Protein changes associated with embryonic stem cell differentiation to vascular smooth muscle cells

PhDEmbryonic stem (ES) cells can differentiate into many different cell lines, including vascular smooth muscle cells (SMCs). The aim of this project is to characterize protein changes during this differentiation process. Mouse ES cells are pre-differentiated by withdrawal of the leukemia inhibitory factor-1 from the culture medium. Subsequently, stem cell antigen-1 positive (Sca-1) cells are sorted by magnetic labelling cell sorting with anti-Sca-1 microbeads and cultured in differentiation medium with or without platelet-derived growth factor (PDGF). Protein extracts of ES cells and Sca-1+ cells are separated by two-dimensional electrophoresis. About 300 protein species of each cell lines are analyzed by mass spectrometry. Proteome maps are available online (http:/ /vwvw.v ascular-proteomicsc. om). After stimulation with PDGF for 5 passages, Sca-1+ cells differentiate into SMCs (esSMCs) with 95% staining positive for SMC markers such as smooth muscle a-actin, calponin, and smooth muscle myosin heavy chain. Protein profiles of esSMCs and mouse aortic SMCs are compared using the difference gel electrophoresis approach. esSMCs display decreased expression of myofilaments but increased oxidation of redox-sensitive proteins due to higher levels of reactive oxgen species (ROS). While immunoblotting reveals an upregulation of numerous antioxidants in esSMCs, enzymatic assays demonstrate lower glutathione concentrations compared to aortic SMCs despite a 3-fold increase in glutathione reductase activity. Mitochondrial superoxide measurement revealed the mitochondria-derived superoxide is the main source of ROS in esSMCs and inhibition of electron transport chain complex III by antimycin A showed remarkable increase of ROS in esSMCs. Moreover, depletion of glutathione by diethyl maleate or inhibition of glutathione reductase by carmustine (BCNU) results in a remarkable loss of cell viability in esSMCs compared to aortic SMCs while adding 2-mercaptoethanol increased esSMCs survival. These results indicate that esSMCs require additional antioxidant protection for survival and consequently, treatment with anti-oxidants could be beneficial for tissue repair from ES cells

Determinantes do crédito malparado das famílias

Este estudo tem como objetivo analisar os determinantes do incumprimento de crédito à habitação e verificar qual o seu impacto na concessão de crédito à habitação. Foi realizado um estudo econométrico sobre os principais determinantes do incumprimento de crédito à habitação - a taxa de desemprego, o rendimento disponível das famílias e a taxa de juro de crédito à habitação, com as variáveis rácio de crédito habitação vencido e crédito à habitação. Os dados do estudo são trimestrais e uma duração do primeiro trimestre de 2003 até ao último trimestre de 2016. Entre as conclusões obtidas nesta investigação, realço que o rácio de crédito à habitação vencido é positivamente afetado pela taxa de desemprego e pela taxa de juro de crédito à habitação, assim como é negativamente afetado pelo rendimento disponível das famílias e pelo crédito à habitação. Isto significa que um aumento da taxa de desemprego ou da taxa de juro contribui para um aumento do incumprimento de crédito à habitação, enquanto uma diminuição do rendimento disponível ou do crédito à habitação leva também um aumento do incumprimento.This dissertation aims to analyze the determinants of housing credit default and verify how it impacts the housing credit. It was developed an econometric study of the main determinants of housing credit default – unemployment rate, disposable income of families and housing credit interest rate, with the variables non-performing loan (NPL) ratio for the housing credit and the housing credit grant. Quarterly data covering the first quarter of 2003 to the last quarter of 2016 were used. The main result from the econometric study provides that the overdue housing credit ratio is positively affected by the unemployment rate and the housing credit interest rate. On the other hand the non-performing loan ratio for housing credit reacts negatively to the disposable income of the families and the housing credit. This leads to the conclusion that an increase of the unemployment rate or the credit interest rate, will result in an increase of the housing credit default, while a decrease of the disposable income or the housing credit, causes an increase of the default

Efficient Text-Guided 3D-Aware Portrait Generation with Score Distillation Sampling on Distribution

Text-to-3D is an emerging task that allows users to create 3D content with infinite possibilities. Existing works tackle the problem by optimizing a 3D representation with guidance from pre-trained diffusion models. An apparent drawback is that they need to optimize from scratch for each prompt, which is computationally expensive and often yields poor visual fidelity. In this paper, we propose DreamPortrait, which aims to generate text-guided 3D-aware portraits in a single-forward pass for efficiency. To achieve this, we extend Score Distillation Sampling from datapoint to distribution formulation, which injects semantic prior into a 3D distribution. However, the direct extension will lead to the mode collapse problem since the objective only pursues semantic alignment. Hence, we propose to optimize a distribution with hierarchical condition adapters and GAN loss regularization. For better 3D modeling, we further design a 3D-aware gated cross-attention mechanism to explicitly let the model perceive the correspondence between the text and the 3D-aware space. These elaborated designs enable our model to generate portraits with robust multi-view semantic consistency, eliminating the need for optimization-based methods. Extensive experiments demonstrate our model's highly competitive performance and significant speed boost against existing methods

Wireless Real-Time Capacitance Readout Based on Perturbed Nonlinear Parity-Time Symmetry

In this article, we report a vector-network-analyzer-free and real-time LC wireless capacitance readout system based on perturbed nonlinear parity-time (PT) symmetry. The system is composed of two inductively coupled reader-sensor parallel RLC resonators with gain and loss respectively. By searching for the real mode that requires the minimum saturation gain, the steady-state frequency evolution as a function of the sensor capacitance perturbation is analytically deduced. The proposed system can work in different modes by setting different perturbation point. In particular, at the exceptional point of PT symmetry, the system exhibits high sensitivity. Experimental demonstrations revealed the viability of the proposed readout mechanism by measuring the steady-state frequency of the reader resonator in response to the change of trimmer capacitor on the sensor side. Our findings could impact many emerging applications such as implantable medical device for health monitoring, parameter detection in harsh environment and sealed food packages, etc

Apolipoprotein Proteomics for Residual Lipid-Related Risk in Coronary Heart Disease

BACKGROUND: Recognition of the importance of conventional lipid measures and the advent of novel lipid-lowering medications have prompted the need for more comprehensive lipid panels to guide use of emerging treatments for the prevention of coronary heart disease (CHD). This report assessed the relevance of 13 apolipoproteins measured using a single mass-spectrometry assay for risk of CHD in the PROCARDIS case-control study of CHD (941 cases/975 controls). METHODS: The associations of apolipoproteins with CHD were assessed after adjustment for established risk factors and correction for statin use. Apolipoproteins were grouped into 4 lipid-related classes [lipoprotein(a), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides] and their associations with CHD were adjusted for established CHD risk factors and conventional lipids. Analyses of these apolipoproteins in a subset of the ASCOT trial (Anglo-Scandinavian Cardiac Outcomes Trial) were used to assess their within-person variability and to estimate a correction for statin use. The findings in the PROCARDIS study were compared with those for incident cardiovascular disease in the Bruneck prospective study (n=688), including new measurements of Apo(a). RESULTS: Triglyceride-carrying ApoC1, ApoC3, and ApoE (apolipoproteins) were most strongly associated with the risk of CHD (2- to 3-fold higher odds ratios for top versus bottom quintile) independent of conventional lipid measures. Likewise, ApoB was independently associated with a 2-fold higher odds ratios of CHD. Lipoprotein(a) was measured using peptides from the Apo(a)-kringle repeat and Apo(a)-constant regions, but neither of these associations differed from the association with conventionally measured lipoprotein(a). Among HDL-related apolipoproteins, ApoA4 and ApoM were inversely related to CHD, independent of conventional lipid measures. The disease associations with all apolipoproteins were directionally consistent in the PROCARDIS and Bruneck studies, with the exception of ApoM. CONCLUSIONS: Apolipoproteins were associated with CHD independent of conventional risk factors and lipids, suggesting apolipoproteins could help to identify patients with residual lipid-related risk and guide personalized approaches to CHD risk reduction

Proteomics of the epicardial fat secretome and its role in post-operative atrial fibrillation.

Aims: Post-operative atrial fibrillation (POAF) is a predictor of morbidity and mortality after cardiac surgery. Latent predisposing factors may reside in the epicardial adipose tissue (EAT) due to its anatomical position and high protein production rate. In order to explore a possible mechanistic link, we characterized proteins secreted by the EAT preceding the onset of POAF. Methods and results: Epicardial adipose tissue samples were collected from 76 consecutive patients with no history of AF undergoing coronary artery bypass surgery, 50 samples for proteomic analysis and 26 for gene expression studies, further divided according to development of POAF. Ten vs. 10 matched samples representing EAT secretome were analysed by two-dimensional difference in-gel electrophoresis (2D-DIGE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to identify differentially expressed proteins (P 1.2 fold). Findings were validated by Western blotting on EAT protein extracts and by gene expression studies via quantitative polymerase chain reaction (qPCR). Proteomics returned 35 differentially expressed proteins. Amongst those, gelsolin was down regulated in POAF. Western blot analysis confirmed a significant reduction in gelsolin in the AF group. Gene expression for gelsolin was significantly reduced in the AF group confirming the proteomics findings. Conclusion: For the first time we describe EAT secretome as a possible substrate for POAF. It contains various proteins differentially expressed in patients who later develop POAF. Amongst those gelsolin, involved in inflammation and ion channel regulation, was associated with maintenance of sinus rhythm. Understanding the role of EAT may offer novel insights into prevention and treatment of AF

Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins.

AIMS: Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. METHODS AND RESULTS: To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. CONCLUSION: The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling

Background Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200–500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and results Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Conclusions Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes

Protein Aggregation Is an Early Manifestation of Phospholamban p.(Arg14del)-Related Cardiomyopathy:Development of PLN-R14del-Related Cardiomyopathy

BACKGROUND: The p.(Arg14del) pathogenic variant (R14del) of the PLN (phospholamban) gene is a prevalent cause of cardiomyopathy with heart failure. The exact underlying pathophysiology is unknown, and a suitable therapy is unavailable. We aim to identify molecular perturbations underlying this cardiomyopathy in a clinically relevant PLN-R14del mouse model. METHODS: We investigated the progression of cardiomyopathy in PLN-R14Δ/Δ mice using echocardiography, ECG, and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 (before the onset of disease), 5 (mild cardiomyopathy), and 8 (end stage) weeks of age. Data were compared with cardiac expression levels of mice that underwent myocardial ischemia-reperfusion or myocardial infarction surgery, in an effort to identify alterations that are specific to PLN-R14del-related cardiomyopathy. RESULTS: At 3 weeks of age, PLN-R14Δ/Δ mice had normal cardiac function, but from the age of 4 weeks, we observed increased myocardial fibrosis and impaired global longitudinal strain. From 5 weeks onward, ventricular dilatation, decreased contractility, and diminished ECG voltages were observed. PLN protein aggregation was present before onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodeling, inflammation, and metabolic dysfunction, in part, similar to ischemic heart disease. Altered protein homeostasis pathways were identified exclusively in PLN-R14Δ/Δ mice, even before disease onset, in concert with aggregate formation. CONCLUSIONS: We mapped the development of PLN-R14del-related cardiomyopathy and identified alterations in proteostasis and PLN protein aggregation among the first manifestations of this disease, which could possibly be a novel target for therapy