The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE4782 | GEO | 2007/09/30
SECONDARY ACCESSION(S): PRJNA95535
REPOSITORIES: GEO
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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 effects of estrogen and testosterone on gene expression in the rat mesenteric arteries
| PRJNA95535 | ENA
Project description:Kidney stone disease is influenced by multiple factors, including but not limited to age, gender, genetic background, hydration status, diet and drug. Regarding the gender, epidemiologic data across the world has shown that females at the reproductive age (15-49 years) have lower incidence/prevalence of kidney stone disease approximately 1.5-2.5 folds as compared to males at the same age. However, this gap is narrower in the postmenopausal age, whereas the postmenopausal females with higher serum estrogen levels are less likely to have kidney stones. Furthermore, female stone formers (patients with kidney stones) are associated with lower estrogen levels. Therefore, estrogen has been proposed to serve as the protective hormone against kidney stone disease. However, the precise mechanisms underlying such protective effects of estrogen remain unclear and require further investigations. This study thus investigated the effects of estradiol (which is the most prevalent and potent form of estrogen in females at the reproductive age) on cellular proteome of renal tubular cells using a proteomics approach.
2019-09-09 | PXD014768 | Pride
Project description:The role of estrogen and testosterone in the regulation of gene expression in the proximal reproductive tract is not completely understood. To address this question, mice were treated with testosterone or estradiol and RNA from the efferent ducts and caput epididymis was processed and hybridized to Affymetrix MOE 430 2.0 microarrays. Analysis of array output identified probe sets in each tissue with altered levels in hormone treated versus control animals. Hormone treatment efficacy was confirmed by determination of serum hormone levels pre- and post-treatment and observed changes in transcript levels of previously reported hormone-responsive genes. Tissue-specific hormone sensitivity was observed with 2867 and 3197 probe sets changing significantly in the efferent ducts after estrogen and testosterone treatment, respectively. In the caput epididymis, 117 and 268 probe sets changed after estrogen and testosterone treatment, respectively, demonstrating a greater response to hormone in the efferent ducts than the caput epididymis. Transcripts sharing similar profiles in the intact and hormone-treated animals compared with castrated controls were also identified. Ontological analysis of probe sets revealed a significant number of hormone-regulated transcripts encode proteins associated with lipid metabolism, transcription and steroid metabolism in both tissues. Real-time RT-PCR was employed to confirm array data and investigate other potential hormone-responsive regulators of proximal reproductive tract function. The results of this work reveal previously unknown responses to estrogen in the caput epididymis and to testosterone in the efferent ducts as well as tissue specific hormone sensitivity in the proximal reproductive tract.
2009-12-31 | GSE17553 | GEO
Project description:The role of estrogen and testosterone in the regulation of gene expression in the proximal reproductive tract is not completely understood. To address this question, mice were treated with testosterone or estradiol and RNA from the efferent ducts and caput epididymis was processed and hybridized to Affymetrix MOE 430 2.0 microarrays. Analysis of array output identified probe sets in each tissue with altered levels in hormone treated versus control animals. Hormone treatment efficacy was confirmed by determination of serum hormone levels pre- and post-treatment and observed changes in transcript levels of previously reported hormone-responsive genes. Tissue-specific hormone sensitivity was observed with 2867 and 3197 probe sets changing significantly in the efferent ducts after estrogen and testosterone treatment, respectively. In the caput epididymis, 117 and 268 probe sets changed after estrogen and testosterone treatment, respectively, demonstrating a greater response to hormone in the efferent ducts than the caput epididymis. Transcripts sharing similar profiles in the intact and hormone-treated animals compared with castrated controls were also identified. Ontological analysis of probe sets revealed a significant number of hormone-regulated transcripts encode proteins associated with lipid metabolism, transcription and steroid metabolism in both tissues. Real-time RT-PCR was employed to confirm array data and investigate other potential hormone-responsive regulators of proximal reproductive tract function. The results of this work reveal previously unknown responses to estrogen in the caput epididymis and to testosterone in the efferent ducts as well as tissue specific hormone sensitivity in the proximal reproductive tract. Adult animals were castrated or sham-castrated, allowed to recover for 14 days, and then treated with 0.015 mg estradiol (castrated), 0.015 mg testosterone propionate (castrated), or vehicle (castrated and sham-castrated as biological controls) in duplicate. Efferent duct and caput epididymis was collected from each sample and analyzed. Duplicates are included in the provided data and numbered 1 or 2 for each treatment regimen.
2009-12-30 | E-GEOD-17553 | biostudies-arrayexpress
Project description:The goal and objective of this study was to identify the transcriptional profiles differentiating the artery, vein, and lymphatic lineages in the adult rat vasculature with particular emphasis on the unique elements of the collecting lymphatic vessel transcriptome. A 2 x 3 experimental design was utilized in which parallel arteries, veins, and lymphatics from two different tissue beds were examined. The rat thoracic duct was selected as a large, post-nodal collecting lymphatic vessel that exhibits excellent conduit-type behavior while the rat mesenteric lymphatic was selected as a smaller, pre-nodal collecting lymphatic vessel that exhibits excellent pump behavior (see Gashev AA, et al. Microcirculation. 2004 Sep;11(6):477-92. [PMID: 15371129]). The axillary artery and vein were selected for comparison to the thoracic duct due to their similar anatomical position distal to the common junction of the lymphatic and venous vascular trees and represent a large artery and large vein, respectively. The mesentery artery and vein were selected for comparison to the mesenteric lymphatic vessels due to their parallel position within the mesenteric vasculature and represent a small atery and small vein, respectively. A 2 x 3, reference-based, experimental design was utilized consisting of both large (thoracic) and small (mesenteric) arteries, veins, and collecting lymphatic vessels for a total of 6 sample groups with n=6 biological replicates present in each group. All vessels acquired from the same donor animal have the same numerical label and were handled in parallel through all experimental steps. Each vessel sample RNA sample was amplified, labeled with Cy5, and compared to the same Rat Universal Reference RNA sample (Stratagene, La Jolla, CA) that was amplified and labeled with Cy3 dye. No dye swaps were utilized.
2012-06-22 | E-GEOD-17015 | biostudies-arrayexpress
Project description:Background: The vascular wall of small arteries is heavily affected by high blood pressure. However, the underlying mechanisms causing vascular changes are not fully elucidated. Using a novel data-independent acquisition mass spectrometry (DIA-MS) approach, we aimed to determine the proteomic changes in small mesenteric arteries during early-onset high blood pressure in a rat model of hypertension. Methods: Snap frozen small mesenteric and renal arteries from the spontaneous hypertension rat (SHR) model and Wistar Kyoto (WKY) control rats were collected from two time points (6- and 12-weels of age) and analyzed by a label free quantitative DIA-MS workflow. Mesenteric arteries from Wister Hannover rats were included as an additional control to clarify genetic drift caused by selective inbreeding. Results: We identified a total of 3956 consistent proteins in the mesenteric artery wall and found that 286 proteins were significantly regulated in 12-weeks old SHRs compared to WKY controls. Comparing to an in silico matrisome database, we identified 38 extracellular matrix-associated proteins that could distinguish SHRs from WKY controls. Furthermore, when comparing the significantly regulated proteins identified in mesenteric and renal arteries, we identified 18 proteins, including Serpina3l, Igg-2a, ENSRNOG00000049829, Acyp2, Enpp3, Lss, Acaa1a, Basp1, an isoform of Basp1, Flot1, Flot2, Gstt1, Nit1, Ppid, Ikbkap, Poglut3, P4ha2 and Usp15, that were changed in both vascular beds. These proteins were associated with vital cellular processes, such as dyslipidemia, protease inhibition, remodeling and generation of reactive oxygen species. Majority of the identified proteins and pathways were associated with hypertension, and mapping the underlying changes help understanding the pathological processes occurring in the arterial wall during early-onset hypertension. Conclusions: Our data provides an in-depth analysis of the proteomic architecture of the mesenteric and renal artery wall from SHRs and WKY control rats. We identified 18 novel candidate proteins that highlights critical changes in small arteries of the SHR.
2022-01-06 | PXD026051 | Pride
Project description:Endothelin-1 (ET-1), an endothelium-derived vasoconstrictor peptide, plays a role in the pathophysiology of cardiovascular disease. Transgenic mice that overexpress human preproET-1 selectively in the endothelium (eET-1) exhibit endothelial dysfunction, hypertrophic remodeling and vascular inflammation of resistance-size arteries in the absence of blood pressure elevation. To understand the mechanisms whereby ET-1 induces vascular damage, vascular gene expression using DNA microarrays was employed. RNA from mesenteric arteries of female and male young (6-7 weeks) and mature (6-8 months) eET-1 and wild type (WT) mice was isolated and changes in gene expression were determined by genome-wide expression profiling using Illumina microarray. This study revealed a set of genes potentially regulated by ET-1, which might be implicated in ET-1 induced-vascular damage. Samples 1-8: Total RNA obtained from the entire mesenteric arterial tree of young (6-7 weeks) female preproendothelin-1 overexpressing mice compared to age and sex matched wild type littermate controls. Samples 9-16: Total RNA obtained from the entire mesenteric arterial tree of mature (6-8 months) female preproendothelin-1 overexpressing mice compared to age and sex matched wild type littermate controls. Samples 17-24: Total RNA obtained from the entire mesenteric arterial tree of young (6-7 weeks) male preproendothelin-1 overexpressing mice compared to age and sex matched wild type littermate controls. Samples 25-32: Total RNA obtained from the entire mesenteric arterial tree of mature (6-8 months) male preproendothelin-1 overexpressing mice compared to age and sex matched wild type littermate controls.
2010-09-01 | E-GEOD-19624 | biostudies-arrayexpress
Project description:The dynamic nature of the microtubule network is regulated in part by post-translational modifications – particularly through acetylation, which stabilizes the microtubule network. Whether post-translational modifications of the microtubule network in vascular smooth muscle cells contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR), and investigate whether sustained acetylation via treatment with a histone deacetlyase-6 (HDAC-6) inhibitor, tubacin, would affect isoprenaline-mediated vasorelaxations in normotensive and hypertensive vessels.Using Western blotting, and mass spectrometry, we show that acetylation of the microtubule network is increased in the mesenteric arteries of the SHR compared to normotensive rats. Using wire myography, we found that tubacin enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young’s Modulus of smooth muscle cells, but found no difference between control, tubacin-treated, or taxol-treated cells.Acetylation of the microtubule network is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation. These findings pave the way for evaluation of HDAC inhibitors in the treatment of hypertension in human clinical studies.
2024-02-21 | PXD045602 | Pride