Patient and Management Variables Associated With Survival After Postcardiotomy Extracorporeal Membrane Oxygenation in Adults: The PELS-1 Multicenter Cohort Study.

Background Extracorporeal membrane oxygenation (ECMO) has been increasingly used for postcardiotomy cardiogenic shock, but without a concomitant reduction in observed in-hospital mortality. Long-term outcomes are unknown. This study describes patients' characteristics, in-hospital outcome, and 10-year survival after postcardiotomy ECMO. Variables associated with in-hospital and postdischarge mortality are investigated and reported. Methods and Results The retrospective international multicenter observational PELS-1 (Postcardiotomy Extracorporeal Life Support) study includes data on adults requiring ECMO for postcardiotomy cardiogenic shock between 2000 and 2020 from 34 centers. Variables associated with mortality were estimated preoperatively, intraoperatively, during ECMO, and after the occurrence of any complications, and then analyzed at different time points during a patient's clinical course, through mixed Cox proportional hazards models containing fixed and random effects. Follow-up was established by institutional chart review or contacting patients. This analysis included 2058 patients (59% were men; median [interquartile range] age, 65.0 [55.0-72.0] years). In-hospital mortality was 60.5%. Independent variables associated with in-hospital mortality were age (hazard ratio [HR], 1.02 [95% CI, 1.01-1.02]) and preoperative cardiac arrest (HR, 1.41 [95% CI, 1.15-1.73]). In the subgroup of hospital survivors, the overall 1-, 2-, 5-, and 10-year survival rates were 89.5% (95% CI, 87.0%-92.0%), 85.4% (95% CI, 82.5%-88.3%), 76.4% (95% CI, 72.5%-80.5%), and 65.9% (95% CI, 60.3%-72.0%), respectively. Variables associated with postdischarge mortality included older age, atrial fibrillation, emergency surgery, type of surgery, postoperative acute kidney injury, and postoperative septic shock. Conclusions In adults, in-hospital mortality after postcardiotomy ECMO remains high; however, two-thirds of those who are discharged from hospital survive up to 10 years. Patient selection, intraoperative decisions, and ECMO management remain key variables associated with survival in this cohort. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03857217.

Cardiomyocyte-targeting exosomes from sulforaphane-treated fibroblasts affords cardioprotection in infarcted rats.

BACKGROUND: Exosomes (EXOs), tiny extracellular vesicles that facilitate cell-cell communication, are being explored as a heart failure treatment, although the features of the cell source restrict their efficacy. Fibroblasts the most prevalent non-myocyte heart cells, release poor cardioprotective EXOs. A noninvasive method for manufacturing fibroblast-derived exosomes (F-EXOs) that target cardiomyocytes and slow cardiac remodeling is expected. As a cardioprotective isothiocyanate, sulforaphane (SFN)-induced F-EXOs (SFN-F-EXOs) should recapitulate its anti-remodeling properties. METHODS: Exosomes from low-dose SFN (3 µM/7 days)-treated NIH/3T3 murine cells were examined for number, size, and protein composition. Fluorescence microscopy, RT-qPCR, and western blot assessed cell size, oxidative stress, AcH4 levels, hypertrophic gene expression, and caspase-3 activation in angiotensin II (AngII)-stressed HL-1 murine cardiomyocytes 12 h-treated with various EXOs. The uptake of fluorescently-labeled EXOs was also measured in cardiomyocytes. The cardiac function of infarcted male Wistar rats intramyocardially injected with different EXOs (1·1012) was examined by echocardiography. Left ventricular infarct size, hypertrophy, and capillary density were measured. RESULTS: Sustained treatment of NIH/3T3 with non-toxic SFN concentration significantly enhances the release of CD81 + EXOs rich in TSG101 (Tumor susceptibility gene 101) and Hsp70 (Heat Shock Protein 70), and containing maspin, an endogenous histone deacetylase 1 inhibitor. SFN-F-EXOs counteract angiotensin II (AngII)-induced hypertrophy and apoptosis in murine HL-1 cardiomyocytes enhancing SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) levels more effectively than F-EXOs. In stressed cardiomyocytes, SFN-F-EXOs boost AcH4 levels by 30% (p < 0.05) and significantly reduce oxidative stress more than F-EXOs. Fluorescence microscopy showed that mouse cardiomyocytes take in SFN-F-EXOs ~ threefold more than F-EXOs. Compared to vehicle-injected infarcted hearts, SFN-F-EXOs reduce hypertrophy, scar size, and improve contractility. CONCLUSIONS: Long-term low-dose SFN treatment of fibroblasts enhances the release of anti-remodeling cardiomyocyte-targeted F-EXOs, which effectively prevent the onset of HF. The proposed method opens a new avenue for large-scale production of cardioprotective exosomes for clinical application using allogeneic fibroblasts.

The hypolipidemic, anti-inflammatory and antioxidant effect of Kavolì® aqueous extract, a mixture of Brassica oleracea leaves, in a rat model of NAFLD.

Herein we characterized the bioactive metabolites of the aqueous extract of Kavolì®, a commercial product composed of a mixture of Brassica oleracea leaves, and assessed its potential ameliorating effects in a rat model of non-alcoholic fatty liver disease (NAFLD). Kavolì® extract showed high levels of bioactive compounds and strong in vitro antioxidant activities. Chlorogenic and neochlorogenic acids were identified as the most representative polyphenols. The administration of brassica extract to steatotic rats significantly ameliorated the levels of blood lipids and transaminases, and lipid content and inflammatory markers in liver. Oxidative stress parameters were significantly improved in both liver and brain of steatotic rats. Moreover, plasma and feces levels of short chain fatty acids (SCFAs) were bring back close to control values by Kavolì® treatment, in spite of high fat diet/streptozotocin (HFD/STZ)-induced alterations. The efficacy of Kavolì® in treating hypercholesterolemia, reducing the level of inflammation and cardiovascular disease biomarkers, steatosis and oxidative stress parameters, as well as the ability in modulating SCFAs levels is probably related to the bioactive compounds of the water extract administered to the rat model of NAFLD. In particular, the ameliorating effects are largely attributable to the high content in polyphenols observed in our study.

Selective perfusion of coronary vasculature in preterm sheep: a methodological innovation undermined by unfavourable operation of the foramen ovale.

Antenatal cardiac intervention affords new prospects for hypoplastic left heart syndrome. Its success, however, may come not only from absence of impediments to blood flow but also from a sufficiently developed cardiac wall. Here, we examined the feasibility to perfuse selectively the fetal coronary circulation for treatment with growth promoting agents. Pregnant sheep (94-114 days gestation, term 145 days) were used. An aortic stop-flow procedure was developed for intracoronary access in the nonexposed fetus and human mesenchymal stem cells and their exosomes served as test agents. We found that aortic stop-flow ensures preferential distribution of fluorescent microspheres to the heart. However, intracoronary administration of stem cells or exosomes was detrimental, with fetal demise occurring around surgery or at variable intervals afterwards. Coincidentally, stop-flow caused by itself a marked rise of intraluminal pressure within the occluded aorta along with histological signs of coronary obstruction. We conclude that it is feasible to perfuse selectively the coronary circulation of the preterm fetus, but treatments are not compatible with survival of the animals. The cause for failure is found in the absence of hemodynamic compensation to stop-flow via a left-to-right shunt. This unexpected event is attributed to a largely membranous foramen ovale, characteristic of sheep, that collapses under pressure.

Ticagrelor Enhances Release of Anti-Hypoxic Cardiac Progenitor Cell-Derived Exosomes Through Increasing Cell Proliferation In Vitro.

Despite the widespread clinical use of cardioprotection by long-term direct antagonism of P2Y12 receptor, underlying mechanisms are unclear. Here, we identify how release of pro-survival exosomes from human cardiac-derived mesenchymal progenitor cells (hCPCs) is regulated by clinically relevant dose of ticagrelor (1 µM), an oral selective and reversible non-thienopyridine P2Y12 inhibitor. Ticagrelor-induced enhancement of exosome levels is related to increased mitotic activity of hCPCs. We show a drug-response threshold above which the effects on hCPCs are lost due to higher dose of ticagrelor and larger adenosine levels. While it is known that pan-Aurora kinase inhibitor halts cell proliferation through dephosphorylation of histone H3 residue Ser10, we demonstrate that it also prevents ticagrelor-induced effects on release of cardiac progenitor cell-derived exosomes delivering anti-apoptotic HSP70. Indeed, sustained pre-treatment of cardiomyocytes with exosomes released from explant-derived hCPCs exposed to low-dose ticagrelor attenuated hypoxia-induced apoptosis through acute phosphorylation of ERK42/44. Our data indicate that ticagrelor can be leveraged to modulate release of anti-hypoxic exosomes from resident hCPCs.

Obese mice exposed to psychosocial stress display cardiac and hippocampal dysfunction associated with local brain-derived neurotrophic factor depletion.

INTRODUCTION: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. METHODS: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. FINDINGS: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. INTERPRETATION: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. FUND: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: "Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities". M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication.

Distribution of Gadolinium in Rat Heart Studied by Fast Field Cycling Relaxometry and Imaging SIMS.

Research on microcirculatory alterations in human heart disease is essential to understand the genesis of myocardial contractile dysfunction and its evolution towards heart failure. The use of contrast agents in magnetic resonance imaging is an important tool in medical diagnostics related to this dysfunction. Contrast agents significantly improve the imaging by enhancing the nuclear magnetic relaxation rates of water protons in the tissues where they are distributed. Gadolinium complexes are widely employed in clinical practice due to their high magnetic moment and relatively long electronic relaxation time. In this study, the behavior of gadolinium ion as a contrast agent was investigated by two complementary methods, relaxometry and secondary ion mass spectrometry. The study examined the distribution of blood flow within the microvascular network in ex vivo Langendorff isolated rat heart models, perfused with Omniscan® contrast agent. The combined use of secondary ion mass spectrometry and relaxometry allowed for both a qualitative mapping of agent distribution as well as the quantification of gadolinium ion concentration and persistence. This combination of a chemical mapping and temporal analysis of the molar concentration of gadolinium ion in heart tissue allows for new insights on the biomolecular mechanisms underlying the microcirculatory alterations in heart disease.

Injection-Molded Microfluidic Device for SERS Sensing Using Embedded Au-Capped Polymer Nanocones.

To enable affordable detection and diagnostic, there is a need for low-cost and mass producible miniaturized sensing platforms. We present a fully polymeric microfluidic lab-on-a-chip device with integrated gold (Au)-capped nanocones for sensing applications based on surface-enhanced Raman spectroscopy (SERS). All base components of the device were fabricated via injection molding (IM) and can be easily integrated using ultrasonic welding. The SERS sensor array, embedded in the bottom of a fluidic channel, was created by evaporating Au onto IM nanocone structures, resulting in densely packed Au-capped SERS active nanostructures. Using a Raman active model analyte, trans-1,2-bis-(4-pyridyl)-ethylene, we found a surface-averaged SERS enhancement factor of ∼5 × 106 with a relative standard deviation of 14% over the sensor area (2 × 2 mm2), and a 18% signal variation among substrates. This reproducible fabrication method is cost-effective, less time consuming, and allows mass production of fully integrated polymeric, microfluidic systems with embedded high-density and high-aspect ratio SERS sensor.

ADAMTS13 Deficiency Shortens the Life Span of Mice With Experimental Diabetes.

In patients with diabetes, impaired activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), the plasma metalloprotease that cleaves highly thrombogenic von Willebrand factor multimers, is a major risk factor of cardiovascular events. Here, using Adamts13-/- mice made diabetic by streptozotocin, we investigated the impact of the lack of ADAMTS13 on the development of diabetes-associated end-organ complications. Adamts13-/- mice experienced a shorter life span than their diabetic wild-type littermates. It was surprising that animal death was not related to the occurrence of detectable thrombotic events. The lack of ADAMTS13 drastically increased the propensity for ventricular arrhythmias during dobutamine-induced stress in diabetic mice. Cardiomyocytes of diabetic Adamts13-/- mice exhibited an aberrant distribution of the ventricular gap junction connexin 43 and increased phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII), and with the consequent CaMKII-induced disturbance in Ca2+ handling, which underlie propensity for arrhythmia. In vitro, thrombospondin 1 (TSP1) promoted, in a paracrine manner, CaMKII phosphorylation in murine HL-1 cardiomyocytes, and ADAMTS13 acted to inhibit TSP1-induced CaMKII activation. In conclusion, the deficiency of ADAMTS13 may underlie the onset of lethal arrhythmias in diabetes through increased CaMKII phosphorylation in cardiomyocytes. Our findings disclose a novel function for ADAMTS13 beyond its antithrombotic activity.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A.

Aims: Cell therapy trials using cardiac-resident progenitor cells (CPCs) and bone marrow-derived mesenchymal stem/progenitor cells (BMCs) in patients after myocardial infarction have provided encouraging results. Exosomes, nanosized extracellular vesicles of endosomal origin, figure prominently in the bioactivities of these cells. However, a head-to-head comparison of exosomes from the two cell types has not been performed yet. Methods and results: CPCs and BMCs were derived from cardiac atrial appendage specimens and sternal bone marrow, respectively, from patients (n = 20; age, 69.9 ± 10.9) undergoing heart surgery for aortic valve disease and/or coronary artery disease. Vesicles were purified from cell conditioned media by centrifugation/filtration and ultracentrifugation. Vesicle preparations were predominantly composed of exosomes based on particle size and marker expression (CD9, CD63, CD81, Alix, and TSG-101). CPC-secreted exosomes prevented staurosporine-induced cardiomyocyte apoptosis more effectively than BMC-secreted exosomes. In vivo, CPC-secreted exosomes reduced scar size and improved ventricular function after permanent coronary occlusion in rats more efficiently than BMC-secreted exosomes. Both types of exosomes stimulated blood vessel formation. CPC-secreted exosomes, but not BMC-derived exosomes, enhanced ventricular function after ischaemia/reperfusion. Proteomics profiling identified pregnancy-associated plasma protein-A (PAPP-A) as one of the most highly enriched proteins in CPC vs. BMC exosomes. The active form of PAPP-A was detected on CPC exosome surfaces. These vesicles released insulin-like growth factor-1 (IGF-1) via proteolytic cleavage of IGF-binding protein-4 (IGFBP-4), resulting in IGF-1 receptor activation, intracellular Akt and ERK1/2 phosphorylation, decreased caspase activation, and reduced cardiomyocyte apoptosis. PAPP-A knockdown prevented CPC exosome-mediated cardioprotection both in vitro and in vivo. Conclusion: These results suggest that CPC-secreted exosomes may be more cardioprotective than BMC-secreted exosomes, and that PAPP-A-mediated IGF-1 release may explain the benefit. They illustrate a general mechanism whereby exosomes may function via an active protease on their surface, which releases a ligand in proximity to the transmembrane receptor bound by the ligand.

Injection molded lab-on-a-disc platform for screening of genetically modified E. coli using liquid-liquid extraction and surface enhanced Raman scattering.

We present the development of an automated centrifugal microfluidic platform with integrated sample pre-treatment (filtration and liquid-liquid extraction) and detection (SERS-based sensing). The platform consists of eight calibration and four assay modules, fabricated with polypropylene using injection molding and bonded with ultrasonic welding. The platform was used for detection of a secondary bacterial metabolite (p-coumaric acid) from bacterial supernatant. The obtained extraction efficiency was comparable to values obtained in batch experiments and the SERS-based sensing showed a good correlation with HPLC analysis.

Long-term Intake of Pasta Containing Barley (1-3)Beta-D-Glucan Increases Neovascularization-mediated Cardioprotection through Endothelial Upregulation of Vascular Endothelial Growth Factor and Parkin.

Barley (1-3)ß-D-Glucan (BBG) enhances angiogenesis. Since pasta is very effective in providing a BBG-enriched diet, we hypothesized that the intake of pasta containing 3% BBG (P-BBG) induces neovascularization-mediated cardioprotection. Healthy adult male C57BL/6 mice fed P-BBG (n = 15) or wheat pasta (Control, n = 15) for five-weeks showed normal glucose tolerance and cardiac function. With a food intake similar to the Control, P-BBG mice showed a 109% survival rate (P < 0.01 vs. Control) after cardiac ischemia (30 min)/reperfusion (60 min) injury. Left ventricular (LV) anion superoxide production and infarct size in P-BBG mice were reduced by 62 and 35% (P < 0.0001 vs. Control), respectively. The capillary and arteriolar density of P-BBG hearts were respectively increased by 12 and 18% (P < 0.05 vs. Control). Compared to the Control group, the VEGF expression in P-BBG hearts was increased by 87.7% (P < 0.05); while, the p53 and Parkin expression was significantly increased by 125% and cleaved caspase-3 levels were reduced by 33% in P-BBG mice. In vitro, BBG was required to induce VEGF, p53 and Parkin expression in human umbelical vascular endothelial cells. Moreover, the BBG-induced Parkin expression was not affected by pifithrin-α (10 uM/7days), a p53 inhibitor. In conclusion, long-term dietary supplementation with P-BBG confers post-ischemic cardioprotection through endothelial upregulation of VEGF and Parkin.

Nanopillar Filters for Surface-Enhanced Raman Spectroscopy.

We present a simple, robust, and automated molecule extraction technique based on a centrifugal microfluidic platform. Fast and facile extraction of a food adulterant (melamine) from a complex sample medium (milk) on a SERS substrate is demonstrated. The unique characteristic of the detection method is the obtained "filter paper/chromatographic" effect which combines centrifugal force and wetting properties of the SERS substrate. The work addresses issues related to SERS-based detection of analytes in complex media, which is important for realizing next generation SERS platforms applicable for a broad variety of real-life applications.

New cardiac expression of two adenosine-2A receptor isoforms in dysfunctioning minipigs.

PURPOSE: Eight A2AR variants are reported in humans while no A2AR isoforms in pigs. The aim of this study was to evaluate potential isoforms presence in cardiac pig tissue to better define possible involvement of A2AR in the cardiovascular pathophysiology. MATERIALS AND METHODS: In adult male minipigs (n = 4) left ventricular dysfunction (LVD) was induced by pacing at 200 bpm in the right ventricular (RV) apex. In these animals and in sham operated pigs (C-SHAM, n = 4) cardiac tissue was collected from LV-septal wall (LV-SW)-close to pacing site-and from lateral (opposite) site (LV-OSW). A2AR specific primers, derived from Sus scrofa AY772412 sequence, were used for Real-Time PCR. The DNA was sequenced using the Sanger method. Histological analysis was also performed. RESULTS: In LV-SW of LVD minipigs the A2AR melting curves were characterized by a sharp peak between 87 and 91 °C (short isoform, 1-94 bp) on the right of the principal peak corresponding to a long A2AR isoform (GenBank: JQ229674.1) 1-213 bp. As for C-SHAM only one peak was observed in LV-OSW region of LVD animals. The short isoform had an alternative promoter region and a specific translated protein. Histology showed in LVD-LV-SW prominent Purkinje cells compared to LV-OSW and C-SHAM. No difference in A2AR expression was observed between LVD animals and C-SHAM although a slight decrease was observed in LVD-LV-OSW. CONCLUSIONS: The presence of two different isoforms in the myocardium close to the insertion of pacing is suggestive of a differential state-specific expression of A2AR in cardiac tissue.

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms.

BACKGROUND: We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp+) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp+ in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp+ or unconditioned stem cells. METHODS: Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. RESULTS: The proteomic remodeling was largely prevented in MSCp+ group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp+. In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp+. CONCLUSIONS: Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. GENERAL SIGNIFICANCE: Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart.

Comparison of Ultrasonic Welding and Thermal Bonding for the Integration of Thin Film Metal Electrodes in Injection Molded Polymeric Lab-on-Chip Systems for Electrochemistry.

We compare ultrasonic welding (UW) and thermal bonding (TB) for the integration of embedded thin-film gold electrodes for electrochemical applications in injection molded (IM) microfluidic chips. The UW bonded chips showed a significantly superior electrochemical performance compared to the ones obtained using TB. Parameters such as metal thickness of electrodes, depth of electrode embedding, delivered power, and height of energy directors (for UW), as well as pressure and temperature (for TB), were systematically studied to evaluate the two bonding methods and requirements for optimal electrochemical performance. The presented technology is intended for easy and effective integration of polymeric Lab-on-Chip systems to encourage their use in research, commercialization and education.

Magnetic resonance imaging of infarct-induced canonical wingless/integrated (Wnt)/ß-catenin/T-cell factor pathway activation, in vivo.

AIMS: Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/ß-catenin/TCF pathway activation, an important regulator of post-ischaemic cardiac remodelling. METHODS AND RESULTS: We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the ß-catenin-responsive TCF/lymphoid-enhancer binding factor (Lef) promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n = 11). By 1.5 T MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, post-mortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n = 6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of ß-catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischaemic remodelling process, as demonstrated by the significant preservation of cardiac function, the 42 ± 1% increase of peri-infarct arteriolar density and 43 ± 3% reduction in infarct scar size compared with untreated animals. CONCLUSIONS: The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/ß-catenin/TCF activation state in response to cardiac injury and therapeutic interventions.

Rapid Voltammetric Measurements at Conducting Polymer Microelectrodes Using Ultralow-Capacitance Poly(3,4-ethylenedioxythiophene):Tosylate.

UNLABELLED: We use a vapor-phase synthesis to generate conducting polymer films with low apparent capacitance and high conductance enabling rapid electrochemical measurements. Specifically, oxidative chemical vapor deposition was used to create thin films of poly(3,4-ethylenedioxythiophene):tosylate ( PEDOT: tosylate). These films had a conductance of 17.1 ± 1.7 S/cm. Furthermore, they had an apparent capacitance of 197 ± 14 µF/cm(2), which is an order of magnitude lower than current commercially available and previously reported PEDOT. Using a multistage photolithography process, these films were patterned into PEDOT: tosylate microelectrodes and were used to perform fast-scan cyclic voltammetry (FSCV) measurements. Using a scan rate of 100 V/s, we measured ferrocene carboxylic acid and dopamine by FSCV. In contrast to carbon-fiber microelectrodes, the reduction peak showed higher sensitivity when compared to the oxidation peak. The adsorption characteristics of dopamine at the polymer electrode were fit to a Langmuir isotherm. The low apparent capacitance and the microlithographic processes for electrode design make PEDOT: tosylate an attractive material for future applications as an implantable biosensor for FSCV measurements. Additionally, the integration of PEDOT: tosylate electrodes on plastic substrates enables new electrochemical measurements at this polymer using FSCV.

Myocardial Expression Analysis of Osteopontin and Its Splice Variants in Patients Affected by End-Stage Idiopathic or Ischemic Dilated Cardiomyopathy.

Osteopontin (OPN) is a phosphoglycoprotein of cardiac extracellular matrix and it is still poorly defined whether its expression changes in failing heart of different origin. The full-length OPN-a and its isoforms (OPN-b, OPN-c) transcriptomic profile were evaluated in myocardium of patients with dilated or ischemic cardiomyopathy (DCM n = 8; LVEF% = 17.5±3; ICM n = 8; LVEF% = 19.5±5.2) and in auricle of valvular patients (VLP n = 5; LVEF%≥50), by Real-time PCR analysis. OPN-a and thrombin mRNA levels resulted significantly higher in DCM compared to ICM patients (DCM:31.3±7.4, ICM:2.7±1.1, p = 0.0002; DCM:19.1±4.9, ICM:5.4±2.2, p = 0.007, respectively). Although both genes' mRNA levels increased in patients with LVEF<50% (DCM+ICM) with respect to VLP with LVEF>50%, a significant increase in OPN (p = 0.0004) and thrombin (p = 0.001) expression was observed only in DCM. In addition, a correlation between OPN-a and thrombin was found in patients with LVEF<50% (r = 0.6; p = 0.003). The mRNA pattern was confirmed by OPN-a cardiac protein concentration (VLP:1.127±0.26; DCM:1.29±0.22; ICM:1.00±0.077 ng/ml). The OPN splice variants expression were detectable only in ICM (OPN-b: 0.357±0.273; OPN-c: 0.091±0.033) and not in DCM patients. A significant correlation was observed between collagen type I, evaluated by immunohistochemistry analysis, and both OPN-a mRNA expression (r = 0.87, p = 0.002) and OPN protein concentrations (r = 0.77, p = 0.016). Concluding, OPN-a and thrombin mRNA resulted dependent on the origin of heart failure while OPN-b and OPN-c highlighted a different expression for DCM and ICM patients, suggesting their correlation with different clinical-pathophysiological setting.