Biomedical subjects

H D Orzechowski

Publications and source records attributed to H D Orzechowski.

At least 19 recordsLinked to original sources

PubMed2001

Impaired hyperpolarization in regenerated endothelium after balloon catheter injury.

Ca(2+)-activated K(+) (K(Ca)) channels control endothelial Ca(2+) homeostasis and the formation of vasodilators. After angioplasty, dysfunction of the regenerated endothelium leads to abnormal vasoregulation. In this study, we tested the expression and function of K(Ca) channels in regenerated endothelium at 6 weeks after balloon catheter injury of rat carotid arteries (CAs) by using single-cell reverse transcription-polymerase chain reaction, patch-clamp techniques, and analysis of vasoreactivity. In single regenerated endothelial cells (ECs), the percentage of ECs expressing the K(Ca) genes, rSK3 (12+/-8%) and rIK1 (22+/-9%), was significantly lower compared with the percentage of native ECs expressing these genes (rSK3 58+/-8%, rIK1 64+/-10%). In patch-clamp experiments, K(Ca) currents and acetylcholine-induced hyperpolarization were markedly reduced in regenerated ECs (shift of membrane potential -6+/-3 mV) compared with those in native ECs (shift of membrane potential -21+/-5 mV). In pressure myograph experiments, acetylcholine-induced dilation was impaired in reendothelialized CAs compared with normal CAs. Intraluminal application of the K(Ca) blocker apamin and charybdotoxin inhibited dilation by 30% in normal CAs but was without effect in reendothelialized CAs. Intraluminal application of 1-ethyl-2-benzimidazolinone (100 micromol/L), an opener of K(Ca) channels, evoked dilation by 29% in normal CAs but had no effect in reendothelialized CAs. In conclusion, the impaired expression of K(Ca) channels in regenerated endothelium results in defective hyperpolarization and impaired dilation. Thus, the impaired K(Ca) channel function contributes to functional alterations of regenerated endothelium after angioplasty.

Acetylcholine↗

PubMed2001

Functional characterization of the human prion protein promoter in neuronal and endothelial cells.

Human prion diseases such as Creutzfeld-Jakob disease and kuru are of major medical and biological importance because of their fatal course, epidemic potential, and unique pathophysiology. Endogenous expression of the normal cellular prion protein (PrP(C)) is necessary for infection and prion replication. However, knowledge of human PrP(C) gene regulation is rudimentary. We therefore cloned1543 bp of the 5' untranslated and promoter region of the PrP gene. Using transient transfection assays, the full-length promoter and serial deletion mutants subcloned in a luciferase reporter vector were analyzed in neuronal (KELLY) and endothelial (EA.hy926) cell lines, which both express PrP(C) as shown by RT/PCR. Analysis of promoter constructs in KELLY cells indicated two activating regions at -131/-284 and -1303/-1543, relative to the 3'-terminal end of exon 1, and also two repressing elements at -254/-567 and -567/-909 in neuronal cells. In EA.hy926 cells, activating elements were identified at -131/-284 and -284/-567, and one repressing region was localized at -567/-909. In addition, transcriptional start sites were determined by 5'-RACE reaction and RNase protection assay, revealing one major transcriptional start site located at -47 (in KELLY cells), -53 (in human thalamus) and at about -55 (in EA.hy926 cells).

5' Untranslated Regions↗

PubMed2001

Increased matrix metalloproteinase 2 concentration and transcript expression in advanced colorectal carcinomas.

Colorectal cancer is one of the most common malignant tumors and entails a relatively poor prognosis. Clinical outcome depends on the extent of local and metastatic tumor spread. Results of in vivo and in vitro studies suggest that the balance between matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases TIMPs) is altered in neoplasia, contributing to the invasive and metastatic properties of malignant tumors. We quantified tissue concentrations of MMP-2 and TIMP-2 in 65 malignant colorectal lesions and corresponding normal mucosa by enzyme-linked immunosorbent assay, western blotting, and in situ hybridization. In situ hybridization and western blot analyses demonstrated a clear increase in both stromal expression of MMP-2 transcripts and protein in primary carcinomas. The protein concentration of MMP-2 was higher in all tumor stages, except stage I tumors, than in normal mucosa and adenomas. MMP-2 concentrations were not related to tumor differentiation or to colonic versus rectal location. Surprisingly, the MMP-2 concentration was not increased in metastases. Interestingly, tissue concentrations and epithelial mRNA expression of TIMP-2 decreased significantly in primary colorectal cancer (UICC stages III and IV) but increased in metastases. Therefore an increased ratio of MMP-2 to TIMP-2 is strongly associated with advanced tumor stages, but a decreased ratio was observed in metastases. These findings suggest that the MMP-2:TIMP-2 ratio may prove useful as a marker of local invasion but not of metastasis in colorectal cancer.

Blotting, Western↗

PubMed2001

Transcriptional mechanism of protein kinase C-induced isoform-specific expression of the gene for endothelin-converting enzyme-1 in human endothelial cells.

Isoform-specific expression of endothelin-converting enzyme (ECE)-1, the major big endothelin-processing enzyme, is controlled by alternative promoters. Signaling pathways and transcriptional mechanisms of ECE-1 mRNA expression are largely unknown. To investigate ECE-1 isoform expression after protein kinase C (PKC) activation, we used phorbol 12-myristate 13-acetate (PMA) to stimulate primary cultured human umbilical vein endothelial cells and the related EA.hy926 cell line. ECE-1a mRNA was up-regulated (approximately 3-fold), whereas mRNA of alternative isoforms (b, c, and d) was unchanged, which was confirmed on the protein level. PMA effects on mRNA expression were suppressed by the PKC inhibitors H-7 and Calphostin C. Because increased ECE-1a expression was preceded by induction of the transcription factor Ets-1, we performed gel shift assays and demonstrated specific DNA/protein interactions involving the ETS binding motif GGAA. Luciferase reporter assays showed that PMA induced ECE-1a promoter activity about 2.5-fold in EA.hy926 cells. Similarly, coexpression of Ets-1 protein resulted in a dose-dependent increase in ECE-1a promoter activity (more than 8-fold). Using gel shift assays and mutation analysis, we identified two tandemly arranged Ets-1 binding sites (EBS) at -638 and -658, respectively, that are involved in transcriptional activation of the ECE-1a promoter by PMA or Ets-1. Moreover, we also found evidence for binding of a transcriptional repressor to EBS -638. The inhibitor of mitogen-activated protein kinase kinase, PD98059, inhibited PMA effects on ECE-1a mRNA expression and promoter activity, respectively. Our results provide the first detailed analysis of signaling pathways and transcriptional mechanisms involved in isoform-specific ECE-1 gene expression.

Antibodies↗

PubMed2001

Characterization of polymorphic structure of cathepsin G gene: role in cardiovascular and cerebrovascular diseases.

Cathepsin G (CTSG), a serine protease released from activated neutrophils, may cause platelet activation, leading to intravascular thrombosis, thus contributing to cardiovascular and cerebrovascular disease. Applying the candidate gene approach, we screened the 5'-flanking region and the entire coding region of the CTSG gene for genetic variation by using polymerase chain reaction/single-strand conformation polymorphism analysis from 96 patients at high risk for myocardial infarction (MI). We identified 4 polymorphisms in the 5'-flanking region (G-618C, G-315A, C-179T, and C-160T) and 1 polymorphism in the coding region (Asn125Ser) of the gene and genotyped the participants in the Etude Cas-Temoins sur l'Infarctus du Myocarde (ECTIM Study), a case-control study for MI, and in the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC Study), a case-control study for brain infarction (BI), for all identified genetic variants. The potential in vitro functionality of the 4 variants in the 5'-flanking region was investigated with transient transfection analyses in U937 cells with different allelic promoter constructs by using a luciferase assay. Our in vitro analyses did not reveal any differences for the investigated allelic constructs with respect to promoter activity, and none of the polymorphisms in the 5'-flanking region was associated with the available phenotypes in either study. Allele and genotype distributions of all identified polymorphisms did not globally differ between cases and controls in the ECTIM Study. However, in patients from the ECTIM Study, the Ser125 allele was significantly associated with elevated plasma fibrinogen levels (P=0.006), but this effect was not seen in controls (case-control heterogeneity, P=0.04). There was a significant interaction between CTSG Asn125Ser and the beta-fibrinogen gene polymorphism G-455A on plasma fibrinogen levels (P=0.04). In the GENIC Study, the odds ratio for BI associated with CTSG Ser125 carrying was 1.82 (95% CI 1.16 to 2.84, P=0.008) in patients without a history of cardiovascular or cerebrovascular diseases. Our results indicate that the CTSG Ser125 allele is associated with plasma fibrinogen levels in MI patients from the ECTIM Study and with BI in the GENIC Study. Further studies should be carried out to define the underlying mechanisms.

Adult↗

PubMed2001

Big endothelin-1 and endothelin-1 plasma levels are correlated with the severity of primary pulmonary hypertension.

STUDY OBJECTIVES: Primary pulmonary hypertension (PPH) is a rare disease of unknown etiology that is characterized by a poor prognosis. This study was undertaken to investigate possible correlations between endothelin (ET)-1 and big ET-1 plasma levels and the severity of PPH. PATIENTS: Sixteen consecutive patients with PPH were included. INTERVENTIONS: Hemodynamics of patients with PPH were measured by right-heart catheterization, and a 6-min walk test was performed. MEASUREMENTS: Plasma levels of the biologically active peptide ET-1 and its precursor big ET-1 were determined in blood samples from the pulmonary artery, peripheral artery, and peripheral vein by radioimmunoassay. RESULTS: A strong correlation was shown between pulmonary vascular resistance, mean pulmonary artery pressure, cardiac output, cardiac index, 6-min walk data, and elevated plasma levels of big ET-1 as well as mature ET-1 plasma levels at all sites of blood sampling (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: Levels of circulating ET-1 might become a prognostic marker for patients with PPH and serve as a tool for the selection of patients who may benefit from treatment with ET-receptor antagonists.

Adult↗

PubMed2000

Cardiac endothelin system impairs left ventricular function in renin-dependent hypertension via decreased sarcoplasmic reticulum Ca(2+) uptake.

BACKGROUND: We evaluated the role of the cardiac endothelin (ET) system in compensated hypertensive left ventricular (LV) hypertrophy (LVH) and after the transition toward LV dysfunction. METHODS AND RESULTS: Hypertensive transgenic rats overexpressing the Ren2 gene (Ren2 rats) were investigated between the ages of 10 and 30 weeks (Ren2-10 and Ren2-30 groups, respectively) and compared with age-matched normotensive Sprague-Dawley (SD) rats (SD-10 and SD-30 groups, respectively). Systolic blood pressure and LV weight were elevated in both Ren2 groups compared with their age-matched SD control groups (P:<0.0001). In Ren2-30 rats, LV end-diastolic pressure increased and -dP/dt(max) decreased compared with the values in SD-30 and Ren2-10 rats (P:<0.05). This was paralleled by an activation of LV mRNA expression of preproET-1 and ET-converting enzyme-1 and ET subtype A (ETA) receptor binding in Ren2-30 compared with Ren2-10 rats (P:<0.001). Cardiac fibrosis was increased and sarcoplasmic reticulum (SR) Ca(2+) reuptake was reduced in Ren2-30 compared with SD-30 and Ren2-10 rats (P:<0.05). Treatment of Ren2 rats with the selective ETA receptor antagonist Lu135252 between 10 and 30 weeks of age did not lower systolic blood pressure, heart weight, or cardiac fibrosis but completely prevented the deterioration of LV end-diastolic pressure and abolished alterations in -dP/dt(max) and SR Ca(2+) reuptake compared with no treatment in Ren2-30 and SD-30 rats (P:<0.05). CONCLUSIONS: Activation of the cardiac ET system accounts at least in part for the LV dysfunction that gradually develops in LVH. The protective effect of ETA antagonism can be attributed to the improvement of diastolic LV function that is due to normalization of impaired SR Ca(2+) uptake.

Animals↗

PubMed2000

Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1).

Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (-142/-240 and -240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5'-RACE revealed multiple transcriptional start sites located at about -110, -140 and -350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities.

Aspartic Acid Endopeptidases↗

PubMed2000

Oxidative stress increases synthesis of big endothelin-1 by activation of the endothelin-1 promoter.

Modulation of the biosynthesis of the vasoconstrictor peptide endothelin-1 by oxygen-derived free radicals generated by xanthine oxidase or hydrogen peroxide was studied in cultured endothelial cells. Endothelin-1 metabolism was investigated at the level of endothelin-1 promoter, preproendothelin-1 mRNA and intracellular big endothelin-1. Endothelin-1 mRNA, as characterized by Northern blotting, was increased both time- and dose-dependently by xanthine oxidase to up to 500% above baseline. Analysis of endothelin-1 promoter activity using a construct containing 1329 bp of the endothelin-1 promoter revealed that promoter activity was increased up to eight-fold by incubation with xanthine oxidase. Specificity was ascertained by co-incubation with superoxide dismutase and catalase leading to inhibition of the effect of xanthine oxidase. A significant contribution of nitric oxide was ruled out, since NOS III-mRNA transcription remained unchanged and l -NAME did not significantly alter endothelin-1 promoter activity. Synthesis of intracellular big endothelin-1 protein was increased dose-dependently by xanthine oxidase. Our results indicate that oxidative stress leads to increased endothelial synthesis of big endothelin-1, which is a previously unknown mechanism and may help to understand the detrimental association of increased oxidative stress and elevated endothelin-1 levels in pathophysiological conditions promoting atherosclerosis.

Animals↗

PubMed2000

Transcriptional control of deformation-induced preproendothelin-1 gene expression in endothelial cells.

Deformation-induced synthesis of endothelin-1 (ET-1) in endothelial cells exposed to high blood pressure may play an important role in vein graft disease and in restenosis following percutaneous transluminal angioplasty. Effective inhibitors of preproendothelin ET-1 (ppET-1) processing to ET-1 are not available, and blockade of ppET-1 expression may therefore emerge as an alternative therapeutic approach. To evaluate this, we investigated deformation-sensitive transcription factors controlling ppET-1 expression in both native (rabbit carotid artery and jugular vein) and cultured endothelial cells (EC; porcine aorta and human umbilical vein). Deformation of both native and cultured endothelial cells for 6 h resulted in a marked increase in ET-1 synthesis which was preceded by a transient (30-60 min) activation of transcription factors activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP) beta and/or delta. A decoy oligodeoxynucleotide directed against AP-1 inhibited deformation-induced ppET-1 expression in the rabbit jugular vein as well as in porcine aorta EC and human umbilical vein EC but not in the rabbit carotid artery. Subsequent reporter gene analyses with different rat ppET-1 promoter-luciferase constructs transiently transfected into porcine aorta EC identified a single AP-1 binding site at -110 to -100 bp as the primary response element for deformation-induced ppET-1 expression. Moreover, a C/EBP-specific decoy oligodeoxynucleotide abolished ppET-1 expression in the endothelium of the rabbit carotid artery, but not in the jugular vein where basal ET-1 synthesis was greatly enhanced instead. These findings suggest that the key transcription factors controlling deformation-induced ppET-1 expression in endothelial cells are blood vessel rather than species-specific. In humans, adjunct treatment with an AP-1-specific decoy oligodeoxynucleotide may prove be an interesting gene therapeutic option for the above cardiovascular interventions.

Animals↗

PubMed2000

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells.

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy 926, p < 0.05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Cloning and functional characterization of the bovine endothelin-converting enzyme-1a promoter.

Endothelin-converting enzyme-1 (ECE-1) mRNA is expressed in three isoforms, termed a, b, and c, originating from alternative promoters. In cultured bovine aortic endothelial cells, we detected mRNA isoform expression of ECE-1a and ECE-1b/c, respectively. Investigating transcriptional mechanisms of bovine endothelial ECE-1a expression in more detail, we identified multiple transcription start sites localized 120-415 nucleotides upstream from the presumptive translation start codon by RNase protection assay and 5' RACE. Using luciferase reporter gene assays we found that 1.4 kb of the 5' untranslated region showed strong promoter activity in endothelial cells. Sequence analysis revealed 71% overall homology of the bovine ECE-1a promoter with its human homologue. The proximal 680 base pair promoter region was shown to contain cis elements that are sufficient for basal and serum-induced transcriptional activation.

Animals↗

PubMed1999

The cardiac endothelin system in established pressure overload left ventricular hypertrophy.

In normal hearts, endothelin-1 (ET-1) has been shown to initiate myocyte growth and to modulate cardiac function. However, regulation of the various components of the system and the functional effects of ET-1 in established left ventricular hypertrophy (LVH) are less clear. We thus studied ET-1, ET(A) receptor, and endothelin converting enzyme (ECE-1) mRNA regulation as well as the effects of ET-1 on coronary resistance, LV contractility and relaxation in hypertrophied rat hearts. Cardiac pressure overload, secondary to banding of the ascending aorta, resulted in a transient increase of cardiac ET-1 and ET(A) receptor mRNAs that reached a maximum at 2 days (+75% and +40%, respectively, P<0.05, each). ET-1 mRNA levels reached a second peak at 84 days of pressure overload (+60%, P<0.05), at the later time point in conjunction with elevated ECE-1 mRNA levels (+20%, P<0.05). The functional implications of ET-1 were examined in a study of isolated perfused hearts. Both hearts with established LVH and sham control hearts responded to ET-1 perfusion (10(-1)] to 10(-9) M) with an increase of coronary perfusion pressure (CPP; +85+/-15 and +75+/-8 mm Hg; P<0.001 each) and a slight decrease of LV systolic pressure (LVP; -12+/-9 and -9+/-7 mm Hg; P = NS). In contrast, ET-1 increased LV end-diastolic pressure (LVEDP) only in LVH hearts (+22+/-7 mm Hg, P<0.05 versus baseline and +20+/-7 mm Hg, P<0.05 versus sham). Direct stimulation of protein kinase C mimicked the effects of ET-1, whereas inhibition of this kinase or the Na+ -H+ exchanger blunted the effects of ET-1 on CPP, LVP, and LVEDP. Interestingly, coadministration of the vasodilator and the nitric oxide (NO) donor nitroglycerin not only prevented the increase of CPP and LVEDP, but also uncovered a slight positive inotropic effect of ET-1 in LVH hearts. Thus, the cardiac expression of ET-1, ET(A), and ECE-1 mRNAs displays a distinct pattern during early and advanced cardiac pressure overload. Furthermore, ET-1 mediates a slight depression of systolic, and a profound depression of diastolic, functional parameters in hearts with established LVH, effects that appear to be secondary to ET-1-related coronary vasoconstriction. The data suggest a functional role of the endothelin system in hearts with established pressure overload hypertrophy.

Amiloride↗

PubMed1999

32/67-kD laminin receptor expression in human colonic neoplasia: elevated transcript levels correlate with the degree of epithelial dysplasia.

OBJECTIVE: The 32/67-kD laminin receptor is thought to be involved in tumor cell migration and metastasis formation, and enhanced expression was observed in human colorectal carcinoma. Our objective was to investigate further the expression of the 32/67-kD laminin receptor RNA in human colonic carcinogenesis. METHODS: We obtained sections of human colonic tissues in various stages of malignant transformation and analyzed them by in situ hybridization. RESULTS: Normal colonic mucosa displayed a gradient between crypt base and surface epithelium with lowest receptor RNA levels in superficial epithelial cells. Increased laminin receptor RNA expression was observed in epithelial cells of adenomas with positive correlation between transcript levels and the degree of epithelial dysplasia. At variance with published results, we did not observe significant differences in 32/67-kD laminin receptor transcripts between adenomas with high-grade dysplasia and invasive adenocarcinoma. However, adenocarcinoma metastases displayed significantly higher laminin receptor RNA levels than high-grade adenomas and primary carcinomas. CONCLUSIONS: We propose a two-step mechanism which controls first, upregulation of laminin receptor RNA before the acquisition of an invasive phenotype in dysplastic epithelial cells, and second, a further upregulation in metastatic cells during the adenoma-carcinoma sequence of the colon.

Adenocarcinoma↗

PubMed1998

Human endothelin-converting enzyme-1 beta mRNA expression is regulated by an alternative promoter.

The central step in endothelin biosynthesis is site-specific cleavage of big endothelins by endothelin-converting enzymes (ECEs). ECE-1 is a membrane-bound metalloprotease, predominantly but not exclusively expressed in endothelial cells. ECE-1 is expressed in two mRNA isoforms, termed alpha and beta, which differ only in the 5'-terminal regions but are functionally very similar when expressed in vitro. The structure of the human ECE-1 gene suggests either alternative splicing or alternative promoters as underlying mechanisms of mRNA isoform expression. We have previously shown that the alpha-upstream region exerts promoter activity in endothelial cells. To clarify whether the 5'-untranslated region upstream of exon 3, which contains the beta-specific sequence, acts as an alternative transcriptional promoter, we sequenced and cloned 1,206 bp upstream of the beta-specific translation initiation codon in a luciferase reporter vector. After transfection, we detected strong promoter activity in primary cultured endothelial cells (HU-VECs, BAECs) but only marginal activity in the endothelial cell line ECV304 and in CHO cells. Maximal promoter activity was observed with the full-length construct, 1206 (136% of the SV40 promoter activity in BAECs). Transfection of serial deletion mutants indicated at least three major regulatory regions within the promoter. Our results are consistent with cell type-restricted action of the beta-promoter and, in conjunction with the previously reported transcriptional start sites, clearly prove the existence of an alternative beta-specific promoter located in intron 2 of the human ECE-1 gene.

Animals↗

PubMed1998

Endothelin-converting enzyme-1 mRNA expression in human cardiovascular disease.

Endothelin-converting enzyme-1 (ECE-1) plays a substantial role in activation of the endothelin (ET) system by cleaving the precursor, big ET-1, to the active peptide ET-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. ECE-1 expression was related to echocardiographic data, drug treatment, age, sex, and NYHA heart failure classification. A quantitative PCR assay (qPCR) was established to measure ECE-1 mRNA in these samples. The ECE-1 measurements were normalized over a simultaneously performed GAPDH qPCR. The results indicate a higher ECE-1 expression level in atrial tissue samples of patients who have experienced a myocardial infarction compared with those who did not (ECE-1/GAPDH: 5.81 +/- 0.76 fg/ng; n = 21 vs. 3.20 +/- 0.51 fg/ng; n = 22; p = 0.007). The transverse diameter of the left atrium over 37 mm was associated with a lower ECE-1 expression (ECE-1/GAPDH: 3.11 +/- 0.69 fg/ng; n = 18 vs. 5.12 +/- 0.65 fg/ng; n = 25; p = 0.044). In assessing the drug treatment, decreased ECE-1 expression could be observed in patients who received a beta-blocker (ECE-1/GAPDH: 3.90 +/- 58 fg/ng; n = 31 vs. 5.81 +/- 0.76 fg/ng; n = 12; p = 0.077). These data suggest an involvement of the ET system is cardiovascular disease that may be clinically important.

Aspartic Acid Endopeptidases↗

PubMed1998

Endothelial expression of endothelin-converting enzyme-1 beta mRNA is regulated by the transcription factor Ets-1.

Cleavage of big endothelins (ETs) by endothelin-converting enzymes (ECEs) represents the final step in the biosynthesis of ETs. ECE-1 is expressed predominantly in endothelial cells and exists in two isoforms, termed alpha and beta, differing in their 5' termini. We have recently shown that isoform-specific mRNA expression is directed by alternative promoters. To investigate possible mechanisms of transcriptional regulation of ECE-1, we stimulated E.A. hy 926 cells with phorbol ester and found a greater than threefold increase in ECE-1 beta mRNA at 12-24 h of stimulation. Because the beta-specific promoter is characterized by multiple consensus sequences for transcription factors of the ETS family, Ets-1 and PEA3, we also analyzed Ets-1 mRNA expression and found at least a fivefold increase in Ets-1 mRNA at 3 h of phorbol ester stimulation. Gel shift analysis revealed a specific interaction of nuclear proteins isolated from E.A. hy 926 cells with an oligonucleotide harboring the Ets-1 consensus sequence. Using a specific anti-Ets-1 antibody, we detected a supershifted band indicating the expression of Ets-1 protein in E.A. hy 926 cells. We conclude that Ets-1 is involved in transcriptional upregulation of ECE-1 beta mRNA in E.A. hy 926 cells induced by phorbol ester.

Aspartic Acid Endopeptidases↗

PubMed1998

Transcriptional regulation of endothelin-1 by erythropoietin in endothelial cells.

Recombinant human erythropoietin (rHuEpo) has been widely used in patients undergoing chronic hemodialysis treatment to correct anemia. In a subgroup of patients, i.v. administration of rHuEpo leads to manifestation or worsening of hypertension. The underlying mechanism of this remains unclear but it has been suggested that it is associated with increased expression of the vasoconstrictor endothelin (ET) in endothelial cells (ECs). There is also evidence for expression of specific rHuEpo receptors on ECs. The aim of this work was to study the time course and mechanisms of ET-1 regulation on the mRNA level in bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) stimulated with pharmacologic doses of rHuEpo (1-10 IU/ml). Compared to vehicle-treated controls, rHuEpo-treatment of ECs increases preproET-1 mRNA expression up to 170%, as shown by Northern blotting. To study the transcriptional regulation of ET-1 expression by rHuEpo, ECs were transfected with a luciferase construct driven by the rat ET-1 promoter and subsequently stimulated with rHuEpo. Compared to controls, luciferase activity increased up to 200% (n = 6; p < 0.05), suggesting transcriptional regulation of preproET-1 mRNA-expression by rHuEpo. Our data support the hypothesis that ET contributes to the hypertensive side effects of rHuEpo treatment and that this interaction occurs at the transcriptional level.

Animals↗