Vitamin K Vitamers Differently Affect Energy Metabolism in IPEC-J2 Cells

The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot de novo synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to in vitro study the effect of compounds at the intestinal level. The acute VK treatments on the bioenergetic features of IPEC-J2 cells were evaluated by Seahorse XP Agilent technology. VK exists in different structurally related forms (vitamers), all featured by a naphtoquinone moiety, but with distinct effects on IPEC-J2 energy metabolism. The VK1, which has a long hydrocarbon chain, at both concentrations (5 and 10 \u3bcM), increases the cellular ATP production due to oxidative phosphorylation (OXPHOS) by 5% and by 30% through glycolysis. The VK2 at 5 \u3bcM only stimulates ATP production by OXPHOS. Conversely, 10 \u3bcM VK3, which lacks the long side chain, inhibits OXPHOS by 30% and glycolysis by 45%. However, even if IPEC-J2 cells mainly prefer OXPHOS to glycolysis to produce ATP, the OXPHOS/glycolysis ratio significantly decreases in VK1-treated cells, is unaffected by VK2, and only significantly increased by 10 \u3bcM VK3. VK1, at the two concentrations tested, does not affect the mitochondrial bioenergetic parameters, while 5 \u3bcM VK2 increases and 5 \u3bcM VK3 reduces the mitochondrial respiration (i.e., maximal respiration and spare respiratory capacity). Moreover, 10 \u3bcM VK3 impairs OXPHOS, as shown by the increase in the proton leak, namely the proton backward entry to the matrix space, thus pointing out mitochondrial toxicity. Furthermore, in the presence of both VK1 and VK2 concentrations, the glycolytic parameters, namely the glycolytic capacity and the glycolytic reserve, are unaltered. In contrast, the inhibition of glycoATP production by VK3 is linked to the 80% inhibition of glycolysis, resulting in a reduced glycolytic capacity and reserve. These data, which demonstrate the VK ability to differently modulate IPEC-J2 cell energy metabolism according to the different structural features of the vitamers, can mirror VK modulatory effects on the cell membrane features and, as a cascade, on the epithelial cell properties and gut functions: balance of salt and water, macromolecule cleavage, detoxification of harmful compounds, and nitrogen recycling

Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project

: The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective

The Impairment of Cell Metabolism by Cardiovascular Toxicity of Doxorubicin Is Reversed by Bergamot Polyphenolic Fraction Treatment in Endothelial Cells

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted

Efficacy of Stem Cell Therapy in Large Animal Models of Ischemic Cardiomyopathies: A Systematic Review and Meta-Analysis

Stem-cell therapy provides a promising strategy for patients with ischemic heart disease. In recent years, numerous studies related to this therapeutic approach were performed; however, the results were often heterogeneous and contradictory. For this reason, we conducted a systematic review and meta-analysis of trials, reporting the use of stem-cell treatment against acute or chronic ischemic cardiomyopathies in large animal models with regard to Left Ventricular Ejection Fraction (LVEF). The defined research strategy was applied to the PubMed database to identify relevant studies published from January 2011 to July 2021. A random-effect meta-analysis was performed on LVEF mean data at follow-up between control and stem-cell-treated animals. In order to improve the definition of the effect measure and to analyze the factors that could influence the outcomes, a subgroup comparison was conducted. Sixty-six studies (n = 1183 animals) satisfied our inclusion criteria. Ischemia/reperfusion infarction was performed in 37 studies, and chronic occlusion in 29 studies; moreover, 58 studies were on a pig animal model. The meta-analysis showed that cell therapy increased LVEF by 7.41% (95% Confidence Interval 6.23–8.59%; p < 0.001) at follow-up, with significative heterogeneity and high inconsistency (I2 = 82%, p < 0.001). By subgroup comparison, the follow-up after 31–60 days (p = 0.025), the late cell injection (>7 days, p = 0.005) and the route of cellular delivery by surgical treatment (p < 0.001) were significant predictors of LVEF improvement. This meta-analysis showed that stem-cell therapy may improve heart function in large animal models and that the swine specie is confirmed as a relevant animal model in the cardiovascular field. Due to the significative heterogeneity and high inconsistency, future translational studies should be designed to take into account the evidenced predictors to allow for the reduction of the number of animals used

Effects of Hydrogen Sulfide Donor NaHS on Porcine Vascular Wall-Mesenchymal Stem Cells

Hydrogen sulfide (H2S) is now considered not only for its toxicity, but also as an endogenously produced gas transmitter with multiple physiological roles, also in maintaining and regulating stem cell physiology. In the present work, we evaluated the effect of a common H2S donor, NaHS, on porcine vascular wall–mesenchymal stem cells (pVW–MSCs). pVW–MSCs were treated for 24 h with increasing doses of NaHS, and the cell viability, cell cycle, and reactive oxygen species (ROS) production were evaluated. Moreover, the long-term effects of NaHS administration on the noteworthy characteristics of pVW–MSCs were analyzed. The MTT test revealed no alteration in cell viability, however, the cell cycle analysis demonstrated that the highest NaHS dose tested (300 μM) determined a block in S phase, which did not depend on the ROS production. Moreover, NaHS (10 μM), continuously administered in culture for 21 days, was able to significantly reduce NG2, Nestin and PDGFR-β expression. The pro-angiogenic attitude of pVW–MSCs was partially reduced by NaHS: the cells maintained the ability to grow in spheroid and sprouting from that, but endothelial markers (Factor VIII and CD31) were reduced. In conclusion, NaHS can be toxic for pVW–MSCs in high doses, while in low doses, it influences cellular physiology, by affecting the gene expression with a slowing down of the endothelial lineage

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by\u2009>\u2009100% and\u2009<\u200950%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements

ConcePTION WP3 task 3.2. Isolation and characterization of Mammary Epithelial Cells from Göttingen minipig (mpMECs): an in vitro study to compare mpMECs vs pMECs (porcine Mammary Epithelial Cells)

This data set contains data related to the characterization of an appropriate in vitro animal model based on primary culture of Göttingen Minipig Mammary Epithelial Cells (mpMECs). The objective of the research was to verify the accuracy of mpMECs as solid translational model for the study of mammary epithelial barrier and compare mpMECs results with those obtained from Mammary Epithelial Cells isolated from hybrid commercial pig (pMECs). The results showed that it was possible to isolate, culture and expand three pure epithelial cell lines obtained from three different animals. The mpMECs maintained until P10 showed a typical cobblestone morphology and a similar doubling time profile (MG9 32.6 ± 4 h, MG11 30.5 ± 3 h and MG12 27.4 ± 2.8 h, as reported in the data file CONCEPTION_WP3_mpMECs_DoublingTime_09032023.xlsx). DNA index was normal for all the three cell populations with a mean value of 0.98 ± 0.01. Regarding the cell cycle, the cell populations MG9, MG11 and MG12 showed the three distinct phases that could be recognized in a proliferating cell population: G0/G1, S and G2/M (see data file CONCEPTION_WP3_mpMECs_CellCycle_DNAIndex_09032023.xlsx). All the three mpMECs cell lines expressed epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18), confirming their epithelial origin (see data file CONCEPTION_WP3_mpMECs_FlowCytometer_E-Cad_CK18_09032023.xlsx). In particular in MG12, the contour of CK18 positive peak showed a shoulder suggesting the presence of a cellular subpopulation with a particularly high positivity. The barrier function of mpMECs was evaluated via TEER and fluorescein sodium (SF) transport, the formation of the monolayer integrity was evaluated in all the three primary mpMECs and in the pre-mixed pool of mpMECs and pMECs. MG9, MG11 and MG12 resulted in a similar TEER profile at the 0.15×10^6 cells, achieving higher TEER and lower SF values at the day 3 and 4 of culture, which was also confirmed in the pre-mixed pool case. Moreover, by comparing the maximum values of TEER reached at the different seeding density tested, a significative difference resulted in MG9 with respect to MG11, MG12 and pool. From a similar comparison with minimum values of SF transport, no difference between the groups was showed. Finally, in pre-mixed pools of mpMECs and pMECs a difference in the ability to form the epithelial barrier was shown: mpMEC barrier resulted less tight compact then that formed by pMECs. (see data file CONCEPTION_WP3_mpMECs_TEER_SF_09032023.xlsx). On pre-mixed pool of mpMECs and pMECs we evaluated the bioenergetic metabolism: no difference in energy production under basal cell culture condition was observed but under stressed state pMECs made more efficient use of mitochondrial oxidative metabolism than mpMECs, conversely, these last ones could more efficiently increased the glycolytic activity (see data file CONCEPTION_WP3_mpMECs_BioenergerticMetabolism_09032023.xlsx). The transcriptional profile of drug transporters evaluated in pre-mixed pool of mpMECs or pMECs cultured MECGM medium. Among the 84 genes, 66 genes were detectable, 18 genes were not detectable or higher than 35 threshold cycle, so considered as negative according to the handbook, in both cell lines. No difference between mpMECs and pMECs drug transporter gene expression levels was observed (see data file CONCEPTION_WP3_mpMECs_RT2ARRAY_09032023.xlsx)

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters

ConcePTION WP3 task 3.2. Isolation of porcine Mammary Epithelial Cells (pMECs). In vitro model for epithelial barrier

The present research aimed to develop a method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study mammary epithelial barrier in vitro. In agreement with the application of the 3Rs, porcine mammary gland tissues were collected at a local slaughterhouse and, after dissociation, the cells were isolated, cultured and characterized by morphology, cell cycle analysis and immunophenotyping. The ability to create a barrier was tested by TEER (Transepithelial/transendothelial electrical resistance) and the gene expression of genes related to drug transporters was evaluated by a PCR array. All the three pMECs cellular lines isolated are able to create a tight barrier and express the main ABC and SLC drug transporters confirming that the method of isolation is efficient to obtain primary pMECs lines to study epithelial barrier functions in the pig model. This activity was carried out as a part of Conception project (GA 821520)

Development of a Pig Mammary Epithelial Cell Culture Model as a Non-Clinical Tool for Studying Epithelial Barrier—A Contribution from the IMI-ConcePTION Project

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model