Circulating microRNAs and therapy-associated cardiac events in HER2-positive breast cancer patients: an exploratory analysis from NeoALTTO.

PURPOSE: The relevance of cardiotoxicity in the context of HER2-positive breast cancer is likely to increase with increasing patient treatment exposure, number of treatment lines, and prolonged survival. Circulating biomarkers to early identify patients at risk of cardiotoxicity could allow personalized treatment and follow-up measures. The aim of this study is to examine the relationship between circulating microRNAs and adverse cardiac events in HER2-positive breast cancer patients. METHODS: We based our work on plasma samples from NeoALTTO trial obtained at baseline, after 2 weeks of anti-HER2 therapy, and immediately before surgery. Eleven patients experienced either a symptomatic or asymptomatic cardiac event. Circulating microRNAs were profiled in all patients presenting a cardiac event (case) and in an equal number of matched patients free of reported cardiac events (controls) using microRNA-Ready-to-Use PCR (Human panel I + II). Sensitivity analyses were performed by increasing the number of controls to 1:2 and 1:3. Normalized microRNA expression levels were compared between cases and controls using the non-parametric Kruskal-Wallis test. RESULTS: Eight circulating microRNAs resulted differentially expressed after 2 weeks of anti-HER2 therapy between patients experiencing or not a cardiac event. Specifically, the expression of miR-125b-5p, miR-409-3p, miR-15a-5p, miR-423-5p, miR-148a-3p, miR-99a-5p, and miR-320b increased in plasma of cases as compared to controls, while the expression of miR-642a-5p decreases. Functional enrichment analysis revealed that all these microRNAs were involved in cardiomyocyte adrenergic signaling pathway. CONCLUSION: This study provides proof of concept that circulating microRNAs tested soon after treatment start could serve as biomarkers of cardiotoxicity in a very early stage in breast cancer patients receiving anti-HER2 therapy.

Carbon nitrides: synthesis and characterization of a new class of functional materials.

Carbon nitride compounds with high N : C ratios and graphitic to polymeric structures are being investigated as potential next-generation materials for incorporation in devices for energy conversion and storage as well as for optoelectronic and catalysis applications. The materials are built from C- and N-containing heterocycles with heptazine or triazine rings linked via sp2-bonded N atoms (N(C)3 units) or -NH- groups. The electronic, chemical and optical functionalities are determined by the nature of the local to extended structures as well as the chemical composition of the materials. Because of their typically amorphous to nanocrystalline nature and variable composition, significant challenges remain to fully assess and calibrate the structure-functionality relationships among carbon nitride materials. It is also important to devise a useful and consistent approach to naming the different classes of carbon nitride compounds that accurately describes their chemical and structural characteristics related to their functional performance. Here we evaluate the current state of understanding to highlight key issues in these areas and point out new directions in their development as advanced technological materials.

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity.

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Cardiac safety of adjuvant pegylated liposomal doxorubicin with concurrent trastuzumab: a randomized phase II trial.

BACKGROUND: The cardiac safety of trastuzumab concurrent with pegylated liposomal doxorubicin (PLD) in an adjuvant breast cancer treatment regimen is unknown. PATIENTS AND METHODS: Women with resected node-positive or intermediate-risk node-negative HER2 overexpressing breast cancer and baseline left ventricular ejection fraction (LVEF)≥55% were randomized (1:2) to doxorubicin 60 mg/m2 (A)+cyclophosphamide 600 mg/m2 (C) every 21 days (q21d) for four cycles or PLD 35 mg/m2+C q21d+trastuzumab 2 mg/kg weekly (H) for 12 weeks. Both groups then received paclitaxel (Taxol, T) 80 mg/m2 with H for 12 weeks followed by H to complete 1 year. The primary end point was cardiac event rate or inability to administer 1 year of trastuzumab. RESULTS: Of 181 randomized patients, 179 underwent cardiac analysis. The incidence of cardiac toxicity or inability to administer trastuzumab due to cardiotoxicity was 18.6% [n=11; 95% confidence interval (CI) 9.7% to 30.9%] with A+C→T+H and 4.2% (n=5; 95% CI 1.4% to 9.5%) with PLD+C+H→T+H (P=0.0036). All events, except one, were asymptomatic systolic dysfunction or mildly symptomatic heart failure. Mean absolute LVEF reduction at cycle 8 was greater with doxorubicin (5.6% versus 2.1%; P=0.0014). CONCLUSION: PLD+C+H→T+H is feasible and results in lower early cardiotoxicity rates compared with A+C→T+H.

Cardiovascular complications of conventional and targeted adjuvant breast cancer therapy.

Adjuvant therapy has improved the survival of women with early breast cancer (BC). Meta-analyses suggest that anthracycline-based regimens reduced the annual BC death rate by ∼40% in women below the age of 50 and 20% in older women. Novel agents designed to modulate abnormal growth factor signaling in and around the BC cell further increase patients' chances of survival. However, both conventional chemotherapeutic agents as well as some of the novel signaling inhibitors can induce important cardiovascular side-effects, potentially attenuating the progress made in recent years. The mechanism of cancer drug-induced cardiovascular complications varies greatly with some compounds inducing irreversible myocardial cell damage, while others lead to temporary cell dysfunction. The challenge of the future will be to prospectively discriminate between irreversible damage which can lead to progressive cardiovascular disease and reversible cardiovascular dysfunctions without further prognostic implications. Since adjuvant therapy for BC is potentially curative, emphasis must be placed on finding treatments combining maximum efficacy with the minimum of long-term side-effects in order to achieve survival with preserved quality of life.

111Indium-trastuzumab visualises myocardial human epidermal growth factor receptor 2 expression shortly after anthracycline treatment but not during heart failure: a clue to uncover the mechanisms of trastuzumab-related cardiotoxicity.

AIM: Trastuzumab can induce cardiotoxicity, particularly when combined with anthracyclines. Myocardial human epidermal growth factor receptor 2 (HER2) expression may be transiently upregulated by a compensatory mechanism following cardiac stress. 111In-DTPA-trastuzumab, scintigraphy can detect HER2 positive tumour lesions, however previously, we found myocardial uptake in only 1 of the 15 anthracycline-pre-treated patients with a median of 11 months after the last anthracycline administration. To evaluate whether myocardial HER2 expression is upregulated by anthracycline-induced cardiac stress or in case of heart failure by chronic pressure or volume overload, we performed 111In-DTPA-trastuzumab scans in patients shortly after anthracyclines and with non-anthracycline-related heart failure. METHODS: Patients within 3 weeks after undergoing 4-6 cycles first-line anthracycline-based chemotherapy and patients with heart failure due to cardiac disease underwent gammacamera imaging 48 and 96 h after 111In-DTPA-trastuzumab intravenously. RESULTS: Myocardial 111In-DTPA-trastuzumab uptake was observed in 5 out of 10 anthracycline-treated patients, who all were without symptomatic cardiac dysfunction. None of the 10 heart failure patients showed myocardial uptake. CONCLUSION: Shortly after completion of anthracycline treatment, myocardial HER2 over-expression was detectable in 50% of the patients. 111In-DTPA-trastuzumab scintigraphy after anthracyclines prior to adjuvant trastuzumab potentially identifies patients susceptible for trastuzumab-related cardiotoxicity and thus may facilitate the optimal timing of trastuzumab therapy.

Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Cardiotoxicity associated with trastuzumab (Herceptin) therapy in the treatment of metastatic breast cancer.

Trastuzumab (Herceptin) is a humanised monoclonal antibody that specifically targets HER2-positive breast cancer cells. Safety data collected from pivotal trials with trastuzumab indicate that this therapy is generally well tolerated. However trials of the combination of trastuzumab plus chemotherapy, and in particular chemotherapy with anthracyclines, have revealed an elevated incidence of cardiotoxicity in some patients, which was not apparent in preclinical or early clinical studies. Analyses of the available data suggest that in most cases the cardiotoxicity observed may reflect an exacerbation of anthracycline-induced cardiotoxicity. The biological mechanism of the cardiotoxicity has been investigated in several studies, and current data indicate that the heregulin/HER2-signalling pathway may have an important role. It is of note that the cardiotoxicity is generally reversible and can usually be managed with standard medical treatment. Improvement in cardiac function is seen both in patients who continue trastuzumab and in those in whom further therapy is withdrawn, indicating that with careful management anticancer therapy can be continued.

Chronic central infusion of cocaine- and amphetamine-regulated transcript (CART 55-102): effects on body weight homeostasis in lean and high-fat-fed obese rats.

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART) is expressed within hypothalamic nuclei implicated in the regulation of feeding behaviour. It is up-regulated by leptin, and CART-derived peptides acutely inhibit food intake. OBJECTIVE: The present study was designed to assess the long-term effects of central CART administration on food intake, body weight, plasma levels of glucose, insulin, leptin, free fatty acids and triglycerides, and on fuel utilisation in normal and high-fat-fed obese rats. DESIGN: Normal and high-fat-fed obese rats were cannulated intracerebroventricularly (i.c.v.) and infused for 6 days with CART (55-102) or its vehicle. At day 4, animals were placed in an indirect calorimeter for a 24 h period during which the respiratory quotient and the energy expenditure were determined hourly. RESULTS: In both normal and obese animals, the chronic i.c.v. infusion of CART (55-102) had marked, sustained inhibitory effects on food intake and body weight gain that were accompanied by decreases in plasma insulin and leptin levels. Using indirect calorimetry, it was observed that CART infusion promoted an increase in lipid oxidation in normal and in obese animals, although this increase reached statistical significance only in the obese group. The hypothalamic CART mRNA expression was found to be higher in obese rats (displaying hyperleptinaemia) than in normal animals. CONCLUSION: The data together show that chronic i.c.v. CART infusion is effective in inhibiting food intake, favouring lipid oxidation and limiting fat storage, both in normal and high-fat-diet-induced obese rats. The CART pathway thus seems to be an important determinant of body weight homeostasis in normal animals as well as in a model of nutritionally induced obesity.

[Chronic heart failure requires a multidisciplinary treatment concept]. / Die chronische Herzinsuffizienz erfordert ein multidisziplinäres Behandlungskonzept.

In the industrialized world, the incidence and prevalence of heart failure are dramatically rising. Characterized by a high mortality and morbidity, 1-2% of Switzerland's total health care expenditure is spent on this disease. Since the hospital admission rate of heart failure patients is extremely high, 70% of the costs result from hospitalizations. New therapeutic measures are urgently needed to reduce morbidity and therefore, hospital admission rate. Both, morbidity and hospitalization rate have been demonstrated to be reduced by multidisciplinary disease management programs. Such programs seem to be practical, efficient, and financially feasible and thus, should be evaluated in Switzerland.

Chronic alpha-adrenergic receptor stimulation modulates the contractile phenotype of cardiac myocytes in vitro.

BACKGROUND: Heart failure is characterized by contractile dysfunction of the myocardium and elevated sympathetic activity. We tested the hypothesis that chronic alpha-adrenergic (alpha-ADR) stimulation modifies the molecular and contractile phenotype of cardiac myocytes. METHODS AND RESULTS: Adult rat ventricular myocytes in culture were exposed to alpha-ADR stimulation (norepinephrine + propranolol) for 48 hours. alpha-ADR stimulation decreased the mRNAs for sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+) release channel by 56% and 52%, respectively, and increased mRNA and protein for the Na(+)-Ca(2+) exchanger by 70% and 39%, respectively. After washout of the alpha-ADR agonist, simultaneous measurement of [Ca(2+)](i) transients with fura 2 and myocyte shortening by video edge-detection showed that [Ca(2+)](i) amplitude and myocyte shortening were decreased in alpha-ADR-treated myocytes, and the time to peak and time from peak to 80% decline of both [Ca(2+)](i) and myocyte shortening were increased. The concentration-response curve for myocyte shortening by the Na(+) channel activator veratridine was shifted leftward in alpha-ADR-stimulated myocytes (EC(50), 21.6+/-4.6 versus 105.8+/-10.5 nmol/L, P:<0.001). CONCLUSIONS: Chronic alpha-ADR stimulation of cardiac myocytes causes decreases in the expression of sarcoplasmic reticulum Ca(2+)-ATPase and the Ca(2+) release channel that are associated with decreases in [Ca(2+)](i) and contractility. alpha-ADR stimulation simultaneously increases Na(+)-Ca(2+) exchanger expression, thereby increasing sensitivity to intracellular Na(+).

Altered beta-adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats.

Desensitization of the beta-adrenergic receptor (beta-AR) response is well documented in hypertrophied hearts. We investigated whether beta-AR desensitization is also present at the cellular level in hypertrophied myocardium, as well as the physiological role of inhibitory G (G(i)) proteins and the L-type Ca(2+) channel in mediating beta-AR desensitization. Left ventricular (LV) myocytes were isolated from hypertrophied hearts of hypertensive Dahl salt-sensitive (DS) rats and nonhypertrophied hearts of normotensive salt-resistant (DR) rats. Cells were paced at a rate of 300 beats/min at 37 degrees C, and myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) were simultaneously measured. In response to increasing concentrations of isoproterenol, DR myocytes displayed a dose-dependent augmentation of cell shortening and the [Ca(2+)](i) transient amplitude, whereas hypertrophied DS myocytes had a blunted response of both cell shortening and the [Ca(2+)](i) transient amplitude. Interestingly, inhibition of G(i) proteins did not restore beta-AR desensitization in DS myocytes. The responses to increases in extracellular Ca(2+) and an L-type Ca(2+) channel agonist were also similar in both DS and DR myocytes. Isoproterenol-stimulated adenylyl cyclase activity, however, was blunted in hypertrophied myocytes. We concluded that compensated ventricular hypertrophy results in a blunted contractile response to beta-AR stimulation, which is present at the cellular level and independent of alterations in inhibitory G proteins and the L-type Ca(2+) channel.

Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes.

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFalpha, and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

Increased calcium loading and inotropy without greater cell death in hypoxic rat cardiomyocytes.

To test whether contractile function in "hypoxic" myocytes treated with high glucose (19.5 mM) can be improved by increasing intracellular Ca2+ without accelerating cell contracture or death, we challenged metabolically inhibited, paced myocytes with high extracellular Ca2+ concentration ([Ca2+]o) and measured simultaneously cell shortening and intracellular Ca2+ concentration ([Ca2+]i). NaCN exposure at a physiological [Ca2+]o level (1.2 mM) caused a decline of contractile function to 58 +/- 8% of the pre-NaCN value (P < 0.001) but increased systolic and diastolic [Ca2+]i by 104 +/- 17 and 37 +/- 9% above baseline (P < 0.01), respectively. Consequent doubling of [Ca2+]o to 2.4 mM, in the presence of NaCN, immediately restored contractile function, and twitch amplitude after 18 min was 123 +/- 14% (P < 0.001) of baseline pre-NaCN values, whereas systolic [Ca2+]i increased further to 225 +/- 63% (P < 0.05) and diastolic [Ca2+]i to 73 +/- 16% above baseline (P < 0.01). This marked increase in [Ca2+]i had no deleterious effect on myocyte diastolic function or survival. These results suggest that if adequate metabolic substrate is provided, contractile function in metabolically inhibited, hypoxic myocytes can be restored by increasing [Ca2+]i without causing short-term cell injury.

Early changes in excitation-contraction coupling: transition from compensated hypertrophy to failure in Dahl salt-sensitive rat myocytes.

OBJECTIVE: The aims were to (1) define the early changes in excitation-contraction coupling during the transition from cardiac hypertrophy to heart failure, and (2) to clarify the causal relationship between mechanical dysfunction and abnormal Ca2+ handling in the Dahl salt-sensitive rat model. METHODS: Myocardial contractile function was assessed in whole heart perfusion studies. In separate experiments, isolated left ventricular myocytes from Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were paced at a physiological rate of 5Hz and cell shortening (CS) and [Ca2+]i measured simulataneously by video-edge detection and fura-2 fluorescence. RESULTS: DS hearts developed hypertrophy after 4 weeks of a high-salt diet (4WHSD), as indicated by a 26% increase (p < 0.01) in the heart to body weight ratio and a 21% increase (p < 0.01) in cell width. Heart failure developed after 12 weeks of a high-salt diet (12WHSD), as indicated by an 11% increase (p < 0.01) in the lung wet to dry weight ratio. Furthermore, in DS-12WHSD hearts, the diastolic pressure-volume relationship had shifted rightward. DR rats did not develop hypertension and seved as age-matched controls. A 31% (p < 0.05) increase in the %CS in DS-4WHSD myocytes compared to DR-4WHSD myocytes with a trend of a parallel increase in Ca2+ transient amplitude was found. There was no difference in the Ca2+ transient parameters between DR and DS at 12WHSD, but an 18% (p < 0.01) decrease occurred in peak [Ca2+]i in DS myocytes between 4WHSD and 12WHSD. In DS-12WHSD, the time to peak shortening and the time from peak shortening to 50% and 90% relaxation was significantly prolonged by 27%, 44%, and 38%, respectively, as compared to the age-matched DR myocytes. CONCLUSION: Our results indicated that: (I) normal Ca2+ homeostasis is preserved at the stage of compensated hypertrophy; (2) the early signs of isolated myocyte dysfunction were a prolongation of the shortening and relaxation time course without an abnormal time course of the Ca2+ transient. Thus, in the hypertensive Dahl salt rat model, abnormal Ca2+ handling appears neither to precede nor initiate the transition to failure.

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.

Exercise-induced vasomotion of angiographically normal and stenotic coronary arteries improves after cholesterol-lowering drug therapy with bezafibrate.

OBJECTIVES: We attempted to determine whether the coronary vasomotor response to exercise improves after cholesterol-lowering drug therapy with bezafibrate. BACKGROUND: Hypercholesterolemia and other coronary risk factors are associated with impaired endothelium-dependent coronary vasomotor response to physiologic or pharmacologic stimuli, even in the absence of overt coronary atherosclerosis. It is still unknown whether the coronary artery vasomotor response to dynamic exercise improves under cholesterol-lowering drug therapy. METHODS: Of 15 male patients (age 51 +/- 7 years [mean +/- SD]) included in the study, 7 had markedly elevated cholesterol levels (> or = 6.5 mmol/liter, therapy group), and 8 had normal or slightly elevated cholesterol levels (< 6.5 mmol/liter, control group). At baseline and after 7 months of cholesterol-lowering therapy with bezafibrate (400 mg/day) in the therapy group, coronary vasomotor response to dynamic exercise (percent change in cross-sectional vascular area at maximal exercise vs. rest [100%]) in normal and stenotic, previously dilated vessels was assessed by quantitative coronary angiography. RESULTS: During follow-up, total serum cholesterol levels in the therapy group decreased from 7.8 +/- 1.1 to 5.8 +/- 1.1 mmol/liter (p = 0.0001) and did not change significantly in the control group (from 5.4 +/- 0.9 to 6.0 +/- 1.2 mmol/liter, p = NS). Exercise-induced vasomotor response (at similar work loads in the therapy and control groups) in both normal and dilated stenotic coronary arteries improved significantly in the therapy group, from 100 +/- 9% to 109 +/- 7% (p = 0.0001, cross-sectional area at rest 100%) and from 80 +/- 11% to 106 +/- 7% (p = 0.0002), respectively, but did not improve during follow-up in the control group. CONCLUSIONS: The present study indicates that cholesterol-lowering drug therapy with bezafibrate for 7 months improves exercise-induced vasomotion of angiographically normal coronary arteries. Seven months after coronary angioplasty, the reduction in serum cholesterol levels is, at least in part, associated with a restoration of the initially disturbed vasomotor response of stenotic vessel segments to exercise.