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A major feature of epithelial and endothelial cells is the creation of biological barriers able to protect the body against stressors that could compromise homeostasis. The ability to characterize biological barriers in vitro is an important study tool especially used for the intestinal barrier, the blood-brain barrier, and the lung barrier. The strength and integrity of biological barriers may be assessed by the measurement of the transepithelial/transendothelial electrical resistance (TEER) that reflects the ionic conductance of the paracellular pathway. The TEER measurement is a quantitative, non-invasive, highly useful, and representative method that must be strictly standardized. Here we describe a quantitative protocol to assess the mammary epithelial barrier integrity by combining the TEER measurement with a test for studying the passage of the sodium fluorescein, that is, a hydrophilic paracellular marker. Being the swine species an excellent translational model, primary cultures of mammary epithelial cells, isolated from hybrid pig tissue collected at slaughterhouse, are used., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

The bergamot polyphenolic fraction (BPF) was evaluated in the F 1 F O -ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F 1 F O -ATPase, dependent on the natural cofactor Mg 2+ , increased by 15%, whereas the enzyme activity in the presence of Ca 2+ was inhibited by 10%. By considering this opposite BPF effect, the F 1 F O -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.

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  μ g/ml); no viability alterations were found in the 10-50  μ g/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  μ g/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  μ g/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25  μ g/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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Irvin Tubon et al.)

Hydrogen sulfide (H 2 S) 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 H 2 S 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.

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  μ g/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.

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.

In the field of cardiovascular research, the pig is considered to be an excellent animal model of human diseases. It is well-known that primary cultures of endothelial cells (ECs) are a powerful tool for the study of vascular physiology and pathology, and, according to the principles of the Three Rs, their use results in a substantial reduction in the numbers of experimental animals required. However, a limitation of EC culture is that the cells are not in their physiological context. Here, we describe and characterise a method for the culture of porcine vessels that overcomes the limitation of EC cultures, with the advantage of reducing the number of animals used for research purposes. The organ cultures were set-up by using an aortic cylinder obtained from the arteries of control pigs sacrificed for other experimental purposes. In order to characterise the method, vascular endothelial growth factor (VEGF) secretion, matrix metalloproteinase (MMP) activation and the vessel's structural features were evaluated during organ culture. These analyses confirm that the culture of aortic cylinder lumen, in a medium specific for ECs, results in a stable system in terms of VEGF and MMP secretion. The ECs do not undergo cell division during the organ culture, which is also the case in vivo, if no stimulation occurs. Overall, we show that this novel system closely resembles the in vivo context. Importantly, porcine aortas can be collected from either veterinary surgeries or slaughterhouses, without having to sacrifice animals specifically for the purposes of this type of research., (2013 FRAME.)

Treponema denticola is an oral spirochete that is associated with periodontal disease and detected occasionally in extraoral lesions associated with systemic disorders such as cardiovascular diseases. The effect of specific bacterial products from oral treponemes on endothelium is poorly investigated. This study analyzed the ability of components of the outer membrane of T. denticola (OMT) to induce apoptosis and heat shock proteins (HO-1 and Hsp70) in porcine aortic endothelial cells (pAECs), compared with results obtained with classical pro-inflammatory lipopolysaccharide (LPS) treatment. Cellular apoptosis was detected when pAECs were treated with either OMT or LPS, suggesting that OMT can damage endothelium integrity by reducing endothelial cell vitality. Stimulation with OMT, similarly to LPS response, increased HO-1 and Hsp-70 protein expression in a time-dependent manner, correlating with a rise in HO-1 and Hsp-70 mRNA. Collectively, these results support the hypothesis that T. denticola alters endothelial cell function. Moreover, our in vitro experiments represent a preliminary investigation to further in vivo study using a pig model to elucidate how T. denticola leaves the initial endodontic site and participates in the development of several systemic diseases.

Increasing evidence indicates that carbon monoxide (CO) may protect against several diseases including sepsis. The ability of CO pre-treatment to provide good pre-conditioning against lipopolysaccharide (LPS)-induced injury was tested using an in vitro model of primary culture of porcine aortic endothelial cells (pAEC). pAEC were exposed to CO (250 ppm) or air for 1 h prior to the addition of LPS (10 microg/ml). Hsp70, HO-1, and Egr-1 protein levels were determined as well as vascular endothelial growth factor (VEGF) secretion after 4, 7, and 15 h. The effect of CO on LPS-induced apoptosis was also detected at 15 h. CO pre-treatment before the addition of LPS, significantly reduced LPS-induced apoptosis. LPS induced an increase in the level of VEGF in culture media after 7 and 15 h, and a larger increase was detected in CO pre-treated cells. In addition, CO pre-treatment reduced LPS-induced Hsp70, HO-1, and Egr-1 protein expression. In conclusion, CO treatment seems to provide a good pre-conditioning for the prevention of LPS-induced endothelial injury.

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.

Simple Summary: It has been widely demonstrated that blood vessels are sources of multipotent progenitor cells, including mesenchymal stem cells. These stem cellular populations persist throughout adulthood and can be isolated from both microvascular and large vessels. Increasing evidence suggests that vascular stem cells, together with other cell populations residing in blood vessels, such as endothelial cells, are involved in physiological and pathological vascular remodeling. In the present paper, we described, for the first time, a new improved method to isolate a pure population of vascular wall cells showing a preserved mesenchymal tri-lineage differentiative potential from thoracic aorta of Göttingen Minipigs, preserving and also collecting endothelial cells. Considering the increasing interest in the use of Göttingen Minipigs as an animal model for cardiovascular diseases, the results obtained in the present research open the way to plan in vitro vascular remodeling experiments by using in co-culture system vascular mesenchymal stem cells and endothelial cells. Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Göttingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Göttingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Göttingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies. [ABSTRACT FROM AUTHOR]

Abstract Background It is well known that nitric oxide (NO) is generated by a family of constitutively (nNOS and eNOS) or inducibly (iNOS) expressed enzymes and takes part in different aspects of the inflammatory response; nevertheless, its effective role in the pathogenesis of multiple organ dysfunction and septic shock is not fully understood. Methods To investigate the Nitric Oxide Synthases (NOSs) expression in endothelial cells during endotoxin exposure and the involvement of NO in lipopolysaccharide (LPS)-induced apoptosis, primary cultures of porcine Aortic Endothelial Cells (pAECs) were exposed to LPS for different time periods (1-24 h) and to LPS + L-NAME (15 h). Results Lipopolysaccharide induced an increase in mRNA and protein iNOS expression; on the contrary, the expression of eNOS was decreased. Furthermore, NOSs localisation was in part modified by LPS treatment. No alteration in the total level of Nitric Oxide was observed. L-NAME (5 mM) addition determined a slight decrease of LPS-induced apoptosis. Conclusions Endotoxin treatment strongly influenced NOS expression with an upregulation of iNOS and a simultaneous down regulation of eNOS. Moreover, in our model, the involvement of NO on LPS-induced apoptosis is very modest, suggesting that different pathways are involved in the regulation of this process.

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 MSCand 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 endothelialcadherin, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1–4h) of LPS (40μg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis. [Copyright &y& Elsevier]

Clusterin (CLU) is a multifunctional protein whose expression has been related to many physiological and pathological processes. The most abundant gene product is the secreted form of CLU (sCLU) described as a prosurvival and cytoprotective protein. A nuclear CLU (nCLU) isoform involved in death and inhibition of cell growth has also been reported. The aims of the present work were to verify the differential presence of sCLU and nCLU in a primary culture of porcine aortic endothelial cells (pAEC) and to evaluate whether lipopolysaccharide (LPS) can influence their expression. LPS-treated pAEC expressed both mRNA isoforms but only sCLU protein was detected. LPS exposure induced transient mRNA and protein increases of sCLU at 1 h of treatment. In conclusion our data showed that in LPS-treated pAEC sCLU is the most abundant form of clusterin and nCLU can be considered irrelevant and is rarely translated. [ABSTRACT FROM AUTHOR]