Abstract: AIMS: Female sex and sex hormones contribute to cardiac remodelling. 17beta-estradiol (E2) is involved in the modulation of extracellular matrix composition and function. Here, we analysed the effect of E2 on matrix metalloproteinase (MMP)-2 gene expression and studied the underlying molecular mechanisms in rat cardiac fibroblasts and in a human fibroblast cell line. METHODS AND RESULTS: In adult rat cardiac fibroblasts, E2 significantly decreased MMP-2 gene expression in an estrogen receptor (ER)-dependent manner. Transient transfection experiments of human MMP-2 (hMMP-2) promoter deletion constructs in a human fibroblast cell line revealed a regulatory region between -324 and -260 bp that is involved in E2/ERalpha-mediated repression of hMMP-2 gene transcription. Electrophoretic mobility shift assays (EMSA) and supershift analysis demonstrated the binding of transcription factor Elk-1 within this promoter region. Elk-1 was phosphorylated by E2 via the mitogen-activated protein kinase (MAPK) signalling pathway as shown by western blotting. Treatment of cells with the MAPK inhibitor PD98059 blocked the E2-dependent repression of hMMP-2 promoter activity as well as the endogenous MMP-2 mRNA levels in both human fibroblast cells and rat cardiac fibroblasts. CONCLUSION: E2 inhibits MMP-2 expression via the ER and the MAPK pathway in rat cardiac fibroblasts and in a human fibroblast cell line. These mechanisms may contribute to sex-specific differences in fibrotic processes that are observed in human heart and other diseases.
Abstract: Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but the regulation of ER expression in the human heart has not yet been analyzed. In various cell lines and tissues, multiple human estrogen receptor alpha (hERalpha) mRNA isoforms are transcribed from distinct promoters and differ in their 5'-untranslated regions. Using PCR-based strategies, we show that in the human heart the ERalpha mRNA is transcribed from multiple promoters, namely, A, B, C, and F, of which the F-promoter is most frequently used variant. Transient transfection reporter assays in a human cardiac myocyte cell line (AC16) with F-promoter deletion constructs demonstrated a negative regulatory region within this promoter. Site-directed mutagenesis and electrophoretic mobility shift assays indicated that NF-kappaB binds to this region. An inhibition of NF-kappaB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-kappaB expression by tumor necrosis factor-alpha reduced the expression of ERalpha, indicating that the NF-kappaB pathway inhibits expression of ERalpha in human cardiomyocytes. Finally, 17beta-estradiol induced the transcriptional activity of hERalpha promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERalpha expression via activation and subsequent binding of NF-kappaB to the ERalpha F-promoter, and 17beta-estradiol/hERalpha may antagonize the inhibitory effect of NF-kappaB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-kappaB.
Abstract: Pressure overload (PO) first causes cardiac hypertrophy and then heart failure (HF), which are associated with sex differences in cardiac morphology and function. We aimed to identify genes that may cause HF-related sex differences. We used a transverse aortic constriction (TAC) mouse model leading to hypertrophy without sex differences in cardiac function after 2 weeks, but with sex differences in hypertrophy 6 and 9 weeks after TAC. Cardiac gene expression was analyzed 2 weeks after surgery. Deregulated genes were classified into functional gene ontology (GO) categories and used for pathway analysis. Classical marker genes of hypertrophy were similarly upregulated in both sexes (alpha-actin, ANP, BNP, CTGF). Thirty-five genes controlling mitochondrial function (PGC-1, cytochrome oxidase, carnitine palmitoyl transferase, acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase) had lower expression in males compared to females after TAC. Genes encoding ribosomal proteins and genes associated with extracellular matrix remodeling exhibited relative higher expression in males (collagen 3, matrix metalloproteinase 2, TIMP2, and TGFbeta2, all about twofold) after TAC. We confirmed 87% of the gene expression by real-time polymerase chain reaction. By GO classification, female-specific genes were related to mitochondria and metabolism and males to matrix and biosynthesis. Promoter studies confirmed the upregulation of PGC-1 by E2. Less downregulation of metabolic genes in female hearts and increased protein synthesis capacity and deregulation of matrix remodeling in male hearts characterize the sex-specific early response to PO. These differences could contribute to subsequent sex differences in cardiac function and HF.
Abstract: BACKGROUND: The interaction of the risk factors of abdominal obesity, disturbed glucose homeostasis, dyslipidemia, and hypertension is believed to represent a distinct entity, termed the metabolic syndrome (MetS), that leads to a greater increase in cardiovascular risk than does the sum of its components. OBJECTIVE: We reviewed currently available information regarding gender differences in the role of the MetS as a risk factor for cardiovascular disease (CVD). METHODS: Using the search terms women, men, sex, gender, sex differences, and gender differences in combination with the metabolic syndrome, we conducted a systematic review of the available literature on sex differences in the MetS. The National Institutes of Health, PubMed, and MEDLINE databases were searched retrospectively from 2007 to 1987. Reference lists of identified articles were also used as a source, and articles were not restricted to the English language. RESULTS: In recent years, the MetS has been more prevalent in men than in women but has risen particularly in young women, where it is mainly driven by obesity. Diagnostic criteria for the MetS vary for the cutoff points and definition of its components in a gender-specific manner. Based on the definition of impaired glucose homeostasis and pathologic abdominal circumference or waist/hip ratio, more or fewer women are included. Glucose and lipid metabolism are directly modulated by estrogen and testosterone, with a lack of estrogen or a relative increase in testosterone inducing insulin resistance and a proatherogenic lipid profile. Hypertension is a strong risk factor in both sexes, but the prevalence of hypertension increases more rapidly in aging women than in men. Menopause and polycystic ovary syndrome contribute to the development of MetS by the direct effects of sex hormones. Some components of the MetS (eg, diabetes and hypertension) carry a greater risk for CVD in women. CONCLUSIONS: Future gender-related clinical and research activities should focus on the identification of sex- and gender-specific criteria for risk management in patients with the MetS. We propose small, focused, mechanistic studies on sex-specific surrogate end points and sex-specific studies in animal models for diabetes and aging.
Abstract: Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estrogen receptor alpha (ERalpha) expression, localization, and association with structural proteins was altered in end stage-failing hearts. We found a 1.8-fold increase in ERalpha mRNA and protein in end-stage human dilated cardiomyopathy (DCM, n=41), as compared with controls (n=25). ERalpha was visualized by confocal immunofluorescence microscopy and localized to the cytoplasm, sarcolemma, intercalated discs and nuclei of cardiomyocytes. Immunofluorescence studies demonstrated colocalization of ERalpha with beta-catenin at the intercalated disc in control hearts and immunoprecipitation studies confirmed complex formation of both proteins. Interestingly, the ERalpha/beta-catenin colocalization was lost at the intercalated disc in DCM hearts. Thus, the ERalpha/beta-catenin colocalization in the intercalated disc may be of functional relevance and a loss of this association may play a role in the progression of heart failure. The increase of total ERalpha expression may represent a compensatory process to contribute to the stability of cardiac intercalated discs.
Abstract: Epidemiological studies identified a higher risk of developing Alzheimer's disease (AD) among subjects with elevated cholesterol levels. This association may be caused by a modulation of the amyloid precursor protein (APP) processing in response to the cellular cholesterol content. High cholesterol levels may favor the amyloidogenic pathway by inhibition of the alpha-secretase probably leading to elevated beta-Amyloid (Abeta) production. The identification of a linkage peak on chromosome 10q using high Abeta as quantitative trait led us to examine polymorphisms of genes located on chromosome 10 involved in cholesterol metabolism, like Lipase A (LIPA), Cholesterol 25 hydroxylase (CH25H), and FLJ22476, a high density lipoprotein binding related protein. Using 286 patients with AD and 162 controls we analyzed several single nucleotide polymorphisms (SNPs) within LIPA, CH25H, and FLJ22476. None of the polymorphisms showed significant association with AD which contradicts recent findings on CH25H. From our results we conclude that the investigated genetic variations do not contribute to the genetic risk of AD.
Abstract: BACKGROUND: Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but ER expression in the human heart and its relation to hypertrophy-mediated gene expression have not yet been analyzed. We therefore investigated sex- and disease-dependent alterations of myocardial ER expression in human aortic stenosis together with the expression of hypertrophy-related genes. METHODS AND RESULTS: ER-alpha and -beta, calcineurin A-beta, and brain natriuretic peptide (BNP) mRNA were quantified by real-time polymerase chain reaction in left ventricular biopsies from patients with aortic valve stenosis (n=14) and control hearts with normal systolic function (n=17). ER protein was quantified by immunoblotting and visualized by immunofluorescence confocal microscopy. ER-alpha mRNA and protein were increased 2.6-fold (P=0.003) and 1.7-fold (P=0.026), respectively, in patients with aortic valve stenosis. Left ventricular ER-beta mRNA was increased 2.6-fold in patients with aortic valve stenosis (P<0.0001). ER-alpha and -beta were found in the cytoplasm and nuclei of human hearts. A strong inverse correlation exists between ER-beta and calcineurin A-beta mRNA in patients with aortic valve stenosis (r=-0.83, P=0.002) but not between ER-alpha or -beta and BNP mRNA. CONCLUSIONS: ER-alpha and -beta in the human heart are upregulated by myocardial pressure load.
Abstract: A transformation / regeneration system was developed for the grain legume Vicia narbonensis. A number of fertile plants were obtained after cocultivation of hypocotyl explants together with Agrobacterium tumefaciens strain C58C1pGV3850hygro. The T-DNA of this construct confers hygromycin resistance and nopalin production. The progeny of three such clones - harbouring a single copy of the T-DNA only - was analyzed. In the first generation both characters were inherited according to Mendelian law. Throughout self pollinations homozygous as well as hemizygous sublines were obtained and further characterized. In order to follow the transmission of the two foreign genes for the hygromycin phosphotransferase (hph) and the nopaline synthase (nos) for a long period, biochemical and molecular analyses were performed up to the seventh generation. The genetic stability as well as the stable expression of both characters was demonstrated for the majority of the homozygous sublines and their progenies. For a small number of hemizygous but also for a few homozygous sublines some unexpected results were obtained regarding the coexpression of both characters. This indicates that in some cases genetic instability and modification of parts of the integrated foreign DNA - especially the hph gene - took place. In a few hemizygous plants various DNA modification patterns of the hph gene seem to occur, leading to a multicellular mosaic in respect to the methylation status of parts of the T-DNA. Two homozygous sublines originating from two different transgenic clones were found to be instable. Detailed analysis of this two subprogenies showed both partial or complete loss of the character âhygromycin resistanceâ as well as a ârestorationâ of the original phenotype too.
