scholarly journals An oxidative DNA “damage” and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression

2015 ◽  
Vol 309 (11) ◽  
pp. L1367-L1375 ◽  
Author(s):  
Viktor Pastukh ◽  
Justin T. Roberts ◽  
David W. Clark ◽  
Gina C. Bardwell ◽  
Mita Patel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Mrna Expression ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Oxidative Dna Damage ◽  
Vegf Mrna ◽  
Base Modifications ◽  
Inducible Genes ◽  
Vegf Mrna Expression

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

scholarly journals Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms

2017 ◽  
Vol 313 (1) ◽  
pp. H179-H189 ◽  
Author(s):  
Adam H. Korayem ◽  
Patricio E. Mujica ◽  
Haruo Aramoto ◽  
Ricardo G. Durán ◽  
Prerna R. Nepali ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Striated Muscle ◽  
Ischemia Reperfusion ◽  
Physiological Mechanism ◽  
Camp Concentration ◽  
Vegf Mrna ◽  
Downstream Effector ◽  
Vegf Mrna Expression ◽  
Exchange Protein

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that 1) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, 2) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and 3) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that 1) different hyperpermeability mechanisms operate after brief and prolonged ischemia and 2) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury. NEW & NOTEWORTHY Here, we demonstrate that 1) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells; 2) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and 3) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Abstract 191: NF-?B p65 Methylation by Protein Arginine Methyltransferase 5 (PRMT5) is Necessary for the Transcriptional Induction of CXCL10 by TNF-a

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Daniel P Harris
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Chromatin Immunoprecipitation ◽  
Proximal Promoter ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Expression Of Genes ◽  
Tnf Α ◽  
Affect Expression ◽  
Transcriptional Induction

TNF-α initiates the expression of genes involved in the recruitment, adhesion, and transmigration of leukocytes to sites of inflammation. Here, we report that the protein arginine methyltransferase PRMT5 is required for the transcriptional induction of the pro-inflammatory chemokine CXCL10 (IP-10) in endothelial cells. Depletion of PRMT5 by siRNA results in significantly diminished TNF-α-induced CXCL10 mRNA expression, but does not affect expression of other chemokines, such as MCP-1 or IL-8. Chromatin immunoprecipitation experiments of the CXCL10 proximal promoter show the presence of symmetrical dimethylated arginine (sDMA)-containing proteins upon exposure to TNF-α. This methylation is completely lost when PRMT5 is removed from cells by siRNA. Using immunoprecipitation, we show that PRMT5 enhances CXCL10 expression by methylating the RelA (p65) subunit of NF-κB. In summary, we have identified that PRMT5 is a novel regulator of CXCL10 expression. Further, we have discovered that PRMT5 methylates NF-κB, a finding which may further knowledge of the post-translational code governing NF-κB regulation and target specificity.

scholarly journals Adenosine upregulates VEGF expression in cultured myocardial vascular smooth muscle cells

1999 ◽  
Vol 277 (2) ◽  
pp. H595-H602 ◽  
Author(s):  
Jian-Wei Gu ◽  
Ann L. Brady ◽  
Vivek Anand ◽  
Michael C. Moore ◽  
Whitney C. Kelly ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Vascular Smooth Muscle Cells ◽  
Vegf Expression ◽  
Vegf Mrna ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Vegf Mrna Expression ◽  
Vegf Protein

We tested whether adenosine has differential effects on vascular endothelial growth factor (VEGF) expression under normoxic and hypoxic conditions, and whether A1 or A2 receptors (A1R; A2R) mediate these effects. Myocardial vascular smooth muscle cells (MVSMCs) from dog coronary artery were exposed to hypoxia (1% O2) or normoxia (20% O2) in the absence and presence of adenosine agonists or antagonists for 18 h. VEGF protein levels were measured in media with ELISA. VEGF mRNA expression was determined with Northern blot analysis. Under normoxic conditions, the adenosine A1R agonists, N 6-cyclopentyladenosine and R(-)- N 6-(2-phenylisopropyl)adenosine did not increase VEGF protein levels at A1R stimulatory concentrations. However, adenosine (5 μM) and the adenosine A2R agonist N 6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)]ethyl adenosine (DPMA; 100 nM) increased VEGF protein levels by 51 and 132% and increased VEGF mRNA expression by 44 and 90%, respectively, in cultured MVSMCs under normoxic conditions. Hypoxia caused an approximately fourfold increase in VEGF protein and mRNA expression, which could not be augmented with exogenous adenosine, A2R agonist (DPMA), or A1R agonist [1,3-diethyl-8-phenylxanthine (DPX)]. The A2R antagonist 8-(3-chlorostyryl)-caffeine completely blocked adenosine-induced VEGF protein and mRNA expression and decreased baseline VEGF protein levels by up to ∼60% under normoxic conditions but only by ∼25% under hypoxic conditions. The A1R antagonist DPX had no effect. These results are consistent with the hypothesis that 1) adenosine increases VEGF protein and mRNA expression by way of A2R. 2) Adenosine plays a major role as an autocrine factor regulating VEGF expression during normoxic conditions but has a relatively minor role during hypoxic conditions. 3) Endogenous adenosine can account for the majority of basal VEGF secretion by MVSMCs under normoxic conditions and could therefore be a maintenance factor for the vasculature.

The relationship of EGFR and VEGF mRNA expression in ovarian carcinoma

2009 ◽  
Vol 6 (2) ◽  
pp. 100-103 ◽  
Author(s):  
Aiping Chen ◽  
Ruirui Yang ◽  
Hongling Zhang ◽  
Hui Song
Keyword(s):  
Ovarian Carcinoma ◽  
Mrna Expression ◽  
Vegf Mrna ◽  
Relationship Of ◽  
The Relationship ◽  
Vegf Mrna Expression

REAL TIME RT-PCR VEGF mRNA EXPRESSION AND ERK1 EXPRESSION AND ACTIVITY IN RENAL TUMORS

1999 ◽  
pp. 148
Author(s):  
Donata Villari ◽  
Giulio Nicita ◽  
Carmela Tricarico ◽  
Astrid Parenti ◽  
Alessandro Della Melina ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Renal Tumors ◽  
Rt Pcr ◽  
Vegf Mrna ◽  
Vegf Mrna Expression ◽  
Expression And Activity

The Role Of PO2, HbO2 and Exercise in Human VEGF mRNA Expression

2004 ◽  
Vol 36 (Supplement) ◽  
pp. S52???s53
Author(s):  
Lesley Lawrenson ◽  
Jennifer G. Poole ◽  
I. Mark Olfert ◽  
Russell S. Richardson
Keyword(s):  
Mrna Expression ◽  
Vegf Mrna ◽  
Vegf Mrna Expression
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