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J Holtz

Publications and source records attributed to J Holtz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Lipid peroxidation and the expressional regulation of the heat-shock response during ischemia-reperfusion of rat kidney.

Because of the continuing shortage of donor organs, 'marginal kidneys' are increasingly being used. The purpose of our experiments was to characterize the extent of lipid peroxidation after ischemia-reperfusion (IR) injury in rat kidney, to analyze the expressional regulation of the heat-shock response and now to discuss the clinical application of these results. After ischemia, xanthine oxidase (XO) is thought to be the main oxygen radical-generating system and malondialdehyde (MDA) is considered to be a marker of LPO. In young rats (10 weeks) a unilateral warm ischemia of 40 and 60 min duration with subsequent reperfusion up to 1 h was conducted. Beside the 'footprints' of oxidative stress, the cytosolic antioxidative capacity, expressed as superoxide anion (SOA) scavenging capacity, was investigated. There was only a moderate and transient increase of renal MDA 5 and 10 min after the onset of reoxygenation (133.57/70.67 and 97.84/91.57 vs. 49.47 nmol/g wet weight (ww) in preischemic controls). ATP breakdown (to 83/65 from 2,947 nmol/g ww) with consecutive accumulation of hypoxanthine (up to 1,105 nmol/g ww) at the end of the ischemic period and the subsequent rapid decline of hypoxanthine by XO during reperfusion were used for an assessment of the SOA-generating capacity of these kidneys. Only 1/25-1/50 of the kidney cytosol was able to scavenge the whole amount of SOA generated by the total XO activity of rat kidney. Thus, it could be analytically and stoichiometrically shown that after IR there is only a moderate oxidative stress in kidneys of young rats; this is due to their high SOA-scavenging capacity compared to their SOA-generating ability. We investigated the time course of HSP70-1 and -2 mRNA expression and its relation to cellular ATP levels in renal cortex after different periods of unilateral warm renal ischemia (10-60 min) and reperfusion (up to 60 min) in 10-week-old male Wistar rats, since IR is known to cause induction of both genes. Immediately after ischemia there was a significant induction of both HSP70i genes. While HSP70-1 expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSP70-2 mRNA - generally being expressed on a far lower level than HSP70-1 mRNA - was strongly induced (3-fold) during reperfusion only after brief periods (10 min) of ischemia. Cellular ATP levels rapidly dropped down to 5% with ischemia and the pattern of recovery during reperfusion significantly depended on the duration of the ischemic period thus showing a good relation to the heat-shock (protein) gene expression. We conclude that the HSP70-2 is the more sensitive gene with a lower threshold activation by mild injury, while the HSP70-1 gene mediates the big response of HSP induction after severe injury. Thus, the measurement of the cytosolic antioxidative capacity and the differential expression of HSP70-1 and -2 mRNA could be promising clinical tools to assess the donor viability.

Adenosine Triphosphate↗
PubMed1999

Myocardial gene expression of regulators of myocyte apoptosis and myocyte calcium homeostasis during hemodynamic unloading by ventricular assist devices in patients with end-stage heart failure.

BACKGROUND: In patients with end-stage heart failure, characterized by an increased susceptibility to cardiomyocyte apoptosis and a labile cardiomyocyte calcium homeostasis, a ventricular assist device (VAD) is implanted for bridging to cardiac transplantation and results in myocardial unloading. Although phenotype changes in the failing heart are assumed to result from hemodynamic overload, the reversibility of these changes under unloading is unknown. METHODS AND RESULTS: By use of quantitative reverse-transcription polymerase chain reaction, mRNA expression analyses were performed on left ventricular specimens obtained from 10 nonfailing donor hearts (from 8 patients with dilated cardiomyopathy and 2 patients with coronary heart disease) at the time of VAD implantation and 36 to 169 days later during VAD removal with subsequent cardiac transplantation. In terminally failing hearts before VAD support, left ventricular mRNA analyses revealed increased Pro-ANP, reduced antiapoptotic Bcl-x(L) and antiapoptotic Fas isoform FasExo6Del, and a decreased ratio of sarcoplasmic reticulum Ca(2+)-ATPase per sarcolemmal Na(+)-Ca(2+) exchanger in comparison with nonfailing ventricles. After VAD unloading, ventricular transcription of Pro-ANP was immediately normalized, and apoptotic DNA fragmentation was attenuated. In patients with dilated cardiomyopathy, mRNAs of Bcl-x(L) and FasExo6Del/Fas were enhanced depending on time on VAD. The Bcl-x(L) mRNA level correlated positively with that of the Bcl-x(L) protein. Transcription of sarcoplasmic reticulum Ca(2+)-ATPase/Na(+)-Ca(2+) exchanger demonstrated recovery in only 4 of 10 patients. CONCLUSIONS: Mechanical support of the failing heart induces a time-dependent change in myocardial gene expression compatible with a decreased susceptibility to apoptosis.

Adult↗
PubMed1999

Angiotensin II induces LOX-1, the human endothelial receptor for oxidized low-density lipoprotein.

BACKGROUND: Oxidatively modified LDL (oxLDL) plays an important role in the development of atherosclerosis. OxLDL effects, eg, foam cell formation, are mediated in part by the classic scavenger receptor, whereas other effects may involve the recently cloned endothelial oxLDL receptor, LOX-1 (lectinlike oxLDL receptor-1), which is distinct from macrophage scavenger receptors. Because the regulation of LOX-1 must still be defined, we investigated whether LOX-1 is regulated by the potentially proatherosclerotic stimulant angiotensin II (Ang II). METHODS AND RESULTS: Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we quantified mRNA expression of LOX-1 in primary cultures of human umbilical vein endothelial cells (HUVECs). After treatment with Ang II for 3 hours (1 nmol/L to 1 micromol/L), LOX-1 mRNA was concentration-dependently induced (from 6.9+/-1.4 to 23.1+/-5.5 relative units [RU] by 1 micromol/L Ang II; P<0.05). The angiotensin II type 1 (AT(1)) receptor antagonist losartan prevented this induction. Incubation of HUVECs with Ang II (100 nmol/L, 3 hours) induced LOX-1 protein expression (212+/-21% of control level; P<0. 01) and uptake of 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled oxLDL (209+/-17% of control level; P<0.05) by an AT(1)-dependent pathway, reaching its maximum after 24 hours (680+/-89%; P<0.05). In internal mammary artery biopsy samples from patients with or without ACE inhibitor treatment before coronary artery bypass surgery, LOX-1 mRNA was downregulated by ACE inhibition (6.4+/-2.0 versus 19.3+/-5. 9 RU; n=12 each; P<0.05). CONCLUSIONS: We conclude that LOX-1 is regulated by Ang II in vitro and in vivo, that induction of LOX-1 is mediated by the AT(1) receptor, and that repression of LOX-1 by long-term ACE inhibitor treatment may contribute to the antiatherosclerotic potential of this therapy.

Angiotensin II↗
PubMed1999

Rapid induction and translocation of Egr-1 in response to mechanical strain in vascular smooth muscle cells.

The effect of mechanical strain on transcription and expression of the immediate-early genes, early growth response gene-1 (Egr-1), c-jun, and c-fos, was investigated in neonatal rat aortic vascular smooth muscle (VSM) cells. Cells grown on silicone elastomer plates were subjected to cyclic mechanical strain (1 Hz) at various durations and magnitudes. Egr-1 mRNA increased rapidly in response to cyclic strain, reached a maximum of 10-fold after 30 minutes, and returned to baseline after 4 hours. c-jun exhibited a similar pattern, whereas c-fos mRNA expression was unaffected by strain. Cycloheximide prolonged the increase in Egr-1 and c-jun mRNA and caused superinduction of both. The threshold level of continuous cyclic strain needed to induce expression was 5% for Egr-1 and c-jun. Even a single cycle of mechanical strain that lasted 1 second was sufficient to induce Egr-1 and c-jun mRNA. Strain also increased expression of a transiently transfected Egr-1 promoter-reporter construct. The effect of varying extracellular matrices on strain-induced Egr-1 and c-jun mRNA was examined. In contrast to collagen type 1- and pronectin-coated plates, strain did not significantly alter expression of Egr-1 and c-jun was less induced on laminin-coated plates. On collagen type 1, strain increased Egr-1 protein levels by 2.1-fold at 60 minutes. Immunofluorescence microscopy revealed translocation of Egr-1 to the nucleus in response to strain. These observations indicate that Egr-1 expression and translocation are sensitive to mechanical perturbation of the cell. c-jun is also induced by strain, but c-fos is not. The signal for this induction may involve specific cell-matrix interactions.

Animals↗
PubMed1999

Quantification of cardioprotective gene expression in porcine short-term hibernating myocardium.

Several cardioprotective proteins are induced during myocardial ischemia, such as heat shock proteins and anti-apoptotic Bcl-2-related proteins which, when experimentally overexpressed, have been shown to prevent ischemia-induced myocyte loss. As this pathophysiological induction is obviously not sufficient to prevent losses of myocytes, we analysed whether it could occur under moderate myocardial ischemia with hibernation, thus potentially contributing to myocyte protection under these conditions. Therefore, using anesthetized pigs with documented myocardial hypoperfusion and short-term hibernation, we investigated the left ventricular mRNA expression of the inducible heat shock protein Hsp70 and of the anti-apoptotic Bcl-XL in comparison with the pro-apoptotic Bak and Fas expression. For transcriptional analyses, the porcine cDNA sequences of Bcl-XL, Bak and Fas were identified by polymerase chain reaction (PCR) or by screening of a porcine heart cDNA library and cloned. Using reverse transcription polymerase chain reaction (RT-PCR), we observed an unchanged mRNA expression of inducible Hsp70, Bcl-XL, Bak and Fas after 85 min of hypoperfusion in the short-term hibernating myocardium, as well as after 30 min of subsequent reperfusion in the stunned myocardium, compared with transcription in a non-hypoperfused control area of the same ventricle. In conclusion, the mRNA expression of inducible Hsp70 and of several apoptosis-modulating proteins is not altered during moderate myocardial ischemia resulting in short-term hibernation of the affected area and during subsequent stunning.

Amino Acid Sequence↗
PubMed1999

[Apoptosis--what is it? Significance in coronary heart disease and myocardial infarct].

Apoptosis is a physiological, highly conserved program of cellular suicide, characterized by nuclear condensation with DNA-fragmentation, by alterations in the distribution of cell membrane phospholipids, and by cellular shrink-age. Apoptotic cellular remnants engulfed by cell membranes are phagocytized largely without activation of inflammatory reactions. The apoptotic program is executed by a cascade of highly specific caspases, activated by complexation of initiatorcaspases in cytosolic signalling complexes at receptors of the TNF family or at impaired mitochondria. In many forms of cellular stress with damage of nuclear DNA and mitochondria, mixed forms of cell death are triggered with regulated activation of the apoptotic program and concomitantly, with induction of catastrophic necrosis. Such a mixed form of myocyte death is observed in myocardial ischemia and reperfusion. Antiapoptotic interventions can delay ischemic myocardial damage in experiments. Therefore, those interventions appear conceivable as future strategy for acutely enhancing the available time interval for therapeutic reperfusion. However, chronic inhibition of apoptosis for ongoing prevention of myocardial ischemic damage may not become a plausible strategy because of disturbances of the immune system, because of putatively infavorable effects on arteriosclerotic lesions and because of likely disturbances in the physiologic elimination of damaged mitochondria.

Apoptosis↗
PubMed1999

Evidence for only a moderate lipid peroxidation during ischemia-reperfusion of rat kidney due to its high antioxidative capacity.

The extent of lipid peroxidation after ischemia-reperfusion (I-R) injury in rat kidney has been controversial. After I, xanthine oxidase (XO) is thought to be the main oxygen radical-generating system and malondialdehyde (MDA) is considered to be a marker of lipid peroxidation (LPO). In young rats (10 weeks old) a unilateral warm I of 40 and 60 min duration with subsequent R up to 1 h was conducted. Beside the "footprints" of oxidative stress, the cytosolic antioxidative capacity, expressed as superoxide anion (SOA) scavenging capacity, and the renal catalase were also investigated. There was only a moderate and transient increase of renal MDA 5 and 10 min after the onset of reoxygenation (133.57/70. 67 and 97.84/91.57 vs. 49.47 nmol/g ww in preischemic controls). ATP breakdown (to 83/65 from 2947 nmol/g ww) with consecutive accumulation of hypoxanthine (up to 1105 nmol/g ww) at the end of ischemic period and the subsequent rapid decline of hypoxanthine by XO during reperfusion were used for an assessment of the SOA-generating capacity of these kidneys. Superoxide dismutase (SOD) activity, glutathione (GSH) and the high activity of catalase (18000 U/g ww) remained nearly unchanged during R. Only 1/25-1/50 of the kidney cytosol was able to scavenge the whole amount of SOA generated by the total XO activity of rat kidney. Thus, it could be analytically and stoichiometrically shown that after IR there is only a moderate oxidative stress in kidneys of young rats; this is due to their high SOA-scavenging capacity compared with their SOA-generating ability.

Adenosine Triphosphate↗
PubMed1999

Differential expression of heat shock proteins 70-1 and 70-2 mRNA after ischemia-reperfusion injury of rat kidney.

Ischemia-reperfusion injury in the kidney is known to cause induction of the inducible form of the 70 kDa heat shock protein HSP70i (or HSP72). However, knowledge of the expressional regulation of the two coding genes for HSP70i - HSP70-1 gene and HSP70-2 gene - is very limited. We investigated the time course of HSP70-1 and -2 mRNA expression and its relation to cellular ATP levels in the renal cortex after different periods of unilateral warm renal ischemia (10-60 min) and reperfusion (up to 60 min) in 10-week-old male Wistar rats. Immediately after ischemia there was a significant induction of both HSP70i genes. While HSP70-1 expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSP70-2 mRNA - which was generally expressed at a far lower level than HSP70-1 mRNA - was strongly induced (3-fold) during reperfusion only after brief periods (10 min) of ischemia. Cellular ATP levels rapidly dropped to 5% with ischemia and the pattern of recovery during reperfusion significantly depended on the duration of the ischemic period, thus showing a good relation with the heat shock (protein) gene expression. We conclude that HSP70-2 is the more sensitive gene with a lower activation threshold by mild injury, while the HSP70-1 gene mediates the major response of heat shock protein induction after severe injury.

Adenosine Triphosphate↗
PubMed1999

Altered levels of mRNA of apoptosis-mediating genes after mid-term mechanical ventricular support in dilative cardiomyopathy--first results of the Halle Assist Induced Recovery Study (HAIR).

BACKGROUND: Ventricular assist devices (VADs) lead to an immediate unloading of the failing heart. Although VADs are used as a bridge to transplant, in some cases patients suffering from dilated cardiomyopathy have been weaned from the VAD without transplantation after a recovery process initiated by the cardiac support. Myocardial apoptosis is associated with inadequate myocardium and might be reverted during VAD support of the failing heart. Therefore we measured transcription of apoptosis-associated genes FasExo6 Del, Fas-receptor, and Bcl-xL as markers of a putative recovery. METHODS: Fas-receptor, its soluble isoform FasExo6Del, and Bcl-xL mRNA were quantified by standard calibrated competitive reverse-transcription polymerase chain reaction (PCR) in 6 patients suffering from dilated cardiomyopathy. RNA standards were prepared by introducing 100 bp deletions into the native cDNA, resulting in truncated PCR products with identical primer-binding sites. Standards were transcribed in vitro and the resulting RNA was quantified. RESULTS: Transcription of apoptosis-inhibiting genes FasExo6 Del and Bcl-xL were upregulated in patients supported for more than 6 weeks. Fas receptor mRNA remained unaffected by VAD support. CONCLUSIONS: Transcriptional upregulation of apoptosis-inhibiting genes might be caused by a desensitization to apoptotic stimuli and might indicate a relaxation of the diseased status of the myocardium. These data outline the first biochemical evidence of a remodelling process occurring in supported ventricular myocardium.

Apoptosis↗
PubMed1999

Evaluation of the circulation of reconstructive flaps using laser-induced fluorescence of indocyanine green.

A new method for evaluating the circulation in surgical flaps using laser-induced fluorescence of indocyanine green (ICG) images is reported. In clinical trials the authors found that ICG imaging demonstrated good circulation accurately in 16 of 21 flaps with no clinical manifestations of compromised circulation. In 3 patients in whom partial discoloration and cyanosis of the flaps were visible, the dye study indicated poor circulation in the identical areas. In 2 other patients in whom flaps appeared clinically satisfactory, the flaps were shown by ICG imaging to have greatly compromised circulation. In a patient in whom the flap was left in place, slough of almost the entire flap resulted. Another flap with questionable circulation was returned to its original location, where it healed. Thus, while it is a still a new approach and under continual evaluation, the use of ICG fluorescence shows promise as a valuable adjunct to current methods of flap evaluation.

Coloring Agents↗
PubMed1999

Enhanced angiotensin-converting enzyme activity and impaired endothelium-dependent vasodilation in aortae from hypertensive rats: evidence for a causal link.

Endothelial vasomotor function is impaired in a variety of disorders representing both early and late stages of atherosclerosis. There is experimental evidence for enhanced vascular angiotensin-converting enzyme (ACE) activity in these disorders. We explored whether enhanced vascular ACE activity accounts for endothelial dysfunction in experimental hypertension. Hypertension was induced in rats by coarctation of the aorta. At 2 weeks post-operation, the animals were randomly divided into groups receiving the ACE inhibitor quinapril (2.0 mg.kg(-1).day(-1)), the angiotensin type-1 receptor antagonist losartan (3.0 mg.kg(-1).day(-1)), the B(2) kinin receptor antagonist icatibant (0.4 mg.kg(-1).day(-1)), quinapril plus icatibant, losartan plus icatibant, or no drug. Analyses were performed 4 weeks post-operation. None of the drug treatments had any significant effect on blood pressure. ACE activity was nearly doubled in aortae from untreated hypertensive rats as compared with sham-operated rats. Quinapril reduced ACE activity in aortae from hypertensive rats by 75%, losartan caused a 40% decrease, and icatibant had no effect. Endothelium-dependent, nitric oxide-mediated vasodilator responses studied in vitro were impaired by 40% in aortae from untreated hypertensive rats as compared with sham-operated rats. Both quinapril and losartan restored endothelial vasomotor function in aortae from hypertensive rats. Co-applied icatibant negated the effects of quinapril, but not those of losartan. The level of endothelial NO synthase (eNOS) mRNA determined by competitive RNA PCR was decreased by half in aortae from untreated hypertensive rats as compared with sham-operated rats. Quinapril induced an increase in the eNOS mRNA level of 350% in aortae from hypertensive rats, which was negated by co-applied icatibant. Losartan restored eNOS mRNA expression in aortae from hypertensive rats to normal levels, and this effect was not modified by co-applied icatibant. These findings suggest that enhanced vascular ACE activity accounts for endothelial vasomotor dysfunction by impairing the bioavailability of endothelium-derived NO. Both enhanced formation of angiotensin II and enhanced metabolism of bradykinin might account for a vascular deficiency of bioactive NO.

Animals↗
PubMed1998

Endotoxin and tumor necrosis factor alpha exert a similar proinflammatory effect in neonatal rat cardiomyocytes, but have different cardiodepressant profiles.

Bolus application of endotoxin to healthy volunteers results in reversible hemodynamic alterations, such as observed in septic cardiomyopathy. Currently, endotoxin-induced cardiodepression is mainly attributed to the endotoxin-induced release of proinflammatory cytokines into the circulation, particularly of tumor necrosis factor alpha and interleukin-1, the serum levels of these cytokines being enhanced in sepsis and septic shock, and also in various heart diseases. In this study, we report a proinflammatory effect of endotoxin (1-10 micrograms/ml, 24-h incubation period) on neonatal rat cardiomyocytes in serum-free culture, evidenced by induction of inducible nitric oxide synthase, enhanced release of nitrite (protein synthesis-dependent) and interleukin-6 into the supernatant, as well as an increase in cell-associated interleukin-1 and a specific cardiodepressant profile: endotoxin disrupts beta-adrenoceptor-mediated increase in pulsation amplitude, but alpha-adrenoceptor-induced increase in pulsation amplitude and arrhythmias are not suppressed. In the presence of dexamethasone (0.1 microM), the endotoxin-mediated blockade of beta-adrenergic responsiveness, as well as induction of inducible nitric oxide synthase, enhanced nitrite release and interleukin-1/-6-production are inhibited. In contrast, tumor necrosis factor alpha at a low concentration (10 U/ml) depresses alpha- and beta-adrenergic responsiveness in the presence of dexamethasone in a nitric oxide-independent manner. These data suggest a stimulatory effect of endotoxin on the cardiomyocyte and a specific proinflammatory and nitric oxide-dependent cardiodepressant profile of endotoxin.

Animals↗
PubMed1998

Role of ACE inhibition or AT1 blockade in the remodeling following myocardial infarction.

Ventricular remodeling following nonfatal myocardial infarction includes an excentric hypertrophy of the surviving myocardium, associated with hemodynamic overload. Disseminated cardiomyocyte apoptosis and phenotype changes of the surviving myocardium, such as a labile calcium homeostasis of the hypertrophied cardiomyocytes, impaired beta-adrenergic signal transduction, interstitial fibrosis, and reduced coronary reserve are typical features of the overloaded, distended ventricular wall. They are considered as relevant for the myocardial dysfunction progressing to overt cardiac failure and for the enhanced mortality risk. Hemodynamic load and trophic angiotensin effects are assumed to cause this excentric hypertrophy, since systemic and local angiotensin formation in the overloaded myocardium is activated. In isolated rat cardiomyocytes, cultured on distensible membranes, overload is mimicked by distension, and causes enhanced formation and release of angiotensin II, which results in AT1 receptor mediated trophic reactions of distended neonatal cardiomyocytes and in AT1-mediated apoptosis in distended adult cardiomyocytes. In experimental models of postinfarct remodeling, therapy with ACE inhibition or with AT1 blockade similarly normalizes myocardial hypertrophy, interstitial fibrosis, and impaired coronary reserve. In patients with terminal heart failure, a reduction of apoptotic signs and a normalization of antiapoptotic gene expression is obtained by myocardial unloading under ventricular assist devices or by treatment with ACE inhibitors, and the latter therapy has been shown to improve survival. In contrast to general assumptions and to predictions from data in vitro, the improvements obtained by AT1 blocker therapy in vivo are mediated by bradykinin, as are those obtained under ACE inhibitors. Under AT1 blockade, bradykinin is probably activated due to stimulation of AT2 and other AT receptors.

Angiotensin II↗
PubMed1998

Expressional analysis of the cardiac Na-Ca exchanger in rat development and senescence.

The cardiac Na-Ca exchanger (NCX) serves as the main calcium extrusion mechanism in heart muscle and is important in maintaining intracellular calcium homeostasis. The accumulations of NCX RNA and protein are known to be regulated in cardiac hypertrophy, by thyroid hormone and during postnatal development. In this study the temporal and spatial patterns of NCX mRNA and protein accumulations were examined, and nuclear run-on assays performed. NCX is highly expressed in late fetal and neonatal rat hearts, decreasing to adult levels by 20 days after birth for RNA (P < 0.05, fetal and 1 neonatal day old (1 ND) versus 20 day old (20 ND)). Maximal protein expression is seen in 19 embryonic day (ED) old hearts, and reaches adult levels sometime after 20 neonatal days. (P < 0.05, fetal versus adult). Spatially, NCX is homogenously expressed in early embryonic and fetal heart, followed by a decline after birth. The protein levels decline more slowly suggesting a long protein half-life. The lowest level of mRNA accumulation is seen in 6 and 18 month old animals (P < 0.05 for all time points before 10 neonatal days). In the 24 month old senescent rat, NCX transcripts are increased by almost 50% above that seen at 6 and 18 months (P < 0.05) but are not different from those at 15 neonatal days. Perinatal NCX expression is regulated transcriptionally: late fetal and neonatal hearts have high transcriptional activity but by 20 postnatal days, no detectable transcriptional activity can be demonstrated. Throughout development, at least five transcription start sites are used, and no significant difference in the 5' untranslated or 3' coding splice sites could be demonstrated, although several new cardiac splicing variants were identified. We also report the cloning of a 3.7 kb fragment containing the cardiac NCX1 promoter which is transcriptionally active in neonatal cardiomyocytes.

Aging↗