Short- and long-term effects of essential oils on swine spermatozoa during liquid phase refrigeration.

The application of essential oils as potential alternatives to antibiotics in swine semen storage is promising, due to their antioxidant and antibacterial properties. However, detrimental effects on spermatozoa should be clarified first. The aim of this study was to evaluate 9 essential oils (EOs; Satureja montana, Pelargonium graveolens, Cymbopogon nardus, Melaleuca leucadendron, Eucaliptus globulus, Citrus limon, Lavandula angustifolia, Lavandula hybrida, Mentha piperita) and a blend (GL mix) on key morpho-functional parameters of swine spermatozoa. Test compounds were firstly chemo-characterized and experimental doses were prepared by suspending a fixed number of spermatozoa with 3 different concentrations (0.1, 0.5, 1 mg/mL) of EOs. Experimental doses were stored at 16 °C and sampled after 3 and 120 h for analysis. Overall, S. montana, P. graveolens and L. angustifolia EOs induced the strongest alterations, with C. nardus and E. globulus EOs being the best tolerated. Swine spermatozoa represent a good preliminary testing platform to screen toxicity and its different patterns. The comprehensive overview on the potential mechanisms of action of some of the most common EOs, despite of the direct aim of the study being swine reproduction, may be exploited in other fields of research within both veterinary and human medicine.

Comparative evaluation of the effects of different activating media and temperatures on European eel (Anguilla anguilla) sperm motility assessed by computer assisted sperm analysis.

The European eel is a critically endangered teleost fish with very poor success rate for captive breeding and artificial reproduction. Therefore, to support its conservation, new strategies are needed to ensure fertilization. Objective analysis of sperm motility may be critical as it potentially represents one of the most important reproductive quality parameters. Spermatozoa acquire motility once in contact with hyperosmotic solutions as saltwater, yet the exact mechanisms and the role of temperature are still to be clarified. The main aim of the study was to assess the effects of 3 activating media (artificial sea water, tank water and commercial Actifish®) at 4 and 20 °C on sperm motility, by means of computer assisted sperm analysis. Secondary aim was to test 2 different concentrations of Actifish® mimicking sea water pH/osmolality, at 4 °C. The results suggested how both temperature and activating media have effects on spermatozoa motility and kinematics, with temperature mainly acting upon interaction with the media type. The samples activated with tank water at 20 °C showed the poorest motility outcomes (mean 38.1%), while the ones activated with Actifish® diluted 1:4 and artificial sea water, at 4 °C, the highest (means 51.8 and 51.5% respectively). Additionally, diluting Actifish® to reach same pH and osmolality of seawater led to worse motility outcomes, suggesting that composition may be the critical factor for activation rather than osmolality itself.

Iron Content, Iron Speciation and Phycocyanin in Commercial Samples of Arthrospira spp.

Cyanobacteria are characterized by high iron content. In this research, we collected ten commercial samples of Arthrospira spp. sold as food supplement to determine iron content and assess whether iron speciation showed variability among samples and changed respect to A. platensis grown in controlled conditions. Particular attention was also paid to phycocyanin, as an iron-binding protein. In six of the ten samples, 14 essential and non-essential trace elements were analysed using ICP-MS. Iron content measured in samples using atomic absorption spectrometry (AAS) varied from 353 (sample S5) to 1459 (sample S7) µg g-1 dry weight and was in the range of those reported by other authors in commercial supplements. Iron speciation was studied using size exclusion chromatography followed by the analysis of the collected fraction for the determination of iron by AAS and for protein separation using SDS-PAGE. Overlapping chromatographic profiles were obtained for total proteins, phycocyanin and iron, although quantitative differences were evidenced among the samples analysed. In most samples, iron was mainly bound to ligands with high molecular mass; however, in four samples iron was also bound to ligands with low molecular mass. In fractions containing the most relevant iron burden, the principal protein was phycocyanin, confirming its role as an iron-binding protein in commercial samples.

Iron Speciation and Iron Binding Proteins in Arthrospira platensis Grown in Media Containing Different Iron Concentrations

Cyanobacteria are characterized by high iron content. This study investigated the effects of varying iron concentrations (1, 5, and 10 mg L-1) in the culture media on the biochemical composition and the iron bioaccumulation and speciation in Arthrospira platensis F&M-C256. Iron content measured in biomasses varied from 0.35 to 2.34 mg g-1 dry weight depending on the iron concentration in the culture media. These biomasses can be considered of interest for the production of spirulina-based supplements with low and high iron content. Iron speciation was studied using size exclusion chromatography followed by atomic absorption spectrometry and proteomic analysis. The role of C-phycocyanin as an iron binding protein was also investigated. Overall, the present results provide a better understanding of iron metabolism in cyanobacteria and a foundation for further studies.

Potential Applications of Essential Oils for Environmental Sanitization and Antimicrobial Treatment of Intensive Livestock Infections.

The extensive use of antibiotics has contributed to the current antibiotic resistance crisis. Livestock infections of Salmonella spp, Clostridium spp. and E. coli antimicrobial-resistant bacteria represent a public threat to human and animal health. To reduce the incidence of these zoonoses, essential oils (EOs) could be effective antibiotic alternatives. This study aims at identifying EOs safe for use, effective both in complementary therapy and in the environmental sanitization of intensive farming. Natural products were chemo-characterized by gas chromatography. Three S. Typhimurium, three C. perfringens and four E. coli strains isolated from poultry and swine farms were used to assess the antimicrobial properties of nine EOs and a modified GR-OLI (mGR-OLI). The toxicity of the most effective ones (Cinnamomum zeylanicum, Cz; Origanum vulgare, Ov) was also evaluated on porcine spermatozoa and Galleria mellonella larvae. Cz, Ov and mGR-OLI showed the strongest antimicrobial activity; their volatile components were also able to significantly inhibit the growth of tested strains. In vitro, Ov toxicity was slightly lower than Cz, while it showed no toxicity on G. mellonella larvae. In conclusion, the study confirms the importance of evaluating natural products to consolidate the idea of safe EO applications in reducing and preventing intensive livestock infections.

Testicular Melatonin and Its Pathway in Roe Deer Bucks (Capreolus capreolus) during Pre- and Post-Rut Periods: Correlation with Testicular Involution.

Roe deer are seasonal breeders with a complete yearly testicular cycle. The peak in reproductive activity is recorded during summer, the rutting period, with the highest levels of androgens and testicular weight. Melatonin plays a pivotal role in seasonal breeders by stimulating the hypothalamus-pituitary-gonads axis and acting locally; in different species, its synthesis within testes has been reported. The aim of this study was to evaluate the physiological melatonin pattern within roe deer testes by comparing data obtained from animals sampled during pre- and post-rut periods. Melatonin was quantified in testicular parenchyma, along with the genetic expression of enzymes involved in its local synthesis (AANAT and ASMT) and function (UCP1). Melatonin receptors, MT1-2, were quantified both at protein and gene expression levels. Finally, to assess changes in reproductive hormonal profiles, testicular dehydroepiandrosterone (DHEA) was quantified and used for a correlation analysis. Melatonin and AANAT were detected in all samples, without significant differences between pre- and post-rut periods. Despite DHEA levels confirming testicular involution during the post-rut period, no correlations appeared between such involution and melatonin pathways. This study represents the first report regarding melatonin synthesis in roe deer testes, opening the way for future prospective studies in the physiology of this species.

Quantification of Hair Corticosterone, DHEA and Testosterone as a Potential Tool for Welfare Assessment in Male Laboratory Mice.

Steroids, providing information regarding several biological patterns including stress and sexual behavior, have been investigated in different matrices in laboratory mice. Data regarding hair quantification, indicative of longer timespans when compared to blood and saliva, are lacking. The aim of the work was to analyze the hormonal hair profile of laboratory male mice and to investigate potential relationships with age and housing, as a potential tool for welfare assessment. Fifty-six adult male C57BL/6J and C57BL/6OlaHsd substrain mice were included in the study, housed in pairs or groups. Testosterone (T) and dehydroepiandrosterone (DHEA) were quantified by radioimmunoassay, corticosterone (CORT) by ELISA. Mean hormone levels were 6.42 pg/mg for T, 23.16 pg/mg for DHEA and 502.1 pg/mg for CORT. Age influenced all hormones by significantly increasing T and DHEA levels and decreasing CORT; only DHEA, significantly higher in grouped mice, was influenced by housing conditions. The influence of age indicates the need for accurate age-related reference intervals, while the higher levels of DHEA in grouped animals suggests that such housing practice may be beneficial for social interactions. In conclusion, it seems that hair hormones quantification may be a good tool for welfare assessment in laboratory mice and may help in refining husbandry.

Clinopodium tomentosum (Kunth) Govaerts Leaf Extract Influences in vitro Cell Proliferation and Angiogenesis on Primary Cultures of Porcine Aortic Endothelial Cells.

Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 µg/ml); no viability alterations were found in the 10-50 µg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 µg/ml of extract caused a significant increase in pAEC's migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions' number. On the other hand, CTEE at 100 µg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 µg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2-0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Uncovering the Physiological Mechanisms Underlying the Roe Deer (Capreolus capreolus) Testicular Cycle: Analyses of Gelatinases and VEGF Patterns and Correlation with Testes Weight and Testosterone.

The roe deer (Capreolus capreolus) represents a spontaneous model of testicular inactivation: During winter, bucks show a suspension of spermatogenesis that starts again in spring and peaks during the breeding season (July-August). The underlying mechanisms to the regulation of the cyclic testicular changes are still not fully clear but seem to be imputable to the spermatogenic cell line since other testicular cell populations remain stable without apoptotic phenomena. The aim of the study was to investigate apoptosis, gelatinases (MMP2 and 9), their inhibiting factors (TIMP 1-2), and two isoforms of vascular endothelial growth factor (VEGF121 and 165) with its receptors (VEGFR1-2) in testes collected during pre- and post-rut periods, and to correlate them with testicular weight (TW) and testosterone (TEST). Testes from 18 adult sexually mature bucks were collected in Bologna Apennines (Italy). Samples were weighed and parenchyma collected. Radioimmunoassay, real-time PCR, and zymography were performed. The results showed a post-rut decrease in TW and TEST and an increase in proMMP2, also highlighting a correlation between the gelatinases and the testicular functionality. The VEGF pattern did not show modifications nor correlation with TW and TEST. Overall, gelatinases and their inhibitors, described herein for the first time in roe deer testes, seem to play an important role in the testicular cycle.

Progesterone and Cortisol Levels in Blood and Hair of Wild Pregnant Red Deer (Cervus Elaphus) Hinds.

The red deer (Cervus elaphus L., 1758) is one of the largest deer species in the world. Females are seasonal polyestrous, with negative photoperiod: the increase of the night peak of melatonin determines the secretion of GnRH and, therefore, LH and FSH. To date there is little information regarding the hormonal control during pregnancy for this species; this could be due to the difficulty of sampling wild subjects, while farmed animals' hormonal concentrations may not reflect the physiology of the animal in a natural state. In this study we evaluated the concentration of cortisol and progesterone, extracted from blood and hair, on 10 wild and pregnant red deer females. Belonging to the population of the Bolognese Apennines (Italy), the hinds were sampled in the January-March 2018 period, according to the regional selective hunting plan. Plasma progesterone (P4) ranged from a minimum of 1.9 to a maximum of 7.48 ng/mL; while hair P4 concentrations varied from 41.68 to 153.57 pg/mg. The plasma and hair cortisol ranges are respectively 0.4-2.97 ng/mL and 0.03-0.55 pg/mg; the only significant correlation was found between hair concentration of P4 and the date of death. The results of this preliminary study represent a small step towards a better knowledge of this species' physiology during pregnancy.

Antimicrobial capabilities of non-spermicidal concentrations of tea tree (Melaleuca alternifolia) and rosemary (Rosmarinus officinalis) essential oils on the liquid phase of refrigerated swine seminal doses.

Antimicrobial resistance is increasing within the porcine industry with consequential high impact on human health, leading to a need for new antimicrobials. Lately, the scientific community has turned its interest towards natural compounds, and different essential oils have been tested on spermatozoa for preliminary assessment of toxicity before considering them as good substitutes for standard antibiotics. The aim of the present work was to investigate the potential antimicrobial effect of Melaleuca alternifolia and Rosmarinus officinalis essential oils, already evaluated for toxicity, on swine artificial insemination doses deprived of spermatozoa and stored at 16 °C for 5 days. This was accomplished by setting up an in vitro model with a standardized quantity of E. coli. Essential oils, previously chemo-characterized by means of gas chromatography, were tested at 0.2 and 0.4 mg/ml. Analyses, performed at 24 and 120 h, included optical density evaluation, bacterial DNA quantification by qPCR, and colony count. The results demonstrate that both Melaleuca alternifolia and Rosmarinus officinalis essential oils, at a concentration of 0.4 mg/ml, are capable of delivering similar effects to ampicillin, used as control, on the experimental samples. At the lower concentration, M. alternifolia essential oil seemed more effective when compared to R. officinalis. Overall, these findings strengthen the hypothesis of the potential use of phyto-complexes as antimicrobial agents for reproductive biotechnologies.

Cytotoxic Effects of Artemisia annua L. and Pure Artemisinin on the D-17 Canine Osteosarcoma Cell Line.

Artemisia annua has been used for centuries in Traditional Chinese Medicine. Although used as an antimalarial drug, its active compound artemisinin and the semisynthetic derivatives have also been investigated for their anticancer properties, with interesting and promising results. The aims of this research were to evaluate (i) the cytotoxicity and the antiproliferative effect of pure artemisinin and a hydroalcoholic extract obtained from A. annua on the D-17 canine osteosarcoma cell line and (ii) the intracellular iron concentration and its correlation with the cytotoxic effects. Both artemisinin and hydroalcoholic extract induced a cytotoxic effect in a dose-dependent manner. Pure artemisinin caused an increase of cells in the S phase, whereas the hydroalcoholic extract induced an evident increase in the G2/M phase. A significant decrease of iron concentration was measured in D-17 cells treated with pure artemisinin and hydroalcoholic extract compared to untreated cells. In conclusion, although preliminary, the data obtained in this study are indicative of a more potent cytotoxic activity of the hydroalcoholic extract than pure artemisinin, indicating a possible synergistic effect of the phytocomplex and a mechanism of action involving iron and possibly ferroptosis. Considering the similarities between human and canine osteosarcomas, progress in deepening knowledge and improving therapeutic protocols will probably be relevant for both species, in a model of reciprocal translational medicine.

In Vitro Anti-Inflammatory Effect of Salvia sagittata Ethanolic Extract on Primary Cultures of Porcine Aortic Endothelial Cells.

The aim of the present research was to study the effects of an ethanolic extract of Salvia sagittata Ruiz & Pav (SSEE), an endemic Ecuadorian plant traditionally used to treat inflammation and different intestinal affections, on primary cultures of porcine aortic endothelial cells (pAECs). pAECs were cultured in the presence of different concentrations (1-200 µg/mL) of SSEE for 24 h, and cytotoxicity was evaluated by the MTT assay. SSEE did not negatively affect cellular viability at any concentration tested. Cell cycle was analyzed and no significant change was observed. Then, the anti-inflammatory effects of SSEE on pAECs were analyzed using a lipopolysaccharide (LPS) as the inflammatory stimulus. Different markers involved in the inflammatory process, such as cytokines and protective molecules, were evaluated by real-time quantitative PCR and Western blot. SSEE showed the ability to restore pAEC physiological conditions reducing interleukin-6 and increasing Heme Oxygenase-1 protein levels. The phytochemical composition of SSEE was also evaluated via HPLC-DAD and spectrophotometric assays. The presence of different phenolic acids and flavonoids was revealed, with rosmarinic acid as the most abundant component. SSEE possesses an interesting antioxidant activity, as assessed through both the Oxygen Radical Absorbance Capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. In conclusion, results suggest that SSEE is endowed with an in vitro anti-inflammatory effect. This represents the initial step in finding a possible scientific support for the traditional therapeutic use of this plant.

Constitutive and LPS-stimulated secretome of porcine Vascular Wall-Mesenchymal Stem Cells exerts effects on in vitro endothelial angiogenesis.

BACKGROUND: MSCs secretome is under investigation as an alternative to whole-cell-based therapies, since it is enriched of bioactive molecules: growth factors, cytokines and chemokines. Taking into account the translational value of the pig model, the leading aim of the present paper was to characterize the secretome of porcine Vascular Wall-Mesenchymal Stem Cells (pVW-MSCs) and its change in presence of LPS stimulation. Moreover, considering the importance of angiogenesis in regenerative mechanisms, we analysed the effect of pVW-MSCs secretome on in vitro angiogenesis. RESULTS: Our results demonstrated that conditioned medium from unstimulated pVW-MSCs contained high levels of IL-8, GM-CSF, IFN-γ and other immunomodulatory proteins: IL-6 IL-18 IL-4 IL-2 IL-10. LPS modulates pVW-MSCs gene expression and secretome composition, in particular a significant increase of IL-6 and IL-8 was observed; conversely, the amount of GM-CSF, IFN-γ, IL-2, IL-4, IL-10 and IL-18 showed a significant transient decrease with the LPS stimulation. Conditioned medium from unstimulated pVW-MSCs induced in vitro endothelial angiogenesis, which is more evident when the conditioned medium was from LPS stimulated pVW-MSCs. CONCLUSIONS: The lines of evidence here presented shed a light on possible future application of secretome derived by pVW-MSCs on research studies in translational regenerative medicine.

Anti-Inflammatory Activity of Boswellia serrata Extracts: An In Vitro Study on Porcine Aortic Endothelial Cells.

This study is aimed at investigating the cytotoxicity, anti-inflammatory, and angiogenic activities of two Boswellia serrata extracts on primary culture of porcine aortic endothelial cells (pAECs). Chemical characterization of a dry extract (extract A) and a hydroenzymatic extract (extract G) of B. serrata was performed by HPLC using pure boswellic acids (BAs) as standard. In cultured pAECs, extract G improved cell viability, following LPS challenge, in a dose-dependent manner and did not show any toxic effect. On the other hand, extract A was toxic at higher doses and restored pAEC viability after LPS challenge only at lower doses. Pure BAs, used at the same concentrations as those determined in the phytoextracts, did not contrast LPS-induced cytotoxicity. Extract A showed proangiogenic properties at the lowest dose, and the same result was observed using pure AKBA at the corresponding concentration, whereas extract G did not show any effect on the migration capacity of endothelial cells. In conclusion, an anti-inflammatory activity of B. serrata extracts on endothelial cells was reported, though cytotoxicity or proliferative stimulation can occur instead of a protective effect, depending on the dose and the formulation.

Water/ethanol extract of Cucumis sativus L. fruit attenuates lipopolysaccharide-induced inflammatory response in endothelial cells.

BACKGROUND: It is widely accepted the key role of endothelium in the onset of many chronic and acute vascular and cardiovascular diseases. In the last decade, traditional compounds utilized in "folk medicine" were considered with increasing interest to discover new bioactive molecules potentially effective in a wide range of diseases including cardiovascular ones. Since ancient times different parts of the Cucumis sativus L. plant were utilized in Ayurvedic medicine, among these, fruits were traditionally used to alleviate skin problem such as sunburn irritation and inflammation. The main purpose of the present research was, in a well-defined in vitro model of endothelial cells, to investigate whether a water/ethanol extract of Cucumis sativus L. (CSE) fruit can attenuate the damaging effect of pro-inflammatory lipopolysaccharide (LPS). METHODS: Cell viability, gene expression of endothelial cell markers, cytokines secretion and in vitro angiogenesis assay were performed on porcine Aortic Endothelial Cells exposed to increasing doses (0.02; 02; 2 mg/ml) of CSE in the presence of pro-inflammatory lipopolysaccharide (LPS 10 µg/ml). RESULTS: CSE reduced LPS-induced cytotoxicity and decreased the cellular detachment, restoring the expression of tight junction ZO-1. The increase of TLR4 expression induced by LPS was counterbalanced by the presence of CSE, while the protective gene Hemeoxygenase (HO)-1 was increased. Cucumis sativus L. inhibited the early robust secretion of inflammatory IL-8 and GM-CSFs, furthermore inhibition of inflammatory IL-6 and IL-1α occurred late at 7 and 24 h respectively. On the contrary, the secretion of anti-inflammatory IL-10, together with IL-18 and IFN-γ was increased. Moreover, the in vitro angiogenesis induced by inflammatory LPS was prevented by the presence of Cucunis sativus L. extract, at any doses tested. CONCLUSIONS: Our results have clearly demonstrated that Cucumis sativus L. extract has attenuated lipopolysaccharide-induced inflammatory response in endothelial cells.

Antioxidant and Ex Vivo Immune System Regulatory Properties of Boswellia serrata Extracts.

Boswellia serrata (BS) is an important traditional medicinal plant that currently represents an interesting topic for pharmaceutical research since it possesses several pharmacological properties (e.g., anti-inflammatory, antimicrobial, and antitumour). The safety and versatility of this dietary supplement should allow for its use in numerous pathological conditions; however the quality of the extracts needs to be standardized to increase the clinical success rate resulting from its use. In the present study, different commercially available B. serrata extracts were employed to compare their AKBA content and in vitro antioxidant power. Furthermore, their ability to modulate the immune system regulatory properties was investigated. Our results showed that the AKBA content varied from 3.83 ± 0.10 to 0.03 ± 0.004%, with one sample in which it was not detectable. The highest antioxidant power and phenolic content were shown by the same extract, which also exhibited the highest AKBA concentration. Finally, the BS extracts showed the ability to influence the regulatory and effector T-cell compartments. Our results suggest that frankincense should be further investigated for its promising potentiality to modulate not only inflammation/oxidative stress but also immune dysregulation, but attention should be paid to the composition of the commercial extracts.

Deleterious effects of tributyltin on porcine vascular stem cells physiology.

The vascular functional and structural integrity is essential for the maintenance of the whole organism and it has been demonstrated that different types of vascular progenitor cells resident in the vessel wall play an important role in this process. The purpose of the present research was to observe the effect of tributyltin (TBT), a risk factor for vascular disorders, on porcine Aortic Vascular Precursor Cells (pAVPCs) in term of cytotoxicity, gene expression profile, functionality and differentiation potential. We have demonstrated that pAVPCs morphology deeply changed following TBT treatment. After 48h a cytotoxic effect has been detected and Annexin binding assay demonstrated that TBT induced apoptosis. The transcriptional profile of characteristic pericyte markers has been altered: TBT 10nM substantially induced alpha-SMA, while, TBT 500nM determined a significant reduction of all pericyte markers. IL-6 protein detected in the medium of pAVPCs treated with TBT at both doses studied and with a dose response. TBT has interfered with normal pAVPC functionality preventing their ability to support a capillary-like network. In addition TBT has determined an increase of pAVPC adipogenic differentiation. In conclusion in the present paper we have demonstrated that TBT alters the vascular stem cells in terms of structure, functionality and differentiating capability, therefore effects of TBT in blood should be deeply explored to understand the potential vascular risk associated with the alteration of vascular stem cell physiology.

In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes.