Biomedical subjects

Dieter Weichenhan

Publications and source records attributed to Dieter Weichenhan.

12 recordsLinked to original sources

PubMed2006

New members of the neurexin superfamily: multiple rodent homologues of the human CASPR5 gene.

Proteins of the Caspr family are involved in cell contacts and communication in the nervous system. We identified and, by in silico reconstruction, compiled three orthologues of the human CASPR5 gene from the mouse genome, four from the rat genome, and one each from the chimpanzee, dog, opossum, and chicken genomes. Obviously, Caspr5 gene duplications have taken place during evolution of the rodent lineage. In the rat, the four paralogues are located in one chromosome arm, Chr 13p. In the mouse, however, the three Caspr5 genes are located in two chromosomes, Chr 1 and Chr 17. RT-PCR shows that all three mouse paralogues are being expressed. Common expression is found in brain tissue but different expression patterns are seen in other organs during fetal development and in the adult stage. Tissue specificity of expression has diverged during evolution of this young rodent gene family.

Animals↗

PubMed2006

Large-scale mutation screening in patients with dilated or hypertrophic cardiomyopathy: a pilot study using DGGE.

Cardiomyopathies are complex myocardial diseases characterized by inappropriate ventricular hypertrophy (HCM) or dilation (DCM). Both disorders may lead to sudden death or progressive heart failure and exhibit familial aggregation with marked genetic heterogeneity. Many candidate genes were identified by linkage analysis, experimental animal studies, and expression analysis. A systematic assessment of the prevalence of different mutations in these genes requires high-throughput analyses. In this paper, we present a simple and reliable protocol for mutation screening by heteroduplex analysis which reduced costs and workload of sequencing. Employing denaturing gradient gel electrophoresis (DGGE), 11 known and 14 potential candidate genes for HCM and DCM were analyzed. DGGE assays allowed analysis of 286 of the 312 protein coding exons, performing only four alternative polymerase chain reaction protocols and only two different DGGE analysis conditions. Sensitivity for the detection of heteroduplexes proved excellent, even for GC-rich DNA fragments, which were analyzed by a combination of DGGE and constant denaturant gel electrophoresis. To confirm DGGE sensitivity in cases where no variants in our human DNA samples could be observed, we generated heteroduplexes from homologous human and chimpanzee DNA. The platform proved a valuable contribution to elucidating the genetic causes of DCM and HCM as demonstrated by the identification of 17 different known and novel mutations and 98 different polymorphisms in our setting.

Adolescent↗

PubMed2006

Fine mapping of Dyscalc1, the major genetic determinant of dystrophic cardiac calcification in mice.

Calcification of severely dystrophic muscle is occasionally observed in targeted mouse models of muscular dystrophy and cardiomyopathy. Intracellular calcium deposition occurs in necrotic myocytes in the absence of plasma calcium and phosphate imbalances. In the heart, this recessive trait is referred to as dystrophic cardiac calcinosis (DCC). We identified previously Dyscalc1, a major genetic determinant of DCC, in a 15.2-Mbp region on proximal chromosome 7. We report now further steps toward the identification of the Dyscalc1 gene by reverse genetics. Transferring the Dyscalc1 locus from susceptible mouse strain C3H/He onto a resistant C57BL/6 strain background, we generated congenic inbred strains B6.C3-(D7Mit56-D7Mit230) and B6.C3-(D7Nds5-D7Mit230). Three days after myocardial freeze-thaw injury, both strains exhibited calcification of necrotic lesions, confirming the pathogenetic relevance of Dyscalc1. Analysis of two (129S1 x C57BL/6) x 129S1 backcrosses allowed mapping of Dyscalc1 more precisely to a region spanning 0.76 Mbp between genes Fgf21 (39.70 Mbp) and Myod1 (40.46 Mbp). This interval contains 31 known and putative genes in three large, ancestral haplotypes shared by susceptible strains C3H/He, 129S1, and DBA/2. Thus we were able to exclude previously proposed candidate genes Bax and Hrc. Instead, a potential candidate may be the gene encoding the ATP-binding cassette C6. Mutations in the orthologous human ABCC6 gene cause pseudoxanthoma elasticum, or Gronblad-Strandberg syndrome, an elastic tissue disorder with cardiovascular calcifications.

ATP-Binding Cassette Transporters↗

PubMed2006

Increased proinflammatory endothelial response to S100A8/A9 after preactivation through advanced glycation end products.

BACKGROUND: Atherosclerosis is an inflammatory disease in which a perpetuated activation of NFkappaB via the RAGE (receptor for advanced glycation end products)-MAPK signalling pathway may play an important pathogenetic role. As recently S100 proteins have been identified as ligands of RAGE, we sought to determine the effects of the proinflammatory heterodimer of S100A8/S100A9 on the RAGE-NFkappaB mediated induction of proinflammatory gene expression. METHODS: Human umbilical vein endothelial cells (HUVEC) were preincubated for 72 h with AGE-albumin or unmodified albumin for control, whereas AGE-albumin induction resulted in an upregulation of RAGE. Following this preactivation, cells were stimulated for 48 h with heterodimeric human recombinant S100A8/S100A9. RESULTS: Heterodimeric S100A8/S100A9 enhanced secretion of IL-6, ICAM-1, VCAM-1 and MCP1 in AGE-albumin pretreated HUVEC in a dose dependent manner. These effects could not be detected after stimulation with the homodimeric proteins S100A8, S100A9, S100A1 and S100B. The effects of heterodimeric S100A8/S100A9 were reduced by inhibition of the MAP-kinase pathways ERK1/2 and p38 by PD 98059 and SB 203580, respectively. CONCLUSION: The heterodimeric S100A8/S100A9 might therefore play a hitherto unknown role in triggering atherosclerosis in diabetes and renal failure, pathophysiological entities associated with a high AGE burden. Thus, blocking heterodimeric S100A8/S100A9 might represent a novel therapeutic modality in treating atherosclerosis.

Calgranulin A↗

PubMed2005

Calcification of myocardial necrosis is common in mice.

Independent of the severity, phenotypes and clinical outcomes of myocardial infarction may vary considerably in patients, suggesting a strong genetic influence on healing and adaptive processes. Since little is known about these genetic determinants, we examined the tissue response to myocardial injury in seven inbred mouse strains, including those employed for gene targeting or transgenic overexpression. Myocardial necrosis was produced by non-ischemic, trans-diaphragmal freeze-thaw injury in strains C57BL/6, C3H/He, DBA/2, BALB/c, 129S1, FVB/n and A/J. Two days after injury, necrotic cardiomyocytes calcified in C3H/He, DBA/2, BALB/c and 129S1, a phenotype known as dystrophic cardiac calcinosis (DCC). The susceptibility to DCC of 129S1 was determined by Dyscalc1, a locus on chromosome 7, which was identified previously in C3H/He and DBA/2. DCC was also observed in C3H/He following ischemic injury by permanent coronary artery ligation, indicating that DCC was independent of the mode of injury. In contrast, strains C57BL/6, FVB and A/J were resistant to DCC, showing formation of a fibrous scar without calcification. The development of DCC was studied in detail in C3H/He and C57BL/6. In both strains, no calcium deposition and only little structural disintegration were noted in necrotic myocardium 24 h after injury upon calcium-sensitive fluorescence staining. Ultrastructural examination revealed calcified mitochondria in C3H/He that may have served later as a nidus for rapid intracellular calcification of cardiomyocytes. We concluded that the susceptibility to calcification of myocardial necrosis may be common among inbred strains and should be recognised as a strong genetic modifier of experimental myocardial injury.

Animals↗

PubMed2005

S100A1 gene therapy preserves in vivo cardiac function after myocardial infarction.

Myocardial infarction (MI) represents an enormous clinical challenge as loss of myocardium due to ischemic injury is associated with compromised left ventricular (LV) function often leading to acute cardiac decompensation or chronic heart failure. S100A1 was recently identified as a positive inotropic regulator of myocardial contractility in vitro and in vivo. Here, we explore the strategy of myocardial S100A1 gene therapy either at the time of, or 2 h after, MI to preserve global heart function. Rats underwent cryothermia-induced MI and in vivo intracoronary delivery of adenoviral transgenes (4 x 10(10) pfu). Animals received saline (MI), the S100A1 adenovirus (MI/AdS100A1), a control adenovirus (MI/AdGFP), or a sham operation. S100A1 gene delivery preserved global in vivo LV function 1 week after MI. Preservation of LV function was due mainly to S100A1-mediated gain of contractility of the remaining, viable myocardium since contractile parameters and Ca(2+) transients of isolated MI/AdS100A1 myocytes were significantly enhanced compared to myocytes isolated from both MI/AdGFP and sham groups. Moreover, S100A1 gene therapy preserved the cardiac beta-adrenergic inotropic reserve, which was associated with the attenuation of GRK2 up-regulation. Also, S100A1 overexpression reduced cardiac hypertrophy 1 week post-MI. Overall, our data indicate that S100A1 gene therapy provides a potential novel treatment strategy to maintain contractile performance of the post-MI heart.

Adenoviridae↗

PubMed2005

Early diagnostic marker panel determination for microarray based clinical studies.

We present a novel, cost efficient two-phase design for predictive clinical gene expression studies: early marker panel determination (EMPD). In Phase-1, genome-wide microarrays are used only for a small number of individual patient samples. From this Phase-1 data a panel of marker genes is derived. In Phase-2, the expression values of these marker panel genes are measured for a large group of patients and a predictive classification model is learned from this data. Phase-2 does not require the use of expensive whole genome microarrays, thus making EMPD a cost efficient alternative for current trials. The expected performance loss of EMPD is compared to designs which use genome-wide microarrays for all patients. We also examine the trade-off between the number of patients included in Phase-1 and the number of marker genes required in Phase-2. By analysis of five published datasets we find that in Phase-1 already 16 patients per group are sufficient to determine a suitable marker panel of 10 genes, and that this early decision compromises the final performance only marginally.

Journal Article↗

PubMed2004

S100A1 gene transfer: a strategy to strengthen engineered cardiac grafts.

BACKGROUND: Cardiac tissue replacement therapy, although a promising novel approach for the potential treatment of heart failure, still suffers from insufficient contractile support to the failing myocardium. Here, we explore a strategy to improve contractile properties of engineered heart tissue (EHT) by S100A1 gene transfer. METHODS: EHTs were generated from neonatal rat cardiomyocytes and transfected (MOI 10 PFU) with the S100A1 adenovirus (AdvS100A1, n = 25) while an adenovirus devoid of the S100A1 cDNA served as a control (AdvGFP, n = 30). Contractile properties of transfected EHTs were measured 7 days following gene transfer. RESULTS: Western blot analysis confirmed a 8.7 +/- 3.6-fold S100A1 protein overexpression in AdvS100A1-transfected EHTs (n = 4; P < 0.01) that increased maximal isometric force (mN; AdvGFP 0.175 +/- 0.03 vs. AdvS100A1 0.47 +/- 0.06; P < 0.05) at 0.4 mmol/L extracellular calcium concentration [Ca(2+)](e). In addition, S100A1 overexpression enhanced both maximal Ca(2+)-stimulated force generation (+81%; P < 0.05) and Ca(2+)-sensitivity of EHTs (EC50% [Ca(2+)](e) mM; AdvGFP 0.33 +/- 0.04 vs. AdvS100A1 0.21 +/- 0.0022; P < 0.05). The S100A1-mediated gain in basal graft contractility was preserved throughout a series of isoproterenol interventions (10(-9) to 10(-6) M). Physiological properties of EHTs resembling intact heart preparations were preserved. CONCLUSIONS: S100A1 gene transfer in EHT is feasible and augments contractile performance, while characteristic physiological features of EHT remain unchanged. Thus, specific genetic manipulation of tissue constructs prior to implantation should be part of an improved tissue replacement strategy in heart failure.

Adenoviridae↗

PubMed2004

A locus on chromosome 7 determines dramatic up-regulation of osteopontin in dystrophic cardiac calcification in mice.

Calcification of necrotic tissue is frequently observed in chronic inflammation and atherosclerosis. A similar response of myocardium to injury, referred to as dystrophic cardiac calcinosis (DCC), occurs in certain inbred strains of mice. We now examined a putative inhibitor of calcification, osteopontin, in DCC after transdiaphragmal myocardial freeze-thaw injury. Strong osteopontin expression was found co-localizing with calcification in DCC-susceptible strain C3H/HeNCrlBr, which exhibited low osteopontin plasma concentrations otherwise. Osteopontin mRNA induction was 20-fold higher than in resistant strain C57BL/6NCrlBr, which exhibited fibrous lesions without calcification and little osteopontin expression. Sequence analysis identified several polymorphisms in calcium-binding and phosphorylation sites in osteopontin cDNA. Their potential relevance for DCC was tested in congenic mice, which shared the osteopontin locus with C57BL/6NCrlBr, but retained a chromosomal segment from C3H/HeNCrlBr on proximal chromosome 7. These mice exhibited strong osteopontin expression and DCC comparable to C3H/HeNCrlBr suggesting that a trans-activator of osteopontin transcription residing on chromosome 7 and not the osteopontin gene on chromosome 5 was responsible for the genetic differences in osteopontin expression. A known osteopontin activator encoded by a gene on chromosome 7 is the transforming growth factor-beta1, which was more induced (3.5x) in C3H/HeNCrlBr than in C57BL/6NCrlBr mice.

Animals↗

PubMed2004

Cardiac adenoviral S100A1 gene delivery rescues failing myocardium.

Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.

Adenoviridae↗

PubMed2003

Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinomas.

A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.

Animals↗

PubMed2002

An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands.

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.

Cloning, Molecular↗