Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain.

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-ß1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-ß1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.

BMP-7 protects male and female rodents against neuropathic pain induced by nerve injury through a mechanism mediated by endogenous opioids.

Neuropathic pain is highly prevalent in pathological conditions such as diabetes, herpes zoster, trauma, etc. The severity and refractoriness to treatments make neuropathic pain a significant health concern. The transforming growth factor (TGF-ß) family of cytokines is involved in pain modulation. Bone morphogenetic proteins (BMPs) constitute the largest subgroup within the TGF-ß family. BMP-7 induces the transcription of genes coding endogenous opioid precursors in vitro. However, a nociception modulatory function for this cytokine remains unexplored in vivo. Herein, we show that BMP-7 and its type I receptors were detected in regions of the nervous system involved in pain transmission, processing, and modulation. BMP-7 haploinsufficiency confers to male and female mice a tactile hyperalgesia phenotype to mechanical stimuli, both at baseline and after sciatic nerve injury (SNI). The administration of recombinant BMP-7 (rBMP-7) reduced the severity of the allodynia after SNI in rodents without sexual dimorphism. Central administration of rBMP-7 delayed allodynia development after SNI and reduced the severity of allodynia. The opioid antagonist naloxone antagonized the antinociceptive effect of rBMP-7 in rats. The analgesic effect of morphine was significantly attenuated in BMP-7+/- mice. The antiallodynic effect of voluntary exercise after SNI, whose mechanism involves the endogenous opioid system, was hampered by BMP-7 deficiency while potentiated by rBMP-7. Our results suggest that BMP-7 may constitute a novel therapeutic target for the treatment of neuropathic pain, which improves the function of the endogenous pain-resolution mechanisms to alleviate chronic pain.

p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling.

Transforming growth factor-ß (TGF-ß) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-ß signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-ß1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-ß1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-ß1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFß-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

TGF-ß and opioid receptor signaling crosstalk results in improvement of endogenous and exogenous opioid analgesia under pathological pain conditions.

Transforming growth factor-ß1 (TGF-ß1) protects against neuroinflammatory events underlying neuropathic pain. TGF-ß signaling enhancement is a phenotypic characteristic of mice lacking the TGF-ß pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-ß1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of µ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of µ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-ß1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-ß1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-ß signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-ß signaling.

Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-ß (TGF-ßs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-ß signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI(-/-)) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-ß. BAMBI deletion led to a gain of myocardial TGF-ß signaling through canonical (Smads) and non-canonical (TAK1-p38 and TAK1-JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI(-/-) mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-ß targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI(-/-) mice involved TGF-ßs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-ß-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-ß remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.

BMP7-based peptide agonists of BMPR1A protect the left ventricle against pathological remodeling induced by pressure overload.

Aortic stenosis (AS) exposes the left ventricle (LV) to pressure overload leading to detrimental LV remodeling and heart failure. In animal models of cardiac injury or hemodynamic stress, bone morphogenetic protein-7 (BMP7) protects LV against remodeling by counteracting TGF-ß effects. BMP receptor 1A (BMPR1A) might mediate BMP7 antifibrotic effects. Herein we evaluated BMP7-based peptides, THR123 and THR184, agonists of BMPR1A, as cardioprotective drugs in a pressure overload model. We studied patients with AS, mice subjected to four-week transverse aortic constriction (TAC) and TAC release (de-TAC). The LV of AS patients and TAC mice featured Bmpr1a downregulation. Also, pSMAD1/5/(8)9 was reduced in TAC mice. Pre-emptive treatment of mice with THR123 and THR184, during the four-week TAC period, normalized pSMAD1/5/(8)9 levels in the LV, attenuated overexpression of remodeling-related genes (Col 1α1, ß-MHC, BNP), palliated structural damage (hypertrophy and fibrosis) and alleviated LV dysfunction (systolic and diastolic). THR184 administration, starting fifteen days after TAC, halted the ongoing remodeling and partially reversed LV dysfunction. The reverse remodeling after pressure overload release was facilitated by THR184. Both peptides diminished the TGF-ß1-induced hypertrophic gene program in cardiomyocytes, collagen transcriptional activation in fibroblasts, and differentiation of cardiac fibroblasts to myofibroblasts. Molecular docking suggests that both peptides bind with similar binding energies to the BMP7 binding domain at the BMPR1A. The present study results provide a preclinical proof-of-concept of potential therapeutic benefits of BMP7-based small peptides, which function as agonists of BMPR1A, against the pathological LV remodeling in the context of aortic stenosis.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-beta family in pain modulation.

Transforming growth factors-beta (TGF-betas) signal through type I and type II serine-threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-beta family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-beta signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI(-/-) mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI(-/-) mice also appeared attenuated through a mechanism involving delta-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI(-/-). Transcript levels of TGF-betas and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-beta family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.

Sex-Specific Regulation of miR-29b in the Myocardium Under Pressure Overload is Associated with Differential Molecular, Structural and Functional Remodeling Patterns in Mice and Patients with Aortic Stenosis.

Pressure overload in patients with aortic stenosis (AS) induces an adverse remodeling of the left ventricle (LV) in a sex-specific manner. We assessed whether a sex-specific miR-29b dysregulation underlies this sex-biased remodeling pattern, as has been described in liver fibrosis. We studied mice with transverse aortic constriction (TAC) and patients with AS. miR-29b was determined in the LV (mice, patients) and plasma (patients). Expression of remodeling-related markers and histological fibrosis were determined in mouse LV. Echocardiographic morpho-functional parameters were evaluated at baseline and post-TAC in mice, and preoperatively and 1 year after aortic valve replacement (AVR) in patients with AS. In mice, miR-29b LV regulation was opposite in TAC-males (down-regulation) and TAC-females (up-regulation). The subsequent changes in miR-29b targets (collagens and GSK-3ß) revealed a remodeling pattern that was more fibrotic in males but more hypertrophic in females. Both systolic and diastolic cardiac functions deteriorated more in TAC-females, thus suggesting a detrimental role of miR-29b in females, but was protective in the LV under pressure overload in males. Clinically, miR-29b in controls and patients with AS reproduced most of the sexually dimorphic features observed in mice. In women with AS, the preoperative plasma expression of miR-29b paralleled the severity of hypertrophy and was a significant negative predictor of reverse remodeling after AVR; therefore, it may have potential value as a prognostic biomarker.

Gender differences of echocardiographic and gene expression patterns in human pressure overload left ventricular hypertrophy.

Gender influence on left ventricular (LV) remodeling associated to aortic valve stenosis (AS) has been long recognized, but underlying myocardial gene expression patterns have not been explored. We studied whether sex differences in echocardiographic LV anatomy and function in AS patients are associated with specific changes in myocardial mRNA expression of remodeling proteins. AS (n=39) and control (n=23)patients were assessed echocardiographically, and LV myocardial mRNA levels were quantified by PCR. AS patients exhibit increased wall thicknesses and LV mass index (LVMI), but only men show chamber dilation.Collagens and fibronectin mRNA levels increased correlatively to transforming growth factor-beta1 (TGF-beta1). In AS women, collagen I upregulation was proportionally higher than other extracellular matrix (ECM)components. No changes in matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 were detected. Gene expressions of sarcomeric proteins (beta-myosin heavy chain and myosin light chain-2) and TGF-beta1 were directly correlated with each other. Myosin light chain-2 mRNA levels increased proportionally to the transvalvular gradient, but women did so in a greater extent than men for a given gradient. In women, the hypertrophic growth response, reflected by LVMI, was proportional to the expression of genes encoding sarcomeric proteins and TGF-beta1. In men, chamber dilation and deterioration of LVEF was proportional to collagens, fibronectin, and TGF-beta1 gene expression levels. We evidenced gender biased gene expression patterns of the intracellular TGF-beta pathways involving the Smad branch, but not the TAK-1 branch, that could contribute to the remodeling differences observed in AS men and women. Based on these findings, a gender specific therapeutic approach of pressure overload LV hypertrophy could be justified.

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.

Supersensitivity to mu-opioid receptor-mediated inhibition of the adenylyl cyclase pathway involves pertussis toxin-resistant Galpha protein subunits.

Sustained administration of opioids leads to antinociceptive tolerance, while prolonged association of L-type Ca2+ channel blockers (e.g. nimodipine) with opioids results in increased antinociceptive response. Herein, we investigated the changes in mu-opioid receptor signalling underlying this shift from analgesic tolerance to supersensitivity. Thus, the interaction of mu-opioid receptors with G proteins and adenylyl cyclase was examined in lumbar spinal cord segments of rats. In control animals, the mu-opioid selective agonists, sufentanil and DAMGO, stimulated [35S]5'-(gamma-thio)-triphosphate ([35S]GTP gamma S) binding and inhibited forskolin-stimulated adenylyl cyclase activity, through a mechanism involving pertussis toxin (PTX) sensitive G alpha(i/o) subunits. Seven days of chronic sufentanil treatment developed antinociceptive tolerance associated with a reduction in mu-agonist-induced [35S]GTP gamma S binding, mu-agonist-induced adenylyl cyclase inhibition, and co-precipitation of G alpha o, G alpha i2 G alpha z and G alpha q11 subunits with mu-opioid receptors. In contrast, combined nimodipine treatment with sufentanil over the same period increased the sufentanil analgesic response. This antinociceptive supersensitivity was accompanied by a significant increase of mu-agonist-induced inhibition of adenylyl cyclase that was resistant to the antagonism by PTX. In good agreement, co-precipitation of the PTX-resistant, G alpha z and G alpha q/11 subunits with mu-opioid receptors was not lowered. On the other hand, the PTX-sensitive subunits, G alpha i2 and G alpha o, as well as agonist-stimulated [35S]GTP gamma S binding were still reduced. Our results demonstrate that mu-opioid analgesic tolerance follows uncoupling of spinal mu-opioid receptors from their G proteins and linked effector pathways. Conversely, the enhanced analgesic response following combined nimodipine treatment with sufentanil is associated with adenylyl cyclase supersensitivity to the opioid inhibitory effect through a mechanism involving PTX-resistant G protein subunits.

Facilitation of avoidance behaviour in mice chronically treated with heroin or methadone.

Although the repercussion of chronic treatment with large amounts of opioids on cognitive performance is a matter of concern, the effects of opioid drugs on passive avoidance learning have been scarcely studied. Here, we analyzed the effects of prolonged administration of heroin and methadone, as well as the impact of suffering repeated episodes of withdrawal on fear-motivated learning using the passive avoidance test. Mice received chronic treatment (39 days) with methadone (10 mg/kg/24 h), associated or not with repeated withdrawal episodes, or with heroin (5 mg/kg/12 h). Our results show that, regardless of the type of treatment received, all mice displayed similar basal thermal nociceptive thresholds during 25 days of treatment. In the hot plate test, both methadone and heroin induced antinociception 30 min after drug administration. The analgesic effect was absent when measured 4 h after heroin and 12 h after methadone. Pain behavioural responses elicited by growing intensities of electric shock, applied on day 28th of treatment, were similar in all groups of mice. Our results indicate that chronic opioid treatment had promnesic effects on passive avoidance behaviour in mice, unrelated to changes in the nociceptive state.

MicroRNA-30c-5p modulates neuropathic pain in rodents.

Neuropathic pain is a debilitating chronic syndrome that is often refractory to currently available analgesics. Aberrant expression of several microRNAs (miRNAs) in nociception-related neural structures is associated with neuropathic pain in rodent models. We have exploited the antiallodynic phenotype of mice lacking the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transforming growth factor-ß (TGF-ß) pseudoreceptor. We used these mice to identify new miRNAs that might be useful for diagnosing, treating, or predicting neuropathic pain. We show that, after sciatic nerve injury in rats, miR-30c-5p was up-regulated in the spinal cord, dorsal root ganglia, cerebrospinal fluid (CSF) and plasma and that the expression of miR-30c-5p positively correlated with the severity of allodynia. The administration of a miR-30c-5p inhibitor into the cisterna magna of the brain delayed neuropathic pain development and reversed fully established allodynia in rodents. The mechanism was mediated by TGF-ß and involved the endogenous opioid system. In patients with neuropathic pain associated with leg ischemia, the expression of miR-30c-5p was increased in plasma and CSF compared to control patients without pain. Logistic regression analysis in our cohort of patients showed that the expression of miR-30c-5p in plasma and CSF, in combination with other clinical variables, might be useful to help to predict neuropathic pain occurrence in patients with chronic peripheral ischemia.

Experimental modelling of cardiac pressure overload hypertrophy: Modified technique for precise, reproducible, safe and easy aortic arch banding-debanding in mice.

Pressure overload left ventricular hypertrophy is a known precursor of heart failure with ominous prognosis. The development of experimental models that reproduce this phenomenon is instrumental for the advancement in our understanding of its pathophysiology. The gold standard of these models is the controlled constriction of the mid aortic arch in mice according to Rockman's technique (RT). We developed a modified technique that allows individualized and fully controlled constriction of the aorta, improves efficiency and generates a reproducible stenosis that is technically easy to perform and release. An algorithm calculates, based on the echocardiographic arch diameter, the intended perimeter at the constriction, and a suture is prepared with two knots separated accordingly. The aorta is encircled twice with the suture and the loop is closed with a microclip under both knots. We performed controlled aortic constriction with Rockman's and the double loop-clip (DLC) techniques in mice. DLC proved superiority in efficiency (mortality and invalid experiments) and more homogeneity of the results (transcoarctational gradients, LV mass, cardiomyocyte hypertrophy, gene expression) than RT. DLC technique optimizes animal use and generates a consistent and customized aortic constriction with homogeneous LV pressure overload morphofunctional, structural, and molecular features.

Chronic methadone treatment and repeated withdrawal impair cognition and increase the expression of apoptosis-related proteins in mouse brain.

OBJECTIVES: This study analyzes the effects of prolonged administration of methadone and withdrawal on sensorimotor and cognitive performance in mice and explores the associated changes in brain expression of proteins regulating the extrinsic (FasL, Fas, and caspase-8) and the mitochondrial (Bcl-2, Bcl-x(L), Bad, and Bax) apoptotic pathways. RESULTS: Our findings indicate that, although acute methadone administration impairs some sensorimotor abilities, tolerance to most of the deleterious effects develops after chronic administration. Cognitive abilities in the Morris water maze were impaired by chronic methadone and, to a greater extent, by exposure to precipitated withdrawal every week in the course of methadone treatment. Both the chronic methadone and repeated withdrawal groups showed up-regulation of several pro-apoptotic proteins (FasL, the active fragment of caspase-8, and Bad) in the cortex and hippocampus, indicating activation of both the death-receptor and mitochondrial apoptotic pathways. In contrast, reduced expression of the apoptosis regulatory proteins FasL and Bad was found after a single administration of methadone. CONCLUSIONS: Our data suggest that neural apoptotic damage could contribute to impairment of the cognitive abilities of mice observed after chronic methadone administration and withdrawal.

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome.

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1-NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

Modulation of brain apoptosis-related proteins by the opioid antagonist naltrexone in mice.

Neuronal loss by apoptosis has been implicated in some neural pathologic disorders. Increasing evidence suggests a neuroprotective effect for opioid antagonists, such as naloxone and naltrexone, in a variety of neural damage experimental models and in the clinic. The purpose of the present study was to analyse the effects of naltrexone on the expression levels of proteins regulating the extrinsic (FasL and Fas) and the mitochondrial (Bcl-2, Bcl-xL, Bad and Bax) apoptotic pathways, as well as the active fragment of the executioner caspase-3 in the mouse brain. Western blotting showed that a single injection of naltrexone (1 mg/kg) induced a down-regulation of the pro-apototic proteins Fas, FasL, Bad and Bax. Our results suggest that naltrexone provides neuronal protection against injuries activating either mitochondrial, or death receptor-apoptotic pathways.

BMP-7 attenuates left ventricular remodelling under pressure overload and facilitates reverse remodelling and functional recovery.

AIMS: TGF-ß regulates tissue fibrosis: TGF-ß promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS: We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-ß and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and ß-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-ß as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-ß-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION: The disequilibrium between BMP-7 and TGF-ß signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.

Extracellular heat shock protein 90 binding to TGFß receptor I participates in TGFß-mediated collagen production in myocardial fibroblasts.

The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor ß (TGFß) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFß signaling by stabilizing the TGFß signaling cascade. We detected extracellular Hsp90 in complex with TGFß receptor I (TGFßRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. This was supported by in silico studies predicting Hsp90 dimers and TGFßRI extracellular domain interaction. Both, Hsp90aa1 and Hsp90ab1 isoforms participate in TGFßRI complex. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFß signaling cascade, and collagen protein synthesis was drastically reduced in Hsp90aa1 KO mice. These observations together with the significant increase in activity of Hsp90 at the plasma membrane pointed to a functional cooperative partnership between Hsp90 and TGFßRI in the fibrotic process. We propose that a surface population of Hsp90 extracellularly binds TGFßRI and this complex behaves as an active participant in collagen production in TGFß-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 and its isoforms during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice.

Opioid tolerance and supersensitivity induce regional changes in the autoradiographic density of dihydropyridine-sensitive calcium channels in the rat central nervous system.

Chronic opioid administration induces adaptations in neurones resulting in opioid tolerance and dependence. The changes in dihydropyridine (DHP)-sensitive Ca(2+) channels (L-type) associated with tolerance and supersensitivity to the antinociceptive effect of the micro-opioid receptor agonist sufentanil were analyzed in the central nervous system (CNS) of rats. Autoradiographic assays were performed with [(3)H]PN-200-110 (isopropyl 4-(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate). Chronic s.c. infusion of sufentanil (2 microg/h) for 7 days, which has been shown to induce tolerance to the opioid antinociceptive effect, produced an up-regulation of DHP binding sites. The highest increases in density were localized in regions involved in nociceptive transmission and perception, such as the dorsal horn of the spinal cord, the dorsal raphe nucleus, the central grey matter, the thalamic nuclei, and the somatosensory cortex. Animals were rendered supersensitive to the antinociceptive effect of sufentanil by chronic and simultaneous infusion of sufentanil (2 microg/h) and nimodipine (1 microg/h) for 7 days. Under these conditions, a greater increase in the number of DHP binding sites was observed in the spinal cord, central grey matter, dorsal raphe nucleus, and somatosensory neocortex, when compared to the sufentanil group. The role of an increased influx through L-type channels in opioid tolerance is reinforced, whereas their persistent blockade is essential for the expression of opioid supersensitivity.