Thomas Thum

Gene expression in distinct regions of the heart

BACKGROUNDnCytochrome P450 mono-oxygenases bring about metabolism of many drugs, including verapamil, but no information is available on the metabolism of such drugs in the human heart.nnnMETHODSnWe investigated the gene expression of major human cytochrome P450 mono-oxygenases in the various regions of explanted hearts from six patients with dilated cardiomyopathy and one with transposition of the arterial trunk. For comparison we also studied samples of liver and lung from three patients and two samples of normal heart. The biological significance of tissue-specific cytochrome mono-oxygenase gene expression was further investigated by study of the microsomal metabolism of verapamil in ventricular tissue.nnnFINDINGSnmRNA for the cytochromes 1A1, 2B6/7, 2C8-19, 2D6, and 4B1 was predominantly expressed in the right ventricle; the unilateral expression of the 2D6 gene in right-venticular tissue is important because of its key role in the metabolism of beta-blockers. A strong correlation between tissue-specific gene expression and enzyme activity was found; microsomal metabolism of verapamil was confined to the right ventricle. By contrast, cytochrome P450 3A genes (which are the major drug-metabolising enzymes in the liver and the lung) were not expressed in any of the human heart tissues, and mRNA transcripts for epoxide hydrolase, cytochrome P450 2E1, and flavin-containing mono-oxygenases 2 and 5 were detected in all regions of the human heart and the great vessels.nnnINTERPRETATIONnThese findings show that expression of genes for cytochrome P450 mono-oxygenases and verapamil metabolism are found predominantly in the right side of the heart. This finding may explain lack of efficacy of certain cardioselective drugs.

Hallmarks of ion channel gene expression in end-stage heart failure

Electrical conductance is greatly altered in end‐stage heart failure, but little is known about the underlying events. We therefore investigated the expression of genes coding for major inward and outward ion channels, calcium binding proteins, ion receptors, ion exchangers, calcium ATPases, and calcium/calmodulin‐dependent protein kinases in explanted hearts (n=13) of patients diagnosed with end‐stage heart failure. With the exception of Kv11.1 and Kir3.1 and when compared with healthy controls, major sodium, potassium, and calcium ion channels, ion transporters, and exchangers were significantly repressed, but expression of Kv7.1, HCN4, troponin C and I, SERCA1, and phospholamban was elevated. Hierarchical gene cluster analysis provided novel insight into regulated gene networks. Significant induction of the transcriptional repressor m‐Bop and the translational repressor NAT1 coincided with repressed cardiac gene expression. The statistically significant negative correlation between repressors and ion channels points to a mechanism of disease. We observed coregulation of ion channels and the androgen receptor and propose a role for this receptor in ion channel regulation. Overall, the reversal of repressed ion channel gene expression in patients with implanted assist devices exemplifies the complex interactions between pressure load/stretch force and heart‐specific gene expression.—Borlak, J., Thum, T. Hallmarks of ion channel gene expression in end‐stage heart failure. FASEB J. 17, 1592–1608 (2003)

Testosterone, cytochrome P450, and cardiac hypertrophy

Cytochrome P450 mono‐oxygenases (CYP) play an essential role in steroid metabolism, and there is speculation that sex hormones might influence cardiac mass and physiology. As CYP mono‐oxygenases activity is frequently altered during disease, we tested our hypothesis that CYP mono‐oxygenase expression and testosterone metabolism are altered in cardiac hypertrophy. We investigate major CYP mono‐oxygenase isoforms and other steroid‐metabolizing enzymes and the androgen receptor in normal, hypertrophic, and assist device‐supported human hearts and in spontaneously hypertensive rats (SHR). We show increased and idiosyncratic metabolism of testosterone in hypertrophic heart and link these changes to altered CYP mono‐oxygenase expression. We show significant induction of 5‐alpha steroid reductase and P450 aromatase gene expression and enhanced production of dihydrotestosterone, which can be inhibited by the 5‐alpha reductase inhibitor finasteride. We show increased gene expression of the androgen receptor and increased levels of lipid peroxidation in diseased hearts, the latter being markedly inhibited by CYP mono‐oxygenase inactivation. We show alpha‐MHC to be significantly repressed in cardiac hypertrophy and restored to normal on testosterone supplementation. We conclude that heart‐specific steroid metabolism is of critical importance in cardiac hypertrophy.—Thum, T., Borlak, J. Testosterone, cytochrome P450, and cardiac hypertrophy. FASEB J. 16, 1537–1549 (2002)

Cellular dedifferentiation of endothelium is linked to activation and silencing of certain nuclear transcription factors: implications for endothelial dysfunction and vascular biology

We investigated the gene expression of the nuclear transcription factors c/EBPa, GATA‐2, and the silencer Oct‐1 in conjunction with the gene expression of all major cytochrome P450 genes and of eNOS in cultures of endothelial cells of the rat. The purity of cultured endothelial cells was also confirmed by flow cytometry measurements of PECAM‐1, a surface antigen of endothelial cells. Taken collectively, the gene expression and flow cytometry studies provide strong evidence for c/EBPa, GATA‐2, and Oct‐1 to play a key role in the cellular dedifferentiation of endothelial cells; gene expression of eight individual CYP genes in conjunction with protein activity could be significantly increased upon treatment with Aroclor 1254, a well‐documented chemical inducer of a battery of genes. Nevertheless, the gene expression of c/EBPa, GATA‐2, and most of the CYP genes was dramatically reduced (up to 90%) in cell cultures lacking PECAM‐1 expression; in strong contrast, expression of the silencer Oct‐1 was massively increased (~ 14‐fold). We thus conclude activation of the silencer Oct‐1 to be strongly correlated with loss of PECAM‐1 and eNOS gene expression, e.g., loss of cellular differentiation and endothelial function; in conjunction, gene expression of all major P450 isoforms was dramatically reduced in cultures of dedifferentiated endothelial cells. This process of cellular dedifferentiation and endothelial dysfunction was accompanied by down‐regulation of endothelial specific transcription factors.—Thum, T., Haverich, A., Borlak, J. Cellular dedifferentiation of endothelium is linked to activation and silencing of certain nuclear transcription factors: implications for endothelial dysfunction and vascular biology. FASEB J. 14, 740–751 (2000)

Mechanistic Role of Cytochrome P450 Monooxygenases in Oxidized Low-Density Lipoprotein–Induced Vascular Injury Therapy Through LOX-1 Receptor Antagonism?

Abstract— Oxidized low-density lipoprotein (oxLDL) is an important risk factor for vascular injury. Its role on coronary vasoconstriction remains speculative. Endothelial monooxygenases (cytochrome P450s [CYPs]) are regulators of vascular tonus through production of epoxy fatty acids. We investigated the effects of oxLDL on CYP monooxygenases in human arterial coronary endothelial cells and explanted healthy and atherosclerotic aortae. We found oxLDL to induce radical oxygen species production via the action of NADPH oxidase NOX4. Intracellular radical oxygen species production prompted reduced protein expression of the transcriptional regulator nuclear factor 1 (NF-1). We identified novel DNA binding sites for NF-1 in promoter regions of CYPs. DNA binding of NF-1 was confirmed by electromobility shift assays. OxLDL repressed DNA binding of NF-1 and diminished transcript level of CYP genes targeted by this factor. The production of endothelial-derived hyperpolarization factor, a key regulator of vascular tonus, was also reduced. Repression of CYP monooxygenases was reversed, and production of endothelial-derived hyperpolarization factor was normalized after treatment of endothelium with the lectin-like oxLDL receptor antagonist &kgr;-carrageenan or blocking of LOX-1 with a specific antibody. This suggests a mechanistic role of CYP monooxygenases in oxLDL-induced vascular injury. Therapy of endothelial dysfunction through LOX-1 receptor antagonism will be an interesting avenue to explore. The full text of this article is available online at http://www.circresaha.org.

Cytochrome P450 mono-oxygenase gene expression and protein activity in cultures of adult cardiomyocytes of the rat.

There are a substantial number of drugs acting either directly or indirectly on the heart, but surprisingly, little is known about the metabolic capacity of heart muscle cells. We therefore investigated the gene expression and protein activity of cytochrome P450 isozymes in cultures of adult cardiomyocytes of the rat. Semi‐quantitative CYP gene expression pattern suggests CYP1A1 and CYP2B1/2 to be key players in cardiomyocytes and upon treatment with Aroclor 1254 approximate 4 fold inductions could be observed for both gene families, when compared with appropriate controls. The mRNA expression of most genes was sustained for prolonged periods of time, e.g. up to 120u2003h in culture and in the case of the CYP3A1 gene an approximate 10 fold induction was observed at the higher Aroclor 1254 dose level (10u2003μM) in 24u2003h old cultures. The constitutively expressed genes, e.g. CYP2C11 and CYP2E1 are expressed throughout the entire culture period (5 days) and did not respond to Aroclor 1254 treatment. CYP4A1 was mainly expressed in freshly isolated cardiomyocytes of control animals and its expression declined rapidly in culture. There was good agreement between gene expression and translated protein activity using 7‐ethoxyresorufin and testosterone as substrates. The data reported herein should foster the routine use of freshly isolated and cultivated cardiomyocytes for drug profiling and toxicity studies.

Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line

Growth hormone deficiency is linked to cardiovascular disease and particularly increased peripheral vascular resistance. Surprisingly, its role in endothelial nitric oxide (NO) synthetase (eNOS) regulation and NO release is basically unknown. We therefore studied the effects of different doses of somatotropin in cultures of a human endothelial cell line (EAhy926). We investigated expression and activity of eNOS, as well as other target genes known to be deregulated in cardiovascular disease including E‐selectin and the lectin‐like oxidized low density lipoprotein receptor. Treatment of cultured human endothelial cells with somatotropin resulted in significant (P<0.05) increases of eNOS gene and protein expression, as well as NO release, whereas production of intracellular reactive oxygen species was significantly reduced, at the highest somatotropin dose level. The enhanced eNOS gene/protein expression and enzyme activity correlate well. Our findings are suggestive for a novel role of growth hormone in endothelial biology, and particularly NO production.

Induction of nuclear transcription factors, cytochrome P450 monooxygenases, and glutathione S-transferase alpha gene expression in Aroclor 1254-treated rat hepatocyte cultures

Aroclor 1254 is a complex mixture of polychlorinated biphenyls and is well known for its potency to induce drug-metabolising enzymes, but little is known about its ability to modulate gene expression of transcription factors, which code for proteins that bind to the regulatory elements of DNA and facilitate transcriptional activation. We therefore investigated the gene expression of the liver-specific transcription factors CCAAT/enhancer-binding protein alpha (c/EBPalpha), hepatic nuclear factor (HNF) 1 and 4, and major cytochrome P450 (CYP) isozymes in addition to glutathione S-transferase alpha 2 (GSTA-2) in cultures of primary rat hepatocytes. We found highly significant and dose-dependent increases of c/EBPalpha (up to 62-fold), HNF-1 (up to 7-fold), HNF-4 (up to 8-fold), and 50- and 4-fold inductions of GSTA-2 and CYP monooxygenases, respectively. Based on the ethoxyresorufin-O-deethylase assay, the gene expression and enzyme activity for CYP1A1 were in good agreement, but for other CYP isozymes similar correlations could not be obtained. In conclusion, the simultaneous induction of liver-specific TFs and of several detoxifying enzymes may point to a coordinate genomic response in cultures of rat hepatocytes upon treatment with Aroclor 1254.

A rapid and simple CYP2D6 genotyping assay—case study with the analgetic tramadol

There is substantial evidence for a causal relationship between genetic variability of the CYP2D6 gene and changes in the pharmacokinetics of drugs. Therefore, knowledge of single-nucleotide polymorphisms (SNPs) prior to drug administration is highly desired for assisting in the development of individualized pharmacotherapy. We therefore developed a robust assay that detects common CYP2D6 alleles within 60 minutes of blood withdrawal and links carriers of the variant CYP2D6*3 and *4 alleles to the pharmacokinetics of tramadol. This new genotyping assay employs fluorescence resonance energy transfer (FRET) analysis, which permits parallel identification of the CYP2D6*3 and CYP2D6*4 alleles within 60 minutes of blood withdrawal. We determined the genotypes of 100 healthy unrelated individuals and studied the pharmacokinetics of tramadol in 24 CYP2D6 genotyped healthy subjects. The total allelic frequencies of homozygote carriers were 0.015 and 0.25 for the CYP2D6*3 and *4 alleles, respectively, and the plasma area under the curve (AUC) was 84% above those of extensive metabolizers (homozygous EM group): 3,941.2 ng/mL.h (95% confidence interval [CI], 2,928.9 ng/mL.h to 4,953.5 ng/mL.h) versus 2,142.6 ng/mL.h (95% CI, 1,829.6 ng/mL.h to 2,455.7 ng/mL.h). Likewise, the AUC for the O-desmethyl-tramadol metabolite (M1) was significantly reduced in poor metabolizers (PMs): 300.2 ng/mL.h (95% CI, 260.3 ng/mL.h to 340.0 ng/mL.h) versus 842,6 ng/mL.h (95% CI, 715.1 ng/mL.h to 970.0 ng/mL.h). We observed a statistically significant correlation between plasma tramadol AUC and production of the O-desmethyl metabolite in CYP2D6 genotyped healthy volunteers. Our assay can be used reliably in clinical pharmacology studies and may be used for dose adjustment.

Verapamil: identification of novel metabolites in cultures of primary human hepatocytes and human urine by LC-MS n and LC-NMR

1. Verapamil is a well-known and world-wide prescribed calcium antagonist, but it suffers from extensive first-pass metabolism. Although it has been marketed for many years, a complete understanding of its biotransformation in humans is still lacking. 2. The metabolism of verapamil was therefore investigated in cultures of primary human hepatocytes and in extracts of human urine after oral dosing. Identification of metabolites was done with LC-MS n and LC-NMR (600 MHz) to obtain in-depth information on its biotransformation products and definitive proof of the proposed chemical structures of metabolites. 3. Hyphenation of LC-MS n and LC-NMR was shown to be a powerful and effective platform for the identification of metabolites. Indeed, 21 Phase I and 16 Phase II metabolites were identified. Basically, all the Phase II metabolites (glucuronides) and 11 of the Phase I (oxidative) metabolites were not reported previously. 4. New insight into verapamils biotransformation pathway is provided as well as evidence about its true complexity of metabolic disposal.