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Since the publication of the book of Russell and Burch in 1959, scientific research has never stopped improving itself with regard to the important issue of animal experimentation. The European Directive 2010/63/EU “On the protection of animals used for scientific purposes” focuses mainly on the animal welfare, fixing the Russell and Burch’s 3Rs principles as the foundations of the document. In particular, the legislator clearly states the responsibility of the scientific community to improve the number of alternative methods to animal experimentation. The swine is considered a species of relevant interest for translational research and medicine due to its biological similarities with humans. The surgical community has, in fact, recognized the swine as an excellent model replicating the human cardiovascular system. There have been several wild-type and transgenic porcine models which were produced for biomedicine and translational research. Among these, the cardiovascular ones are the most represented. The continuous involvement of the porcine animal model in the biomedical research, as the continuous advances achieved using swine in translational medicine, support the need for alternative methods to animal experimentation involving pigs. The main purpose of the present work was to develop and characterize novel porcine alternative methods for cardiovascular translational biology/medicine. The work was mainly based on two different models: the first consisted in an ex vivo culture of porcine aortic cylinders and the second consisted in an in vitro culture of porcine aortic derived progenitor cells. Both the models were properly characterized and results indicated that they could be useful to the study of vascular biology. Nevertheless, both the models aim to reduce the use of experimental animals and to refine animal based-trials. In conclusion, the present research aims to be a small, but significant, contribution to the important and necessary field of study of alternative methods to animal experimentation.

OBJECTIVE: Vascular Stem Cells (VSCs) are recently defined as cells reside within the vessel wall, which show the typical mesenchymal characteristics and can differentiate into smooth muscle cells (SMCs) and endothelial cells (ECs), the cell types that constitute a functional blood vessel. Recently, we described the isolation of vascular precursor cells from the tunica media of porcine thoracic aortic (porcine Aortic Vascular Precursor cells, pAVPCs). These cells have mesenchymal phenotypic characteristics, pericytes typical functional properties and are able to differentiate spontaneously into smooth muscle cells. The aim of this study was to assess the differentiation potential of pAVPCs toward the endothelial lineage. MATERIALS AND METHODS: pAVPCs were seeded in a 24 well plate (5000 cells/cm2) and cultured in Endothelial Differentiation Medium (EDM) (human Endothelial Serum Free Medium supplemented with 5% FBS and 50 ng/mL of human recombinant Vascular Endothelial Growth Factor [hVEGF]) or in Perycite Growth Medium (PGM) as control. After 21 days of culture, the expression level of differentiated endothelial cells markers, CD31, VE-Cadherin, von Willebrand Factor (vWF) and endothelial Nitric Oxide Synthase (eNOS) was analyzed by qPCR. Expression of CD31 was also tested by immunofluorescence. RESULTS: The gene expression analysis revealed a significant increase of expression levels of all the endothelial markers in cells cultured in EDM respect to the control (PGM), with VE-Cadherin and CD31 showing an increase of 33 and 22 times, respectively. Immunofluorescence analysis of CD31 revealed that only cells cultured in EDM expressed the protein with the classical membrane localization. CONCLUSIONS: We demonstrated that pAVPCs, grown in specific endothelial medium and stimulated with VEGF, are able to differentiate toward the endothelial cells phenotype. Therefore, according to the recent definition of VSC, the pAVPCs could be considered a population of VSC-like cells.

In the last 10 years, several populations of vascular progenitor cells (VPCs) were isolated from vessel wall. In particular, it seems that some of these cells could be considered the in vivo counterpart of mesenchymal stromal cells (MSCs). In this work we describe a new method to isolate VPCs from porcine aorta tunica media. The internal lumen of arteries (n=3) was digested with Collagenase I solution, firstly for 40 minutes, to eliminate endothelial cells, and then for further 4 hours. The cell suspension obtained after 4 hours was cultured overnight in high concentration (10%) antibiotic/antimycotic culture medium (DMEM + 10% FBS). After 3 days cells were overnight serum starved and then recovered with DM medium (10% FBS + 1% antibiotic/antimycotic in DMEM:M199-1:1). Histological analysis revealed that after 4 hours of Collagenase I treatment 1/3 of the tunica media was digested. Progenitor cells derived from media layer, cultured in DM medium, display, when sub-confluent, a polygonal morphology and elongated thin arms at their ends. When confluent, cells showed a spindle shape. Moreover, cells display, an increasing doubling time from P1 to P6 (from 36.5±8.0 to 60.8±2.1 hours). At P3, cells were characterized for immunophenotype, antigen expression and differentiation potential. Immunophenotype analysis showed that VPCs derived from porcine aorta media layer grown in DM were positive for CD105 and CD90 and negative (

The aim of this study is to develop a porcine aortic organ culture that preserve phenotypic features of vascular cell types. In particular, we analyzed factors, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), that are associated with endothelial cells activation. VEGF secretion analysis demonstrate that using a specific medium for endothelial cells our system is stable for 14 days. MMPs zymographic analysis reveal a significant activation of MMP2 in culture medium but this is mainly due to the edges of the aortic ring. MMP2 is slightly activated in tissue, but is greatly activated if tissue is mechanically damaged. Histological analysis reveal no structural modification in tissue. These data suggest that our system represents a powerful tool to study vascular physiopathology because it preserves viability and stability of all cellular types, in particular endothelial cells.

Since proteomic differences between male X/Y chromosome-bearing gametes have recently been described, a question has been raised: could these differences be responsible for different behavior between X and Y chromosome-bearing spermatozoa during the binding and internalization of exogenous DNA in the swine species? In order to investigate this hypothesis, our group studied the process of the uptake and internalization of exogenous DNA in X and Y chromosome-bearing sperm sub-populations. No significant differences were found between sperm types in both the uptake and internalization of exogenous DNA. The quantity of internalized exogenous DNA was significantly lower than that of the uptaken DNA. In conclusion, our results showed that X and Y chromosomes-bearing spermatozoa have the same binding capacity and internalization of DNA, and the proteomic differences between them do not seem to interfere with these complex processes.