Porcine Iliac Endothelial Cells Research Articles

TNF-\u03b1 promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression

BackgroundThe major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated.MethodsWe assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus.ResultsRecombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs.ConclusionOur data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.Graphical abstract

A potential role of TLR2 in xenograft rejection of porcine iliac endothelial cells: An in vitro study.

Porcine vascular endothelial cells are a major participant in xenograft rejection. The Toll-like receptor 2 (TLR2) pathway plays an important role in both innate and adaptive immunity. The specific role of TLR2 in the response to a xenograft has not been reported. Whether the TLR2 pathway in pig vascular endothelial cells is involved in acute rejection needs to be investigated, and the mechanism is explored. We used a modified antibody-dependent complement-mediated cytotoxicity (ADCC) assay to conduct in vitro experiments. In porcine iliac artery endothelial cells (PIECs), siRNA was used to knock down the expression of TLR2, CXCL8, and CCL2. The effect of human serum or inactivated human serum on the expression of TLR2 was analyzed by real-time PCR and Western blotting, and transwell assays were used to assess the chemotactic efficiency of PIECs on human monocyte-macrophages (THP-1 cells) and human neutrophils. The downstream signaling pathways activated by human serum were detected by Western blotting, and the regulation of proinflammatory chemokines and cytokines by TLR2 signaling was assessed by real-time PCR and ELISA. TLR2 was significantly upregulated in PIECs after exposure to human serum, and porcine proinflammatory chemokines, CXCL8 and CCL2, were induced, at least partially, in a TLR2-dependent pattern; the upregulated chemokines participated in the chemotaxis of human neutrophils and THP-1 cells across the species barrier. (i) TLR2 is significantly upregulated in PIECs by human serum, (ii) the elevated TLR2 participates in the chemotaxis of inflammatory cells through the secretion of chemokine CCL2 and CXCL8, and (iii) blockade of TLR2 would be beneficial for xenograft survival.

Angiogenesis Potential of Bladder Acellular Matrix Hydrogel by Compounding Endothelial Cells.

Rapid vascularization is very important in tissue engineering. Bladder acellular matrix (BAM) with inherent bioactive factors, a natural extracellular matrix (ECM) derived biomaterial, has been widely used as a scaffold to facilitate the repair and reconstruction of urinary tissues. However, the application of the traditional BAM scaffold has been limited due to the dense structure. To investigate the angiogenic potential of BAM, BAM hydrogels with tailored porous structures were prepared in this study by tuning BAM concentrations (4, 6, and 8 mg/mL). The 6 mg/mL BAM hydrogel was loaded with porcine iliac endothelial cells (PIECs), and their angiogenic potential was analyzed in vitro and in vivo. The mechanical strength and gelation speed of the BAM hydrogels increased, while their pore size decreased as concentration increased. Commercially available collagen hydrogel (2.5 mg/mL) showed weaker mechanical properties than BAM hydrogels but similar gelation speed and pore size as 6 mg/mL BAM hydrogel. To ensure a similar three-dimensional microenvironment for the PIECs, 6 mg/mL BAM and collagen hydrogels were selected for the in vitro and in vivo experiments. A significantly higher density of viable, fusiform PIECs of average length ∼50 μm was observed in the BAM hydrogel, while those inside the collagen hydrogel were spherical and ∼30 μm long. In addition, the PIECs/BAM hydrogel resulted in significantly higher revascularization compared with the PIECs/collagen and unloaded BAM hydrogels. The higher angiogenic potential of the PIECs/BAM hydrogel is due to the growth factors that promote PIEC proliferation and therefore vascularization.

Nanosecond pulsed electric fields promoting the proliferation of porcine iliac endothelial cells: An in vitro study.

Currently, nanosecond pulsed electric fields (nsPEFs) with short pulse duration and non-thermal effects have various potential applications in medicine and biology, especially in tumor ablation. Additionally, there are a few investigations on its proliferative effects in the normal cell. Clinically, proliferation of endothelial cells can perhaps accelerate the stent endothelialization and reduce the risk of acute thrombosis. To explore the feasibility using nsPEFs to induce proliferation of endothelial cells, in this study, porcine iliac endothelial (PIEC) cell line was cultured and tested by CCK-8 assay after nsPEFs treatment. The results reflected that nsPEFs with low field strength (100ns, 5 kV/cm, 10 pulses) had a significant proliferative effect with an increase in the PIEC cell growth of 16% after a 48 hour’ post-treatment. To further understand the mechanism of cell proliferation, intracellular Ca2+ concentration was measured through fluo-4 AM and reactive oxygen species assay was applied to estimate the level of intracellular reactive oxygen species (ROS). Finally, the total nitric oxide assay for NO production in the cultured medium was evaluated. An enhanced concentration of intracellular Ca2+ and ROS were observed, while the concentration of extracellular NO also increased after nsPEFs treatment. Such experimental results demonstrated that nsPEFs with appropriate pulse parameters could effectively enhance cell proliferation on PIEC cells, and the cell proliferation associated strongly with the changes of intracellular Ca2+ concertation, ROS and NO production induced by nsPEFs treatment. This in vitro preliminary study indicates that as a novel physical doping, the nsPEFs have potential in stimulating endothelial cells to accelerate stent endothelialization.

Angiopoietin-1 and angiopoietin-2 protect porcine iliac endothelial cells from human antibody-mediated complement-dependent cytotoxicity through phosphatidylinositide 3-kinase/AKT pathway activation.

Cytokines play crucial roles in inflammation, but their role in xenotransplantation remains elusive. We assessed the role of several cytokines using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Recombinant human angiopoietin-1 (Ang-1) protected porcine iliac endothelial cells (PIECs) from human antibody-mediated CDC. Interestingly, human angiopoietin-2 (Ang-2) had a similar protective effect on PIECs. By flow cytometry analysis, the extent of human IgM and IgG binding to PIECs did not decrease when PIECs were exposed to Ang-1/Ang-2. The mRNA level of complement regulators (CD46, CD55, CD59) was not upregulated in PIECs treated with Ang-1/Ang-2, both of which activated the PI3K/AKT pathway in PIECs. Wortmannin, which inhibits phosphatidylinositide 3-kinase (PI3K), suppressed Ang-1/Ang-2-induced AKT phosphorylation and consequent Ang-1/Ang-2-mediated protection of PIECs in human antibody-mediated CDC model. Moreover, dominant negative AKT also suppressed Ang-1/Ang-2-mediated protection of PIECs in this model. In conclusion, our data suggest that human Ang-1/Ang-2 induces the protection of PIECs from human antibody-mediated CDC by activating the PI3K/AKT pathway. Ang-1/Ang-2 is likely to protect porcine endothelial cells and may be beneficial in xenotransplantation research.

Characterization of bladder acellular matrix hydrogel with inherent bioactive factors

Bladder acellular matrix (BAM) hydrogel may have great potential in tissue engineering due to outstanding biocompatibility and the presence of inherent bioactive factors in BAM. In this study, we prepared the BAM hydrogel by the method of enzymatic solubilization with pepsin and characterize the microrheological properties of the BAM precursor solution. The structures of the BAM hydrogel were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Furthermore, the growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor B (PDGF-BB), keratinocyte growth factor (KGF) were quantified by ELISA. The biological performances of the hydrogels were evaluated by cultivating porcine iliac endothelial cells (PIECs) in vitro. Lyophilized BAM showed porous structure with pore diameter ranging from 50 to 100μm. BAM 4-G hydrogel (4mg/mL) with a short gelation time of 3.95±0.07min presents better thermal stability than BAM 6-G hydrogel (6mg/mL). Growth factors in the BAM hydrogel maintain valuable biological activity even after digestion process. The BAM hydrogel supported the adhesion and growth of PIECs well and has great potential for further tissue engineering.

In vitro cytotoxicity evaluation of graphene oxide from the peroxidase-like activity perspective

In this study, PEGylated graphene oxide (PEG-GO)-hemin composite structure was constructed. Hemin in the form of nanoscaled aggregates were immobilized on PEG-GO sheets by the π-π stacking super-molecular interaction. Via catalyzing the oxidation of chromogenic substrates, we elicited the obtained PEG-GO-Hemin composite sheets have much higher peroxidase-like activity compared to hemin or PEG-GO alone, which is due to the introduction of enzyme active center of hemin with high dispersity, the excellent affinity to organic substrate through π-π stacking and/or electrostatic adsorption and the rapid electron transfer capability of PEG-GO. Similarly, PEG-GO-Hemin was found to be able to catalyze the oxidation of low density lipoprotein (LDL) by H2O2, resulting in toxicity to porcine iliac endothelial cells (PIECs) in vitro. Furthermore, we also demonstrated that PEG-GO sheets showed enhanced peroxidase activity when met hemin containing proteins including hemoglobin and cytochrome c. High glucose level (HG) in human umbilical vein endothelial cells (HUVECs) can induce cytochrome c to release from the respiratory chain, thus applying PEG-GO under HG condition could cause a much higher peroxidase-like activity, resulting in the production of hydroxyl radical (OH) and cytochrome c radical (cytochrome c), which eventually enhance the apoptosis. These results suggest GO has potential hazard for biomedical applications in some pathophysiological conditions.

Bladder Acellular Matrix Graft Reinforced Silk Fibroin Composite Scaffolds Loaded VEGF with Aligned Electrospun Fibers in Multiple Layers.

Bombyx mori silk is of great interest to people for its outstanding mechanical and biological properties. However, the traditional electrospun regenerated silk fibroin (RSF) scaffolds from aqueous solution were weak and had limited applications. This study was to fabricate reinforced scaffolds with well-aligned RSF fibers electrospun on a layer of native extracellular matrix, bladder acellular matrix graft (BAMG). The silk fibroin fibers were well-aligned as a grill in multiple layers. Both the BAMG and the grill structure significantly improved the tensile properties and suture retention of the composite scaffolds, which can be sutured well with tissue during implantation. In vitro assay indicates that the scaffolds had a good biocompatibility. Porcine iliac endothelial cells (PIECs) attached and proliferated well on the vascular endothelial growth factor (VEGF) loaded scaffolds compared with those without VEGF. Moreover, the grill-like structure guides PIECs well along the aligned fiber.

Silk fibroin tissue engineering scaffolds with aligned electrospun fibers in multiple layers

Silk-based materials have great potentials in medical applications because of the proper mechanical properties and outstanding biocompatibility. This paper reports a novel method to prepare multi-layered silk fibroin (SF) tissue engineering scaffolds with well-aligned fibers oriented in different angles by an electrospinning process. The resulted aligned fibers construct the scaffolds with a grill-like structure, large area and improved mechanical properties, which have been demonstrated by scanning electron microscopy and tensile test. Biocompatibility analysis proves that the grill-like structure well guides the adhesion, proliferation and differentiation of porcine iliac endothelial cells along the aligned SF fibers in different directions.

Fabrication and Characterization of Electrospun Polycaprolactone Blended with Chitosan-Gelatin Complex Nanofibrous Mats

Design and fabrication of nanofibrous scaffolds should mimic the native extracellular matrix. This study is aimed at investigating electrospinning of polycaprolactone (PCL) blended with chitosan-gelatin complex. The morphologies were observed from scanning electron microscope. As-spun blended mats had thinner fibers than pure PCL. X-ray diffraction was used to analyze the degree of crystallinity. The intensity at two peaks at 2θof 21° and 23.5° gradually decreased with the percentage of chitosan-gelatin complex increasing. Moreover, incorporation of the complex could obviously improve the hydrophilicity of as-spun blended mats. Mechanical properties of as-spun nanofibrous mats were also tested. The elongation at break of fibrous mats increased with the PCL content increasing and the ultimate tensile strength varied with different weight ratios. The as-spun mats had higher tensile strength when the weight ratio of PCL to CS-Gel was 75/25 compared to pure PCL. Both as-spun PCL scaffolds and PCL/CS-Gel scaffolds supported the proliferation of porcine iliac endothelial cells, and PCL/CS-Gel had better cell viability than pure PCL. Therefore, electrospun PCL/Chitosan-gelatin nanofibrous mats with weight ratio of 75/25 have better hydrophilicity mechanical properties, and cell proliferation and thus would be a promising candidate for tissue engineering scaffolds.

Cross-Linking of Gelatin and Chitosan Complex Nanofibers for Tissue-Engineering Scaffolds

The aim of this study is to investigate cross-linked gelatin–chitosan nanofibers produced by means of electrospinning. Gelatin and chitosan nanofibers were electrospun and then cross-linked by glutaraldehyde (GTA) vapor at room temperature. Scanning electron microscopy (SEM) images showed that the cross-linked mats could keep their nanofibrous structure after being soaked in deionized water at 37° C. The cross-linking mechanism was discussed based on FT-IR results. The two main mechanisms of cross-linking for chitosan and gelatin–chitosan complex are Schiff base reaction and acetalization reaction. For gelatin, the mechanism of cross-linking was Schiff base reaction. The mechanical properties of nanofibrous mats were improved after cross-linking. The biocompatibility of electrospun nanofibrous mats after cross-linking was investigated by the viability of porcine iliac endothelial cells (PIECs). The morphologies of PIECs on the cross-linked nanofibrous mats were observed by SEM. In addition, proliferation of PIECs was tested with the method of methylthiazol tetrazolium (MTT) assay. The results indicate that gelatin–chitosan nanofibrous mats could be a promising candidate for tissue-engineering scaffolds.

Effects of intermittent high glucose on oxidative stress in endothelial cells

The objective of this study is to explore the mechanism of oxidative stress induced by intermittent high glucose in porcine iliac endothelial cells (PIECs). The PIECs were exposed to intermittent or constant high glucose for 3 or 6 days, and the mean fluorescent intensity (MFI) was measured via intracellular reactive oxygen species (ROS) captured by flow cytometry. The NADPH oxidase activity was measured by chemiluminescence with lucigenin. Intermittent high glucose induced a greater over-production of ROS than constant high glucose in PIECs; the NADPH oxidase activity was increased under both constant and intermittent high glucose conditions, being more marked in the latter (P < 0.05). In conclusion, intermittent high glucose induced more ROS in PIECs than constant high glucose, this effect seemed to be, at least in part related to the enhanced activation of NADPH oxidase. Glucose fluctuation may be involved in the development of vascular complications.