Profibrotic Role of Inducible Heat Shock Protein 90α Isoform in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease that affects skin and multiple internal organs. TGF-ß, a central trigger of cutaneous fibrosis, activates fibroblasts with the involvement of the stress-inducible chaperone heat shock protein 90 isoform α (Hsp90α). Available evidence supports overexpression and secretion of Hsp90α as a feature in profibrotic pathological conditions. The aim of this work is to investigate the expression and function of Hsp90α in experimental models of skin fibrosis such as human fibroblasts, C57BL/6 mice, and in human SSc. For this purpose, we generated a new experimental model based on doxorubicin administration with improved characteristics with respect to the bleomycin model. We visualized disease progression in vivo by fluorescence imaging. In this work, we obtained Hsp90α mRNA overexpression in human skin fibroblasts, in bleomycin- and doxorubicin-induced mouse fibrotic skin, and in lungs of bleomycin- and doxorubicin-treated mice. Hsp90α-deficient mice showed significantly decreased skin thickness compared with wild-type mice in both animal models. In SSc patients, serum Hsp90α levels were increased in patients with lung involvement and in patients with the diffuse form of SSc (dSSc) compared with patients with the limited form of SSc. The serum Hsp90α levels of patients dSSc were correlated with the Rodnan score and the forced vital capacity variable. These results provide new supportive evidence of the contribution of the Hsp90α isoform in the development of skin fibrosis. In SSc, these results indicated that higher serum levels were associated with dSSc and lung fibrosis.

Epigenetic alterations of TGFß and its main canonical signaling mediators in the context of cardiac fibrosis.

Cardiac fibrosis is a pathological process that presents a continuous overproduction of extracellular matrix (ECM) components in the myocardium, which negatively influences the progression of many cardiac diseases. Transforming growth factor ß (TGFß) is the main ligand that triggers the production of pro-fibrotic ECM proteins. In the cardiac fibrotic process, TGFß and its canonical signaling mediators are tightly regulated at different levels as well as epigenetically. Cardiac fibroblasts are one of the most important TGFß target cells activated after cardiac injury. TGFß-driven fibroblast activation is subject to epigenetic modulation and contributes to the progression of cardiac fibrosis, mainly through the expression of pro-fibrotic molecules implicated in the disease. In this review, we describe epigenetic regulation related to canonical TGFß signaling in cardiac fibroblasts.

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.

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.

Fibroblast-derived extracellular vesicles as trackable efficient transporters of an experimental nanodrug with fibrotic heart and lung targeting.

The discovery of extracellular vesicles (EVs) as efficient exogenous biotransporters of therapeutic agents into cells across biological membranes is an exciting emerging field. Especially the potential of EVs as targeted delivery systems for diseases with selective treatments, such as fibrosis, whose treatment causes side effects in other organs not involved in the disease. Methods: In this study, we collected embryonic fibroblast-derived EVs from two different centrifugation fractions, 10 K g and 100 K g fractions from a NIH-3T3 cell line loaded with an experimental drug. Mice with fibrotic hearts and lungs were obtained by administration of angiotensin II. We generated fluorescent EVs and bioluminescent drug to observe their accumulation by colocalization of their signals in fibrotic heart and lung. The biodistribution of the drug in various organs was obtained by detecting the Au present in the drug nanostructure. Results: The drug-loaded EVs successfully reduced fibrosis in pathological fibroblasts in vitro, and modified the biodistribution of the experimental drug, enabling it to reach the target organs in vivo. We described the pre-analytical characteristics of EVs related to physical variables, culture and harvesting conditions, crucial for their in vivo application as nanotransporters using a previously validated protein-based antifibrotic drug. The results showed the colocalization of EVs and the experimental drug in vivo and ex vivo and the efficient reduction of fibrosis in vitro. This work demonstrates that 10K-EVs and 100K-EVs derived from fibroblasts can act as effective biotransporters for targeted drug delivery to profibrotic fibroblasts, lungs, or heart. Conclusion: We observed that fibroblast-derived 10K-EVs and 100K-EVs are useful biotransporters encapsulating a new generation drug leading to a reduction of fibrosis in profibrotic fibroblasts in vitro. In addition, drug containing EVs were shown to reach fibrotic heart and lungs in vivo, enhancing free drug biodistribution.

Engineering multifunctional metal/protein hybrid nanomaterials as tools for therapeutic intervention and high-sensitivity detection.

Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine.

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.

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop.

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

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.

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.

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-ß-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.

Circulating levels of miR-133a predict the regression potential of left ventricular hypertrophy after valve replacement surgery in patients with aortic stenosis.

BACKGROUND: Myocardial microRNA-133a (miR-133a) is directly related to reverse remodeling after pressure overload release in aortic stenosis patients. Herein, we assessed the significance of plasma miR-133a as an accessible biomarker with prognostic value in predicting the reversibility potential of LV hypertrophy after aortic valve replacement (AVR) in these patients. METHODS AND RESULTS: The expressions of miR-133a and its targets were measured in LV biopsies from 74 aortic stenosis patients. Circulating miR-133a was measured in peripheral and coronary sinus blood. LV mass reduction was determined echocardiographically. Myocardial and plasma levels of miR-133a correlated directly (r=0.46, P<0.001) supporting the myocardium as a relevant source of plasma miR-133a. Accordingly, a significant gradient of miR-133a was found between coronary and systemic venous blood. The preoperative plasma level of miR-133a was higher in the patients who normalized LV mass 1 year after AVR than in those exhibiting residual hypertrophy. Logistic regression analysis identified plasma miR-133a as a positive predictor of the hypertrophy reversibility after surgery. The discrimination of the model yielded an area under the receiver operator characteristic curve of 0.89 (P<0.001). Multiple linear regression analysis revealed plasma miR-133a and its myocardial target Wolf-Hirschhorn syndrome candidate 2/Negative elongation factor A as opposite predictors of the LV mass loss (g) after AVR. CONCLUSIONS: Preoperative plasma levels of miR-133a reflect their myocardial expression and predict the regression potential of LV hypertrophy after AVR. The value of this bedside information for the surgical timing, particularly in asymptomatic aortic stenosis patients, deserves confirmation in further clinical studies.

Chronic treatment with the opioid antagonist naltrexone favours the coupling of spinal cord µ-opioid receptors to Gαz protein subunits.

Sustained administration of opioid antagonists to rodents results in an enhanced antinociceptive response to agonists. We investigated the changes in spinal µ-opioid receptor signalling underlying this phenomenon. Rats received naltrexone (120 µg/h; 7 days) via osmotic minipumps. The antinociceptive response to the µ-agonist sufentanil was tested 24 h after naltrexone withdrawal. In spinal cord samples, we determined the interaction of µ-receptors with Gα proteins (agonist-stimulated [(35)S]GTPγS binding and immunoprecipitation of [(35)S]GTPγS-labelled Gα subunits) as well as µ-opioid receptor-dependent inhibition of the adenylyl cyclase (AC) activity. Chronic naltrexone treatment augmented DAMGO-stimulated [(35)S]GTPγS binding, potentiated the inhibitory effect of DAMGO on the AC/cAMP pathway, and increased the inverse agonist effect of naltrexone on cAMP accumulation. In control rats, the inhibitory effect of DAMGO on cAMP production was antagonized by pertussis toxin (PTX) whereas, after chronic naltrexone, the effect became resistant to the toxin, suggesting a coupling of µ-receptors to PTX-insensitive Gα(z) subunits. Immunoprecipitation assays confirmed the transduction switch from Gα(i/o) to Gα(z) proteins. The consequence was an enhancement of the antinociceptive response to sufentanil that, in consonance with the neurochemical data, was prevented by Gα(z)-antisense oligodeoxyribonucleotides but not by PTX. Such changes in opioid receptor signalling can be a double-edged sword. On the one hand, they may have potential applicability to the optimisation of the analgesic effects of opioid drugs for the control of pain. On the other hand, they represent an important homeostatic dysregulation of the endogenous opioid system that might account for undesirable effects in patients chronically treated with opioid antagonists. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Androgens contribute to sex differences in myocardial remodeling under pressure overload by a mechanism involving TGF-ß.

BACKGROUND: In clinical studies, myocardial remodeling in aortic valve stenosis appears to be more favorable in women than in men, even after menopause. In the present study, we assessed whether circulating androgens contribute to a less favorable myocardial remodeling under pressure overload in males. We examined sex-related differences in one-year-old male and female mice. Whereas male mice at this age exhibited circulating androgen levels within the normal range for young adults, the circulating estrogens in females were reduced. The contribution of gonadal androgens to cardiac remodeling was analyzed in a group of same-age castrated mice. METHODOLOGY/PRINCIPAL FINDINGS: Animals were subjected to transverse aortic constriction (TAC). Echocardiography was performed 2 weeks after TAC and myocardial mRNA levels of TGF-ßs, Smads 2 and 3, collagens, fibronectin, ß-myosin heavy chain and α-myosin heavy chain were determined by q-PCR. Protein detection of p-SMAD2/3 was performed by Western Blot. Histological staining of fibrosis was performed with picrosirius red and Masson's trichrome. Compared with females, males developed more severe tissue fibrosis, LV dilation and hemodynamic dysfunction. TAC-males showed higher myocardial expression levels of TGF-ßs and the treatment with a neutralizing antibody to TGF-ß prevented myocardial fibrosis development. Orchiectomy diminished TAC-induced up-regulation of TGF-ßs and TGF-ß target genes, and it also reduced fibrosis and hemodynamic dysfunction. The capability of androgens to induce TGF-ß expression was confirmed in NIH-3T3 fibroblasts and H9C2 cardiomyocytes exposed to dihydrotestosterone. CONCLUSIONS/SIGNIFICANCE: Our results indicate that circulating androgens are responsible for the detrimental effects in the myocardium of older male mice subjected to pressure overload through a mechanism involving TGF-ßs.

Myocardial gene expression of microRNA-133a and myosin heavy and light chains, in conjunction with clinical parameters, predict regression of left ventricular hypertrophy after valve replacement in patients with aortic stenosis.

BACKGROUND: Left ventricular (LV) reverse remodelling after valve replacement in aortic stenosis (AS) has been classically linked to the hydraulic performance of the replacement device, but myocardial status at the time of surgery has received little attention. OBJECTIVE: To establish predictors of LV mass (LVM) regression 1 year after valve replacement in a surgical cohort of patients with AS based on preoperative clinical and echocardiographic parameters and the myocardial gene expression profile at surgery. METHODS: Transcript levels of remodelling-related proteins and regulators were determined in LV intraoperative biopsies from 46 patients with AS by RT-PCR. Using multiple linear regression analysis, an equation was developed (adjusted R²=0.73; p<0.0001) that included positive [preoperative LVM, microRNA-133a, serum response factor (SRF, which is known to be a transactivator of miR-133) and age] and negative [body mass index (BMI), Wolf-Hirschhorn syndrome candidate-2 (WHSC2, which is a target for repression by miR-133a), ß-myosin heavy chain, myosin light chain-2, diabetes mellitus, and male gender] independent predictors of LVM reduction. RESULTS: Aortic valve area gain or the reduction in transvalvular gradient maintained no significant relationships with the dependent variable. Logistic regression analysis identified microRNA-133a as a significant positive predictor of LVM normalisation, whereas ß-myosin heavy chain and BMI constituted negative predictors. CONCLUSIONS: Hypertrophy regression 1 year after pressure overload release is related to the preoperative myocardial expression of remodelling-related genes, in conjunction with the patient's clinical background. In this scenario, miR-133 emerges as a key element of the reverse remodelling process. Postoperative improvement of valve haemodynamics does not predict the degree of hypertrophy regression or LVM normalisation. These results led us to reconsider the current reverse remodelling paradigm and (1) to include criteria of hypertrophy reversibility in the decision algorithm used to decide timing for the operation; and (2) to modify other prevailing factors (overweight, diabetes, etc) known to maintain LV hypertrophy.

Plasma levels of transforming growth factor-beta1 reflect left ventricular remodeling in aortic stenosis.

BACKGROUND: TGF-beta1 is involved in cardiac remodeling through an auto/paracrine mechanism. The contribution of TGF-beta1 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-beta1 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-beta1 was determined by ELISA. Under pressure overload, TGF-beta1 plasma levels were significantly increased both in AS patients and TAC mice. In AS patients, plasma TGF-beta1 correlated directly with aortic transvalvular gradients and LV mass surrogate variables, both preoperatively and 1 year after surgery. Plasma TGF-beta1 correlated positively with the myocardial expression of genes encoding extracellular matrix (collagens I and III, fibronectin) and sarcomeric (myosin light chain-2, beta-myosin heavy chain) remodelling targets of TGF-beta1, in TAC mice and in AS patients. CONCLUSIONS/SIGNIFICANCE: A circulating TGF-beta1-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-beta1 as a marker reflecting preoperative myocardial remodeling status in AS patients deserves further analysis in larger patient cohorts.

Modulation of PI-specific phospholipase C by membrane curvature and molecular order.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus has been assayed on large and small unilamellar vesicles consisting of PI, either pure or in mixtures with other lipids. Vesicle diameter (in the 50-300 nm range) influences PI-PLC activity, enzyme rates increasing with decreasing curvature radii. With sonicated unilamellar vesicles of pure PI, two apparent K(s) values are observed, one in the 0-2 mM concentration range and the other in the 2-12 mM concentration range. The latter ( approximately 4.2 mM) corresponds to previously published values, while the low-concentration K(s) is on the same order of magnitude as the single apparent K(m) value found with large unilamellar liposomes ( approximately 0.30 mM). PI-PLC appears to be very sensitive to bilayer composition. Certain nonsubstrate lipids, e.g., galactosylceramide or cholesterol, inhibit PI-PLC in a dose-dependent way, at least up to 33 mol % in the bilayers, under conditions with a constant PI concentration. Simultaneous measurements of enzyme activity, interfacial enzyme binding, and fluorescence of different probes, on a variety of bilayer compositions, reveal that both the level of enzyme binding and activity decrease with increasing lipid order, as measured by the fluorescence polarization of the hydrophobic probe diphenylhexatriene. In contrast, no correlation is found for enzyme activity with fluorescence changes of probes, e.g., laurdan, that report on phenomena occurring mainly at the lipid-water interface. Sphingomyelin has a dual effect. Up to 40 mol %, it increases PI-PLC activity, with little effect on bilayer molecular order. At higher proportions, the increased lipid chain order causes a decrease in enzyme activity. The same effects are observed for distearoylphosphatidylcholine when added to PI bilayers. These results support the "two-stage model" for binding of PI-PLC to lipid bilayers, and underline the significance of the enzyme partial penetration into the membrane hydrophobic matrix for its catalytic activity.

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts.

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.