Evaluation of pulmonary GLUT1 and VEGF mRNA levels in relation to lung weight in medicolegal autopsy cases

Background: Bone Morphogenetic Proteins (BMPs) regulate diverse cellular functions during foetal development and postnatal life. Growth Differentiation Factor 5 (GDF5 a.k.a. BMP-14) is a BMP, which is expressed in a variety of tissues including heart. We previously showed that cardiac GDF5 mRNA levels are elevated after experimental myocardial infarction (MI) caused by permanent left anterior descending coronary artery (LAD) ligation. However, the significance of this finding was not known. Methods & Results: GDF5 knock-out (KO; n = 18 for MI) and wild-type (WT; n = 18 for MI) littermate controls were subjected to chronic LAD ligation in order to investigate the consequences resulting from the loss of GDF5 signalling following MI. At 28 days post-LAD ligation or sham (n = 12 for KO; n = 10 for WT), invasive hemodynamic parameters of cardiac function were examined just prior to sacrifice. Histopathology was assessed by morphometric analyses of perfusion fixed hearts and subsequent immunostaining. At 28 days post-MI, GDF5-KO mice exhibited decreased left ventricular systolic pressure and peak positive- and negative- dP/dt , and increased heart rate as compared to WT littermates ( P < 0.005 for each parameters). GDF5-KO mice also exhibited a significant increase in the area, length and transmural expansion of the infarct, scar thinning and cardiac dilatation ( P < 0.05 for each parameter). In addition, GDF5-KO mice displayed significantly fewer myocardial vessels in the infarct and peri-infarct regions as compared to WT littermates ( P < 0.05) . To explore mechanisms underlying this phenotype, we assessed gene expression levels of relevant potential downstream targets of GDF5. At 7d post-MI, quantitative RT-PCR revealed a significant reduction (35%) in VEGF mRNA levels in hearts of KO (n = 6) as compared to WT mice (n = 5, P = 0.033). Summary & Conclusion: These data suggest that increased GDF5 expression observed in hearts after MI plays an important role in cardiac remodelling. Absence of GDF5 expression in KO mice confers detrimental effects on healing and repair of myocardial and vascular tissues after MI. Regulated levels of GDF5, a BMP family member, play an important role in the repair process following cardiac injury.

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Hypoxia Inducible Factor-1α Is over Expressed in CLL B Cells Because of an Impaired Proteasome Pathway Associated with Defective Interaction with von Hippel-Landau Protein.

Blood ◽

10.1182/blood.v106.11.2115.2115 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2115-2115 ◽

Cited By ~ 1

Author(s):

Yean K. Lee ◽

Ann K. Strege ◽

Nancy D. Bone ◽

Linda E. Wellik ◽

D. A. Chan ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Nuclear Extract ◽

Degradation Pathway ◽

Mrna Levels ◽

Apoptosis Resistance ◽

Vegf Mrna ◽

Prolyl Hydroxylases

Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

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Muscle microvascular adaptation and angiogenic gene induction in response to exercise training are attenuated in middle-aged rats

Comparative Exercise Physiology ◽

10.3920/cep150007 ◽

2015 ◽

Vol 11 (1) ◽

pp. 23-33

Author(s):

J. Suzuki

Keyword(s):

Exercise Training ◽

Acute Exercise ◽

Young Group ◽

Treadmill Running ◽

Mrna Levels ◽

Aged Rats ◽

Middle Aged ◽

Vegf Mrna ◽

Aged Group ◽

Exercise Induced

This study was designed to investigate exercise-induced changes in muscle capillarisation, the mRNA expression of angiogenic genes, and microRNA levels in young and middle-aged rats. Rats in the training groups were subjected to treadmill running 5 days a week for 3 weeks. The exercise protocol for the young (12-week old) group was 20-25 m/min, 40-60 min/day with a gradient of 15%, and for the middle-aged (12-month old) group was 18-20 m/min, 40-60 min/day with a gradient of 5%. The enzyme histochemical identification of capillary profiles was performed on cross-sections of gastrocnemius muscle. Total RNA was isolated, reverse transcription was performed, and mRNA and microRNA levels were determined by real-time PCR. The capillary-to-fibre ratio was significantly increased by exercise training in the young group (by 10%), but only slightly in the middle-aged (by 5%) group. Vascular endothecial growth factor (VEGF) mRNA levels were at significantly higher values after acute exercise (1.6-fold) and the 3-week training protocol (1.9-fold) in the young group, but not in the middle-aged group. VEGF protein expression levels were significantly increased after training in the young group only. Endothelial nitric oxide synthase, VEGF-R2 and thrombospondin-1 mRNA levels were significantly lower in the middle-aged group than in the young group. Anti-angiogenic miR-195 levels were significantly enhanced by exercise training in the middle-aged group only. These results indicated that the exercise-induced adaptation of muscle capillarity was attenuated in middle-aged rats, possibly by the lower induction of VEGF and up-regulation of anti-angiogenic miRNA expression.

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Effect of acute exercise and exercise training on VEGF splice variants in human skeletal muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00071.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. R397-R402 ◽

Cited By ~ 45

Author(s):

Lotte Jensen ◽

Henriette Pilegaard ◽

P. Darrell Neufer ◽

Ylva Hellsten

Keyword(s):

Skeletal Muscle ◽

Exercise Training ◽

Human Skeletal Muscle ◽

Acute Exercise ◽

Splice Variants ◽

Knee Extensor ◽

Vastus Lateralis Muscle ◽

Mrna Levels ◽

Vegf Mrna ◽

Exercise Induced

The present study investigated the effect of an acute exercise bout on the mRNA response of vascular endothelial growth factor (VEGF) splice variants in untrained and trained human skeletal muscle. Seven habitually active young men performed one-legged knee-extensor exercise training at an intensity corresponding to ∼70% of the maximal workload in an incremental test five times/week for 4 wk. Biopsies were obtained from the vastus lateralis muscle of the trained and untrained leg 40 h after the last training session. The subjects then performed 3 h of two-legged knee-extensor exercise, and biopsies were obtained from both legs after 0, 2, 6, and 24 h of recovery. Real-time PCR was used to examine the expression of VEGF mRNA containing exon 1 and 2 (all VEGF isoforms), exon 6 or exon 7, and VEGF165mRNA. Acute exercise induced an increase ( P < 0.05) in total VEGF mRNA levels as well as VEGF165and VEGF splice variants containing exon 7 at 0, 2, and 6 h of recovery. The increase in VEGF mRNA was higher in the untrained than in the trained leg ( P < 0.05). The results suggest that in human skeletal muscle, acute exercise increases total VEGF mRNA, an increase that appears to be explained mainly by an increase in VEGF165mRNA. Furthermore, 4 wk of training attenuated the exercise-induced response in skeletal muscle VEGF165mRNA.

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Regulation of VEGF Gene Expression in Mouse Macrophages by Cottonseed Extracts, Gossypol and Lipopolysaccharides (P06-035-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz031.p06-035-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Heping Cao ◽

Kandan Sethumadhavan

Keyword(s):

Gene Expression ◽

Equal Opportunity ◽

Angiogenic Factor ◽

Mrna Levels ◽

Vegf Gene ◽

Vegf Mrna ◽

Funding Sources ◽

Raw264.7 Macrophages ◽

Mouse Macrophages ◽

Vegf Protein

Abstract Objectives Vascular endothelial growth factor (VEGF) is a key mediator of adipogenesis and a mitogenic and angiogenic factor involved in inflammation, tumor progression, collateral vessel formation, and diabetic retinopathy. VEGFa and VEGFb play a balance role in adipose differentiation and gene expression. Plant extracts and chemical compounds that can regulate VEGF gene expression may have positive effect on nutrition and health. The objective was to investigate the regulation of VEGF gene expression by cottonseed extracts, gossypol and lipopolysaccharides (LPS) in mouse RAW264.7 macrophages. Methods Mouse RAW264.7 macrophages were treated with various concentrations of cottonseed extracts, gossypol and LPS for 2, 8 and 24 h. qPCR and immunoblotting were used to detect the expression of VEGF mRNA and protein. Results qPCR assay showed that cottonseed extracts exhibited modest effects on VEGF gene expression with significant increases in VEGFa mRNA by glanded coat extract and VEGFb mRNA by glanded kernel and glandless coat extracts. Immunoblotting showed that only glandless seed extracts modestly increased VEGF protein. Gossypol stimulated VEGFa and VEGFb mRNA levels by 30- and 4-fold, respectively, and increased VEGF protein in macrophages. LPS increased VEGFa mRNA by 6-fold but decreased VEGFb mRNA under higher concentration for longer treatment. LPS increased VEGF protein in 2–4 h but decreased in 8–24 h. Conclusions These results demonstrate that cottonseed extracts have modest effect but gossypol and LPS have strong effect on VEGF gene expression in mouse macrophages. Funding Sources This work was supported by the USDA-ARS Quality and Utilization of Agricultural Products National Program 306 through CRIS 6054–41,000-103–00-D. USDA is an equal opportunity provider and employer.

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ACE Inhibitor and At1-Receptor Blocker Attenuate the Production of Vegf in Mesothelial Cells

Peritoneal Dialysis International ◽

10.1177/089686080702700213 ◽

2007 ◽

Vol 27 (2) ◽

pp. 167-172 ◽

Cited By ~ 22

Author(s):

Matthias Sauter ◽

Clemens D. Cohen ◽

Markus Wörnle ◽

Thomas Mussack ◽

Roland Ladurner ◽

...

Keyword(s):

Angiotensin Ii ◽

Ace Inhibitors ◽

Mesothelial Cells ◽

At1 Receptor ◽

Mrna Levels ◽

Vegf Mrna ◽

At1 Antagonist ◽

Tnf Α ◽

Ultrafiltration Failure

Objective Human mesothelial cells (HMC) are a major source of intraperitoneal vascular endothelial growth factor (VEGF) and by that are presumably involved in complications of long-term peritoneal dialysis (PD), such as ultrafiltration failure. This prompted us to look for agents that reduce basic mesothelial VEGF production and abrogate VEGF-overproduction induced by proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1α (IL-1α). Angiotensin-converting enzyme (ACE) inhibition was found to preserve peritoneal function and ameliorate morphologic changes in a rat PD model. The present in vitro study was designed to investigate the effect of the ACE inhibitors captopril and enalapril, and the angiotensin II type 1-receptor (AT1) antagonist losartan on mesothelial VEGF synthesis. Methods HMC were isolated from omental tissue and taken into culture. VEGF antigen concentrations were measured in the cell supernatant by ELISA. VEGF mRNA levels were determined by real-time polymerase chain reaction. Results Incubation of HMC with captopril (100 - 1000 μmol/L) resulted in a concentration-dependent attenuation of VEGF synthesis. Incubation with captopril (500 - 1000 – μmol/L), enalapril (100 - 1000 μmol/L), and losartan (1 100 μmol/L) significantly decreased inflammatory mediator (TNF-α, IL-1α)-induced mesothelial VEGF overproduction. Conclusion The results indicate that ACE inhibitors and AT1-receptor blockers are capable of effectively attenuating the overproduction of VEGF due to proinflammatory cytokine stimuli. These data suggest that locally produced angiotensin II in the peritoneal cavity may be a potential therapeutic target in ultrafiltration failure during long-term PD.

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C-Abl Is Required for the Development of Hyperoxia-Induced Retinopathy

Journal of Experimental Medicine ◽

10.1084/jem.193.12.1383 ◽

2001 ◽

Vol 193 (12) ◽

pp. 1383-1392 ◽

Cited By ~ 10

Author(s):

Irene Nunes ◽

Rosemary D. Higgins ◽

Lucia Zanetta ◽

Peter Shamamian ◽

Stephen P. Goff

Keyword(s):

Mrna Expression ◽

Retinal Neovascularization ◽

Mrna Levels ◽

Vascular Endothelial ◽

Wild Type ◽

Vegf Mrna ◽

Nonreceptor Tyrosine Kinase ◽

Kinase C ◽

Endothelial Growth Factor ◽

Inspired Oxygen

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl–deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

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Intrauterine Exposure to Cadmium Reduces HIF-1 DNA-Binding Ability in Rat Fetal Kidneys

Toxics ◽

10.3390/toxics6030053 ◽

2018 ◽

Vol 6 (3) ◽

pp. 53 ◽

Cited By ~ 2

Author(s):

Tania Jacobo-Estrada ◽

Mariana Cardenas-Gonzalez ◽

Mitzi Santoyo-Sánchez ◽

Frank Thevenod ◽

Olivier Barbier

Keyword(s):

Dna Binding ◽

Kidney Development ◽

Protein Extraction ◽

Hypoxia Inducible Factor ◽

Isotonic Saline ◽

Mrna Levels ◽

Vegf Mrna ◽

Binding Ability ◽

Protein Levels ◽

Cd Exposure

During embryonic development, some hypoxia occurs due to incipient vascularization. Under hypoxic conditions, gene expression is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The activity of this transcription factor can be altered by the exposure to a variety of compounds; among them is cadmium (Cd), a nephrotoxic heavy metal capable of crossing the placenta and reaching fetal kidneys. The goal of the study was to determine Cd effects on HIF-1 on embryonic kidneys. Pregnant Wistar rats were exposed to a mist of isotonic saline solution or CdCl2 (DDel = 1.48 mg Cd/kg/day), from gestational day (GD) 8 to 20. Embryonic kidneys were obtained on GD 21 for RNA and protein extraction. Results show that Cd exposure had no effect on HIF-1α and prolyl hydroxylase 2 protein levels, but it reduced HIF-1 DNA-binding ability, which was confirmed by a decrease in vascular endothelial growth factor (VEGF) mRNA levels. In contrast, the protein levels of VEGF were not changed, which suggests the activation of additional regulatory mechanisms of VEGF protein expression to ensure proper kidney development. In conclusion, Cd exposure decreases HIF-1-binding activity, posing a risk on renal fetal development.

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Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.6.h2247 ◽

1999 ◽

Vol 277 (6) ◽

pp. H2247-H2252 ◽

Cited By ~ 58

Author(s):

R. S. Richardson ◽

H. Wagner ◽

S. R. D. Mudaliar ◽

R. Henry ◽

E. A. Noyszewski ◽

...

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Acute Exercise ◽

Work Rate ◽

Northern Analysis ◽

Endothelial Cell Proliferation ◽

Vastus Lateralis Muscle ◽

Mrna Levels ◽

Vegf Mrna ◽

Exercise Induced

Vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation, both of which are precursors to new capillary growth. Angiogenesis is a vital adaptation to exercise training, and the exercise-induced reduction in intracellular[Formula: see text] has been proposed as a stimulus for this process. Thus we studied muscle cell[Formula: see text] [myoglobin[Formula: see text]([Formula: see text])] during exercise in normoxia and in hypoxia (12% O2) and studied the mRNA levels of VEGF in six untrained subjects after a single bout of exercise by quantitative Northern analysis. Single-leg knee extension provided the acute exercise stimulus: a maximal test followed by 30 min at 50% of the peak work rate achieved in this graded test. Because peak work rate was not affected by hypoxia, the absolute and relative work rates were identical in hypoxia and normoxia. Three pericutaneous needle biopsies were collected from the vastus lateralis muscle, one at rest and then the others at 1 h after exercise in normoxia or hypoxia. At rest (control), VEGF mRNA levels were very low (0.38 ± 0.04 VEGF/18S). After exercise in normoxia or hypoxia, VEGF mRNA levels were much greater (16.9 ± 6.7 or 7.1 ± 1.8 VEGF/18S, respectively). In contrast, there was no measurable basic fibroblast growth factor mRNA response to exercise at this 1-h postexercise time point. Magnetic resonance spectroscopy of myoglobin confirmed a reduction in[Formula: see text] in hypoxia (3.8 ± 0.3 mmHg) compared with normoxia (7.2 ± 0.6 mmHg) but failed to reveal a relationship between [Formula: see text] during exercise and VEGF expression. This VEGF mRNA increase in response to acute exercise supports the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis but questions the importance of a reduced cellular [Formula: see text]as a stimulus for this response.

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