Angela De Luca

Identification and characterization of the nano‐sized vesicles released by muscle cells

Several cell types secrete small membranous vesicles that contain cell‐specific collections of proteins, lipids, and genetic material. The function of these vesicles is to allow cell‐to‐cell signaling and the horizontal transfer of their cargo molecules. Here, we demonstrate that muscle cells secrete nano‐sized vesicles and that their release increases during muscle differentiation. Analysis of these nanovesicles allowed us to characterize them as exosome‐like particles and to define the potential role of the multifunctional protein Alix in their biogenesis.

Cardiac Stem Cell Research: An Elephant in the Room?

Heart disease is the leading cause of death in the industrialized world, and stem cell therapy seems to be a promising treatment for injured cardiac tissue. To reach this goal, the scientific community needs to find a good source of stem cells that can be used to obtain new myocardium in a very period range of time. Since there are many ethical and technical problems with using embryonic stem cells as a source of cells with cardiogenic potential, many laboratories have attempted to isolate potential cardiac stem cells from several tissues. The best candidates seem to be cardiac “progenitor” and/or “stem” cells, which can be isolated from subendocardial biopsies from the same patient or from embryonic and/or fetal myocardium. Regardless of the technique used to isolate and characterize these cells, it appears that the different cells isolated from adult myocardium to date are all phenotypic variations of a unique cell type that expresses several markers, such as c‐Kit, CD34, MDR‐1, Sca‐1, CD45, nestin, or Isl‐1, in various combinations. Anat Rec, 292:449–454, 2009.

Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells

The use of three‐dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patients life. Silk fibroin‐based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly. Copyright

HSP90 and eNOS partially co‐localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).

MicroRNAs: Novel Crossroads between Myeloma Cells and the Bone Marrow Microenvironment

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulate in the bone marrow, where a complex microenvironment made by different cell types supports proliferation, survival, and drug resistance of tumor cells. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at posttranscriptional level. Emerging evidence indicates that miRNAs are aberrantly expressed or functionally deregulated in MM cells as the result of multiple genetic or epigenetic mechanisms and that also the tumor microenvironment regulates MM cell functions by miRNAs. Consistently, modulation of miRNA levels in MM cells has been demonstrated to impair their functional interaction with the bone marrow microenvironment and to produce significant antitumor activity even able to overcome the protective bone marrow milieu. This review will describe the most recent findings on miRNA function in the context of MM bone marrow microenvironment, focusing on the therapeutic potential of miRNA-based approaches.

Human Recombinant Vasostatin‐1 May Interfere with Cell–Extracellular Matrix Interactions

Abstract:  Vasostatin‐1 (VS‐1), the N‐terminal fragment derived from the cleavage of chromogranin A (CgA), has been shown to exert several biological activities on several tissues and organs. Recently, it has been reported that human recombinant VS‐1 (STA‐CGA1‐78) may alter myocardial contractility in eel, frog, and rat hearts. In this article we have explored if STA‐CGA1‐78 can induce intracellular cascades interacting both with adhesion molecules and/or extracellular matrix (ECM), components, that is, involvement of the heat shock protein 90 (HSP90) and the endothelial NOS (eNOS), known to be implicated in signal transduction mechanisms affecting myocardial contractility. We used 3D cultured adult rat cardiomyocytes cultivated over fibronectin or fibroblasts or embedded in matrigel or collagen type I. Aurion‐conjugated VS‐1 (Au‐STA‐CGA1‐78) has been used to identify possible sites of interaction of this molecule with the cell membrane. We found that in our 3D culture, cell–ECM interactions played a crucial role in the cellular localization of HSP90 as well as in the expression of eNOS. VS‐1 appeared to modulate cell–ECM interactions, thereby remarkably leading to a different cellular localization of HSP90. Moreover, Au‐STA‐CGA1‐78 was never detected inside the cell nor overlapping the plasma membrane, but nearby the outer side of the cardiomyocyte plasmalemma, at a particular distance, typical of integrins. On the whole, these data suggest that VS‐1 does not have a classic receptor on the membrane but that integrins may represent a nonconventional VS‐1 receptor modulating eNOS signaling pathway.

Adult stem cells, scaffolds for in vivo and in vitro myocardial tissue engineering

The main goal in the last few years in cardiac research has been to isolate cardiac potential stem cells from adult myocardium and to demonstrate their differentiation potential. We have previously demonstrated that c-Kit positive cardiac stem cells are able to organize themselves into a tissue-like cell mass. In this 3D mass, they can produce a high concentration of natural extracellular matrix, can create vessels, a capsule and, with the help of an Open-pore Polylactic Acid scaffold, many cells can organize an elementary myocardium. Drawing from this background, we decided to design and use poly-lactic scaffolds and the model of the athymic Nude-Foxn1(nu) mouse to evaluate the extent of the myogenic vs endothelial differentiation in vivo, and to evaluate the presence or the absence of a foreign body reaction.

Circulating biomarkers in osteosarcoma: new translational tools for diagnosis and treatment

Osteosarcoma (OS) is a rare primary malignant bone tumour arising from primitive bone-forming mesenchymal cells, with high incidence in children and young adults, accounting for approximately 60% of all malignant bone tumours. Currently, long-term disease-free survival can be achieved by surgical treatment plus chemotherapy in approximately 60% of patients with localized extremity disease, and in 20–30% of patients with metastatic lung or bone disease. Diagnosis of primary lesions and recurrences is achieved by using radiological investigations and standard tissue biopsy, the latter being costly, painful and hardly repeatable for patients. Therefore, despite some recent advances, novel biomarkers for OS diagnosis, prediction of response to therapy, disease progression and chemoresistance, are urgently needed. Biological fluids such as blood represent a rich source of non-invasive cancer biomarkers, which allow to understand what is really happening inside the tumour, either at diagnosis or during disease progression. In this regard, liquid biopsy potentially represents an alternative and non-invasive method to detect tumour onset, progression and response to therapy. In this review, we will summarize the state of the art in this novel area, illustrating recent studies on OS. Although the data reported in literature seem preliminary, liquid biopsy represents a promising tool with the potential to be rapidly translated in the clinical practice.

Relevance of 3d culture systems to study osteosarcoma environment

Osteosarcoma (OS) is the most common primary malignant tumor of bone, which preferentially develops lung metastasis. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for patients with metastatic or recurrent OS remains dramatically poor. Novel therapies are therefore required to slow progression and eradicate the disease. Furthermore, to better understand the cellular and molecular mechanisms responsible for OS onset and progression, the development of novel predictive culture systems resembling the native three-dimensional (3D) tumor microenvironment are mandatory. ‘Tumor engineering’ approaches radically changed the previous scenario, through the development of advanced and alternative 3D cell culture in vitro models able to tightly mimic the in vivo tumor microenvironment.In this review, we will summarize the state of the art in this novel area, illustrating the different methods and techniques employed to realize 3D OS cell culture models and we report the achieved results, which highlight the efficacy of these models in reproducing the tumor milieu. Although data need to be further validated, the scientific studies reviewed here are certainly promising and give new insights into the clinical practice.

Osteogenic commitment and differentiation of human mesenchymal stem cells by low-intensity pulsed ultrasound stimulation.

Low‐intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm2 LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7, 14, 21 days). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21 days CD73+/CD90+: 6%; OCT‐3/4+/NANOG+/SOX2+: 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation.