA indicates compensatory adaptation to decreased PLGF-associated placental and fetal growth restriction. Future studies are needed to determine the signaling mechanism underlying Ca2 + influx reduction- induced placental dysfunction and IUGR. Funding SourcesUSDA Agricultural Research Service Project #3062–51,000-054–00D.

Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway.

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3 , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3 -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T3 . Therefore, it was concluded that T3 stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.

The Effects of Deep Brain Stimulation of the Subthalamic Nucleus on Vascular Endothelial Growth Factor, Brain-Derived Neurotrophic Factor, and Glial Cell Line-Derived Neurotrophic Factor in a Rat Model of Parkinson’s Disease

Objective: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has evolved as a powerful therapeutic alternative for the treatment of Parkinson’s disease (PD). Despite its clinical efficacy, the mechanisms of action have remained poorly understood. In addition to the immediate symptomatic effects, long-term neuroprotective effects have been suggested. Those may be mediated through neurotrophic factors (NFs) like vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF). Here, the impact of DBS on the expression of NFs was analysed in a rat model of PD. Methods: Unilateral 6-hydroxydopamine (6-OHDA) lesioned rats received DBS in the STN using an implantable microstimulation system, sham DBS in the STN, or no electrode placement. Continuous unilateral STN-DBS (current intensity 50 µA, frequency 130 Hz, and pulse width 52 µs) was conducted for 14 days. Rats were then sacrificed and brains shock frozen. Striata and motor cortices were dissected with a cryostat. Levels of VEGF, BDNF, and GDNF were analysed, both by quantitative PCR and colorimetric ELISA. Results: PCR revealed a significant upregulation of only BDNF mRNA in the ipsilateral striata of the DBS group, when compared to the sham-stimulated group. There was no significant increase in VEGF mRNA or GDNF mRNA. ELISA analysis showed augmentations of BDNF, VEGF, as well as GDNF protein in the ipsilateral striata after DBS compared to sham stimulation. In the motor cortex, significant increases after DBS were observed for BDNF only, not for the other 2 NFs. Conclusions: The upregulation of trophic factors induced by STN-DBS may participate in its long-term therapeutic efficacy and potentially neuroprotective effects.

β-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a β-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Blue-violet light decreases VEGFa production in an in vitro model of AMD.

Blue light is an identified risk factor for age-related macular degeneration (AMD). The production of vascular endothelial growth factor (VEGF), leading to neovascularization, is a major complication of the wet form of this disease. We investigated how blue light affects VEGF expression and secretion using A2E-loaded retinal pigment epithelium (RPE) cells, a cell model of AMD. Incubation of RPE cells with A2E resulted in a significant increase in VEGF mRNA and, intracellular and secreted VEGF protein levels, but not mRNA levels of VEGFR1 or VEGFR2. Blue light exposure of A2E-loaded RPE cells resulted in a decrease in VEGF mRNA and protein levels, but an increase in VEGFR1 levels. The toxicity of 440 nm light on A2E-loaded RPE cells was enhanced by VEGF supplementation. Our results suggest that age-related A2E accumulation may result in VEGF synthesis and release. This synthesis of VEGF, which enhances blue light toxicity for the RPE cells, is itself suppressed by blue light. Anti-VEGF therapy may therefore improve RPE survival in AMD.

The effect of seminal plasma \u03b2-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

BackgroundNerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas.MethodsExperiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment.ResultsExperiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment.ConclusionsSystemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Ureaplasma species modulate cell adhesion molecules and growth factors in human brain microvascular endothelial cells

Ureaplasma species (spp.) are considered commensals of the adult urogenital tract, but may cause chorioamnionitis and preterm birth as well as sepsis and meningitis in neonates. Pathomechanisms in Ureaplasma-driven neuroinflammation are largely unknown. This study addressed mRNA and protein expression of intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1), granulocyte-colony stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) in native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) exposed to Ureaplasma (U.) urealyticum or U. parvum. Ureaplasma spp. reduced G-CSF mRNA (p < 0.05) and protein expression (p < 0.01) and increased VEGF mRNA levels (p < 0.01) in native HBMEC. Upon co-stimulation, Ureaplasma isolates enhanced LPS-evoked VEGF and ICAM-1 mRNA expression (p < 0.05), but mitigated G-CSF and VCAM-1 mRNA responses (p < 0.05). In line with previous findings, our results indicate an ability of Ureaplasma spp. to compromise blood-brain barrier integrity, mitigate immune defense, and subdue neuroprotective mechanisms. This may facilitate intracerebral inflammation, allow chronic infections, and promote brain injury. More pronounced effects in co-stimulated cells may indicate an immunomodulatory capacity of Ureaplasma spp.

BMAL1 Links Bevacizumab Resistance in Colorectal Cancer to Circadian Rhythm and Heme Receptor REVERBA

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

A Mesenchymal stem cell line (B10) increases angiogenesis in a rat MCAO model

A human mesenchymal stem cell line (B10) transplantation has been shown to improve ischemia-induced neurological deficits in animal stroke models. To understand the underlying mechanism, we have investigated the effects of B10 transplantation on cerebral angiogenesis in a rat middle cerebral artery occlusion (MCAO) model. B10 cells were transplanted intravenously 24 h after MCAO. Immunofluorescence staining results showed that compared to PBS-groups, vWF positive vessel and endoglin positive new vessels were increased in B10-transplanted MCAO groups in the lesion areas. The mRNA of angiogenesis factors including placental growth factor and hypoxia inducible factor (HIF)-1α were increased 3 days after MCAO in the core and IBZ areas of B10-transplanted group. Angiopoetin1 mRNA was increased only in the IBZ. Western blotting results showed that HIF-1α and vascular endothelial growth factor (VEGF) proteins were increased in B10-transplanted group. Both HIF-1α and VEGF were expressed in macrophage/microglia in the core area. In the IBZ, however, HIF-1α was expressed both in astrocytes and macrophage/microglia, while VEGF was expressed only in macrophage/microglia. Moreover, TGFβ protein levels were found to be increased in B10-transplanted group in the core and IBZ regions. Cell culture experiments using a human microglia cell line (HMO6) and B10 showed that IL-1β induced VEGF mRNA expression in both cell types. IL-1β was found to be highly expressed in B10 cells, and its co-culture with HMO6 further increased that in B10. Co-culture increased VEGF mRNA in both B10 and HMO6. In the rat brains, IL-1β was expressed in macrophage/microglia and transplanted-B10 cells in the core. IL-1β positive cell number was increased slightly, but significantly in B10-transplanted rats. To explore further, IL-1β expression was silenced in B10 cells by transfecting mRNA specific siRNA, and then transplanted in MCAO rats. Immunostaining result showed that endoglin positive area was decreased in IL-1β-silenced B10 transplanted groups compared to nonsilenced-B10 transplanted groups. Interestingly, vessel-like structure appeared as early as 3 days after MCAO in IL-1β-silenced B10-transplanted group. Thus our results demonstrated that B10 cells increased angiogenesis in MCAO rat model, through the regulation of HIF-1α and VEGF expression, where IL-1β might play a role.

Involvement of vascular endothelial growth factor (VEGF) and mitogen-activated protein kinases (MAPK) in the mechanism of neuroleptic drugs.

Recent evidence suggests that the mitogen activated protein kinase (MAPK)-associated signaling pathway in the frontal cortical areas demonstrates abnormal activity in cases of schizophrenia. Moreover, schizophrenia patients often display alterations in the regional cellular energy metabolism and blood flow of the brain; these are shown to parallel changes in angiogenesis primarily mediated by vascular endothelial growth factor (VEGF). The present study examines the differential effects of time-dependent treatment with haloperidol, olanzapine and amisulpride (20μM) on VEGF and MAPK mRNA expression and VEGF level, using the T98 cell line as an example of nerve cells. For the purposes of comparison, the effect of neuroprotective pituitary adenylate cyclase-activating polypeptide (PACAP) on the expression of VEGF mRNA and secretion were also evaluated in this cell model. RT-PCR analysis revealed that all the tested neuroleptics increased VEGF mRNA expression after 72-h incubation; however, only haloperidol and olanzapine also increased the level of VEGF detected by ELISA, and they demonstrated significantly stronger effects than PACAP. Haloperidol and olanzapine, but not amisulpride, decreased MAPK14 mRNA expression in T98G cells after 72-h incubation. The obtained results suggest that haloperidol and olanzapine can trigger the MAPK and VEGF signaling pathway, which may contribute to their neuroprotective mechanism of action.

Endogenous acetylcholine regulates neuronal and astrocytic vascular endothelial growth factor expression levels via different acetylcholine receptor mechanisms

Vascular endothelial growth factor (VEGF), a signaling molecule involved in angiogenesis, plays an important role in neuroprotection and neurogenesis. In the present study, we aimed to elucidate the mechanisms underlying endogenous acetylcholine (ACh)-induced VEGF expression in neurons and astrocytes, and identify the neuronal cells contributing to its expression in the medial septal area, a nuclear origin of cholinergic neurons mainly projecting to the hippocampus. The mRNA expression and secretion of VEGF were measured by RT-PCR and ELISA using mouse primary cultured cortical neurons and astrocytes. VEGF expression in the medial septal area was assessed by RT-PCR and immunostaining using mice treated with tacrine [9-amino-1,2,3,4-tetrahydro-acridine HCl (THA); 2.5 mg/kg, i.p.] once daily for 7 days. The THA treatment increased VEGF mRNA expression in neurons in a manner that was reversed by mecamylamine, a nicotinic ACh receptor (AChR) antagonist, whereas in mouse primary cultured astrocytes, carbachol, but not THA dose-dependently increased VEGF mRNA expression and secretion in a manner that was inhibited by scopolamine, a muscarinic AChR inhibitor. In in vivo studies, the administration of THA significantly increased the expression of VEGF in medial septal cholinergic neurons and the effects of THA were significantly blocked by mecamylamine. THA also significantly increased the expression levels of a phosphorylated form of VEGF receptor 2 (p-VEGFR2), an activated form of VEGFR2. The present results suggest that endogenous ACh plays an up-regulatory role for VEGF expression in neurons and astrocytes via different mechanisms. Moreover, endogenous ACh-induced increases in VEGF levels appear to activate VEGFR2 on medial septal cholinergic neurons via an autocrine mechanism.

SFRP2 Is Associated with Increased Adiposity and VEGF Expression.

AimsThe aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance.MethodsExpression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed.ResultssFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed.ConclusionsCirculating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression.

Reduction of prostaglandin transporter predicts poor prognosis associated with angiogenesis in gastric adenocarcinoma.

Prostaglandin (PG) E2 promotes gastrointestinal carcinogenesis and tumor progression. The total amount of biologically active PGE2 in tissues is determined by a balance of PG biosynthesis and degradation pathways, which involve the PG transporter (PGT). We investigated PGT in gastric adenocarcinoma by determining its expression pattern and examining associations of PGT with prognosis and tumor angiogenesis. PGT expression was determined by immunohistochemistry in advanced gastric adenocarcinoma specimens obtained from 96 patients who underwent surgical resection. Correlations between PGT expression level and clinicopathological factors were statistically analyzed. Angiogenesis in the tumor tissue was evaluated by counting the number of microvessels. The role of PGT in mRNA and protein expression of vascular endothelial growth factor (VEGF) was examined in gastric cancer cells stimulated by PGE2 . Based on multivariate and Kaplan-Meier analyses, negativity for PGT expression was an independent poor prognostic factor. There were more microvessels in PGT-negative tumors than in PGT-positive tumors. Transfection of AGS and MKN7 gastric cancer cells with PGT-specific siRNA led to increased VEGF mRNA and protein expression accompanied by increased PGE2 in the culture media. PGT expression is an independent predictor of poor survival and is associated with tumor angiogenesis in gastric adenocarcinoma.

Application of recombinant peroxisome proliferator-activated receptor-γ coactivator-1α mediates neovascularization in the retina.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is able to induce the expression of vascular endothelial growth factor (VEGF), promoting the formation of new blood vessels in skeletal muscle. The aim of the current study was to determine whether PGC-1α is able to regulate angiogenesis in human retinal vascular endothelial cells (hRVECs) in vitro and in retinas in vivo. hRVECs treated with recombinant PGC-1α were incubated for 24 h and then placed into a normoxic (20% O2) or hypoxic (1% O2) environment for a further 16 h. Following this, VEGF mRNA and protein levels were significantly increased. Cellular proliferation was enhanced by treatment with recombinant PGC-1α in normoxic and hypoxic conditions. At 24 h following recombinant PGC-1α treatment, hRVECs were plated into Matrigel-coated plates and cultured under normoxic (20% O2) or hypoxic (1% O2) conditions for a further 24 h. Recombinant PGC-1α-treated cells were observed to form significantly greater numbers of tubes. In a C57BL/6J mouse model of ischemic retinopathy, mice received an intravitreal injection of recombinant PGC-1α, resulting in a significant increase in VEGF mRNA and protein levels in the retina. Retinal neovascular tufts and neovascular nuclei were investigated by angiographic and cross-sectional analysis and were observed to be significantly increased in the PGC-1α group compared with the control group. These results indicate that PGC-1α is able to induce angiogenesis in hRVECs and retinas, and suggests that PGC-1α is a potential anti-angiogenic target in retinal neovascularization.

Effects of HIF prolyl-hydroxylase-2 gene silencing on HCG-induced vascular endothelial growth factor expression in luteal cells.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis plays a crucial role in corpus luteum formation and its functional maintenance in mammalian ovaries. We recently reported that activation of hypoxia-inducible factor (HIF)-1α signaling contributes to the regulation of VEGF expression in luteal cells (LCs) in response to human chorionic gonadotropin (HCG). We examined whether HIF prolyl-hydroxylase (PHD)-2 gene silencing induces VEGF expression in LCs and enhanced its expression induced by HCG in LCs. Using real-time polymerase chain reaction and western blot analysis, we measured the expression of PHD-2 to confirm plasmid PHD-2 shRNA transfection and protein expression and investigated the changes in HIF-1a and VEGF expression after treatment with HCG and PHD-2 shRNA transfection. After PHD-2 shRNA transfection, PHD-2 expression was significantly lower than that in control groups with or without HCG treatment, while a significant increase in VEGF mRNA was observed compared to in controls, indicating that PHD-2 plays an important role in VEGF regulation. Additionally, changes in VEGF mRNA expression were consistent with the expression levels of HIF-1a protein, not HIF- 1a mRNA, which is regulated by HIF prolyl-hydroxylase-mediated degradation. Our results indicate that PHD-2 gene silencing can induce VEGF expression in LCs and HCG-induced VEGF expression can be further enhanced by PHD-2 gene silencing through an HIF-1a-mediated mechanism in LCs. This PHD-2-mediated transcriptional activation may be important for regulating VEGF expression through HIF-1a signaling in LCs during corpus luteum development in mammals.

A vascular endothelial growth factor activating transcription factor increases the endothelial progenitor cells population and induces therapeutic angiogenesis in a type 1 diabetic mouse with hindlimb ischemia

Background Therapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. However, it has side effects that limit its therapeutic utility in vivo, especially at high concentrations. This study aimed to investigate whether an intramuscular injection of a genetically engineered zinc finger VEGF-activating transcription factor modulates the endothelial progenitor cells (EPC) and promotes therapeutic angiogenesis in a hindlimb ischemia model with type 1 diabetes. Methods Alloxan (intravenous injection) was used to induce type I diabetes in C57BL/6 mice (n=58). The ischemic limb received ZFP-VEGF (125 μg ZFP-VEGF plasmid in 1% poloxamer) or placebo (1% poloxamer) intramuscularly. Mice were sacrificed 3, 5, 10, or 20 days post-injection. Limb blood flow was monitored using laser Doppler perfusion imaging. VEGF mRNA and protein expression were examined using real-time PCR and ELISA, respectively. Capillary density, proliferation, and apoptosis were examined using immunohistochemistry techniques. Flow cytometry was used to detect the EPC population in bone marrow. Two-tailed Student's paired t test and repeated-measures analysis of variance were used for statistical analysis. Results ZFP-VEGF increased VEGF mRNA and protein expression at 3 and 10 days post-injection, and increased EPC in bone marrow at day 5 and 20 post-injection compared with controls (P&lt;0.05). ZFP-VEGF treatment resulted in better perfusion recovery, a higher capillary density and proliferation, and less apoptosis compared with controls (P&lt;0.05). Conclusions Intramuscular ZFP-VEGF injection promotes therapeutic angiogenesis in an ischemic hindlimb model with type 1 diabetes. This might be due to the effects of VEGF on cell survival and EPC recruitment.

THU0553 Gliostatin Regulates Vascular Endothelial Growth-Factor Production in Human Fibroblast-Like Synoviocytes

BackgroundAngiogenesis, which is the growth of new capillaries from pre-existing blood vessels, is an essential feature of rheumatoid arthritis (RA). Neovascularisation might be triggered by several factors, such as cytokines,...