David de Gonzalo-Calvo

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study

BackgroundThe metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas.MethodsWe classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis.ResultsGroup-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported.ConclusionsIntratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.

Genetic Basis of Dilated Cardiomyopathy

Dilated cardiomyopathy is a rare cardiac disease characterized by left ventricular dilatation and systolic dysfunction leading to heart failure and sudden cardiac death. Currently, despite several conditions have been reported as aetiologies of the disease, a large number of cases remain classified as idiopathic. Recent studies determine that nearly 60% of cases are inherited, therefore due to a genetic cause. Progressive technological advances in genetic analysis have identified over 60 genes associated with this entity, being TTN the main gene, so far. All these genes encode a wide variety of myocyte proteins, mainly sarcomeric and desmosomal, but physiopathologic pathways are not yet completely unraveled. We review the recent published data about genetics of familial dilated cardiomyopathy.

microRNA expression profile in human coronary smooth muscle cell-derived microparticles is a source of biomarkers

INTRODUCTION microRNA (miRNA) expression profile of extracellular vesicles is a potential tool for clinical practice. Despite the key role of vascular smooth muscle cells (VSMC) in cardiovascular pathology, there is limited information about the presence of miRNAs in microparticles secreted by this cell type, including human coronary artery smooth muscle cells (HCASMC). Here, we tested whether HCASMC-derived microparticles contain miRNAs and the value of these miRNAs as biomarkers. METHODS HCASMC and explants from atherosclerotic or non-atherosclerotic areas were obtained from coronary arteries of patients undergoing heart transplant. Plasma samples were collected from: normocholesterolemic controls (N=12) and familial hypercholesterolemia (FH) patients (N=12). Both groups were strictly matched for age, sex and cardiovascular risk factors. Microparticle (0.1-1μm) isolation and characterization was performed using standard techniques. VSMC-enriched miRNAs expression (miR-21-5p, -143-3p, -145-5p, -221-3p and -222-3p) was analyzed using RT-qPCR. RESULTS Total RNA isolated from HCASMC-derived microparticles contained small RNAs, including VSMC-enriched miRNAs. Exposition of HCASMC to pathophysiological conditions, such as hypercholesterolemia, induced a decrease in the expression level of miR-143-3p and miR-222-3p in microparticles, not in cells. Expression levels of miR-222-3p were lower in circulating microparticles from FH patients compared to normocholesterolemic controls. Microparticles derived from atherosclerotic plaque areas showed a decreased level of miR-143-3p and miR-222-3p compared to non-atherosclerotic areas. CONCLUSIONS We demonstrated for the first time that microparticles secreted by HCASMC contain microRNAs. Hypercholesterolemia alters the microRNA profile of HCASMC-derived microparticles. The miRNA signature of HCASMC-derived microparticles is a source of cardiovascular biomarkers.

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes.

The maintenance of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca(2+)-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low‐density lipoprotein receptor‐related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet‐free plasma by enzyme‐linked immunosorbent assay. Plasma‐derived EVs were extracted by size‐exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo‐transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin‐3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9‐ and CD81‐positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin‐3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

Signature of subclinical femoral artery atherosclerosis in peripheral blood mononuclear cells.

Peripheral arterial disease is a relevant public health problem associated with increased risk of morbimortality. Most of the patients with this condition are asymptomatic. Therefore, the development of accessible biochemical markers seems to be necessary to anticipate diagnosis. Our hypothesis is that asymptomatic subjects with objectively confirmed femoral artery atherosclerosis could be distinguished from control subjects by gene expression analysis in peripheral blood mononuclear cells (PBMC).

Non-coding RNAs and exercise: pathophysiological role and clinical application in the cardiovascular system

There is overwhelming evidence that regular exercise training is protective against cardiovascular disease (CVD), the main cause of death worldwide. Despite the benefits of exercise, the intricacies of their underlying molecular mechanisms remain largely unknown. Non-coding RNAs (ncRNAs) have been recognized as a major regulatory network governing gene expression in several physiological processes and appeared as pivotal modulators in a myriad of cardiovascular processes under physiological and pathological conditions. However, little is known about ncRNA expression and role in response to exercise. Revealing the molecular components and mechanisms of the link between exercise and health outcomes will catalyse discoveries of new biomarkers and therapeutic targets. Here we review the current understanding of the ncRNA role in exercise-induced adaptations focused on the cardiovascular system and address their potential role in clinical applications for CVD. Finally, considerations and perspectives for future studies will be proposed.

Relationship among LRP1 expression, Pyk2 phosphorylation and MMP-9 activation in left ventricular remodelling after myocardial infarction

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low‐density lipoprotein receptor‐related protein 1 (LRP1) and MMP‐9 and MMP‐2 spatiotemporal expression after MI. Real‐time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri‐infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri‐infarct and infarct areas. LRP1 also colocalized with proline‐rich tyrosine kinase 2 (pPyk2) and MMP‐9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP‐9 activity in fibroblasts, without significant changes in MMP‐2 activity. MMP‐9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP‐9 up‐regulation associated with ventricular remodelling after MI.

Plasma microRNAs as biomarkers for Lamin A/C-related dilated cardiomyopathy

Lamin A/C gene (LMNA)-related familial dilated cardiomyopathy (fDCM) is an aggressive heart disease that often leads to transplantation and sudden death. The aim of our study was to evaluate the circulating microRNA (miRNA) profiles of patients with LMNA pathogenic mutations. The study population (N = 75) included (i) patients with pathogenic LMNA mutations responsible for fDCM (LMNAMUT), (ii) age- and sex-matched LMNA wild-type controls (LMNAWT control), and (iii) LMNA wild-type idiopathic DCM (iDCM) patients (LMNAWT iDCM). Detailed clinical information was obtained from each participant. A panel of 179 plasma miRNAs was evaluated using RT-qPCR. An initial screening study was performed in LMNAMUT carriers and age-matched LMNAWT controls (N = 16). Forty-four miRNAs were specifically deregulated in LMNAMUT carriers. Ten miRNA candidates were selected for subsequent validation after coexpression analyses and filtered for expression levels and statistical significance. Among the candidates, let-7a-5p, miR-142-3p, miR-145-5p and miR-454-3p levels were significantly increased in LMNAMUT carriers compared to LMNAWT controls and iDCM patients (P < 0.050). These circulating miRNAs, and their combination, were also associated with the presence of pathogenic mutations in regression and ROC analyses. This signature also discriminates between LMNAWT healthy subjects and LMNAMUT carriers who are phenotypically negative for DCM and between LMNAWT iDCM and LMNA-related DCM patients. Correlation and functional enrichment analyses supported their association with the pathophysiology of the disease. We demonstrated for the first time that a specific miRNA signature could serve as a novel non-invasive tool to assist in the diagnosis of patients with fDCM caused by LMNA pathogenic mutations.Key messagesLet-7a-5p, miR-142-3p, miR-145-5p and miR-454-3p are differentially expressed in LMNAMUT carriers.A composite score based on these miRNAs is a biomarker of mutations in the LMNA gene.This miRNA signature can be associated with the pathophysiology of familial DCM.The circulating miRNA profile can assist in the diagnosis of familial DCM.

Identification of new biophysical markers for pathological ventricular remodelling in tachycardia-induced dilated cardiomyopathy

Our aim was to identify biophysical biomarkers of ventricular remodelling in tachycardia‐induced dilated cardiomyopathy (DCM). Our study includes healthy controls (N = 7) and DCM pigs (N = 10). Molecular analysis showed global myocardial metabolic abnormalities, some of them related to myocardial hibernation in failing hearts, supporting the translationality of our model to study cardiac remodelling in dilated cardiomyopathy. Histological analysis showed unorganized and agglomerated collagen accumulation in the dilated ventricles and a higher percentage of fibrosis in the right (RV) than in the left (LV) ventricle (P = .016). The Fourier Transform Infrared Spectroscopy (FTIR) 1st and 2nd indicators, which are markers of the myofiber/collagen ratio, were reduced in dilated hearts, with the 1st indicator reduced by 45% and 53% in the RV and LV, respectively, and the 2nd indicator reduced by 25% in the RV. The 3rd FTIR indicator, a marker of the carbohydrate/lipid ratio, was up‐regulated in the right and left dilated ventricles but to a greater extent in the RV (2.60‐fold vs 1.61‐fold, P = .049). Differential scanning calorimetry (DSC) showed a depression of the freezable water melting point in DCM ventricles – indicating structural changes in the tissue architecture – and lower protein stability. Our results suggest that the 1st, 2nd and 3rd FTIR indicators are useful markers of cardiac remodelling. Moreover, the 2nd and 3rd FITR indicators, which are altered to a greater extent in the right ventricle, are associated with greater fibrosis.