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H D Orzechowski

Publications and source records attributed to H D Orzechowski.

27 records · Page 2Linked to original sources
PubMed1998

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

Endothelin-1 converting-enzyme (ECE-1) cleaves the precursor, big-endothelin-1, to the active peptide endothelin-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. Tissue samples from the left human atrium were analyzed for ECE-1 expression and related to different clinical parameters. A quantitative PCR assay (qPCR) with competitive and non-competitive standards was established. The ECE-1 measurements were normalized by a GAPDH qPCR. Patients who suffered from a myocardial infarction had elevated ECE-1 levels when compared to controls (5.81+/-0.76 vs. 3.20+/-0.51 fg ECE-1, ng GAPDH, p<0.05). Drug treatment with the beta-blocker metoprolol was associated with a decreased ECE-1 expression level (3.90+/-0.58 vs. 5.81+/-0.76 fg ECE-1, ng GAPDH, p<0.1). We conclude that the expression of ECE-1 is altered in the atrial tissue depending on the physiological status of the heart. This suggests a differential role of ECE-1 in cardiovascular diseases.

Adrenergic beta-Antagonists↗
PubMed1997

Evidence of alternative promoters directing isoform-specific expression of human endothelin-converting enzyme-1 mRNA in cultured endothelial cells.

The endothelins, a family of closely related vasoactive and mitogenic peptides, are thought to play an important role in cardiovascular pathophysiology. The conversion of the inactive precursor "big endothelin" to the biologically active peptide is catalyzed in vitro and in vivo by endothelin-converting enzymes (ECE). Recently the cDNA cloning of two homologous proteins, termed ECE-1 and ECE-2, has been reported. ECE-1 may play a key role in the activation and regulation of the cardiovascular endothelin proteolytic cascade. ECE-1 mRNA is expressed in two isoforms, termed alpha and beta, which are identical except for the 5'-terminal regions. To investigate the transcriptional regulation of isoform-specific ECE-1 mRNA expression we isolated phage clones from a human genomic library and identified the alpha- and beta-specific exons of ECE-1. The exon/intron organization of the 5'-terminal region of the human ECE-1 gene in conjunction with putative transcription initiation start sites suggests the existence of two alternative promoters, each directing the expression of either isoform. A reverse transcription/polymerase chain reaction assay indicated differential mRNA expression of ECE-1 isoforms. Using a luciferase reporter gene assay, we found that the genomic region upstream of exon 1 alpha confers strong promoter activity in the human endothelial cell line ECV 304, which was previously shown to express predominantly ECE-1 alpha mRNA. Transfection of serial deletion mutants in ECV304 cells indicated the existence of three positive and also one negative regulating element within 2 kb of the alpha-promoter region. Luciferase reporter gene studies also revealed that the genomic region upstream of exon 3, which encodes the putative ECE-1 beta specific N-terminus, was able to direct luciferase expression in primary cultured bovine aortic endothelial cells, indicating the existence of an alternative promoter. Transfection of nested deletions spanning 1.2 kb upstream of the putative translation initiation codon of ECE-1 beta suggested the existence of three positive regulating regions within the beta-specific promoter. Both ECE-1 promoters lack TATA or CAAT boxes, and the two show different patterns of consensus sequences for transcription factors, suggesting a differential transcriptional regulation of isoform-specific ECE-1 mRNA expression.

Aspartic Acid Endopeptidases↗
PubMed1997

Vectors based on Semliki Forest virus for rapid and efficient gene transfer into non-endothelial cardiovascular cells: comparison to adenovirus.

OBJECTIVE: Replication-deficient, recombinant adenovirus is used as a carrier for gene transfer, but it is unspecific and the onset of transgene expression is relatively late. Here, we evaluated the efficiency and selectivity of gene transfer mediated by recombinant Semliki Forest virus (SFV). METHODS: We compared the efficiency of a SFV-based vector with an adenoviral vector, using LacZ as a reporter gene. Firstly, the affinity for vascular smooth muscle cells, endothelial cells and cardiac myocytes was assessed. Secondly, we compared the time course of LacZ expression and cytotoxicity in vascular smooth muscle cells. RESULTS: The SFV-based vector infects vascular smooth muscle cells and cardiomyocytes as efficiently as adenovirus. In contrast to adenovirus, SFV hardly transfers LacZ to endothelial cells (2.6% or less). SFV-mediated expression was visible after 1 h, reaching a maximum after 6 h. In contrast, adenovirus-mediated expression became visible after 6 h, and reached a maximum after 48-72 h. Both vectors were cytotoxic. CONCLUSIONS: We demonstrate that SFV efficiently transfers LacZ to vascular smooth muscle cells and cardiomyocytes, but not to endothelial cells. In contrast, adenovirus causes efficient transgene expression in all cell types tested. Furthermore, SFV-mediated expression is faster than adenovirus-mediated expression. Therefore, SFV-mediated gene transfer may be a suitable alternative to adenovirus, providing a fast expression in non-endothelial cardiovascular cell types.

Adenoviridae↗
PubMed1997

Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis.

The remodeling of extracellular matrix during chronic liver disease may partially be attributed to altered activity of matrix metalloproteinases and their tissue inhibitors (TIMPs). Expression of TIMP-1 and -2 was studied by in situ hybridization combined with immunohistochemistry in rat (acute and chronic carbon tetrachloride intoxication and secondary biliary fibrosis) and human livers and on isolated rat hepatic stellate cells. TIMP-1 and -2 transcripts appeared in rat livers within 1 to 3 hours after intoxication, pointing to a role in the protection against accidental activation of matrix metalloproteinases, and were present at high levels in all fibrotic rat and human livers predominantly in stellate cells. TIMP-2 RNA distribution largely matched with previously reported patterns of matrix metalloproteinase-2 (72-kd gelatinase) expression, suggesting generation of a TIMP-2/matrix metalloproteinase-2 complex (large inhibitor of metalloproteinases). Isolated stellate cells expressed TIMP-1 and -2 RNA. Addition of transforming growth factor-beta 1 enhanced TIMP-1 and matrix metalloproteinase-2 RNA levels in vitro, whereas TIMP-2-specific signals were reduced, likely to result in a stoichiometric excess of matrix-metalloproteinase-2 over TIMP-2. In the context of previous demonstrations of transforming growth factor-beta 1 and matrix metalloproteinase-2 in vivo, these patterns suggest an intrahepatic environment permitting only limited matrix degradation, ultimately resulting in redistribution of extracellular matrix with relative accumulation of collagen type 1.

Adult↗
PubMed1996

Characterization of the renal phenotype of transgenic rats expressing the human endothelin-2 gene.

We have previously established a transgenic rat model termed TGR(hET-2)37 overexpressing the human endothelin-2 (ET-2) gene with high renal transgene expression. This renal overexpression is of pathophysiological interest because a long-term activated paracrine renal endothelin system has been implicated in chronic renal failure due to progressive glomerular injury. Therefore, our aim in the present study was to analyze renal transgene expression in detail and address the question of whether transgene expression causes phenotypic and functional changes in the kidney. We used reverse transcription-polymerase chain reaction and in situ hybridization techniques for transgene expression analysis. Tissue ET-2 concentrations were measured with a specific radioimmunoassay. For histological evaluation of renal tissue, all samples were subjected to hematoxylin-eosin and periodic acid-Schiff staining. Renal tissue ET-2 concentrations were significantly increased in TGR(hET-2)37 rats. Using in situ hybridization, we found that the human ET-2 gene was almost exclusively expressed within the glomeruli. The glomerular transgene expression resulted in a significantly increased glomerular injury score and likewise in a significantly increased protein excretion, whereas glomerular filtration rate was not altered. Blood pressure was similar in TGR(hET-2)37 rats and age-matched controls, suggesting that the local changes in the kidney were correlated with paracrine endothelin actions. In conclusion, our study revealed that the major renal expression site of the human ET-2 transgene in TGR(hET-2)37 rats was within the glomeruli and caused the development of glomerulo-sclerosis with significantly increased protein excretion that is independent of blood pressure. We suggest that TGR(hET-2)37 rats are a new monogenetic animal model for study of the paracrine renal endothelin system and its involvement in renal pathophysiology.

Animals↗
PubMed1995

Expression of CD44v6 is associated with cellular dysplasia in colorectal epithelial cells.

There is increasing evidence that the expression of variants of the glycoprotein CD44 is related to the invasive and metastatic potential of tumour cells. By in situ hybridisation, we analysed the cellular expression of human homologues of a rat metastasis-associated CD44 variant v6 in invasive and non-invasive colorectal neoplasia and normal colonic mucosa. No specific hybridisation signals could be detected in epithelial cells of the normal crypt (n = 10). In contrast, we found moderate epithelial hybridisation signals in adenomatous polyps of mild dysplasia (n = 6). Adenoma cells of moderate or severe dysplasia (n = 7) showed increased hybridisation signals compared to mildly dysplastic adenomas (P < or = 0.01). We could not demonstrate significant differences in CD44v6 transcript levels between cells of dysplastic adenoma and primary adenocarcinoma (n = 11) (P > or = 0.05). Furthermore, we were not able to demonstrate a significant difference between primary and metastatic tumours (n = 7) (P > or = 0.05). However, there was a significant difference between metastatic carcinoma and adenomas with advanced dysplasia (P < or = 0.01). Our data demonstrate that significant transcriptional expression of CD44v6 is not confined to invasive tumour cells, but is already detectable in cells of adenomatous polyps showing mild dysplasia. The results of this study show a close relationship between cellular dysplasia and steady state levels of CD44 variant v6 transcripts in colorectal neoplasms.

Adenocarcinoma↗
PubMed1994

Increased fibronectin-receptor expression in colon carcinoma-derived HT 29 cells decreases tumorigenicity in nude mice.

BACKGROUND/AIMS: Following malignant transformation, epithelial cells of colorectal carcinomas, unlike normal colonic epithelial cells, no longer express the alpha 5 beta 1 fibronectin receptor. We hypothesized that the loss of alpha 5 beta 1 expression might facilitate the tumorigenicity of transformed colonic cells. METHODS: To examine this hypothesis, we established subclones of the human colon adenocarcinoma cell line HT 29, which differ in their fibronectin receptor expression and tested their tumorigenicity in nude mice. RESULTS: Our data indicate that the capacity to form tumors in nude mice after subcutaneous injection was significantly lower for alpha 5-positive than for alpha 5-negative cell clones. In addition, tumors from clones expressing no detectable levels of alpha 5 beta 1 grew rapidly, whereas tumors expressing elevated levels of fibronectin receptor grew slowly. Despite similar rates of adhesion to fibronectin for alpha 5-positive and alpha 5-negative cell clones in vitro, deposition of fibronectin in tumor-surrounding stroma was increased in tumors derived from alpha 5-positive cells. CONCLUSIONS: Our results indicate that an increase of the alpha 5 beta 1-mediated interaction of malignant cells with the extracellular matrix may be responsible for decreased tumorigenicity of malignant transformed cells in colorectal carcinomas.

Adenocarcinoma↗
PubMed1993

[Loss of fibronectin receptor expression in malignant transformation of the colon results in increased tumorigenicity of epithelial cells].

Following malignant transformation, epithelial cells of colorectal carcinomas unlike normal colonic epithelial cells do not any longer express the classical alpha 5 beta 1 fibronectin receptor. We speculated that the loss of alpha 5 beta 1 expression may facilitate the tumorigenicity of transformed colonic cells. To examine this hypothesis, we established subclones of the human colon adenocarcinoma cell line HT29 which differ in their fibronectin receptor expression and tested their tumorigenicity in nude mice. Our data indicate that the capacity to form tumors in nude mice after subcutaneous injection was significantly lower for alpha 5 beta 1-positive than for alpha 5 beta 1-negative cell clones. In addition, tumors from clones expressing to detectable levels of alpha 5 beta 1 grew rapidly, while tumors expressing elevated levels of fibronectin receptor grew slowly. Deposition of fibronectin in tumor-surrounding stroma was increased in tumors derived from alpha 5 beta 1-positive cells compared to tumors derived from alpha 5 beta 1-negative cells. Our results indicate that a reduction of the alpha 5 beta 1-mediated interaction of epithelial cells with the extracellular matrix may be responsible for increased tumorigenicity of malignant transformed cells in colorectal carcinomas.

Cell Line↗