Fatty acid-based monolayer culture to promote in vitro neonatal rat cardiomyocyte maturation.

The development of functional and reliable in vitro cardiac models composed of fully mature cardiomyocytes is essential for improving drug screening test quality, therefore, the success of clinical trial outcomes. In their lifespan, cardiomyocytes undergo a dynamic maturation process from the fetal to adult stage, radically changing their metabolism, morphology, contractility and electrical properties. Before employing cells of human origin, in vitro models often use neonatal rat cardiomyocytes (NRCM) to obtain key proof-of-principles. Nevertheless, NRCM monolayers are prone to de-differentiate when maintained in culture. Supplementation of free fatty acids (FFA), the main energy source for mature cardiomyocytes, and co-culture with fibroblasts are each by itself known to promote the shift from fetal to adult cardiomyocytes. Using a co-culture system, our study investigates the effects of FFA on the cardiomyocyte phenotype in comparison to glucose as typical fetal energy source, and to 10% serum used as standard control condition. NRCM decreased their differentiation status and fibroblasts increased in number after 7days of culture in the control condition. On the contrary, both glucose- and FFA-supplementation better preserved protein expression of myosin-light-chain-2v, a marker of mature cardiomyocytes, and the fibroblast number at levels similar to those found in freshly isolated NRCM. Nevertheless, compared to glucose, FFA resulted in a significant increase in sarcomere striation and organization. Our findings constitute an important step forward towards the definition of the optimal culture conditions, highlighting the possible benefits of a further supplementation of specific FFA to promote CM maturation in a co-culture system with FB.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment.

Rapamycin impairs endothelial cell function in human internal thoracic arteries.

BACKGROUND: Definitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model. METHODS: After exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed. RESULTS: Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 µM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 µM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM-1 µM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 µM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged. CONCLUSIONS: The present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

Integrated epigenetics of human breast cancer: synoptic investigation of targeted genes, microRNAs and proteins upon demethylation treatment.

BACKGROUND: The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. METHODS AND FINDINGS: Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. CONCLUSIONS: In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.

Effects of the novel polymer gel LeGoo on human internal thoracic arteries.

PURPOSE: Established hemostatic devices can injure vessel wall integrity. LeGoo (Pluromed, Woburn, MA), a novel poloxamer gel with reverse thermosensitive properties, is a new device for temporary occlusion of blood vessels. The present study investigated the effects of LeGoo on vascular function and morphology. DESCRIPTION: The distal end of the human internal thoracic artery was used to assess vascular function of LeGoo-applied segments in organ bath experiments and by scanning electron microscopy. EVALUATION: After LeGoo application, both maximal contractile responses to noradrenaline and endothelium-dependent relaxant responses to acetylcholine were significantly reduced. Scanning electron microscopy showed areas of injured endothelium with exposure of subendothelial structures being in line with the functional changes. CONCLUSIONS: Data suggested that application of LeGoo induced significant endothelial injury and deterioration of the smooth muscle in human internal thoracic arteries.

Proteome analysis in cardiovascular pathophysiology using Dahl rat model.

Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.

Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

Quantitative proteome analysis in cardiovascular physiology and pathology. I. Data processing.

Methodological evaluation of the proteomic analysis of cardiovascular-tissue material has been performed with a special emphasis on establishing examinations that allow reliable quantitative analysis of silver-stained readouts. Reliability, reproducibility, robustness and linearity were addressed and clarified. In addition, several types of normalization procedures were evaluated and new approaches are proposed. It has been found that the silver-stained readout offers a convenient approach for quantitation if a linear range for gel loading is defined. In addition, a broad range of a 10-fold input (loading 20-200 microg per gel) fulfills the linearity criteria, although at the lowest input (20 microg) a portion of protein species will remain undetected. The method is reliable and reproducible within a range of 65-200 microg input. The normalization procedure using the sum of all spot intensities from a silver-stained 2D pattern has been shown to be less reliable than other approaches, namely, normalization through median or through involvement of interquartile range. A special refinement of the normalization through virtual segmentation of pattern, and calculation of normalization factor for each stratum provides highly satisfactory results. The presented results not only provide evidence for the usefulness of silver-stained gels for quantitative evaluation, but they are directly applicable to the research endeavor of monitoring alterations in cardiovascular pathophysiology.

"Turnover proteome" of human atrial trabeculae.

Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.

Proteomics of ascending aortic aneurysm with bicuspid or tricuspid aortic valve.

Bicuspid aortic valve is often associated with lesions of the ascending aorta, which differ histologically from those in tricuspid valve patients. We undertook proteomic analyses to assess differences at the proteome level. Aortic samples were collected from 20 patients undergoing aortic valve and/or ascending aortic replacement; 9 had a bicuspid valve: 5 with aortic aneurysm (diameter > 50 mm) and 4 without dilation; 11 had a tricuspid valve: 6 with aortic aneurysm and 5 without dilation. Patients with histologically proven connective tissue disorders were excluded. Samples were dissected, solubilized, and subjected to 2-dimensional gel electrophoresis. Gel patterns showed an average of 580 protein spots in samples from bicuspid valve patients, and 564 spots in those with tricuspid valves. Comparative analysis revealed a correlation coefficient of 0.93 for protein expression in the bicuspid valve group compared to the tricuspid group. Three protein spots were significantly over-expressed and 4 were significantly down-regulated in the bicuspid group compared to the tricuspid group. The lowest correlation in protein expression was between non-dilated aortic tissues. These differences between aortic tissues of bicuspid and tricuspid valve patients suggest that mechanisms of aortic dilation might differ, at least in part, between such patients.

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits.

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.

SAP deficiency results in a striking alteration of the protein profile in activated CD4 T cells.

Deficiency in a protein called signaling lymphocytic activation molecule-associated protein (SAP) causes X-linked lymphoproliferative disease (XLP) and helper T cell-dependent antibody defects. To identify proteins regulated by SAP, we performed proteomic analyses of SAP deficient vs wild type T cells. Our results reveal protein species whose abundances are profoundly altered by SAP. Our work therefore identifies candidate cellular mediators of SAP-dependent T cell help.

Proteomics strategies in cardiovascular research.

Cardiovascular research of the past decades dealt with classical pathophysiological descriptions, then shifted toward the identification of relevant receptors, and then proceeded to the analysis of signal transduction pathways. Most recently, hand in hand with the achievements of the human genome project, the research has gone down the road toward molecular biological "disease gene(s) mapping". The application of proteome research will attempt to close the gap between genomic (and genetic) analysis and the physiological research. The rich source of heart surgery specimens represents an excellent starting point in data acquisition of proteomic context. Furthermore, animal models of cardiovascular diseases and deficiencies are considered, and will be explored. Examples of results from feasibility studies are given, with the emphasis on quantitative evaluation of proteomic components, hoping to discover co-regulated sets of proteins that are involved in any particular disease state. Identification of new, not yet discovered proteins will be pursued, though the emphasis of this work will be on the definition of characteristic sets of expressed proteins, which in turn might be able to delimit the state of disease and prognosis of therapy outcome. Besides the systematic issues, this paper refers to a number of methodological questions, like the comparison of the proteins detected by staining procedures and proteins detected in models in which biosynthetic labeling is applicable.