Abstract 637: Role of Nuclear Smooth Muscle Alpha-Actin in the Differentiation of Vascular Smooth Muscle Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Shuangtao Ma ◽  
Dianna M Milewicz
Keyword(s):  
Smooth Muscle ◽  
Chromatin Remodeling ◽  
Vascular Diseases ◽  
Aortic Disease ◽  
Serum Starvation ◽  
Nuclear Fraction ◽  
Missense Mutations ◽  
Differentiation Markers ◽  
Two Dimensional Gel Electrophoresis ◽  
Protein Levels

Although the function of the cytoplasmic smooth muscle (SM) α-actin in contractile units has been well characterized in vascular SMCs, whether the SM α-actin is present in the nucleus and its nuclear function are yet to be determined. Our previous study discovered the mutations in ACTA2 , which encodes SM α-actin, predispose to both thoracic aortic disease and occlusive vascular diseases (stroke and coronary artery disease). We hypothesize that nuclear SM α-actin is critical for differentiation of SMCs. The cytoplasmic and nuclear fractions of mouse SMCs were isolated and the SM α-actin was detected in both cytosol and nucleus by immunoblotting. Moreover, both cytoplasmic and nuclear SM α-actin were dramatically upregulated during the differentiation of SMCs stimulated with TGF-β. Two-dimensional gel electrophoresis, which can separate actin isoforms based on distinct isoelectric points, was used to show that the ratio of SM α-actin to β-actin was increased in nuclear fraction when compared to cytoplasmic fraction with differentiation of SMCs, driven by serum starvation and TGF-β treatment. Furthermore, differentiation was associated with a significantly higher nuclear to cytoplasmic ratio of SM α-actin, indicating SM α-actin accumulates in the nucleus during differentiation. Co-immunoprecipitation experiments identified that nuclear SM α-actin is a component of the chromatin remodeling complex, Ino80, and the immunofluorescence confirmed co-localization of SM α-actin and Ino80 in the nucleus. To investigate the transcriptional function of nuclear SM α-actin, we performed the chromatin immunoprecipitation quantitative PCR, which revealed that the SM α-actin, but not β-actin, accumulated to the promoter region of SMC differentiation markers, including Myh11 , Cnn1 , and Tagln , during the differentiation of SMC. As expected, the Acta2 mutant SMCs (pArg149Cys) disrupt the differentiation of SMCs based on decreased contractile protein levels and increased proliferation. Furthermore, specific Acta2 missense mutations disrupt the ability for SM α-actin to localize to the nucleus. Taken together, our study found the SM α-actin concentrates into the nucleus of SMCs and may play a role in chromatin remodeling required for differentiation of SMCs.

Abstract 17084: Nuclear Smooth Muscle α-actin Participates in Chromatin Remodeling at Smooth Muscle Contractile Gene Promoters

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Callie Kwartler ◽  
Shuangtao Ma ◽  
Caroline Kernell ◽  
Xue-yan Duan ◽  
Charis Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cardiac Muscle ◽  
Chromatin Remodeling ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Nuclear Localization Sequence ◽  
Serum Starvation ◽  
Cell Fractionation ◽  
Gene Promoters ◽  
Chromatin Remodeling Complexes

Actin genes encode for cytoskeletal proteins that polymerize to function in cellular motility, adhesion, and contraction. In mammalian cells, ubiquitously expressed β-actin also moves into the nucleus and associates with chromatin remodeling complexes, however a nuclear function of muscle-specific α-actins has not been previously assessed. We hypothesized that smooth muscle α-actin (SMA) plays a role in chromatin remodeling during the differentiation of smooth muscle cells (SMCs) to enable cell fate specification of SMCs. In explanted SMCs from human and mouse ascending aortas, cell fractionation and 2D gel electrophoresis identify both SMA and β-actin in the nuclear lysates. Nuclear SMA but not β-actin accumulates with SMC differentiation driven by serum starvation and transforming growth factor-β1 treatment. SMA accumulates into the nucleus early in the differentiation of SMCs from neural crest progenitor cells, prior to cytosolic accumulation. Immunoprecipitation studies show that SMA binds specifically to the INO80 and the SWI/SNF chromatin remodeling complexes, and this binding increases with SMC differentiation. Chromatin immunoprecipitation reveals that SMA is bound to the promoters of SMC-specific genes, including Acta2 , Cnn1, and Myh11 and that SMA is enriched over β-actin at these promoters with SMC differentiation. Finally, overexpression of SMA tagged with a nuclear localization sequence (NLS) in multiple cell types increases expression of SMC markers, whereas NLS-tagged β-actin localizes to the nucleus to the same extent but does not increase SMC marker expression in any cell type. Finally, we assessed whether skeletal muscle α-actin (SKA) and cardiac muscle α-actin (CMA) may play a similar role in skeletal and cardiac muscle cells. Both SKA and CMA translocate into the nucleus. CMA accumulates into the nucleus early in the differentiation of cardiomyocytes from pluripotent stem cells. Immunoprecipitation reveals that SKA binds to the SWI/SNF complex in differentiated C2C12 myotube cell cultures. These data support that nuclear SMA enriches with and participates in SMC differentiation, and suggest a potential nuclear role for other muscle specific α-actins in developing muscle cells.

Abstract 67: A Rare Variant in Myh11, R247C, Affects Smooth Muscle Cell Phenotype and Acts as a Modifier for Aortic Disease

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Callie Kwartler ◽  
Shao-Qing Kuang ◽  
Siddharth Prakash ◽  
Katerina Byanova ◽  
John Pham ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Rare Variant ◽  
Focal Adhesions ◽  
Aortic Disease ◽  
Aortic Aneurysms ◽  
Cell Phenotype ◽  
Mendelian Disease ◽  
Missense Mutations ◽  
Degree Relative ◽  
Gene And Protein Expression

Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-fifth of TAAD patients have a first-degree relative with the disease, suggesting a genetic component to its pathogenesis. Missense mutations in a number of genes lead to TAAD, including MYH11 ,which encodes the smooth muscle isoform of myosin heavy chain. However, potential genetic contributors in the four-fifths of patients without affected relatives have not been explored. Variants of unknown significance in MYH11 are prevalent in the population, and are enriched in patients with TAAD and other vascular diseases. The goal of this study was to evaluate one of these variants as a potential contributor to TAAD. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11 R247C/R247C mice also have increased neointimal proliferation following carotid ligation and increased proliferation of vascular smooth muscle cells (SMCs) in vitro. Myh11 R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, adhesions are smaller in Myh11 R247C/R247C SMCs, and there is increased Rac1 and focal adhesion kinase (FAK) activation with decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11 R247C/R247C SMCs, and FAK inhibition reduces proliferation. Since this rare variant occurs in the general population, we sought to determine if it acts as a modifier gene for aortic disease. The Myh11 R247C allele was bred into Acta2 -/- mice, which develop aortic aneurysms, and into Acta2 +/- mice, which do not. In both cases, the presence of the R247C allele increases aortic diameter. These preliminary data suggest that the MYH11 R247C rare variant may be a genetic contributor to TAAD in patients without an inherited mutation, or may affect the clinical presentation of TAAD in patients with Mendelian disease.

scholarly journals 6A3-5/Osa2 is an Early Activated Gene Implicated in the Control of Vascular Smooth Muscle Cell Functions

2006 ◽  
Vol 2006 ◽  
pp. 1-17
Author(s):  
Gwenaele Garin ◽  
Kazem Zibara ◽  
Frederick Aguilar ◽  
Ming Lo ◽  
Adam Hurlstone ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Nuclear Localization ◽  
Molecular Mechanisms ◽  
Vascular Diseases ◽  
Interaction Domain ◽  
Protein Levels ◽  
Cell Functions ◽  
New Family ◽  
Brain Cdna Library

Vascular smooth muscle cells (VSMC) growth plays a key role in the pathophysiology of vascular diseases. However, the molecular mechanisms controlling gene transcription in VSMC remain poorly understood. We previously identified, by differential display, a new gene (6A3-5) overexpressed in proliferating rat VSMC. In this study, we have cloned the full-length cDNA by screening a rat foetal brain cDNA library and investigated its functions. The 6A3-5 protein shows 4 putative conserved functional motifs: a DNA binding domain called ARID (AT-rich interaction domain), two recently described motifs (Osa Homology Domain), and a nuclear localization signal. The deduced protein sequence was observed to be 85% identical to the recently described human Osa2 gene. Immunolabelling, using an anti-6A3-5/Osa2 monoclonal antibody, showed a nuclear localization of the 6A3-5/Osa2 protein. In addition, PDGF upregulated 6A3-5/Osa2 expression at both the transcript and protein levels in a dose and time-dependent fashion. The pattern of upregulation by PDGF was reminiscent of the early responsive gene c-fos. The PDGF-induced upregulation of 6A3-5/Osa2 and proliferation of VSMC were significantly inhibited in a dose and sequence-dependent fashion by an antisense, but not by sense, scrambled or mismatched oligonucleotides directed against 6A3-5/Osa2. In VSMC of aortas derived from hypertensive (LH) rats, 6A3-5/Osa2 is overexpressed as compared to that in normotensive (LL) rats. The 6A3-5/Osa2-gene expression is downregulated by an ACE inhibitor and upregulated by exogenous AngiotensinII in LH rats. In summary, these results indicate that 6A3-5/Osa2 is an early activated gene that belongs to a new family of proteins involved in the control of VSMC growth.

scholarly journals 7-O-methylpunctatin, a Novel Homoisoflavonoid, Inhibits Phenotypic Switch of Human Arteriolar Smooth Muscle Cells

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 716 ◽  
Author(s):  
Fardoun ◽  
Iratni ◽  
Dehaini ◽  
Eid ◽  
Ghaddar ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Migration And Invasion ◽  
Phenotypic Switch ◽  
Differentiation Markers ◽  
Protein Levels ◽  
Anti Inflammatory ◽  
Inflammatory Action ◽  
Synthetic Phenotype

Remodeling of arterioles is a pivotal event in the manifestation of many inflammation-based cardio-vasculopathologies, such as hypertension. During these remodeling events, vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. The latter is characterized by increased proliferation, migration, and invasion. Compounds with anti-inflammatory actions have been successful in attenuating this phenotypic switch. While the vast majority of studies investigating phenotypic modulation were undertaken in VSMCs isolated from large vessels, little is known about the effect of such compounds on phenotypic switch in VSMCs of microvessels (microVSMCs). We have recently characterized a novel homoisoflavonoid that we called 7-O-methylpunctatin (MP). In this study, we show that MP decreased FBS-induced cell proliferation, migration, invasion, and adhesion. MP also attenuated adhesion of THP-1 monocytes to microVSMCs, abolished FBS-induced expression of MMP-2, MMP-9, and NF-κB, as well as reduced activation of ERK1/2 and FAK. Furthermore, MP-treated VSMCs showed an increase in early (myocardin, SM-22α, SM-α) and mid-term (calponin and caldesmon) differentiation markers and a decrease in osteopontin, a protein highly expressed in synthetic VSMCs. MP also reduced transcription of cyclin D1, CDK4 but increased protein levels of p21 and p27. Taken together, these results corroborate an anti-inflammatory action of MP on human microVSMCs. Therefore, by inhibiting the synthetic phenotype of microVSMCs, MP may be a promising modulator for inflammation-induced arteriolar pathophysiology.

scholarly journals European reference network for rare vascular diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Ingrid M. B. H. van de Laar ◽  
Eloisa Arbustini ◽  
Bart Loeys ◽  
Erik Björck ◽  
Lise Murphy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Health Care Professionals ◽  
Consensus Statement ◽  
Vascular Diseases ◽  
Aortic Disease ◽  
Current Data ◽  
Reference Network ◽  
Multicenter Studies ◽  
Pathogenic Variants ◽  
European Reference Network

AbstractThe ACTA2 gene encodes for smooth muscle specific α-actin, a critical component of the contractile apparatus of the vascular smooth muscle cell. Pathogenic variants in the ACTA2 gene are the most frequently encountered genetic cause of non-syndromic hereditary thoracic aortic disease (HTAD). Although thoracic aortic aneurysm and/or dissection is the main clinical manifestation, a variety of occlusive vascular disease and extravascular manifestations occur in ACTA2-related vasculopathy. Current data suggest possible mutation-specific manifestations of vascular and extra-aortic traits.Despite its relatively high prevalence, comprehensive recommendations on the care of patients and families with pathogenic variants in ACTA2 have not yet been established. We aimed to develop a consensus document to provide medical guidance for health care professionals involved in the diagnosis and treatment of patients and relatives with pathogenic variants in ACTA2.The HTAD Working Group of the European Reference Network for Rare Vascular Diseases (VASCERN) convened to review current literature and discuss expert opinions on clinical management of ACTA2 related vasculopathy. This consensus statement summarizes our recommendations on diagnosis, monitoring, treatment, pregnancy, genetic counselling and testing in patients with ACTA2-related vasculopathy. However, there is a clear need for additional prospective multicenter studies to further define proper guidelines.

Effect of vitamin A deficiency on cardiovascular function in the rat

2002 ◽  
Vol 80 (1) ◽  
pp. 1-7 ◽  
Author(s):  
G L Wright ◽  
S Wang ◽  
M E Fultz ◽  
I Arif ◽  
K Matthews ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vitamin A ◽  
Vitamin A Deficiency ◽  
Cardiovascular Function ◽  
Differentiation Markers ◽  
High Potassium ◽  
Protein Levels ◽  
Relaxant Response

Selected parameters of cardiovascular function were evaluated in vitamin A-deficient rats at 70 days of age. Resting heart rate was increased by an average of 100 bpm (21.4 ± 2.7%), whereas resting systolic blood pressure was normal in vitamin A-deficient animals. The maximal contractile force developed per milligram weight of tissue by aortic rings excised from vitamin A-deficient animals was reduced in response to high potassium (–25.0 ± 8.7%) and phorbol 12,13-dibutyrate (–36.8 ± 8.4%) but was only slightly reduced in response to norepinephrine (–17.8 ± 11.1%). Intimal rubbing to remove the endothelium had no effect on the loss in contractile responsiveness, and the relaxant response to acetylcholine was similar between control and vitamin A-deficient tissue groups. This suggests that the decrease in contractility of vascular smooth muscle from the vitamin A-deficient rats did not involve altered release of endothelium-derived vasoactive factors. Western blot analysis suggested a reduction in the protein levels of several differentiation markers including α-actin (–22%), calponin (–37%), desmin (–37%), and vinculin (–40%), whereas the level of PKCα was unchanged from control values. Our findings indicate a significant decrease in contractile responsiveness of aortic smooth muscle of the vitamin A-deficient rat that may be associated with a down regulation in the expression of contractile-related proteins.Key words: vitamin A, retinoids, in vivo, cardiovascular, contractility, blood pressure.

Abstract 169: Smad3 Drives Cross-Talk Between TGFß and Wnt/ß-Catenin Signaling Pathways in Smooth Muscle Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Daniel M DiRenzo ◽  
Xu Dong Shi ◽  
Lian-Wang Guo ◽  
K Craig Kent
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Wnt Signaling ◽  
Vascular Injury ◽  
Cross Talk ◽  
Intimal Hyperplasia ◽  
Muscle Cells ◽  
Arterial Injury ◽  
Protein Levels ◽  
Qrt Pcr

Restenosis (neo-intimal hyperplasia) occurs in approximately 25-50% of patients undergoing arterial interventions, primarily due to the proliferation and migration of arterial smooth muscle cells (SMCs) into the peri-luminal area. Recently, Wnt/β-catenin signaling has been shown to promote SMC proliferation and enhance neo-intimal hyperplasia but its mechanism of activation is unclear. Interestingly, Wnt/β-catenin has been shown to be activated by TGFβ in mesenchymal stem cells and fibroblasts. We have shown that TGFβ and its downstream signaling protein, Smad3, are upregulated following vascular injury and that Smad3 overexpressing SMCs display enhanced proliferation, migration, and neo-intimal hyperplasia. These results led us to hypothesize that TGFβ, through Smad3, activates Wnt/β-catenin to regulate SMC behavior following arterial injury . In primary rat SMCs, TGFβ (5ng/mL) led to β-catenin activation and relocalization from the plasma membrane to the cytoplasm / nucleus within 24 hours. Furthermore, qRT-PCR results demonstrated that expression of Wnt11 (22 fold) and Wnt9a (3.9 fold) were significantly upregulated after 24 hours of TGFβ stimulation (p<0.05, n=3). In addition, 24 hours of TGFβ stimulation in SMCs overexpressing Smad3 (TGFβ/Smad3) further enhanced the gene expression of Wnt11 (>300 fold) and Wnt9a (14 fold) and also stimulated significant increases in Wnt2b (41 fold), Wnt5a (2.9 fold), and Wnt4 (3.2 fold) (p<0.05, n=3) as measured by qRT-PCR. Western blot results demonstrated that the combined TGFβ/Smad3 stimulation increased β-catenin protein levels, suggesting that TGFβ activates canonical Wnt signaling leading to stabilization of β-catenin protein. In normal rat carotid arteries, β-catenin protein was undetectable via immunohistochemistry but could be seen in SMCs of the vessel media at 3 days post-balloon angioplasty and in neo-intimal cells at 7 and 14 days. Smad3 was also expressed in neo-intimal cells at 7 and 14 days post-angioplasty suggesting that TGFβ, through Smad3, is responsible for Wnt/β-Catenin activation during vascular injury. In conclusion, this work describes a novel cross-talk in SMCs between TGFβ and Wnt signaling which may provide a viable target for future anti-restenotic treatments.

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells

2000 ◽  
Vol 279 (1) ◽  
pp. C248-C256 ◽  
Author(s):  
Liu Hua Wei ◽  
Aaron T. Jacobs ◽  
Sidney M. Morris ◽  
Louis J. Ignarro
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Growth ◽  
Muscle Cells ◽  
Protein Levels ◽  
Ifn Γ ◽  
Arginase Ii ◽  
Arginase I

The objectives of this study were to determine whether rat aortic smooth muscle cells (RASMC) express arginase and to elucidate the possible mechanisms involved in the regulation of arginase expression. The results show that RASMC contain basal arginase I (AI) activity, which is significantly enhanced by stimulating the cells with either interleukin (IL)-4 or IL-13, but arginase II (AII) expression was not detected under any condition studied here. We further investigated the signal transduction pathways responsible for AI induction. AI mRNA and protein levels were enhanced by addition of forskolin (1 μM) and inhibited by H-89 (30 μM), suggesting positive regulation of AI by a protein kinase A pathway. Genistein (10 μg/ml) and sodium orthovanadate (Na3VO4; 10 μM) were used to investigate the role of tyrosine phosphorylation in the control of AI expression. Genistein inhibited, whereas Na3VO4enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon (IFN)-γ were investigated for their effects on AI induction. Dex (1 μM) and IFN-γ (100 U/ml) alone had no effect on basal AI expression in RASMC, but both reduced AI induction by IL-4 and IL-13. In combination, Dex and IFN-γ abolished AI induction by IL-4 and IL-13. Finally, both IL-4 and IL-13 significantly increased RASMC DNA synthesis as monitored by [3H]thymidine incorporation, demonstrating that upregulation of AI is correlated with an increase in cell proliferation. Blockade of AI induction by IFN-γ, H-89, or genistein also blocked the increase in cell proliferation. These observations are consistent with the possibility that upregulation of AI might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

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.

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