Maria Carmela Cerra

In vivo detection of multidrug-resistant (MDR1) phenotype by technetium-99m sestamibi scan in untreated breast cancer patients

Technetium-99m sestamibi is a transport substrate recognised by the multidrug-resistant P-glycoprotein (Pgp). To test whether99mTc-sestamibi efflux is enhanced in breast carcinomas overexpressing Pgp, we determined the efflux rates of99mTc-sestamibi and Pgp levels in tumours from 30 patients with untreated breast carcinoma. Patients were intravenously injected with 740 MBq of99mTc-sestamibi and underwent a 15-min dynamic study followed by the acquisition of static planar images at 0.5, 1, 2 and 4 h. Tumour specimens were obtained from each patient 24 h after99mTc-sestamibi scan and Pgp levels were determined using125I-MRK16 monoclonal antibody and in vitro quantitative autoradiography. All breast carcinomas showed high uptake of99mTc-sestamibi and data from region of interest analysis on sequential images were fitted with a monoexponential function. The efflux rates of99mTc-sestamibi, calculated from decay-corrected time-activity curves, ranged between 0.00121 and 0.01690 min−1 and were directly correlated with Pgp levels measured in the same tumours (r=0.62;P<0.001). Ten out of 30 breast carcinomas (33%) contained 5 times more Pgp than benign breast lesions and showed a mean concentration of 5.73±1.63 pmol/g of tumour (group A). The remaining 20 breast carcinomas had a mean Pgp concentration of 1.29±0.64 pmol/g (group B), equivalent to that found in benign breast lesions.99mTc-sestamibi efflux from tumours of group A was 2.7 times higher than that observed in tumours of group B (0.00686±0.00390 min−1 vs 0.00250±0.00090 min−1,P<0.001). The in vivo functional test with99mTc-sestamibi showed a sensitivity and a specificity of 80% and 95%, respectively. In conclusion, the efflux rate of99mTc-sestamibi may be used for the in vivo identification of the multidrug resistant (MDR1) phenotype in untreated breast cancer patients.

The Antihypertensive Chromogranin A Peptide Catestatin Acts as a Novel Endocrine/Paracrine Modulator of Cardiac Inotropism and Lusitropism

Circulating levels of catestatin (Cts; human chromogranin A352-372) decrease in the plasma of patients with essential hypertension. Genetic ablation of the chromogranin A (Chga) gene in mice increases blood pressure and pretreatment of Chga-null mice with Cts prevents blood pressure elevation, indicating a direct role of Cts in preventing hypertension. This notable vasoreactivity prompted us to test the direct cardiovascular effects and mechanisms of action of wild-type (WT) Cts and naturally occurring human variants (G364S-Cts and P370L-Cts) on myocardial and coronary functions. The direct cardiovascular actions of WT-Cts and human variants were determined using the Langendorff-perfused rat heart. WT-Cts dose-dependently increased heart rate and coronary pressure and decreased left ventricular pressure, rate pressure product and both positive and negative LVdP/dt. WT-Cts not only inhibited phospholamban phosphorylation, but also the inotropic and lusitropic effects of WT-Cts were abolished by chemical inhibition of beta2-adrenergic receptors, Gi/o protein, nitric oxide or cGMP, indicating involvement of beta2-adrenergic receptors-Gi/o protein-nitric oxide-cGMP signaling mechanisms. In contrast, G364S-Cts did not affect basal cardiac performance but abolished isoproterenol-induced positive inotropism and lusitropism. P370L-Cts decreased rate pressure product and inhibited only isoproterenol-induced positive inotropism and lusitropism by 70%. Cts also inhibited endothelin-1-induced positive inotropism and coronary constriction. Taken together, the cardioinhibitory influence exerted on basal mechanical performance and the counterregulatory action against beta-adrenergic and endothelin-1 stimulations point to Cts as a novel cardiac modulator, able to protect the heart against excessive sympathochromaffin overactivation, e.g. hypertensive cardiomyopathy.

Recombinant N-terminal fragments of chromogranin-A modulate cardiac function of the Langendorff-perfused rat heart.

AbstractIn this study we tested the hypothesis that vasostatins could act as myocardial modulators in the mammalian heart. Using the Langendorff–perfused rat heart, the cardiac effects of the two recombinant human CGA N–terminal fragments STA–CGA1–78 and STA–CGA1–115, containing the vasostatin–1 (CGA 1–76) and vasostatin–2 (CGA 1–113) sequences, respectively, were evaluated at concentrations of 11 ÷ 165 nM. Cardiac performance was evaluated by analyzing left ventricular pressure (LVP) and the rate pressure product (RPP: HR × LVP), used as indexes of contractile activity and cardiac work, respectively. Under basal conditions, STA–CGA1–78 at all concentrations tested elicited a dose–dependent negative inotropism (LVP variations ranging from –9.6% ± 2 to –23% ± 2.9) without affecting coronary pressure (CP). In contrast, STA–CGA1–115 increased CP at 110 and 165 nM without affecting inotropism. Both STA–CGA1–78 and STA–CGA1–115 counteracted the cardio–stimulatory effects of isoproterenol (ISO). The ISO–dependent positive chronotropism was unaffected by STA–CGA1–78, while being reduced by STA–CGA1–115. Both peptides abolished the ISO–induced positive inotropism without modifying either the β–adrenergic–dependent coronary dilation or the ouabain–induced positive inotropism. The analysis of the percentage of variations of RPP in terms of EC50 values of ISO alone (–8.5 ± 0.3; r2 = 0.88) and in presence of STA–CGA1–78 (11, or 33, or 65 nM: –7.7 ± 0.15, r2 = 0.97; –7.7 ± 0.15, r2 = 0.97; –7.8 ± 0.78, r2 = 0.55, respectively) revealed a non–competitive type of antagonism of STA–CGA1–78. Taken together, these data suggest vasostatins as novel cardioregulatory peptides in mammals.

The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor α-Mediated Signaling in Cancer Cells and in Cardiomyocytes

Background The hydroxylated derivatives of cholesterol, such as the oxysterols, play important roles in lipid metabolism. In particular, 25-hydroxycholesterol (25HC) has been implicated in a variety of metabolic events including cholesterol homeostasis and atherosclerosis. 25HC is detectable in human plasma after ingestion of a meal rich in oxysterols and following a dietary cholesterol challenge. In addition, the levels of oxysterols, including 25HC, have been found to be elevated in hypercholesterolemic serum. Methodology/Principal Findings Here, we demonstrate that the estrogen receptor (ER) α mediates gene expression changes and growth responses induced by 25HC in breast and ovarian cancer cells. Moreover, 25HC exhibits the ERα-dependent ability like 17β-estradiol (E2) to inhibit the up-regulation of HIF-1α and connective tissue growth factor by hypoxic conditions in cardiomyocytes and rat heart preparations and to prevent the hypoxia-induced apoptosis. Conclusions/Significance The estrogen action exerted by 25HC may be considered as an additional factor involved in the progression of breast and ovarian tumors. Moreover, the estrogen-like activity of 25HC elicited in the cardiovascular system may play a role against hypoxic environments.

Nesfatin-1 as a novel cardiac peptide: identification, functional characterization, and protection against ischemia/reperfusion injury

Nesfatin-1 is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial KATP channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.

Catecholamines, cardiac natriuretic peptides and chromogranin A: evolution and physiopathology of a ‘whip-brake’ system of the endocrine heart

Summary In the past 50 years, extensive evidence has shown the ability of vertebrate cardiac non-neuronal cells to synthesize and release catecholamines (CA). This formed the mindset behind the search for the intrinsic endocrine heart properties, culminating in 1981 with the discovery of the natriuretic peptides (NP). CA and NP, co-existing in the endocrine secretion granules and acting as major cardiovascular regulators in health and disease, have become of great biomedical relevance for their potent diagnostic and therapeutic use. The concept of the endocrine heart was later enriched by the identification of a growing number of cardiac hormonal substances involved in organ modulation under normal and stress-induced conditions. Recently, chromogranin A (CgA), a major constituent of the secretory granules, and its derived cardio-suppressive and antiadrenergic peptides, vasostatin-1 and catestatin, were shown as new players in this framework, functioning as cardiac counter-regulators in ‘zero steady-state error’ homeostasis, particularly under intense excitatory stimuli, e.g. CA-induced myocardial stress. Here, we present evidence for the hypothesis that is gaining support, particularly among human cardiologists. The actions of CA, NP and CgA, we argue, may be viewed as a hallmark of the cardiac capacity to organize ‘whip-brake’ connection-integration processes in spatio-temporal networks. The involvement of the nitric oxide synthase (NOS)/nitric oxide (NO) system in this configuration is discussed. The use of fish and amphibian paradigms will illustrate the ways that incipient endocrine-humoral agents have evolved as components of cardiac molecular loops and important intermediates during evolutionary transitions, or in a distinct phylogenetic lineage, or under stress challenges. This may help to grasp the old evolutionary roots of these intracardiac endocrine/paracrine networks and how they have evolved from relatively less complicated designs. The latter can also be used as an intellectual tool to disentangle the experimental complexity of the mammalian and human endocrine hearts, suggesting future investigational avenues.

Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling.

Catestatin (CST) limits myocardial ischaemia/reperfusion (I/R) injury with unknown mechanisms. Clearly phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) isoforms, including intra-mitochondrial PKCε, mitochondrial KATP (mitoKATP) channels and subsequent reactive oxygen species (ROS)-signalling play important roles in postconditioning cardioprotection, preventing mitochondrial permeability transition pore (mPTP) opening. Therefore, we studied the role of these extra- and intra-mitochondrial factors in CST-induced protection. Isolated rat hearts and H9c2 cells underwent I/R and oxidative stress, respectively. In isolated hearts CST (75nM, CST-Post) given in early-reperfusion significantly reduced infarct size, limited post-ischaemic contracture, and improved recovery of developed left ventricular pressure. PI3K inhibitor, LY-294002 (LY), large spectrum PKC inhibitor, Chelerythrine (CHE), specific PKCε inhibitor (εV1-2), mitoKATP channel blocker, 5-Hydroxydecanoate (5HD) or ROS scavenger, 2-mercaptopropionylglycine (MPG) abolished the infarct-sparing effect of CST. Notably the CST-induced contracture limitation was maintained during co-infusion of 5HD, MPG or εV1-2, but it was lost during co-infusion of LY or CHE. In H9c2 cells challenged with H2O2, mitochondrial depolarization (an index of mPTP opening studied with JC1-probe) was drastically limited by CST (75nM). Our results suggest that the protective signalling pathway activated by CST includes mitoKATP channels, ROS signalling and prevention of mPTP opening, with a central role for upstream PI3K/Akt and PKCs. In fact, all inhibitors completely abolished CST-infarct-sparing effect. Since CST-anti-contracture effect cannot be explained by intra-mitochondrial mechanisms (PKCε activation and mitoKATP channel opening) or ROS signalling, it is proposed that these downstream signals are part of a reverberant loop which re-activates upstream PKCs, which therefore play a pivotal role in CST-induced protection.

β3‐Adrenoceptors modulate left ventricular relaxation in the rat heart via the NO‐cGMP‐PKG pathway

Aims:  Using a model of isolated and Langendorff‐perfused rat heart we analysed whether activation of β3‐adrenergic receptors (β3‐ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism.

Beta3-adrenoceptor in the eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism.

SUMMARY Neuroendocrine regulation of cardiac function involves a population of three types of β-adrenoceptors (ARs). In various mammalian species,β 1- and β2-AR stimulation produces an increase in contractility; whereas β3-AR activation mediates negative inotropic effects. At the moment, nothing is known about the physiological role of β3-AR in fish. Using an isolated working heart preparation, we show that a β3-AR selective agonist BRL37344 (0.1-100 nmol l-1) elicits a dose-dependent negative inotropism in the freshwater eel Anguilla anguilla. This effect was insensitive to the β1/β2-AR inhibitor nadolol (10 μmol l-1), but was blocked by theβ 3-AR-specific antagonist SR59230 (10 nmol l-1). The analysis of the percentage of stroke work (SW) variations, in terms of EC50 values, induced by BRL37344 alone (10 nmol l-1), and in presence of SR59230 (10 nmol l-1), indicated a competitive antagonism of SR59230. In addition to the classic positive inotropism, the non-specific β agonist isoproterenol (100 nmol l-1) induced, in 30% of the preparations, a negative inotropic effect that was abrogated by pre-treatment with SR59230, pointing to a β3-mediated pathway. The BRL37344-induced negative inotropic effect was abolished by exposure to a Gi/o proteins inhibitor pertussis toxin (PTx; 0.01 nmol l-1), suggesting a Gi/o-dependent mechanism. Using L-N5(l-imino-ethyl)ornithine (L-NIO; 10 μmol l-1), as a nitric oxide (NO) synthase (NOS) blocker and haemoglobin (Hb; 1 μmol l-1), as a NO scavenger, we demonstrated that NO signalling is involved in the BRL37344-induced response. Pre-treatment with either an inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4) oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ; 10 μmol l-1), or an inhibitor of the cGMP-activated protein kinase (PKG) KT5823 (100 nmol l-1), abolished the β3-dependent negative inotropism, indicating the cGMP-PKG component as a crucial target of NO signalling. Taken together, our findings provide functional evidence for the presence ofβ 3-like adrenoceptors in the eel Anguilla anguilla heart identifying, for the first time in a working fish heart, theβ 3-AR-dependent negative inotropy discovered in mammals.

Angiotensin II-induced inotropism requires an endocardial endothelium-nitric oxide mechanism in the in-vitro heart of Anguilla anguilla.

SUMMARY Using an isolated working heart preparation we show that angiotensin II (ANG II), at concentrations of 10-10–10-7 mol l-1, elicits negative chronotropism and inotropism in the freshwater eel Anguilla anguilla. The negative inotropism was insensitive to losartan and CGP42112 (AT1 and AT2 ANG II receptor antagonists, respectively), and was abrogated by the AT1 receptor antagonist CV11974, the G protein blocker pertussis toxin (PTx) and the muscarinic antagonist atropine. In contrast, it was not affected by the adrenoceptor antagonists propanolol, sotalol and phentolamine. Using donors (l-arginine) and inhibitors [NG-monomethyl-L-arginine (l-NMMA), l-N5(1-iminoethyl)ornithine (L-NIO)] of nitric oxide synthase (NOS), and haemoglobin as NO scavenger, we demonstrate that NO signalling is involved in ANG II-mediated inotropism. Pretreatment with Triton X-100, a detergent that damages the endocardial endothelium (EE), or with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, or with the cGMP-activated protein kinase (PKG) inhibitor KT5328, abolished ANG II-mediated inotropism. Thus, ANG II-mediated inotropism occurs via an EE-NO-cGMP-PKG mechanism. ANG II did not affect the mechanical performance influenced by preload changes (i.e. the Frank–Starling response), which in the eel heart is modulated by NO. This EE-paracrine-mediated cardio-suppressive action of endoluminal ANG II suggests that the hormone plays an important intracardiac role in the fish heart.