“Anatomia scientiæ dux est aditumque ad dei agnitionem præbet”

  from: “ Rehearsing anatomies . The visceral lecture delivered by Barber-Surgeon John Banister ”, anonymous, 1581. By permission of the Glasgow University Library, Special Collections.
(John Banister, 1533-1610, was one of the major English anatomist and teacher )

HSP60 INVOLVEMENT IN CANCER DEVELOPMENT AND PROGRESSION
INTERACTIONS BETWEEN THE CHAPERONING AND THE IMMUNE SYSTEMS
TISSUE ENGINEERING APPROACH TO STUDY THE EPITHELIAL MESENCHYMAL TROPHIC UNIT IN CHRONIC LUNG INFLAMMATORY DISEASES.
CARDIAC STEM CELL-LOADED POLY-LACTIC ACID AND FIBRINOIN SCAFFOLDS AS DEVICES FOR CARDIAC MUSCLE TISSUE REGENERATION
BIOLOGY AND DIFFERENTIATIVE POTENTIAL OF MESENCHYMAL STROMAL STEM CELLS ISOLATED FROM HUMAN UMBILICAL CORD WHARTON'S JELLY
MULTIPLE EFFECTS OF OXIDATIVE STRESS ON ENDOTHELIAL CELLS

HSP60 INVOLVEMENT IN CANCER DEVELOPMENT AND PROGRESSION
(in collaboration with Prof. Alberto J. L. Macario and Prof. Everly Conway de Macario, Department of Microbiology and Immunology, School of Medicine, and IMET, University of Maryland, Baltimore (MD), USA)

Quantitative changes in Hsp60 during the development of some tumors suggest that this chaperonin plays a role in carcinogenesis. A description of the specific role(s) of Hsp60 in tumor-cell growth and proliferation is still incomplete, but it is already evident that monitoring its levels and distribution in tissues and fluids has potential for diagnosis and staging, and for assessing prognosis and response to treatment. Although Hsp60 is considered an intramitochondrial protein, it has been demonstrated in the cytosol, cell membrane, vesicles, cell surface, extracellular space, and blood. The knowledge that Hsp60 occurs at all these locations opens new avenues for basic and applied research. It is clear that elucidating the mechanisms by which the chaperonin arrives at these various locations, and characterizing its functions in each of them will provide information useful for understanding carcinogenesis and for developing diagnostic and therapeutic tools for clinical oncology.

INTERACTIONS BETWEEN THE CHAPERONING AND THE IMMUNE SYSTEMS
(in collaboration with Prof. Alberto J. L. Macario and Prof. Everly Conway de Macario)

Aging entails progressive deterioration of molecules and supramolecular structures, including Hsp chaperones and their complexes, paralleled by functional decline. Recent research has changed our views on Hsp chaperones. They work inside and outside cells in many locations, alone or forming teams, interacting with cells, receptors, and molecules that are not chaperones, in roles that are not typically attributed to chaperones, such as protein folding. Hsp chaperones form a physiological system with a variety of functions and interactions with other systems, for example, the immune system. We propose that chaperone malfunctioning due to structural damage or gene dysregulation during aging has an impact on the immune system, creating the conditions for an overall malfunction of both systems. Pathological chaperones cannot interact with the immune system as normal ones do, and this leads to an overall readjustment of the interactions that is apparent during senescence and is likely to cause many of its manifestations

TISSUE ENGINEERING APPROACH TO STUDY THE EPITHELIAL MESENCHYMAL TROPHIC UNIT IN CHRONIC LUNG INFLAMMATORY DISEASES
(in collaboration with Prof. Donna Davies, The Brooke Laboratories, University of Southampton, UK)

One of the factors involved with the progressive decline in lung function in severe and prolonged cases of asthma is airway remodelling. The structural alterations that result in airway remodelling in asthmatic patients involve collagen deposition within the lamina reticularis, matrix deposition in the submucosa, smooth muscle hyperplasia and microvascular and neuronal proliferation. Previous research work carried out in collaboration with the University of Southampton, has pointed out that epithelial susceptibility to injury could consequently cause the activation of subepithelial myofibroblasts to promote airway remodelling in asthma.
The hypothesis that epithelial susceptibility to environmental injury and a prolonged tissue repair response result in activation of the epithelial mesenchymal trophic unit (EMTU) to promote airway wall remodelling, is supported by the fact that this communication between the epithelium and mesenchyme is reminiscent of the process that regulates airway growth and branching during embryogenesis where the lung epithelium and mesenchyme act as a trophic unit.
The main aim of our investigations is to investigate epithelial mesenchymal signalling in in vitro models of asthma. For this purpose we have developed a novel three-dimensional outgrowth model, obtained from bronchial biopsies and composed of both human bronchial epithelial cells (goblet and columnar cells) and fibroblasts with their basement membrane. Our experiments conducted mainly by Scanning Electron Microscopy and Transmission Electron Microscopy show that cells and matrices within the three-dimensional structures are well differentiated, presenting all the normal features of human bronchial mucosa such as microvilli, cilia, mucus, tight junctions, collagens etc.

CARDIAC STEM CELL-LOADED POLY-LACTIC ACID AND FIBRINOIN SCAFFOLDS AS DEVICES FOR CARDIAC MUSCLE TISSUE REGENERATION
(in collaboration with IZS - Istituto Zooprofilattico Sperimentale della Sicilia and BioTech Laboratories - Università degli Studi di Trento)

The rapid translation of preclinical cell-based therapy to restore damaged myocardium has raised questions concerning the best cell type as well as the best delivery route, and the best time of cell injection into the myocardium. Intramyocardial injection of stem cells is by far the most-used delivery technique in preclinical studies. We have recently demonstrated that c-Kit positive cardiac progenitor cells are able to organize themselves into a tissue-like cell mass in three-dimensional cultures, and with the help of an OPLA scaffold, many cells can create an organized elementary myocardium. Our hypothesis is that synthetic scaffolds designed to deliver cardiac progenitor cells in the infarcted region of the heart may induce a better differentiation into cardiomyocytes.
Scaffolds for in vivo implantations are synthesized by Dr. Antonella Motta from the University of Trento. For the synthesis of PDLLA scaffolds, the Poly (D,L lactic acid) (RESOMER® 207, MW = 252 kDa) polimer is used (6.7%) in Dicloromethane/Dimetilformamide (DCM/DMF) 70/30 (v/v). The three-dimensional structure are obtained by salt-leaching, using NaCl crystals as porosity agent (NaCl < 224 μm and <150 μm). For the synthesis of fibrinoin scaffolds, degummed silk fibres are dried and dissolved into 9.3 m LiBr water solution (20% w/v) at 65°C for 3h. Scaffolds with different porosities, pore size, and properties are made by freeze-drying and salt-leaching. Scaffolds embedded with collagen I and cardiac progenitor cells are implanted in the subcutaneous dorsal region of athymic Nude-Foxn1^nu mice.
Cardiac progenitor cells can differentiate into cardiomyocytes in vitro into PDLLA scaffolds in M-199 medium supplemented with 20% FBS within 21 days, while a foreign body reaction is observed in vivo. Some fibrinoin scaffolds do not induce a foreign body reaction. In conclusion, these three-dimensional cultures may be used in the future as a biodegradable patch for the surgical repair of the heart wall or the infarcted myocardium.

BIOLOGY AND DIFFERENTIATIVE POTENTIAL OF MESENCHYMAL STROMAL STEM CELLS ISOLATED FROM HUMAN UMBILICAL CORD WHARTON'S JELLY
(in collaboration with Dr A. Di Stefano, Fondazione "S. Maugeri", Istituto di Veruno, Novara and with Prof. P.G. Conaldi, ISMETT - Istituto Mediterraneo dei Trapienti e Terapie di Alta Specializzazione, Palermo)

Stem cells can be found in embryonic and extraembryonic tissues as well as in adult organs. In recent years, human mesenchymal stem cells (MSC) have been extensively studied. Their key characteristics of long-term self-renewal and a capacity to differentiate into diverse mature tissues favor their use in regenerative medicine applications. Several reports indicate that cells of Wharton's jelly (WJ), the main component of umbilical cord extracellular matrix, are multipotent stem cells, expressing markers of bone marrow mesenchymal stem cells (BM-MSC), and giving rise to different cellular types of both connective and nervous tissues. WJ-MSC further emerge as promising hypoimmunogenic cells, due to the expression of molecules able to modulate NK cells and expand regulatory T-cell populations. Moreover, it is now accepted that the differentiative capacities of such cells span all the mesoderm-derived tissues, extending to neuroectodermal as well as endodermal lineages. Literature data, together with ones from our laboratory, strongly suggest that WJ-MSC can differentiate into diverse cell types, showing a unique ability to cross lineage borders. This, together with their in vitro proliferative potential and their immunoregulatory features, renders these cells extremely promising for regenerative medicine applications in different pathological settings. Therefore the study of the basic biological properties of these cells and the definition of proper trans-differentiation conditions, together with the establishment of copnditions favoring their regulation of the host immune system are key aims for the development of successful cellular therapy applications.

MULTIPLE EFFECTS OF OXIDATIVE STRESS ON ENDOTHELIAL CELLS
(in collaboration with Dr A. Di Stefano and Dr. P. Giannuzzi, Fondazione "S. Maugeri" IRCCS, Centro Medico di Veruno (NO), Italy)

The increase of oxidative stress is indicated as a key element in the development of cardiovascular pathologies such as hypertension, atherosclerosis and cardiac pathologies. The increase of the reactive oxygen species (ROS) can be due to various factors, among which the establishment of local flogistic processes at vascular level, which may have as main target the endothelial cells. Several literature data suggest the possibility that hydrogen peroxide may act as a mediator of activation of endothelial gene expression, causing for example the well known increase in vascular permeability and adhesiveness for leukocytes. More in general, ROS have been implicated in the pathophysiology of a number of cardiovascular diseases. Hydrogen peroxide is reactive oxygen species which is normally present in endothelial cells at concentrations higher than in other cell types. Moreover, at the low micromolar range it has a proliferative effect on HUVEC cells. Our working hypothesis rises from the fact that in conditions of oxidative and/or inflammatory stress the endothelial cells are in a key position to activate enzymatic responses in order to eliminate the sources of stress. In last analysis this process can be transformed in damage, independently from the participation of the activated neutrophils to the general process of inflammation. In order to investigate the multiple effects of oxidative and/or inflammatory processes on endothelial cells, we will proceed to the isolation of primary endothelial cells from the vein human umbilical cords (HUVEC). After the initial typing to ensure a pure cellular population, we will perform experiments of oxidative stress by exposing HUVEC to hydrogen peroxide, using both sublethal and proapoptotic concentrations. Cellular responses will be characterized by analyzing variations in cell viability, differential expression of endothelial markers as well as molecules involved in the response to the stress (as endothelial isoform of NOS). The protein profile of control and treated cells will be analysed by means of 2D-IPG in order to determine the proteomic profile of cells and its variations following the application of the stimulus. The present project will enrich the present knowledge about the physiopathologic response of endothelium to oxidative stress and the ways by which this tissue may be capable itself to actively participate in the inflammatory process.