Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets

2000 ◽  
Vol 279 (2) ◽  
pp. H817-H824 ◽  
Author(s):  
Petra Koehne ◽  
Carsten Willam ◽  
Evelyn Strauss ◽  
Ralf Schindler ◽  
Kai-Uwe Eckardt ◽  
...  
Keyword(s):  
Tissue Injury ◽  
Polymorphonuclear Neutrophils ◽  
Protein Synthesis Inhibitor ◽  
Low Oxygen ◽  
Vegf Mrna ◽  
Synthesis Inhibitor ◽  
Hypoxic Conditions ◽  
Endothelial Growth Factor ◽  
Vegf Release ◽  
Stimulation Of

Low oxygen (O2) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O2). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.

Desensitization and resensitization of lutropin receptors expressed in transfected Y-1 adrenal cells

1999 ◽  
Vol 163 (2) ◽  
pp. 289-297 ◽  
Author(s):  
GA Ulaner ◽  
J Chuang ◽  
W Lin ◽  
D Woodbury ◽  
RV Myers ◽  
...  
Keyword(s):  
Receptor Occupancy ◽  
Untranslated Regions ◽  
Protein Synthesis Inhibitor ◽  
Receptor Recycling ◽  
Synthesis Inhibitor ◽  
Early Step ◽  
Hormonal Stimulation ◽  
Adrenal Cells ◽  
Metallothionein Promoter ◽  
Stimulation Of

Stimulation of gonadal cells by lutropins such as human chorionic gonadotropin (hCG) is often transient and followed by down-regulation and/or desensitization of lutropin receptors (LHR). Here we describe desensitization/resensitization of LHR in Y-1 adrenal cell lines (termed Y-1L) expressing a rat cDNA lacking most 5' and 3' LHR untranslated regions under the control of a metallothionein promoter. Using a simple morphological assay in which stimulated cells are round and unstimulated cells are flat, we identified clones that rounded and remained round and others that became insensitive to lutropin stimulation and reverted to their flat appearance within 2-4 h. Flattened cells were insensitive to further hormonal stimulation but rounded after treatments with cholera toxin, forskolin, or cyclic AMP, showing that loss of responsiveness was associated with an early step in signal transduction, not loss of rounding potential. Removing the lutropin stimulus for at least 90-120 min reversed hormone insensitivity, even in the presence of the protein synthesis inhibitor puromycin. The number of surface bound receptors did not change during a cycle of rounding/flattening and hCG bound to rounded or flattened cells was replaced equally by radioiodinated hCG during incubations at 4 degrees C. Thus, desensitization/resensitization of LHR in Y-1L cells occurred in the absence of new receptor synthesis, receptor degradation, or receptor recycling. These observations suggest that LHR desensitization/resensitization in Y-1L cells was closely coupled to receptor occupancy and that this cell line may be useful for identifying factors that modulate the activities of occupied receptors.

scholarly journals Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis

2007 ◽  
Vol 28 (2) ◽  
pp. 772-783 ◽  
Author(s):  
Frank Vumbaca ◽  
Kathryn N. Phoenix ◽  
Daniel Rodriguez-Pinto ◽  
David K. Han ◽  
Kevin P. Claffey
Keyword(s):  
Breast Cancer ◽  
Mrna Stability ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Vascular Endothelial ◽  
Vegf Mrna ◽  
Double Stranded Rna ◽  
Hypoxic Conditions ◽  
Endothelial Growth Factor

ABSTRACT Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo.

scholarly journals Control of amylase biosynthesis and release in the parotid gland of the rat

1978 ◽  
Vol 176 (3) ◽  
pp. 855-863 ◽  
Author(s):  
Margaret A. McPherson ◽  
C. Nicholas Hales
Keyword(s):  
Total Protein ◽  
Leucine Incorporation ◽  
Protein Synthesis Inhibitor ◽  
Pulse Labelling ◽  
Synthesis Inhibitor ◽  
Physiological Concentration ◽  
Amylase Release ◽  
Cholinergic Agents ◽  
Rat Parotid ◽  
Stimulation Of

1. Amylase biosynthesis and release in the rat parotid were studied under various conditions. Incorporation of [3H]leucine into amylase, extracted from the tissue by immunoadsorbent, was measured and found to be time-dependent and totally inhibited by the protein synthesis inhibitor puromycin. 2. Adrenaline, at a concentration (10μm) that gave maximum stimulation of release, inhibited [3H]leucine incorporation into both total protein and amylase. This effect was reversed by phentolamine. 3. Adrenaline (1μm) and isoproterenol (10μm) stimulated biosynthesis of total protein and amylase. These effects were blocked by propranolol, as were the effects on release. Dibutyryl cyclic AMP (2mm) mimicked the effects of isoproterenol and adrenaline (1μm) on both amylase biosynthesis and release. All the above stimulatory effects on amylase biosynthesis were only observed if the tissue was pretreated with effector before pulse-labelling with [3H]leucine. 4. Insulin (625μunits/ml initial concentration, 150μunits/ml final concentration) stimulated incorporation of [3H]leucine into total protein and amylase when added to the tissue at the same time as the leucine. 5. Carbamoylcholine (10μm) decreased [3H]leucine incorporation into total protein and amylase when both were added to the tissue simultaneously, but this effect was prevented by removal of effector and washing the tissue before addition of [3H]leucine. 6. Stimulation of β-adrenergic receptors increased both amylase release and biosynthesis, but stimulation of α-receptors can inhibit biosynthesis without inhibiting release. Cholinergic agents can also inhibit amylase biosynthesis, but stimulate release. Insulin at approximately physiological concentration can increase incorporation of leucine into amylase without stimulating release. The system described therefore provides an excellent model for the further investigation of the mechanisms of these diverse effects.

scholarly journals A Fairy Chemical Suppresses Retinal Angiogenesis as a HIF Inhibitor

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1405
Author(s):  
Deokho Lee ◽  
Yukihiro Miwa ◽  
Jing Wu ◽  
Chiho Shoda ◽  
Heonuk Jeong ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Inhibitory Effect ◽  
Retinal Neovascularization ◽  
Vascular Endothelial ◽  
Vegf Mrna ◽  
Oxygen Induced Retinopathy ◽  
Hypoxic Conditions ◽  
Chronic Therapy ◽  
Retinal Angiogenesis ◽  
Endothelial Growth Factor

Neovascular retinal degeneration is a leading cause of blindness in advanced countries. Anti-vascular endothelial growth factor (VEGF) drugs have been used for neovascular retinal diseases; however, anti-VEGF drugs may cause the development of chorioretinal atrophy in chronic therapy as they affect the physiological amount of VEGF needed for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcription factor inducing VEGF expression under hypoxic and other stress conditions. Previously, we demonstrated that HIF was involved with pathological retinal angiogenesis in murine models of oxygen-induced retinopathy (OIR), and pharmacological HIF inhibition prevented retinal neovascularization by reducing an ectopic amount of VEGF. Along with this, we attempted to find novel effective HIF inhibitors. Compounds originally isolated from mushroom-forming fungi were screened for prospective HIF inhibitors utilizing cell lines of 3T3, ARPE-19 and 661W. A murine OIR model was used to examine the anti-angiogenic effects of the compounds. As a result, 2-azahypoxanthine (AHX) showed an inhibitory effect on HIF activation and suppressed Vegf mRNA upregulation under CoCl2-induced pseudo-hypoxic conditions. Oral administration of AHX significantly suppressed retinal neovascular tufts in the OIR model. These data suggest that AHX could be a promising anti-angiogenic agent in retinal neovascularization by inhibiting HIF activation.

Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment

2001 ◽  
Vol 280 (1) ◽  
pp. C72-C80 ◽  
Author(s):  
Jason A. Spector ◽  
Babak J. Mehrara ◽  
Joshua A. Greenwald ◽  
Pierre B. Saadeh ◽  
Douglas S. Steinbrech ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Lactate Concentration ◽  
Vascular Endothelial ◽  
Acidic Ph ◽  
Vegf Mrna ◽  
Hypoxic Conditions ◽  
Extracellular Microenvironment ◽  
Endothelial Growth Factor ◽  
Elevated Lactate

Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions ( P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH ( P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.

scholarly journals Short-Term Stimulation of Lipogenesis by 3,5-l-Diiodothyronine in Cultured Rat Hepatocytes

Endocrinology ◽  
2005 ◽  
Vol 146 (9) ◽  
pp. 3959-3966 ◽  
Author(s):  
Anna M. Giudetti ◽  
Monica Leo ◽  
Math J. H. Geelen ◽  
Gabriele V. Gnoni
Keyword(s):  
Protein Synthesis ◽  
Fatty Acid ◽  
Cholesterol Synthesis ◽  
Rat Hepatocytes ◽  
Experimental Period ◽  
Protein Synthesis Inhibitor ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Lipid Fractions ◽  
Stimulation Of

Abstract Short-term effects of 3,5-l-diiodothyronine (T2) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T3 was routinely carried out. After T2 addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-14C]acetate into these lipid fractions, was observed. The T2 dose-dependent effect on both metabolic pathways, already detectable at 10−8-10−9m, reached a 2-fold stimulation at 10−5m T2. At this concentration, the stimulatory effect was evident within 1 h of T2 addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T2 stimulation of lipogenesis was also confirmed by incubating hepatocytes with [3H]H2O, used as an independent index of lipogenic activity. The effects of T2 are rather specific as 3,3′,5,5′-tetraiodo-d-thyronine and 3,5-diiodo-l-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T2 addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T2 with those seen in the presence of T3, it appeared that T2 was able to mimic T3 effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T2 stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.

scholarly journals Sanguinarine Inhibits Vascular Endothelial Growth Factor Release by Generation of Reactive Oxygen Species in MCF-7 Human Mammary Adenocarcinoma Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Xian-zhe Dong ◽  
Miao Zhang ◽  
Kun Wang ◽  
Ping Liu ◽  
Dai-hong Guo ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Mammary Adenocarcinoma ◽  
Vascular Endothelial ◽  
Oxygen Species ◽  
Vegf Mrna ◽  
Adenocarcinoma Cells ◽  
Endothelial Growth Factor ◽  
Vegf Release ◽  
Mcf 7

The inhibitory action and the possible mechanism of anticancer compound Sanguinarine (SAN) on vascular endothelial growth factor (VEGF) in human mammary adenocarcinoma cells MCF-7 were evaluated in this study. We exposed MCF-7 to SAN for 24 h, then cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Human VEGF was measured using a paired antibody quantitative ELISA kit, relative expression of VEGF mRNA was calculated using the real-time PCR studies, and the effect of SAN on the reactive oxygen species (ROS) level was detected by the flow cytometer. Treatment with SAN remarkably inhibited growth of MCF-7 cells and induced cell apoptosis. We found that VEGF release was stimulated by subtoxic concentrations of SAN and inhibited by high dose of SAN, SAN-evoked VEGF release was mimicked by low concentration of H2O2, and SAN-regulated VEGF inhibition was accompanied by increasing of ROS; these changes were abolished by antioxidant. High concentration of SAN inhibited VEGF mRNA expression in MCF-7 cultures, suggesting an effect at transcriptional level, and was also abolished by antioxidant. The present findings indicated that the regulation of VEGF expression and release from MCF-7 cells were possibly through reactive oxygen species evoked by SAN.

Hypotonicity stimulates renal epithelial sodium transport by activating JNK via receptor tyrosine kinases

2007 ◽  
Vol 293 (1) ◽  
pp. F128-F138 ◽  
Author(s):  
Akiyuki Taruno ◽  
Naomi Niisato ◽  
Yoshinori Marunaka
Keyword(s):  
Protein Synthesis ◽  
Tyrosine Kinase ◽  
P38 Mapk ◽  
Egf Receptor ◽  
Late Phase ◽  
Pi3 Kinase ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Hypotonic Stress ◽  
Stimulation Of

We previously reported that hypotonic stress stimulated transepithelial Na+ transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63–77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of Na+ transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of Na+ transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK was diminished by coapplication of AG1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of Na+ transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced Na+ transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated Na+ transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of Na+ transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of Na+ transport.

scholarly journals Up-regulation of phospholipid hydroperoxide glutathione peroxidase in rat casein-induced polymorphonuclear neutrophils

2005 ◽  
Vol 389 (2) ◽  
pp. 279-287 ◽  
Author(s):  
Hiroyuki Hattori ◽  
Hirotaka Imai ◽  
Akiharu Hanamoto ◽  
Kazuhisa Furuhama ◽  
Yasuhito Nakagawa
Keyword(s):  
Glutathione Peroxidase ◽  
Polymorphonuclear Neutrophils ◽  
Protein Synthesis Inhibitor ◽  
Polymorphonuclear Leucocytes ◽  
Mrna Levels ◽  
Phospholipid Hydroperoxide Glutathione Peroxidase ◽  
Synthesis Inhibitor ◽  
Regulation Of Expression ◽  
Phospholipid Hydroperoxide ◽  
Cultivation In Vitro

Antioxidant enzymes play key roles in the protection of cells from oxidative damage. Little is known, however, about the expression of antioxidants and/or their roles in PMNs (polymorphonuclear leucocytes), which are thought to suffer from oxidative stress in an inflammation site. In the present paper, we report on the regulation of expression of PHGPx (phospholipid hydroperoxide glutathione peroxidase) and cGPx (cytosolic glutathione peroxidase) in rat PMNs in the inflammation site. PHGPx mRNA levels were much lower in casein-induced peritoneal and carrageenan-induced pleural PMNs just after their collection than in peripheral PMNs. cGPx mRNA was also reduced in the casein-induced PMNs, but not in carrageenan-induced PMNs. Both enzymes with decreased levels in the casein-induced PMNs were up-regulated during further 24 h cultivation in vitro and in vivo, with elevation of their protein levels and activities, and reduction of intracellular peroxides. Up-regulation of PHGPx mRNA was attenuated by cycloheximide, a protein synthesis inhibitor, and this effect was cancelled by culturing the cells in the conditioned medium of the cultured casein-induced PMNs. This latter effect was attenuated by pre-treatment with anti-GRO (growth-regulated oncogene) antibody. Recombinant rat GRO could also induce the up-regulation in the presence of cycloheximide, demonstrating that GRO may play an important role in the PHGPx up-regulation of casein-induced PMNs. Production of the lipid mediators leukotriene B4 and 5-HETE (5-hydroxyeicosatetraenoic acid) was decreased in the cultured casein-induced PMNs exhibiting PHGPx up-regulation. The evidence obtained indicates that PHGPx activity in the activated PMNs would be related to the appearance of the intrinsic function of PMNs in the inflammatory site.

scholarly journals Hypoxia inducible factor signaling in macrophages promotes lymphangiogenesis in Leishmania major infection.

2021 ◽  
Author(s):  
Anne Bowlin ◽  
Hayden Roys ◽  
Humphrey Wanjala ◽  
Manjunath Bettadapura ◽  
Gopinath Venugopal ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Hypoxia Inducible Factor ◽  
Inflammatory Cells ◽  
Low Oxygen ◽  
Cutaneous Lesions ◽  
Aryl Hydrocarbon ◽  
Hypoxic Conditions ◽  
Local Site ◽  
Lymphatic Network ◽  
Endothelial Growth Factor

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Furthermore, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

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