Radiation dosimetry of (68)Ga-PSMA-11 (HBED-CC) and preliminary evaluation of optimal imaging timing.

PURPOSE: The clinical introduction of (68)Ga-PSMA-11 ("HBED-CC") ligand targeting the prostate-specific membrane antigen (PSMA) has been regarded as a significant step forward in the diagnosis of prostate cancer (PCa). In this study, we provide human dosimetry and data on optimal timing of PET imaging after injection. METHODS: Four patients with recurrent PCa were referred for (68)Ga-PSMA-11 PET/CT. Whole-body PET/CTlow-dose scans were conducted at 5 min, and 1, 2, 3, 4 and 5 h after injection of 152-198 MBq (68)Ga-PSMA-11. Organs of moderate to high uptake were used as source organs; their total activity was determined at all measured time points. Time-activity curves were created for each source organ as well as for the remainder. The radiation exposure of a (68)Ga-PSMA-11 PET was identified using the OLINDA-EXM software. In addition, tracer uptake was measured in 16 sites of metastases. RESULTS: The highest tracer uptake was observed in the kidneys, liver, upper large intestine, and the urinary bladder. Mean organ doses were: kidneys 0.262 ± 0.098 mGy/MBq, liver 0.031 ± 0.004 mGy/MBq, upper large intestine 0.054 ± 0.041 mGy/MBq, urinary bladder 0.13 ± 0.059 mGy/MBq. The calculated mean effective dose was 0.023 ± 0.004 mSv/MBq (=0.085 ± 0.015 rem/mCi). Most tumor lesions (n = 16) were visible at 3 h p.i., while at all other time points many were not qualitatively present (10/16 visible at 1 h p.i.). CONCLUSIONS: The mean effective dose of a (68)Ga-PSMA-11 PET is 0.023 mSv/MBq. A 3-h delay after injection was optimal timing for (68)Ga-PSMA-11 PET/CT in this patient cohort.

Mediastinal emphysema after long-distance flight with ketoacidosis and underlying diabetes mellitus type 1.

A 21-year old female with diabetes mellitus type 1 presented to our hospital's emergency department having suffered from shortness of breath, mild chest pain, and vomiting following her arrival after a long-distance flight two days earlier. Symptoms had since subsided and physical examination was normal. Blood analysis revealed increased D-dimers and diabetic ketoacidosis. Computed tomography (CT) examination excluded pulmonary embolism but demonstrated significant mediastinal emphysema. After conservative treatment including nasal oxygen and adjustment of insulin therapy, follow-up low-dose CT after four days confirmed full regression of the emphysema. The patient was discharged feeling well, with a recommendation for improved diabetes treatment. Spontaneous pneumomediastinum is a rare condition occurring in younger patients without trauma or pulmonary disease. Over-inflation and/or pulmonary vasoconstriction have been proposed as major physiological contributors and were likely evoked in the present case by increased respiratory drive due to ketoacidosis and hypoxic vasoconstriction during long distance flight.

Pulmonary hypertension associated with left heart disease: Updated Recommendations of the Cologne Consensus Conference 2018.

In the summer of 2016, delegates from the German Society of Cardiology (DGK), the German Respiratory Society (DGP), and the German Society of Pediatric Cardiology (DGPK) met in Cologne, Germany, to define consensus-based practice recommendations for the management of patients with pulmonary hypertension (PH). These recommendations were built on the 2015 European Pulmonary Hypertension guidelines, aiming at their practical implementation, considering country-specific issues, and including new evidence, where available. To this end, a number of working groups was initiated, one of which was specifically dedicated to PH associated with left heart disease. In this context, the European Guidelines point out that the drugs currently approved to treat patients with PAH (prostanoids, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, sGC stimulators) have not sufficiently been investigated in other forms of PH. However, despite the lack of respective efficacy data, an uncritical use of targeted PAH drugs in patients with PH associated with left heart disease is currently observed at an increasing rate. This development is a matter of concern. On the other hand, PH is a frequent problem that is highly relevant for morbidity and mortality in patients with left heart disease. In that sense, the distinction between isolated post-capillary pulmonary hypertension (IpcPH) and combined post- and pre-capillary pulmonary hypertension (CpcPH) and their proper definition may be of particular relevance. The detailed results and recommendations of the working group on PH associated with left heart disease, which were last updated in the spring of 2018, are summarized in this article.

Lactate accumulation rather than ATP depletion predicts ischemic myocardial necrosis: implications for the development of lethal myocardial injury.

In ischemia, the myocardial metabolic status determines the expansion of necrosis. Decreased ATP levels and increased lactate contents in ischemic myocardium undergoing lethal injury are known to be related to the expansion of irreversible damage. However, their individual contributions have not yet been firmly established. Using two differently effective protocols of ischemic preconditioning (IP short and IP long), ischemic cardioplegic arrest (CP) and their combination (IP+CP) to directly influence the metabolic status of porcine myocardium, graded preservations in ATP content and decreases in lactate accumulation during 45 min ischemia could be achieved (control: ATP, 0.15+/-0.03; lactate, 60.53+/-4.89 micromol/g wet weight; IP short, 0.33+/-0.10/27.42+/-3.90; IP long, 0.60+/-0.10/17.49+/-2.14; CP, 0.98+/-0.12/11.82+/-0.96; IP+CP, 2.24+/-0.28/10.88+/-0.89; all P<0.001 vs. control). At the same time, a graded reduction of myocardial necrosis was observed (90.0+/-3.1 vs. 31.7+/-4.55 vs. 5.05+/-2.1 vs. 0.0 [isolated patchy necroses] vs. none). Regression analysis revealed only a weak correlation of infarct size and ATP preservation (r=0.567). In fact, there was a biphasic relation: with ATP levels above 1 micromol/g wet weight, no infarction occurred. ATP levels below this threshold value were associated with steep increase in infarct size. However, even for this latter range, the regression coefficient remained low (r=0.654). Instead, over the entire range, there was a close, rectilinear correlation of infarct size and lactate accumulation (r=0.939). These data indicate that lactate accumulation rather than ATP depletion determines the development of lethal myocardial injury. However, the biphasic relation between ATP depletion and infarct size suggests the latter to play a permissive role, since above a threshold value of 1 micromol/g wet weight neither substantial lactate accumulation nor infarction was observed. Below this threshold, however, infarct size increased as lactate accumulated.

Impact of beta-blocker treatment on the prognostic value of currently used risk predictors in congestive heart failure.

OBJECTIVES: This prospective study tested the impact of beta-blocker treatment on currently used risk predictors in congestive heart failure (CHF). BACKGROUND: Given the survival benefit obtained by beta-blockade, risk stratification by factors established in the "pre-beta-blocker era" may be questioned. METHODS: The study included 408 patients who had CHF with left ventricular ejection fraction (LVEF) <45%, all treated with an angiotensin-converting enzyme inhibitor or angiotensin type 1 receptor antagonist, who were classified into those receiving a beta-blocker (n = 165) and those who were not (n = 243). In all patients, LVEF, peak oxygen consumption (peakVO(2)), plasma norepinephrine (NE) and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined. RESULTS: Although the New York Heart Association functional class (2.2 +/- 0.7 vs. 2.3 +/- 0.7), peakVO(2) (14.4 +/- 5.2 ml/min per kg vs. 14.4 +/- 5.5 ml/min per kg) and NT-proBNP (337 +/- 360 pmol/l vs. 434 +/- 538 pmol/l) were similar in the groups with and without beta-blocker treatment, the group with beta-blocker treatment had a lower heart rate (68 +/- 30 beats/min vs. 76 +/- 30 beats/min), lower NE (1.7 +/- 1.2 nmol/l vs. 2.5 +/- 2.2 nmol/l) and higher LVEF (24 +/- 10% vs. 21 +/- 9%; all p < 0.05). Within one year, 34% of patients without beta-blocker treatment, but only 16% of those with beta-blocker treatment (p < 0.001), reached the combined end point, defined as hospital admission due to worsening CHF and/or cardiac death. A beneficial effect of beta-blocker treatment was most obvious in the advanced stages of CHF, because the end-point rates were markedly lower (all p < 0.05) in the group with beta-blocker treatment versus the group without it, as characterized by peakVO(2) <10 ml/min per kg (26% vs. 64%), LVEF < or = 20% (25% vs. 45%), NE >2.24 nmol/l (18% vs. 40%) and NT-proBNP >364 pmol/l (27% vs. 45%), although patients with beta-blocker treatment received only 37 +/- 21% of the maximal recommended beta-blocker dosages. CONCLUSIONS: The prognostic value of variables used for risk stratification of patients with CHF is markedly influenced by beta-blocker treatment. Therefore, in the beta-blocker era, a re-evaluation of the selection criteria for heart transplantation is warranted.

Severe energy deprivation of human hibernating myocardium as possible common pathomechanism of contractile dysfunction, structural degeneration and cell death.

OBJECTIVES: We tested the hypothesis that severe alterations in myocardial energy metabolism play an important role in the pathophysiology of human hibernating myocardium (HHM). BACKGROUND: It is well established that a disturbed myocardial energy metabolism results in impairments of contractile performance, structure and viability. All of these are important characteristics of HHM. METHODS: In 16 patients with documented coronary artery disease and impaired left ventricular function, HHM was preoperatively detected by thallium-201 scintigraphy, radionuclide ventriculography and low-dose dobutamine echocardiography. These regions were validated as HHM by their recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed both biochemically and morphologically. These findings were compared to normal human myocardium. All metabolite contents given were normalized for the degree of fibrosis (control: 9.8 +/- 0.5%; HHM: 28.1 +/- 3.0%; p < 0.05), providing myocellular contents. RESULTS: In HHM, decreased contents (micromol/g wet weight) in adenosine triphosphate (ATP) (control: 4.17 +/- 0.26; HHM: 1.72 +/- 0.25; p < 0.001), creatine phosphate (5.67 +/- 0.70 vs. 0.84 +/- 0.13; p < 0.001) and creatine (27.6 +/- 3.19 vs. 11.2 +/- 1.56; p < 0.0001) were found, but contents in lactate (2.22 +/- 0.26 vs. 25.38 +/- 3.53; p < 0.001), purine bases (0.58 +/- 0.09 vs. 1.26 +/- 0.13; p < 0.001) and protons (pH units: 7.199 +/- 0.01 vs. 6.59 +/- 0.07; p < 0.001) were increased. Levels in adenosine diphosphate, adenosine monophosphate and inorganic phosphate remained unchanged. Energy depletion in HHM was reflected by decreases in the free energy of ATP hydrolysis and in energy charge. CONCLUSIONS: These data confirm our hypothesis that HHM is energy-depleted myocardium, exhibiting signs of chronic reduction in resting blood flow and a downregulation of energy turnover. The alterations in energy metabolism observed may become operative in triggering and maintaining contractile dysfunction, continuous tissue degeneration and cardiomyocyte loss.

Primary pulmonary hypertension may be a heterogeneous disease with a second locus on chromosome 2q31.

OBJECTIVES: The aim of our study was to identify genetic causes of primary pulmonary hypertension (PPH), to estimate the proportion of families with mutations in the BMPR2 (bone morphogenetic protein receptor type 2) gene, and to examine whether genetic heterogeneity might play a role. BACKGROUND: The BMPR2 mutations have been identified in a substantial portion of patients with familial or sporadic PPH. However, the genetic cause of PPH remains unclear in at least 45% of families. METHODS: We investigated 130 members of 10 families with at least 1 PPH patient, recruited without selection for familial disease. Manifest PPH was documented in 21 individuals. An increase in pulmonary artery systolic pressure (PASP) above 40 mm Hg during supine bicycle exercise was found in 46 healthy individuals. Their PASP increased from 21.0 +/- 4.6 mm Hg at rest to 54.0 +/- 9.8 mm Hg during exercise. In 51 relatives, PASP values were normal at rest and during exercise, and 12 members were classified as status unknown. RESULTS: Two families showed a mutation in the BMPR2 gene. Three families with no BMBR2 mutation showed evidence for linkage to a more proximal location on chromosome 2q31 (odds ratio [OR] for linkage 1.1.10(6):1). This locus, designated PPH2, maps in-between the markers D2S335 and D2S2314. We obtained significant support for heterogeneity in PPH with an OR of 2.8.10(11). CONCLUSIONS: We conclude that PPH may be a genetically heterogeneous disorder with at least two-and possibly more-causative genes.

Monocyte chemoattractant protein-1 induces proliferation and interleukin-6 production in human smooth muscle cells by differential activation of nuclear factor-kappaB and activator protein-1.

Inflammatory response and chemotaxis of vascular wall cells play an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for monocytes. Besides the induction of monocyte recruitment, it has been suggested that MCP-1 may directly activate smooth muscle cells. We investigated whether MCP-1 affects the proliferation and cytokine production of human vascular smooth muscle cells (VSMCs) and determined the underlying signal transduction pathways. Stimulation of VSMCs with MCP-1 induced proliferation and resulted in a concentration- and time-dependent release of the proinflammatory cytokine interleukin-6 (IL-6). Pretreatment with pertussis toxin, GF109203X, and pyrrolidine dithiocarbamate inhibited MCP-1-dependent IL-6 release, suggesting the involvement of G(i) proteins, protein kinase C, and nuclear factor-kappaB (NF-kappaB). MCP-1 also induced extracellular signal-regulated kinase, which, along with IL-6 release, was inhibited by pertussis toxin. PD98059 prevented MCP-1-induced extracellular signal-regulated kinase activation and cell proliferation. MCP-1 stimulated the binding activity of NF-kappaB and of activator protein-1 (AP-1). As demonstrated by cis element double-stranded (decoy) oligodeoxynucleotides, NF-kappaB was involved in IL-6 release by MCP-1, whereas proliferation was dependent on AP-1. The results clearly demonstrate that MCP-1 induces differential activation of NF-kappaB and AP-1 in VSMCs. Thus, our data propose a new mechanism for the proatherogenic effect of MCP-1.

The Theranostic PSMA Ligand PSMA-617 in the Diagnosis of Prostate Cancer by PET/CT: Biodistribution in Humans, Radiation Dosimetry, and First Evaluation of Tumor Lesions.

UNLABELLED: PET imaging with the prostate-specific membrane antigen (PSMA)-targeted radioligand (68)Ga-PSMA-11 is regarded as a significant step forward in the diagnosis of prostate cancer (PCa). More recently, a PSMA ligand was developed that can be labeled with (68)Ga, (111)In, (177)Lu, and (90)Y. This ligand, named PSMA-617, therefore enables both diagnosis and therapy of PCa. The aims of this evaluation were to clinically investigate the distribution of (68)Ga-PSMA-617 in normal tissues and in PCa lesions as well as to evaluate the radiation exposure by the radioligand in PET imaging. METHODS: Nineteen patients, most of them with recurrent PCa, were referred for (68)Ga-PSMA-617 PET/CT. The quantitative assessment of tracer uptake of several organs and of 53 representative tumor lesions was performed in 15 patients at 1 and 3 h after injection. In 4 additional patients, the same procedure was conducted at 5 min, 1 h, 2 h, 3 h, 4 h, and 5 h after injection. On the basis of the data for these 4 patients (mean injected dose, 231 MBq), the radiation exposure of a (68)Ga-PSMA-617 PET/CT was identified. RESULTS: Intense tracer uptake was observed in the kidneys and salivary glands. In 14 of 19 patients (73.7%), at least 1 lesion suspected of being a tumor was detected at 3 h after injection. Of 53 representative tumor lesions selected at 3 h after injection, 47 lesions were visible at 1 h after injection. The mean tumor-to-background ratio for maximum standardized uptake value was 20.4 ± 17.3 (range, 2.3-84.0) at 1 h after injection and 38.2 ± 38.6 (range, 3.6-154.3) at 3 h after injection. The average radiation exposure (effective dose) was approximately 0.021 mSv/MBq. CONCLUSION: Within healthy organs, the kidneys and salivary glands showed the highest (68)Ga-PSMA-617 uptake. The radiation exposure was relatively low. (68)Ga-PSMA-617 shows PCa lesions with high contrast. Images obtained between 2 and 3 h after injection seem to be the best option with regard to radiotracer uptake and tumor contrast. Later images can help to clarify unclear lesions.

Metabolic control analysis of anaerobic glycolysis in human hibernating myocardium replaces traditional concepts of flux control.

Myocardial hibernation represents an adaptation to sustained ischemia to maintain tissue vitality during severe supply-demand imbalance which is characterized by an increased glucose uptake. To elucidate this adaptive protective mechanism, the regulation of anaerobic glycolysis was investigated using human biopsies. In hibernating myocardium showing an increase in anaerobic glycolytic flux metabolizing exogenous glucose, the adjustment of flux through this pathway was analyzed by flux:metabolite co-responses. By this means, a previously unknown pattern of regulation using multisite modulation was found which largely differs from traditional concepts of metabolic control of the Embden-Meyerhof pathway in normal and diseased myocardium.

Increased MIBG uptake after transfer of the human norepinephrine transporter gene in rat hepatoma.

UNLABELLED: The transport of MIBG by the human norepinephrine transporter (hNET) seems to be the critical step in the treatment of MIBG-concentrating tumors. Therefore, we investigated whether the accumulation of MIBG may be induced by retroviral transfection of the hNET gene in Morris hepatoma cells. METHODS: A bicistronic retroviral vector for the transfer of the hNET coding sequence and the hygromycin resistance gene was generated. Morris hepatoma cells (MH3924A) were infected with the respective retroviral particles, and hNET-expressing cell lines MHhNEThyg1 to MHhNEThyg9 were obtained through hygromycin selection. The uptake of (3)H-norepinephrine or (131)I-MIBG and the efflux of (131)I-MIBG were determined in transfected and wild-type cells. In addition, the (131)I-MIBG distribution was monitored in nude mice and rats bearing wild-type and hNET-expressing hepatomas. RESULTS: hNET-expressing hepatoma cell lines accumulated up to 36 times more norepinephrine than did wild-type cells and 8 times more than did hNET-expressing neuroblastoma cell line SK-N-SH. The addition of nisoxetine, a selective inhibitor of noradrenaline uptake, inhibited norepinephrine uptake. Maximal (131)I-MIBG accumulation was observed 2 h after incubation and was followed by 43% efflux within 4 h after the (131)I-MIBG-containing medium had been removed. In vivo experiments performed with nude mice bearing both hNET-expressing and wild-type tumors showed a 10-fold-higher accumulation of (131)I-MIBG in transfected tumors than in wild-type tumors. The ex vivo calculations revealed doses of 605 and 75 mGy in hNET-expressing and wild-type tumor tissues, respectively. CONCLUSION: Transduction of the hNET gene enables Morris hepatoma cells to accumulate norepinephrine and MIBG. However, the retention of MIBG is brief; therefore, the absorbed dose of radiation in vivo is not expected to be therapeutically effective.

Iodide kinetics and dosimetry in vivo after transfer of the human sodium iodide symporter gene in rat thyroid carcinoma cells.

UNLABELLED: Transfer of the human sodium iodide symporter (hNIS) has been proposed as a new principle of cancer gene therapy. This study evaluates the iodide kinetics and dosimetry of iodide in hNIS-expressing thyroid carcinoma cells under optimized conditions. METHODS: Using a bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance gene, hNIS-expressing rat thyroid carcinoma cell lines were generated. Afterward, Na(125)I uptake and efflux were determined in genetically modified and wild-type cells in the presence or absence of modulators of iodide transport. In addition, the (131)I distribution in thyroid-ablated nude mice bearing wild-type and genetically modified thyroid carcinomas was monitored after intraperitoneal administration of (131)I with and without coadministration of lithium carbonate. RESULTS: hNIS-expressing cell lines accumulated up to 49 times more iodide than did noninfected cells, with a maximal iodide uptake after 30 min of incubation. However, a 90% efflux of the radioactivity occurred 20 min after replacement of the medium. In mice, the hNIS-expressing tumors accumulated up to 23 and 19.5 times more iodide than did the wild-type tumors in lithium-treated and control animals, respectively. However, efflux of the radioactivity was also observed in vivo: After 24 h, hNIS-expressing tumors lost 82.5% and 80.4% of the initial activity. Dosimetric calculations showed that 1,650 MBq of (131)I per square meter resulted in 5.4 and 5.2 Gy in hNIS-expressing tumors and 0.24 and 0.26 in wild-type tumors. CONCLUSION: Transduction of the hNIS gene in rat thyroid carcinoma cells induces iodide transport, which is associated with rapid efflux. Application of (131)I in clinically relevant amounts did not result in therapeutically useful absorbed doses in hNIS-expressing tumors in vivo, even under optimized conditions of thyroid ablation and treatment with lithium carbonate.

Prognostic value of serial cardiac assessment and familial screening in patients with dilated cardiomyopathy.

OBJECTIVES: This prospective study was performed to analyse whether routine clinical follow-up investigations at 12+/-6 months add to risk stratification and improve survival rates in patients with a first diagnosis of dilated cardiomyopathy (DCM). METHODS: Four hundred and eighty consecutive patients (mean age 53.4+/-12.3 years, 369 males, mean NYHA class 2.4+/-0.8) with invasively confirmed DCM were included and followed for 3.9+/-3.5 years. Patients were requested to adhere to a follow up investigation within 6-18 months either at the referring physicians or at our out patient department. Two hundred and eighty-one of the 480 patients presented for follow up which consisted of a detailed evaluation of symptoms, standardized physical examination, 12-lead-electrocardiogram recording and echocardiography. Seventeen patients were lost for follow up, 182 did not seek specialized medical follow up. Patients outcome was assessed by structured telephone interviews. RESULTS: Independent predictors of death or transplantation at initial diagnosis were LV-ejection fraction <30% (P=0.0001, risk ratio 2.25), LV-end diastolic pressure >or=15 mmHg (P=0.002, risk ratio 2.0), age >or=54 years, (P=0.04, risk ratio 1.55), and presence of left bundle branch block (P=0.046, risk ratio 1.53). On follow up investigations only deterioration of clinical status by at least one NYHA-class (P=0.001, risk ratio 2.6) and new onset or worsening of mitral regurgitation (P=0.02, risk ratio 1.8), remained independent prognostic factors for cardiac death. Patients who presented for routine follow up revealed significant better 5-year survival rates (n=281, 70%) than those who did not (n=153, 55%, P=0.005). CONCLUSIONS: Routine clinical follow up investigations within 6-18 months after first diagnosis of DCM adds to risk stratification and improves survival rates.

Contribution of stress echocardiography to clinical decision making in unselected ambulatory patients with known or suspected coronary artery disease.

BACKGROUND: Due to its higher diagnostic accuracy stress echocardiography (SE) has been advocated as a substitute for stress ECG to detect coronary heart disease (CAD). However, its contribution to clinical decision-making in unselected patients presenting to the ambulatory care centre for known or suspected coronary artery disease is unclear. METHODS: To evaluate the clinical value of SE in unselected patients, we prospectively obtained SE and stress ECG in 221 consecutive patients (142 males; mean age 58+/-12 years) presenting to the ambulatory care centre with known or suspected CAD. Patients with acute coronary syndrome were not included. RESULTS: Results of stress ECG and SE were concordant in 181 (82%) and discordant in 40 patients (18%). The clinical decision to recommend or to currently withhold coronary angiography was possible solely on the basis of clinical criteria and stress ECG findings in 191 (86.4%) patients but was guided by the results of SE in 30 patients (13.6%). Left heart catheterization and coronary angiography were conducted in 61 patients. In this population SE was more accurate (82.6%) than stress ECG (65.6%) in indicating significant coronary artery stenosis. CONCLUSION: Despite its higher accuracy, SE adds little to the information derived from dynamic stress ECG and symptom evaluation in unselected outpatients with known or suspected CAD. Thus, SE should not in general replace stress ECG as a screening method for detecting significant coronary artery disease, for both clinical and economic reasons.

Myocardial preconditioning and remote renal preconditioning--identifying a protective factor using proteomic methods?

It is still unknown whether remote ischemic preconditioning is mediated by a humoral or a neurogenic mechanism from the preconditioning to the preconditioned tissue. The purpose of the following study was to identify a possible humoral trigger of ischemic myocardial preconditioning and remote renal preconditioning. Open chest rats were subjected to a coronary artery occlusion period of 45 min followed by 2 h of reperfusion (Control animals; n = 6). The coronary preconditioned group (IPC, n = 6) was subjected to a preceding preconditioning period of 5 min coronary artery occlusion followed by 5 min of reperfusion, repeated three times. The renal preconditioned group (IPR, n = 6) was subjected to a preceding renal artery occlusion period of 10 min followed by 20 min of reperfusion. Area at risk (AAR) and infarcted area (IA) were determined at the end of each protocol. Blood samples were taken at the end of the preconditioning protocols from parallel experiments for proteomic analysis using two-dimensional gel electrophoresis (2-DE), matrix assisted laser desorption and ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS). IA/AAR was 87.8 +/- 10.7% in the control group. IPC and IPR significantly reduced IA/AAR (58.2 +/- 9.3% and 56.9 +/- 9.0%, p < 0.001). Proteomic analyses detected four protein spots which were either up- (n = 3) or down-regulated in the preconditioned groups vs. the control group. The three up-regulated protein spots were identified as albumin fragments, whereas the down-regulated spot was identified as liver regeneration-related protein (LRRG03). Interestingly, albumin modification by brief ischemia has been recently shown and evaluated for the clinical diagnosis of sublethal myocardial ischemia. However, no differentially abundant proteins which possess a known signaling function could be found. Hence, though there is a differential protein expression in blood following IPC and IPR, our data are not in favor of a humoral mediator of remote preconditioning with a molecular weight of more than 8 kDa. Our results rather suggest either a neurogenic pathway or a mediator smaller than 8 kDa.

Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: a subcellular correlate to ischemic preconditioning?

BACKGROUND: There is increasing evidence that mitochondria - owning a high degree of autonomy within the cell - might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K (ATP)) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia. METHODS AND RESULTS: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K (ATP) opener diazoxide (30 micromol/l) and was completely inhibited by the K (ATP) blocker 5-hydroxydecanoic acid (300 micromol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia. CONCLUSION: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K(ATP) but is independent of ATP preservation.

Myocardial energy metabolism in ischemic preconditioning and cardioplegia: a metabolic control analysis.

For both, cardioplegia (CP) and ischemic preconditioning (IP), increased ischemic tolerance with reduction in infarct size is well documented. These cardioprotective effects are related to a limitation of high energy phosphate (HEP) depletion. As CP and IP have to be assumed to act by different mechanisms, their effects on myocardial HEP metabolism cannot be assumed to be identical. Therefore, a systematic analysis of myocardial HEP metabolism for both procedures and their combination was performed, addressing the question whether there are different effects on myocardial HEP metabolism by IP and CP. In this study, metabolic control analysis was used to analyze the regulation of HEP metabolism. In open chest pigs subjected to 45 min LAD occlusion (index ischemia), CP and IP preserved myocardial ATP (control (C) 0.14 +/- 0.05 micromol/g wwt; CP: 0.95 +/- 0.14, IP: 0.61 +/- 0.12; p<0.05 C vs. CP and IP) and reduced myocardial necrosis (infarct size IA/RA: C: 90.0 +/- 3.0%; CP: 0.0 +/- 0.0% but patchy necroses; IP: 5.05 +/- 2.1%; p<0.05 C vs. CP and IP). The effects on HEP metabolism, however, were different: CP acted predominantly by slowing down the breakdown of phosphocreatine (PCr) during early phases of ischemia (C: DeltaPCr 0-2 min: 5.24 +/- 0.32 micromol/g wwt; CP: DeltaPCr 0-2 min: 3.38 +/- 0.23 micromol/g wwt, p<0.05 vs. C), leaving ATP breakdown during later stages unaffected (C: DeltaATP 5-45 min: 1.77 +/- 0.11 micromol/g wwt CP: DeltaATP 5-45 min: 1.59 +/- 0.28 micromol/g wwt, n.s. vs. C). In contrast to CP, in IP PCr breakdown was even increased (IP: DeltaPCr 0-2 min: 7.06 +/- 0.34 micromol/g wwt, p<0.05 vs. C), but ATP depletion greatly attenuated (IP: DeltaATP 5-45 min: 0.48 +/- 0.10 micromol/g wwt, p<0.05 vs. C and CP). Combining IP and CP yielded an additive effect with slowing down the breakdown of both PCr (IP+CP: DeltaPCr 0-2 min: 5.09+/- 0.35 micromol/g wwt, p<0.05 vs. C and IP) and ATP (IP+CP: DeltaATP 5-45 min: 0.56 +/- 0.48 micromol/g wwt, p<0.05 vs. C and CP), resulting in a higher ATP content at the end of index ischemia (1.86 +/- 0.46 micromol/g wwt, p<0.05 vs. C, CP and IP). Compared to IP, combining IP+CP achieved also a further reduction in infarct size (IA/RA: 0.0 +/- 0.0%, p<0.05 vs IP) and--compared to CP--a disappearance of the patchy necroses. The concept of major differences in myocardial HEP metabolism during CP and IP is further supported at a molecular level by metabolic control analysis. CP but not IP slowed down the CK reaction velocity at high PCr levels. In contrast to CP exerting a continuous decline in vATPase for any given ATP level, in IP myocardium ATPase reaction velocity was even increased at higher ATP contents, whereas a marked decrease in ATPase reaction velocity was found if ATP levels decreased. The equilibrium of the CK-reaction remained unchanged following CP, whereas IP induced a changing CK equilibrium, which was the more shifted towards PCr the more myocardial HEP content decreased. The data demonstrate different effects of CP and IP on myocardial HEP metabolism, i.e. PCr and ATP breakdown as well as the apparent equilibrium of the creatine kinase (CK)-reaction. For these reasons the combination of the two protective interventions has an additive effect.

Ionic radiocontrast media disrupt intercellular contacts via an extracellular calcium-independent mechanism.

Direct cytotoxic effects of radiocontrast (RC) agents have been implicated in radiocontrast nephropathy (RCIN). The interaction between extracellular calcium, which plays a central role in intercellular contacts, and the in vitro toxicity of RC was tested in Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports. Cell viability was determined by trypan blue exclusion. The function of intercellular junctions was assessed by measuring the electrical transmonolayer resistance (TMR). The cell contacts were examined with indirect immunofluorescence microscopy using antibodies against the junctional proteins E-cadherin, ZO-1 and occludin. The ionic RC agents diatrizoate and ioxaglate (74 mg iodine/ml), but not the nonionic compounds iohexol or iodixanol, decreased ionized calcium (Ca2+) in the incubation media from 1.48 +/- 0.04 mM (control) to 0.89 +/- 0.06 mM (diatrizoate), respectively to 1.05 +/- 0.08 mM (ioxaglate). Diatrizoate, and to a lesser extent ioxaglate, reduced the number of viable MDCK cells and showed a redistribution of the E-cadherin, ZO-1 and occludin immunofluorescence signal with a parallel decrease of the TMR indicating an impaired monolayer integrity. A similar reduction of extracellular Ca2+ through EGTA failed to reproduce these effects. Conversely, raising Ca2+ in diatrizoate-containing media to control levels did not abrogate its toxicity. In conclusion, the ionic RC agents diatrizoate and ioxaglate, but not the nonionic compounds iohexol or iodixanol, reduce extracellular Ca2+ in vitro. However, this reduction of Ca2+ does not explain their cytotoxic effects which could contribute to the pathogenesis of RCIN in vivo by opening intercellular junctions.