Search Results (17)

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein. Full article

The introduction of single-cell RNA sequencing (scRNA-seq) technology has spurred additional advancements in analyzing the cellular composition of tissues. The longissimus dorsi (LD) in pigs serves as the primary skeletal muscle for studying meat quality in the pig industry. However, the single-cell profile of porcine LD is still in its infancy stage. In this study, we profiled the transcriptomes of 16,018 cells in the LD of a newborn Suhuai pig at single-cell resolution. Subsequently, we constructed a cellular atlas of the LD, identifying 11 distinct cell populations, including endothelial cells (24.39%), myotubes (18.82%), fibro-adipogenic progenitors (FAPs, 18.11%), satellite cells (16.74%), myoblasts (3.99%), myocytes (5.74%), Schwann cells (3.81%), smooth muscle cells (3.22%), dendritic cells (2.99%), pericytes (1.86%), and neutrophils (0.33%). CellChat was employed to deduce the cell–cell interactions by evaluating the gene expression of receptor–ligand pairs across different cell types. The results show that FAPs and pericytes are the primary signal contributors in LD. In addition, we delineated the developmental trajectory of myogenic cells and examined alterations in the expression of various marker genes and molecular events throughout various stages of differentiation. Moreover, we found that FAPs can be divided into three subclusters (NR2F2-FAPs, LPL-FAPs, and TNMD-FAPs) according to their biological functions, suggesting that the FAPs could be associated with the differentiation of tendon cell. Taken together, we constructed the cellular atlas and cell communication network in LD of a newborn Suhuai pig, and analyzed the developmental trajectory of myogenic cells and the heterogeneity of FAPs subpopulation cells. This enhances our comprehension of the molecular features involved in skeletal muscle development and the meat quality control in pigs. Full article

Acute Liver Failure (ALF) is a life-threatening illness characterized by the rapid onset of abnormal liver biochemistries, coagulopathy, and the development of hepatic encephalopathy. Extracorporeal bioengineered liver (BEL) grafts could offer a bridge therapy to transplant or recovery. The present study describes the manufacture of clinical scale BELs created from decellularized porcine-derived liver extracellular matrix seeded entirely with human cells: human umbilical vein endothelial cells (HUVECs) and primary human liver cells (PHLCs). Decellularized scaffolds seeded entirely with human cells were shown to adhere to stringent sterility and safety guidelines and demonstrated increased functionality when compared to grafts seeded with primary porcine liver cells (PPLCs). BELs with PHLCs were able to clear more ammonia than PPLCs and demonstrated lower perfusion pressures during patency testing. Additionally, to determine the full therapeutic potential of BELs seeded with PHLCs, longer culture periods were assessed to address the logistical constraints associated with manufacturing and transporting a product to a patient. The fully humanized BELs were able to retain their function after cold storage simulating a product transport period. Therefore, this study demonstrates the manufacture of bioengineered liver grafts and their potential in the clinical setting as a treatment for ALF. Full article

The blood–brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models. Full article

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H₂O₂. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research. Full article

Fucoidans are polysaccharides and constituents of cell walls of brown algae such as Laminaria hyperborea (LH). They exhibit promising effects regarding age-related macular degeneration (AMD). However, the safety of this compound needs to be assured. The focus of this study lies on influences of an LH fucoidan on the retinal pigment epithelium (RPE). The high-molecular weight LH fucoidan Fuc1 was applied to primary porcine RPE cells, and a tetrazolium (MTT) cell viability assay was conducted. Further tests included a scratch assay to measure wound healing, Western blotting to measure expression of retinal pigment epithelium-specific 65 kDa protein (RPE65), as well as immunofluorescence to measure uptake of opsonized fluorescence beads into RPE cells. Lipopolysaccharide was used to proinflammatorily activate the RPE, and interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion was measured. RPE/choroid cultures were used to assess vascular endothelial growth factor (VEGF) secretion. Real-time polymerase chain reaction (real-time PCR) was performed to detect the gene expression of 91 different genes in a specific porcine RPE gene array. Fuc1 slightly reduced wound healing, but did not influence cell viability, phagocytosis or RPE65 expression. Fuc1 lowered IL-6, IL-8 and VEGF secretion. Furthermore, Fuc1 did not change tested RPE genes. In conclusion, Fuc1 does not impair RPE cellular functions and shows antiangiogenic and anti-inflammatory activities, which indicates its safety and strengthens its suitability concerning ocular diseases. Full article

Ommochromes are pigments of invertebrates that exhibit oxidative stress protection. The aim of this study was to investigate ommochromes extracted from cephalopod’s skin for their ability to inhibit age-related-macular degeneration (AMD)-related factors such as H₂O₂-induced and iron-dependent oxidative stress (ferroptosis and erastin), accumulation of advanced glycation end-products (AGEs), as well as vascular endothelial growth factor (VEGF), and inflammatory cytokines (interleukin 6 and interleukin 8) secretion. As cell systems, we used primary porcine retinal pigment epithelium (RPE), human retinal pigment epithelium cell line ARPE-19 and uveal melanoma cell line OMM-1. In vitro, ommochromes produced an antiglycation effect by the inhibition of fructosylation reaction. The ommochromes showed protective effects against erastin- induced cell death in ARPE-19. In addition, in long-term stimulation (7 days) ommochromes decreased constitutively secreted VEGF, as well as interleukin 6 and interleukin 8 induced by Poly I:C in primary RPE. No relevant effects were detected in OMM-1 cells. The effects are dependent on the cell system, time of exposition, and concentration. This substance is of interest for further research concerning age-related macular degeneration. Full article

Endothelial cells (ECs), lining blood vessels’ lumen, play an essential role in regulating vascular functions. As multifunctional components of vascular structures, pluripotent stem cells (PSCs) are the promising source for potential therapeutic applications in various vascular diseases. Our laboratory has previously established an approach for differentiating porcine epiblast stem cells (pEpiSCs) into ECs, representing an alternative and potentially superior cell source. However, the condition of pEpiSCs-derived ECs growth has yet to be determined, and whether pEpiSCs differentiate into functional ECs remained unclear. Changes in morphology, proliferation and functional endothelial marker were assessed in pEpiSCs-derived ECs in vitro. pEpiSCs-derived ECs were subjected to magnetic-activated cell sorting (MACS) to collect CD-31+ of ECs. We found that sorted ECs showed the highest proliferation rate in differentiation media in primary culture and M199 media in the subculture. Next, sorted ECs were examined for their ability to act as typical vascular ECs through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and three-dimensional spheroid sprouting. Consequently, pEpiSCs-derived ECs function as typical vascular ECs, indicating that pEpiSC-derived ECs might be used to develop cell therapeutics for vascular disease. Full article

The porcine reproductive and respiratory syndrome virus (PRRSV), especially the highly pathogenic strains, can cause serious acute lung injury (ALI), characterized by extensive hemorrhage, inflammatory cells and serous fluid infiltration in the lung vascular system. Meanwhile, the pulmonary microvascular endothelial cells (PMVECs) are essential for forming the air–blood barrier and keeping the water–salt balance to prevent leakage of circulating nutrients, solutes, and fluid into the underlying tissues. As well, they tightly regulate the influx of immune cells. To determine the possible relationship between the PMVECs’ function changes and lung vascular permeability during PRRSV infection, the PMVECs were co-cultured with HP-PRRSV-inoculated primary pulmonary alveolar macrophages (PAMs) in transwell model, and then the RNA sequencing (RNA-seq) and comprehensive bioinformatics analysis were carried out to characterize the dynamic transcriptome landscapes of PMVECs. In total, 16,489 annotated genes were identified, with 275 upregulated and 270 downregulated differentially expressed genes (DEGs) were characterized at both 18 and 24 h post PRRSV inoculation. The GO terms and KEGG pathways analysis indicated that the immune response, metabolic pathways, cell death, cytokine–cytokine receptor interaction, viral responses, and apoptotic process are significantly regulated upon co-culture with PRRSV-infected PAMs. Moreover, according to the TERR and dextran flux assay results, dysregulation of TJ proteins, including CLDN1, CLDN4, CLDN8, and OCLN, is further confirmed to correlate with the increased permeability of PMVECs. These transcriptome profiles and DEGs will provide valuable clues for further exploring the roles of PMVECs in PRRSV-induced ALI in the future. Full article

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations. Full article

Both short- and long-term exposure to fine dust (FD) from air pollution has been linked to various cardiovascular diseases (CVDs). Endothelial cell (EC) senescence is an important risk factor for CVDs, and recent evidence suggests that FD-induced premature EC senescence increases oxidative stress levels. Hop plant (Humulus lupulus) is a very rich source of polyphenols known to have nutritional and therapeutic properties, including antioxidant behavior. The aims of this study were to evaluate whether Humulus lupulus extract prevents FD-induced vascular senescence and dysfunction and, if so, to characterize the underlying mechanisms and active components. Porcine coronary arteries and endothelial cells were treated with FD in the presence or absence of hop extract (HOP), and the senescence-associated-beta galactosidase (SA-β-gal) activity, cell-cycle progression, expression of senescence markers, oxidative stress level, and vascular function were evaluated. Results indicated that HOP inhibited FD-induced SA-β-gal activity, cell-cycle arrest, and oxidative stress, suggesting that HOP prevents premature induction of senescence by FD. HOP also ameliorated FD-induced vascular dysfunction. Additionally, xanthohumol (XN) and isoxanthohumol (IX) were found to produce the protective effects of HOP. Treatment with HOP and its primary active components XN and IX downregulated the expression of p22^phox, p53, and angiotensin type 1 receptor, which all are known FD-induced redox-sensitive EC senescence inducers. Taken together, HOP and its active components protect against FD-induced endothelial senescence most likely via antioxidant activity and may be a potential therapeutic agent for preventing and/or treating air-pollution-associated CVDs. Full article

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood–brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the α_S1-casein peptide ¹⁸⁵PIGSENSEKTTMPLW¹⁹⁹, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals. Full article

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm² and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs). Full article

Background: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. Methods and Results: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-β-gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-β and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. Conclusions: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence. Full article

Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development. Full article