María A. Hurlé

Myocardial and circulating levels of microRNA-21 reflect left ventricular fibrosis in aortic stenosis patients

BACKGROUND Various human cardiovascular pathophysiological conditions associate aberrant expression of microRNAs (miRNAs) and circulating miRNAs are emerging as promising biomarkers. In mice, myocardial miR-21 overexpression is related to cardiac fibrosis elicited by pressure overload. This study was designed to determine the role of myocardial and plasmatic miR-21 in the maladaptive remodeling of the extracellular matrix induced by pressure overload in aortic stenosis (AS) patients and the clinical value of miR-21 as a biomarker for pathological myocardial fibrosis. METHODS In left ventricular biopsies from 75 AS patients and 32 surgical controls, we quantified the myocardial transcript levels of miR-21, miR-21-targets and ECM- and TGF-β-signaling-related elements. miR-21 plasma levels were determined in 25 healthy volunteers and in AS patients. In situ hybridization of miR-21 was performed in myocardial sections. RESULTS The myocardial and plasma levels of miR-21 were significantly higher in the AS patients compared with the controls and correlated directly with the echocardiographic mean transvalvular gradients. miR-21 overexpression was confined to interstitial cells and absent in cardiomyocytes. Using bootstrap validated multiple linear regression, the variance in myocardial collagen expression was predicted by myocardial miR-21 (70% of collagen variance) or plasma miR-21 (52% of collagen variance), together with the miR-21 targets RECK and PDCD4, and effectors of TGF-ß signaling. CONCLUSIONS Our results support the role of miR-21 as a regulator of the fibrotic process that occurs in response to pressure overload in AS patients and underscore the value of circulating miR-21 as a biomarker for myocardial fibrosis.

Enhancement of opiate analgesia by nimodipine in cancer patients chronically treated with morphine: a preliminary report.

The ability of nimodipine, a calcium-channel blocker, to enhance morphine analgesia and/or modify the development of tolerance was studied in patients with cancer pain who had needed successive increments of morphine for periods ranging from 21 to 780 days. Assessment of daily morphine consumption was the primary effect parameter. Nimodipine succeeded in reducing the daily dose of morphine in 16 of 23 patients (oral, n = 13; intrathecal, n = 3), and failed to modify it in 2 patients. Total oral daily dose was reduced by nimodipine (120 mg/day) from 282.6 +/- 47.7 mg to 158.7 +/- 26.2 mg (n = 15, P < 0.001). Intrathecal morphine was also reduced by 1-5 mg/day. Nimodipine was withdrawn in 5 patients during the first week of treatment due to intolerance (n = 3) or aggravation of the disease (n = 2). These preliminary results support experimental findings showing that pharmacological interference with Ca(2+)-related events may modify chronic opioid effects, including the expression of tolerance.

Calcium channel modulation by dihydropyridines modifies sufentanil-induced antinociception in acute and tolerant conditions.

SummaryThe study was aimed at elucidating the possible participation of l-type Ca2+ channel in the acute analgesic effect of an opiate and the development of tolerance to this action. Sufentanil, a selective p agonist, and two dihydropyridines, the Ca2+ antagonist nimodipine and the Ca2+ agonist Bay K 8644, were selected. The tail-flick test was used to assess the nociceptive threshold. In naive rats, nimodipine (200 μg/kg) potentiated the analgesic effect of sufentanil reducing the ED50 from 0.26 to 0.08 μg/kg. Similar results were observed with its (−)-enantiomer Bay N 5248, while the (+) enantiomer Bay N 5247 was ineffective. Tolerance to the opiate was induced by chronic subcutaneous administration of sufentanil with minipumps (2 μg/h, 7 days). In these conditions the dose-response curve to sufentanil was displaced to the right and the ED50 was increased to 1.49 μg/kg. In tolerant rats, nimodipine preserved its potentiating ability and prevented the displacement to the right of the sufentanil dose response-curve (ED50 = 0.48 μg/kg). When nimodipine was pumped (1 μg/h, 7 days) concurrently with sufentanil, the development of tolerance to the opioid was not disturbed. However, the expression of tolerance was abolished and even the effect of acutely administered sufentanil was markedly potentiated (ED50 = 0.03 μg/kg). Similar experiments were performed with Bay K 8644. In naive rats, Bay K 8644 at a low dose (20 μg/kg) that behaves as a calcium agonist, antagonized the analgesic effect of sufentanil (ED50 = 0.58 μg/kg), whereas at a high dose (200 μg/kg) it potentiated this action (ED50 = 0.15 μg/kg). In tolerant rats, Bay K 8644 (20 μg/kg) preserved its antagonizing ability inducing a displacement to the right of the sufentanildose-response curve (ED50 = 4.2 μg/kg). When Bay K 8644 was pumped (1 μg/h, 7 days) concurrently with sufentanil, it enhanced the expression of tolerance to the opiate (ED50 = 3.8 μg/kg). These results suggest that the calcium fluxes through the l-type channel in neurones are functionally linked to the activation of the μ opiate receptor: the blockade of the channel increased the potency of sufentanil, whereas its activation reduced the potency of the opiate. In chronic experiments, DHPs concurrently administered with sufentanil did not affect the development of tolerance to the opiate. However, nimodipine prevented the expression of this phenomenon. Even more, the animals became hypersensitive to the opiate suggesting that the adaptative mechanisms induced by chronic opiate could be affected by chronic nimodipine.

Modulation of apoptosis in the mouse brain after morphine treatments and morphine withdrawal

We have examined the effects of acute or chronic morphine and naltrexone‐precipitated withdrawal on mouse brain apoptotic cell death. The associated changes in the expression of apoptosis regulatory proteins were also analyzed. After a single dose of morphine, no apoptotic cells were detected by TUNEL or active caspase‐3 immunocytochemistry. Concurrently, a down‐regulation of the proapoptotic proteins FasL and Bad was detected in cortical lysates. On the other hand, the brains of chronic‐morphine‐treated mice and abstinent mice exhibited scattered apoptotic neurons and astrocytes throughout the brain. This neurotoxic effect was accompanied by up‐regulation of the proapoptotic proteins FasL, Fas, and Bad and the active fragments of caspases‐8 and ‐3 in cortical and hippocampal lysates. Abstinent mice also displayed a reduced expression of the antiapoptotic protein Bcl‐2. No changes on t‐Bid expression were detected under any experimental condition. These results suggest a neurotoxic effect exerted by chronic, but not acute, morphine and its withdrawal by activating both the intrinsic and the extrinsic apoptotic pathways. The possible clinical implications of our findings are discussed.

Changes in the expression of G protein-coupled receptor kinases and β-arrestin 2 in rat brain during opioid tolerance and supersensitivity

We previously demonstrated that chronic treatment of rats with the µ‐opioid receptor agonist sufentanil induced pharmacological tolerance associated with µ‐opioid receptor desensitization and down‐regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented µ‐opioid receptor down‐regulation, induced down‐stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein‐coupled receptor kinases (GRKs) and β‐arrestin 2 in agonist‐induced µ‐opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7‐day chronic infusion of sufentanil (2 µg/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 µg/h) and nimodipine (1 µg/h) for 7 days. Antinociception was evaluated by the tail‐flick test. GRK2, GRK3, GRK6 and β‐arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up‐regulation of GRK2, GRK6, and β‐arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and β‐arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and β‐arrestin 2 are involved in the short‐ and long‐term adaptive changes in µ‐opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and β‐arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.

Plasma Levels of Transforming Growth Factor-β1 Reflect Left Ventricular Remodeling in Aortic Stenosis

Background TGF-β1 is involved in cardiac remodeling through an auto/paracrine mechanism. The contribution of TGF-β1 from plasmatic source to pressure overload myocardial remodeling has not been analyzed. We investigated, in patients with valvular aortic stenosis (AS), and in mice subjected to transverse aortic arch constriction (TAC), whether plasma TGF-β1 relates with myocardial remodeling, reflected by LV transcriptional adaptations of genes linked to myocardial hypertrophy and fibrosis, and by heart morphology and function. Methodology/Principal Findings The subjects of the study were: 39 patients operated of AS; 27 healthy volunteers; 12 mice subjected to TAC; and 6 mice sham-operated. Myocardial samples were subjected to quantitative PCR. Plasma TGF-β1 was determined by ELISA. Under pressure overload, TGF-β1 plasma levels were significantly increased both in AS patients and TAC mice. In AS patients, plasma TGF-β1 correlated directly with aortic transvalvular gradients and LV mass surrogate variables, both preoperatively and 1 year after surgery. Plasma TGF-β1 correlated positively with the myocardial expression of genes encoding extracellular matrix (collagens I and III, fibronectin) and sarcomeric (myosin light chain-2, β-myosin heavy chain) remodelling targets of TGF-β1, in TAC mice and in AS patients. Conclusions/Significance A circulating TGF-β1-mediated mechanism is involved, in both mice and humans, in the excessive deposition of ECM elements and hypertrophic growth of cardiomyocytes under pressure overload. The possible value of plasma TGF-β1 as a marker reflecting preoperative myocardial remodeling status in AS patients deserves further analysis in larger patient cohorts.

Regulation of μ-opioid receptors, G-protein-coupled receptor kinases and β-arrestin 2 in the rat brain after chronic opioid receptor antagonism

The aim of this study was to analyse the biochemical and behavioural consequences of chronic treatment with opioid receptor antagonists in rats. We have evaluated the respiratory depressant and antinociceptive effects of the mu-opioid agonist sufentanil, the density of brain mu-opioid receptors, and the expression of G-protein-coupled receptor kinases and beta-arrestin 2 in cerebral cortex and striatum, following sustained opioid receptor blockade. Our results demonstrate that 24 h after interruption of 7 days chronic infusion of naltrexone (120 microg/h), the respiratory depressant potency of the mu-opioid receptor agonist sufentanil was increased to a similar extent as the antinociceptive potency (about three-fold). This was accompanied by mu-opioid receptor up-regulation in several areas of the rat brain associated with opioid control of pain perception and breathing. Moreover, chronic treatment with either naltrexone (120 microg/h) or naloxone (120 microg/h) caused significant increases in the expression levels of G-protein-coupled receptor kinases types 2, 3, and 6, and of beta-arrestin 2 in brain cortex and striatum. Together our data suggest an increased constitutive receptor activity secondary to mu-opioid receptor up-regulation following chronic antagonist treatment.

Nimodipine-enhanced opiate analgesia in cancer patients requiring morphine dose escalation : a double-blind, placebo-controlled study

&NA; The ability of nimodipine, a dihydropyridine calcium antagonist, to reduce the daily dose of oral morphine in cancer patients who had developed dose escalation, was tested in 54 patients under randomized, double‐blind, placebo‐controlled conditions. We selected patients that required at least two successive increments of morphine to maintain pain relief. A possible pharmacokinetic interaction between nimodipine and morphine was also studied in 14 patients by assaying steady‐state serum levels of morphine and its 3‐ and 6‐glucuronides. A total of 30 patients completed the study, 14 and 16 in the nimodipine and placebo groups, respectively. Nimodipine controlled the escalation of the morphine dose in 9 patients (65%), and placebo in 4 (28%), the difference being statistically significant (P=0.03). The dose of morphine was reduced from 313±52 to 174±33 mg/day (P<0.001) in the nimodipine group, and from 254±26 to 218±19 mg/day (not significant) in the placebo group. The percentages of reduction in the daily dose of morphine also showed significant differences between both groups (P=0.02). One week after introducing nimodipine or placebo, while the dose of morphine remained similar to that of the pre‐test week, the serum levels of morphine and its glucuronides were not modified significantly. We conclude that the introduction of nimodipine in patients chronically treated with morphine may be a safe alternative to reduce the daily requirements of the opioid. It is suggested that interference with Ca2+‐related events may attenuate the development and/or expression of tolerance to morphine in a clinically relevant way.

Chronic administration of heroin to mice produces up-regulation of brain apoptosis-related proteins and impairs spatial learning and memory

Several studies open up the possibility that chronic exposure to opioid drugs in the CNS would interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Here, we have analyzed the effects of prolonged heroin administration on sensorimotor and cognitive performance in mice, as well as the associated changes in brain expression of proteins regulating the extrinsic (FasL and Fas) and the mitochondrial (Bcl-2, Bcl-X(L), Bad and Bax) apoptotic pathways. Our findings indicate that chronic heroin did not interfere with mice performance in a battery of sensorimotor tests. On the other hand, cognitive ability in the Morris water maze and cognitive flexibility-related performance were strongly impaired by chronic heroin. These effects were associated with up-regulation of pro-apoptotic proteins such as Fas, FasL and Bad, in the cortex and hippocampus, indicating the activation of both the death receptor and the mitochondrial apoptotic pathways. Another indicator of apoptosis was the presence of TUNEL (TdT-mediated dUTP nick-end labeling) positive cells scattered throughout the brain.

Differential respiratory patterns induced by opioids applied to the ventral medullary and dorsal pontine surfaces of cats

The purpose of the present study was to make a functional dissection of the respiratory action of opioids, by their restricted application to the ventral surface of the medulla oblongata and to the rostro-dorsal surface of the pons in cats. The effects were compared to those induced after intracerebroventricular (i.c.v.) injection. Two mu-agonists, morphine and D-Ala2-Me-Phe4-Met(O)ol5-enkephalin (FK-33824), and the delta-agonist D-Ala2-D-Leu5-enkephalin (DADLE) were used. When applied to the ventral medullary surface, the opioids selectively depressed the generating mechanisms for tidal volume and the response to CO2, whereas the frequency was increased. The application to the rostral dorsal surface of the pons was followed by a selective depression of the respiratory frequency. By intracerebroventricular administration, the opioids depressed both the tidal volume and frequency generating mechanisms. The effects were always reversed by naloxone. The pontine structures were more sensitive to the action of the opioids than were the medullary centres. These findings suggest that the opioids can interact with different populations of respiratory neurones and that the respiratory output differs depending on the group of neurones selectively affected and the function they subserve in regulating respiratory activity.