Nadia Ucciferri

Allometric scaling and cell ratios in multi-organ in vitro models of human metabolism

Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell–cell or cell–tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.

A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation

BackgroundThe use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before.ResultsThis paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude.ConclusionsIn our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.

Sensing scaffolds to monitor cellular activity using impedance measurements

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Hypothesis-free secretome analysis of thoracic aortic aneurysm reinforces the central role of TGF-β cascade in patients with bicuspid aortic valve

BACKGROUND Ascending thoracic aortic aneurysm (ATAA) is a major cause of morbidity and mortality worldwide. The pathogenesis of medial degeneration of the aorta remains undefined. High-throughput secretome analysis by mass spectrometry may be useful to elucidate the molecular mechanisms involved in aneurysm formation as well as to identify biomarkers for early diagnosis or targets of therapy. The purpose of the present study was to analyze the secreted/released proteins from ATAA specimens of both tricuspid aortic valve (TAV) and bicuspid aortic valve (BAV) patients. METHODS Aortic specimens were collected from patients undergoing elective surgery and requiring graft replacement of the ascending aorta. Each sample of the ascending aortic aneurysm, 4 BAV (3 males; aged 53.5±11.4 years) and 4 TAV (1 male; 78±7.5 years), was incubated for 24h in serum-free medium. Released proteins were digested with trypsin. Peptide mixtures were fractioned by nano-high performance liquid chromatography and analyzed by mass spectrometry. Following identification of differentially expressed proteins, quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed. RESULTS The comparison between the proteins released from BAV and TAV aneurysmatic tissues showed significantly diverging expression fingerprints in the two groups of patients. Bioinformatics analysis revealed 38 differentially released proteins; in particular 7 proteins were down-regulated while 31 were up-regulated in BAV with respect to TAV. Most of the proteins that were up-released in BAV were related to the activation of transforming growth factor (TGF)-β signaling. Latent TGF-β binding protein 4 (LTBP4) exhibited one of the highest significant under-expressions (10-fold change) in BAV secretomes with respect to TAV. qRT-PCR analysis validated this significant difference at LTBP4 gene level (BAV: 1.03±0.9 vs TAV: 3.6±3.2; p<0.05). CONCLUSION Hypothesis-free secretome profiling clearly showed diverging expression fingerprints in the ATAA of TAV and BAV patients, confirming the crucial role of TGF-β signaling in modulating ATAA development in bicuspid patients.

Proteomics Techniques for the Detection of Translated Pseudogenes

Increasing evidence indicates that pseudogenes can reach the translational process. Translated pseudogene products have in fact been found in various organisms, confuting the original definition of pseudogenes as genes without any coding potential. Proteomics is the main technology allowing the study of proteins and, when integrated with genomics, is defined as proteogenomics. In proteogenomics, the peptide-genome alignment drives the identification and annotation of gene products and allows for a better understanding of their function. In this chapter, we give a brief overview of the proteomic techniques applied to pseudogenes. In particular, we discuss peptide spectrum acquisition, mass data analysis, and genome database matching.

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells

BackgroundEmbryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role.ResultsWe performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells.ConclusionsWe propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

Low T3 State Is Correlated with Cardiac Mitochondrial Impairments after Ischemia Reperfusion Injury: Evidence from a Proteomic Approach

Mitochondria are major determinants of cell fate in ischemia/reperfusion injury (IR) and common effectors of cardio-protective strategies in cardiac ischemic disease. Thyroid hormone homeostasis critically affects mitochondrial function and energy production. Since a low T3 state (LT3S) is frequently observed in the post infarction setting, the study was aimed to investigate the relationship between 72 h post IR T3 levels and both the cardiac function and the mitochondrial proteome in a rat model of IR. The low T3 group exhibits the most compromised cardiac performance along with the worst mitochondrial activity. Accordingly, our results show a different remodeling of the mitochondrial proteome in the presence or absence of a LT3S, with alterations in groups of proteins that play a key role in energy metabolism, quality control and regulation of cell death pathways. Overall, our findings highlight a relationship between LT3S in the early post IR and poor cardiac and mitochondrial outcomes, and suggest a potential implication of thyroid hormone in the cardio-protection and tissue remodeling in ischemic disease.

Site-Specific Secretome Map Evidences VSMC-Related Markers of Coronary Atherosclerosis Grade and Extent in the Hypercholesterolemic Swine

A major drawback in coronary atherosclerosis (ATS) research is the difficulty of investigating early phase of plaque growth and related features in the clinical context. In this study, secreted proteins from atherosclerotic coronary arteries in a hypercholesterolemic swine model were characterized by a proteomics approach and their expression was correlated to site-specific ATS stage and extent. A wide coronary artery map of secreted proteins has been obtained in high fat (HF) diet induced ATS swine model and a significantly different expression of many proteins related to vascular smooth muscle cell (VSMC) activation/migration has been identified. Significant associations with ATS stage of HF coronary lesions were found for several VSMC-derived proteins and validated for chitinase 3 like protein 1 (CHI3L1) by tissue immunoexpression. A direct correlation (R 2 = 0.85) was evidenced with intima to media thickness ratio values and ELISA confirmed the higher blood concentrations of CHI3L1 in HF cases. These findings confirmed the pivotal role of VSMCs in coronary plaque development and demonstrated a strong site-specific relation between VSMC-secreted CHI3L1 and lesion grade, suggesting that this protein could be proposed as a useful biomarker for diagnosing and staging of atherosclerotic lesions in coronary artery disease.

Endothelial progenitor cell secretome delivered by novel polymeric nanoparticles in ischemic hindlimb

&NA; Endothelial progenitor cells (EPCs) contribute to ischemic tissue repair by paracrine secretion up‐regulated by hypoxia. In this study we use novel nanoparticles (NPs) as carriers for a controlled release of EPC secretome (CM) to improve their angiogenic properties. The in vivo effect in ischemic hindlimb rat model was evaluated, comparing hypoxic EPC‐CM‐NPs with hypoxic EPC‐CM alone. A proteomic characterization of hypoxic CM and the in vitro effect on endothelial cells (HUVECs) were also performed. Up to 647 protein, 17 of which with angiogenic properties, were upregulated by hypoxia. Moreover, hypoxic EPC‐CM significantly promoted capillary‐like structures on Matrigel. A significant increase of blood perfusion in ischemic limbs at 2 weeks with EPC‐CM‐loaded NPs as compared to both EPC‐CM and control and a significant increase of capillary formation were observed. The use of EPC‐CM‐NPs significantly improved neoangiogenesis in vivo, underlining the advantages of controlled release in regenerative medicine.