The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries.
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ABSTRACT:
SUBMITTER: Eyster KM
PROVIDER: S-EPMC2080788 | biostudies-literature | 2007 Oct
REPOSITORIES: biostudies-literature
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The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries.
Eyster Kathleen M KM Mark Connie J CJ Gayle Richard R Martin Douglas S DS
Vascular pharmacology 20070703 4
A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of this gender difference are the sex steroids, estrogen and testosterone. The vasculature is considered to be a site of action of these steroids. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 ...[more]
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Project description:Goal of the experiment: To examine the effects of estrogen and testosterone on gene expression in the rat mesenteric arteries. Brief description of the experiment: A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of gender differences in cardiovascular disease are the sex steroids, estrogen and testosterone. The effect of estrogen on the cardiovascular system has been expected to be protective but this concept has become controversial. The effect of testosterone on the cardiovascular system is considered to be deleterious. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. Since estrogen and testosterone exert many of their effects through genomic mechanisms, the DNA microarray is an excellent tool for assessing their effects in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day). The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays (GE) were used to identify differential gene expression. Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR. The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone. 3alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone. The aryl hydrocarbon nuclear translocator-like was reduced by testosterone. These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature. Keywords: hormone treatment, vasculature, gender differences Experimental factors: hormone treatment Keywords: hormone treatment
2007-09-30 | GSE4782 | GEO
Project description:Estrogen has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Gene targets of estrogen and SERMs in the vasculature are not well-known. Thus, the present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation.
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Project description:BACKGROUND AND PURPOSE:PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone. EXPERIMENTAL APPROACH:In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings. KEY RESULTS:In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with l-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSIONS AND IMPLICATIONS:Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.
| S-EPMC5659990 | biostudies-literature
Project description:Deep sequencing of RNA samples from rat small mesenteric arteries (MA) and aorta (AO) identified common and unique features of their gene programs. ~5% of mRNAs were quantitatively differentially expressed in MA versus AO. Unique transcriptional control in MA smooth muscle is suggested by the selective or enriched expression of transcription factors Nkx2-3, HAND2, and Tcf21 (Capsulin). Enrichment in AO of PPAR transcription factors and their target genes of mitochondrial function, lipid metabolism, and oxidative phosphorylation is consistent with slow (oxidative) tonic smooth muscle. In contrast MA was enriched in contractile and calcium channel mRNAs suggestive of components of fast (glycolytic) phasic smooth muscle. Myosin phosphatase regulatory subunit paralogs Mypt1 and p85 were expressed at similar levels, while smooth muscle MLCK was the only such kinase expressed, suggesting functional redundancy of the former but not the latter in accordance with mouse knockout studies. With regard to vaso-regulatory signals, purinergic receptors P2rx1 and P2rx5 were reciprocally expressed in MA versus AO, while the olfactory receptor Olr59 was enriched in MA. Alox15, which generates the EDHF HPETE, was enriched in MA while eNOS was equally expressed, consistent with the greater role of EDHF in the smaller arteries. mRNAs that were not expressed at a level consistent with impugned function include skeletal myogenic factors, IKK2, nonmuscle myosin, and Gnb3. This screening analysis of gene expression in the small mesenteric resistance arteries suggests testable hypotheses regarding unique aspects of small artery function in the regional control of blood flow.
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Project description:Obesity hypertension is driven by sympathetic neurotransmission to the heart and blood vessels. We tested the hypothesis that high-fat diet (HFD)-induced hypertension is driven by sympathetic neurotransmission to mesenteric arteries (MA) in male but not female Dahl salt-sensitive (Dahl ss) rat. Rats were fed a control diet (CD; 10?kcal% from fat) or HFD (60?kcal% from fat) beginning at 3?weeks (wk) of age; measurements were made at 10-, 17- and 24-wk. Body weight increased with HFD, age and sex. Mean arterial pressure (MAP) was higher in HFD versus CD rats from both sexes at 17- and 24-wk. MA constriction measured using pressure myography, and electrical field stimulation (EFS, 0.2-30?Hz) was greater in HFD versus CD in males at 17-wk; this was not due to changes in ?2 autoreceptor or norepinephrine transporter (NET) function. Prazosin (?1-AR antagonist) and suramin (P2 receptor antagonist) inhibited neurogenic MA constriction equally in all groups. Arterial reactivity to exogenous norepinephrine (NE; 10-8 - 10-5?M) was lower in HFD versus CD at 10-wk in males. Female MA reactivity to exogenous ATP was lower at 24-weeks compared to earlier time points. HFD did not affect tyrosine hydroxylase (TH) or the vesicular nucleotide transporter (VNUT) nerve density in MA from both sexes. NE content was lower in MA but higher in plasma at 24-wk compared to 10- and 17-wk in both sexes. In conclusion, HFD-induced hypertension is not driven by increased sympathetic neurotransmission to MA in male and female Dahl ss rats.
| S-EPMC6931401 | biostudies-literature
Project description:Background and purposeNuciferine, a constituent of lotus leaf, is an aromatic ring-containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanismExperimental approachNuciferine-induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca(2+) level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging.Key resultsNuciferine induced relaxations in arterial segments pre-contracted by KCl or phenylephrine. Nuciferine-elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L-NAME or the NO-sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser(1177) and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca(2+) influx. Under endothelium-free conditions, nuciferine attenuated CaCl2-induced contraction in Ca(2+)-free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl2. Nuciferine also suppressed Ca(2+) influx in Ca(2+)-free K(+)-containing solution in VSMCs.Conclusions and implicationsNuciferine has a vasorelaxant effect via both endothelium-dependent and -independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction.
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Project description:B. monnieri extract (BME) is an abundant source of bioactive compounds, including saponins and flavonoids known to produce vasodilation. However, it is unclear which components are the more effective vasodilators. The aim of this research was to investigate the vasorelaxant effects and mechanisms of action of saponins and flavonoids on rat isolated mesenteric arteries using the organ bath technique. The vasorelaxant mechanisms, including endothelial nitric oxide synthase (eNOS) pathway and calcium flux were examined. Saponins (bacoside A and bacopaside I), and flavonoids (luteolin and apigenin) at 0.1-100 µM caused vasorelaxation in a concentration-dependent manner. Luteolin and apigenin produced vasorelaxation in endothelial intact vessels with more efficacy (Emax 99.4 ± 0.7 and 95.3 ± 2.6%) and potency (EC50 4.35 ± 1.31 and 8.93 ± 3.33 µM) than bacoside A and bacopaside I (Emax 83.6 ± 2.9 and 79.9 ± 8.2%; EC50 10.8 ± 5.9 and 14.6 ± 5.4 µM). Pretreatment of endothelial intact rings, with L-NAME (100 µM); an eNOS inhibitor, or removal of the endothelium reduced the relaxant effects of all compounds. In K+-depolarised vessels suspended in Ca2+-free solution, these active compounds inhibited CaCl2-induced contraction in endothelial denuded arterial rings. Moreover, the active compounds attenuated transient contractions induced by 10 µM phenylephrine in Ca2+-free medium containing EGTA (1 mM). Thus, relaxant effects occurred in both endothelial intact and denuded vessels which signify actions through both endothelium and vascular smooth muscle cells. In conclusion, the flavonoids have about twice the potency of saponins as vasodilators. However, in the BME, there is ~20 × the amount of vaso-reactive saponins and thus are more effective.
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Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation.
2011-07-06 | GSE23037 | GEO
Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation. Goal of the experiment: To examine the effects of three formulations of estrogen (ethinyl estradiol, equilin, and estradiol benzoate) and of the selective estrogen receptor modulators, tamoxifen and raloxifene, on gene expression in the mesenteric arteries of the rat. Keywords: rat, artery, gene expression, mesentery, estrogen, tamoxifen, raloxifene Experimental factors: hormone treatment, drug treatment Experimental design: Prepubertal female Sprague Dawley rats (Harlan, Indianapolis, IN) were ovariectomized at 4 weeks of age, prior to the initiation of regular ovarian cycles. The animals were allowed to recover for 2 weeks before the experimental protocol was initiated. The rats were randomly divided into 6 groups (n=3-4/group) and treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After 3 weeks of treatment, the rats were euthanized and the mesentery obtained. Total RNA was extracted from each individual sample and used for the synthesis of labeled cRNA for microarray analysis. After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated and harvested and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Mesenteric artery RNA was extracted and purified using an RNeasy Mini Kit (Qiagen). Any potentially contaminating DNA was removed by treatment with RNase-free DNase (Qiagen) on the RNeasy column. Total RNA was quantified and RNA integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA). Following analysis, the RNA was stored at -80ºC until it was used for gene expression studies. DNA microarray: CodeLink Whole Rat Genome Bioarrays (Applied Microarrays, Tempe, AZ) were used. Five μg total RNA was used from each mesenteric arterial sample for the microarray analysis. Complementary RNA was synthesized per company protocol (Ambion). A set of bacterial RNAs was added to each total RNA sample and carried through the synthesis and labeling reactions as controls and for nor¬malization of the data. The microarrays were scanned with the GenePix 4000B (Axon Instruments, Union City, CA) with GenePix Pro 5.0 software (Axon Instruments). Analysis of the microar¬rays was performed with the CodeLink Expression Analysis software (Applied Microarrays) and Acuity 3.1 software (Axon), and statistical analysis was performed with GeneSpring 7.1 software (Agilent, Santa Clara, CA). A sample of n=3 was completed with the microarrays for each treatment. Quality control steps: The cRNA that was synthesized from each mesenteric artery treatment sample was used for hybridization to a single CodeLink (Applied Microarrays) whole rat genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were used. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were isolated mesenteric arteries from individual Sprague Dawley rats that had been treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). Manipulations of biological samples and protocols used: Ovariectomized female rats were treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated, harvested, and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Experimental factor: hormone treatment, drug treatment Technical protocols: The mesenteric arteries were minced with scalpels and TRI reagent (MRC) was added. The tissue was homogenized with a Polytron homogenizer. Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labeled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 5.0 microgram of rat mesenteric artery total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides (CodeLink Whole Rat Genome Bioarrays), and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2011-07-06 | E-GEOD-23037 | biostudies-arrayexpress
Project description:The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated myogenic constriction and vascular function of mesenteric arteries of rats chronically treated with FTY720. Wistar rats received FTY720 1mg/kg/daily for six weeks. At termination, blood pressure was recorded and small mesenteric arteries collected for vascular studies in a perfusion set up. Myogenic constriction to increased intraluminal pressure was low, but a sub-threshold dose of S1P profoundly augmented myogenic constriction in arteries of both controls and animals chronically treated with FTY720. Interestingly, endothelial denudation blocked the response to S1P in arteries of FTY720-treated animals, but not in control rats. In acute experiments, presence of FTY720 significantly augmented the contractile response to S1P, an effect that was partially abolished after the inhibition of cyclooxygenase (COX-)-derived prostaglandins. FTY720 down regulated S1P1 but not S1P2 in renal resistance arteries and in cultured human endothelial cells. This study therefore demonstrates the endothelium is able to compensate for the complete loss of responsiveness of the smooth muscle layer to S1P after long term FTY720 treatment through a mechanism that most likely involves enhanced production of contractile prostaglandins by the endothelium.
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