Porcine Aortic Endothelial Cells Research Articles

Protective Effects of Polysaccharide of Atractylodes Macrocephala Koidz against Porcine Aortic Valve Endothelial Cells Damage Induced by di (2-ethylhexyl) Phthalate.

The activation, injury, and dysfunction of endothelial cells are considered to be the initial key events in the development of atherosclerosis. Di (2-ethylhexyl) phthalate (DEHP), a prevalent organic pollutant, can cause damage to multiple organs. Polysaccharide of Atractylodes macrocephala Koidz (PAMK) is a bioactive compound extracted from A. macrocephala Koidz with various biological activities. This study investigates the protective effects of PAMK on porcine aortic valve endothelial cells (PAVEC) damaged by DEHP. PAVECs treated with DEHP alone or with PAMK showed reduced cell apoptosis and death in PAMK-pretreated cells. PAMK up-regulated Bcl-2 expression and down-regulated Bax protein, suppressing apoptosis. Flow cytometry analysis demonstrated that PAMK protected PAVECs from DEHP-induced damage. These findings suggest that PAMK inhibits cell apoptosis and protects against DEHP damage in endothelial cells.

Baicalin inhibited PANX-1/P2Y6 signaling pathway activation in porcine aortic vascular endothelial cells infected by Glaesserella parasuis

Glaesserella parasuis can induce endothelial barrier damage in piglets, although the mechanism by which this pathogen triggers inflammatory damage remains unclear. Baicalin possesses anti-inflammatory and anti-oxidant activities. However, whether baicalin can relieve endothelial barrier damage caused by Glaesserella parasuis infection has not yet been studied. Hence, we evaluated the ability of baicalin to counteract the changes induced by Glaesserella parasuis in porcine aortic vascular endothelial cells. The results showed that Glaesserella parasuis could upregulate the expression of pannexin 1 channel protein and promote the release of adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate in porcine aortic vascular endothelial cells. The expression level of purinergic receptor P2Y6 was upregulated in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. In addition, Glaesserella parasuis could activate phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells. Baicalin could inhibit pannexin 1 channel protein expression, reduce adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate release, and attenuate the expression level of P2Y6 in porcine aortic vascular endothelial cells induced by Glaesserella parasuis. Baicalin could also reduce the activation of phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. Our study report that Glaesserella parasuis could promote pannexin 1 channel protein expression, induce nucleosides substance release, and P2Y6 expression in porcine aortic vascular endothelial cells and baicalin could inhibit the expression levels of pannexin 1, nucleosides substance, and P2Y6 in the porcine aortic vascular endothelial cells induced by Glaesserella parasuis, which might be served as some targets for treatment of inflammation disease caused by Glaesserella parasuis.

Ischemia-Reperfusion Injury in Porcine Aortic Valvular Endothelial and Interstitial Cells.

Ischemia-reperfusion injury (IRI) in the myocardium has been thoroughly researched, especially in acute coronary syndrome and heart transplantation. However, our understanding of IRI implications on cardiac valves is still developing. This knowledge gap becomes even more pronounced given the advent of partial heart transplantation, a procedure designed to implant isolated human heart valves in young patients. This study aims to investigate the effects of IRI on aortic valvular endothelial cells (VECs), valvular interstitial cells (VICs), and whole leaflet cultures (no separation of VECs and VICs). We employed two conditions: hypoxic cold storage reperfusion (HCSR) and normothermia (NT). Key markers, secreted protein acidic and cysteine rich (SPARC) (osteonectin), and inducible nitric oxide synthase (iNOS2) were evaluated. In the isolated cells under HCSR, VICs manifested a significant 15-fold elevation in SPARC expression compared to NT (p = 0.0016). Conversely, whole leaflet cultures exhibited a 1-fold increment in SPARC expression in NT over HCSR (p = 0.0011). iNOS2 expression in VECs presented a marginal rise in HCSR, whereas, in whole leaflet settings, there was a 1-fold ascent in NT compared to HCSR (p = 0.0003). Minor escalations in the adhesion molecules intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), E-selection, and P-selection were detected in HCSR for whole leaflet cultures, albeit without statistical significance. Additionally, under HCSR, VICs released a markedly higher quantity of IL-6 and IL-8, with respective p-values of 0.0033 and <0.0001. Interestingly, the IL-6 levels in VECs remained consistent across both HCSR and NT conditions. These insights lay the groundwork for understanding graft IRI following partial heart transplantation and hint at the interdependent dynamic of VECs and VICs in valvular tissue.

Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Smooth muscle cell-targeted RNA ligand promotes accelerated reendothelialization in a swine peripheral injury model

The local delivery of antiproliferative agents to inhibit neointimal growth is not specific to vascular smooth muscle cells (VSMC) and delays reendothelialization and vascular healing. This investigation was intended to evaluate the effect of luminal delivery of a VSMC-specific aptamer on endothelial healing. The impact of an RNA aptamer (Apt 14) was first examined on the migration and proliferation of primary cultured porcine aortic endothelial cells (ECs) in response to invitro scratch wound injury. We further evaluated the impact of Apt 14 on reendothelialization when delivered locally in a swine iliofemoral injury model. Although Apt 14 did not affect EC migration and proliferation, invitro results confirmed that paclitaxel significantly inhibited EC migration and proliferation. En face scanning electron microscopy demonstrated confluent endothelium with elongated EC morphology in Apt 14-treated arteries 14 and 28days post-treatment. In contrast, vessels treated with paclitaxel-coated balloons displayed a cobblestone morphology and significant platelet and fibrin attachment at cell junctions. These results provide the first evidence of the efficacy of a cell-targeted RNA aptamer to facilitate endothelial healing in a clinically relevant large animal model.

Application of microphysiologic system to assess neutrophil extracellular trap in xenotransplantation

Transplantation of organs, cells, or tissues from one species to another, known as xenotransplantation, has the potential to alleviate organ donor shortages and enhance the success of organ transplantation. However, the possibility of immunological rejection by the recipient is one of the biggest difficulties associated with xenotransplantation. The creation of neutrophil extracellular traps (NETs), also known as NETosis, is hypothesized as a mechanism of rejection.Innovations in microfluidics and co-culturing techniques have provided access to several classes of microengineered model systems in experimental models, connecting animal research and traditional in vitro methods such as organoids, microphysiological systems, and organs-on-chip.To achieve this goal, we established a perfusable 3D Xeno vessel chip using a porcine aortic endothelial cell line and examined how NETs grow when isolated human and primate neutrophils were used. Neutrophils from both humans and monkeys displayed the usual NETosis phases, including nuclear decondensation, enlargement, and rounding of DNA, occupying the entire cytoplasm, and discharge of fragmented DNA after cell membrane rupture. Using confocal fluorescence imaging of DNA and citrullinated histone colocalization and DNA histone complex formation in supernatants from xeno vessel chips, we confirmed NETs generation by human and monkey neutrophils when cocultured in a xeno-vessel chip.

Human and porcine aortic valve endothelial and interstitial cell isolation and characterization.

Calcific aortic valve stenosis (AVS) is defined by pathological changes in the aortic valve (AV) and their predominant cell types: valvular interstitial (VICs) and endothelial cells (VECs). Understanding the cellular and molecular mechanisms of this disease is a prerequisite to identify potential pharmacological treatment strategies. In this study, we present a unique aortic valve cell isolation technique to acquire specific human and porcine cell populations and compared VICs and VECs of these species with each other for the first time. AV cells were isolated from tissue obtained from human patients undergoing surgical aortic valve replacement (SAVR) or from porcine hearts. Functional analysis and in vitro experiments revealed that endothelial-to-mesenchymal transition (EndMT) can be induced in hVECs, leading to a significant increase in mesenchymal markers. In vitro calcification experiments of VICs demonstrated pronounced expression of calcification markers and visible calcific deposits in Alizarin Red staining in both species after incubation with pro-calcific media. Cells isolated from patient-derived AVs showed mesenchymal and endothelial-specific gene signatures (VIC and VEC, respectively). For instance, von Willebrand factor (vWF) and platelet endothelial adhesion molecule-1 (PECAM1) were upregulated in VECs, while the myofibroblastic markers alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were downregulated in VECs compared to VICs. Analysis of cell function by migration revealed that VECs are more migratory than VICs. Induction of EndMT in vitro in VECs displayed increased expression of EndMT markers and decreased expression of endothelial markers, confirming their mesenchymal transdifferentiation ability. In vitro calcification of VICs revealed upregulation of alkaline phosphatase (ALPL), a hallmark of calcification. In addition, other calcification-related genes such as osteocalcin (BGLAP) and runt-related factor 2 (RUNX2) were upregulated. Alizarin red staining of calcified cells provided a further layer of confirmation that the isolated cells were VICs with osteoblastic differentiation capacity. This study aims to take a first step towards standardizing a reproducible isolation technique for specific human and porcine VEC and VIC populations. A comparison of human and porcine aortic valve cells demonstrated that porcine cells may serve as an alternative cellular model system in settings where human tissue is difficult to obtain.

[Embedded 3D printing of porous silicon orbital implants and its surface modification].

To prepare customized porous silicone orbital implants using embedded 3D printing and assess the effect of surface modification on the properties of the implants. The transparency, fluidity and rheological properties of the supporting media were tested to determine the optimal printing parameters of silicone. The morphological changes of silicone after modification were analyzed by scanning electron microscopy, and the hydrophilicity and hydrophobicity of silicone surface were evaluated by measuring the water contact angle. The compression modulus of porous silicone was measured using compression test. Porcine aortic endothelial cells (PAOECs) were co-cultured with porous silicone scaffolds for 1, 3 and 5 days to test the biocompatibility of silicone. The local inflammatory response to subcutaneous porous silicone implants was evaluated in rats. The optimal printing parameters of silicone orbital implants were determined as the following: supporting medium 4% (mass ratio), printing pressure 1.0 bar and printing speed 6 mm/s. Scanning electron microscopy showed that the silicone surface was successfully modified with polydopamine and collagen, which significantly improved hydrophilicity of the silicone surface (P < 0.05) without causing significant changes in the compression modulus (P > 0.05). The modified porous silicone scaffold had no obvious cytotoxicity and obviously promoted adhesion and proliferation of PAOECs (P < 0.05). In rats bearing the subcutaneous implants, no obvious inflammation was observed in the local tissue. Poprous silicone orbital implants with uniform pores can be prepared using embedded 3D printing technology, and surface modification obviously improves hydrophilicity and biocompatibility of the silicone implants for potential clinical application.

Resistin secreted by porcine alveolar macrophages leads to endothelial cell dysfunction during Haemophilus parasuis infection

ABSTRACT Haemophilus parasuis (H. parasuis) causes exudative inflammation, implying endothelial dysfunction during pathogen infection. However, so far, the molecular mechanism of endothelial dysfunction caused by H. parasuis has not been clarified. By using the transwell-based cell co-culture system, we demonstrate that knocking out resistin in porcine alveolar macrophages (PAMs) dramatically attenuated endothelial monolayer damage caused by H. parasuis. The resistin secreted by PAMs inhibited the expression of the tight junction proteins claudin-5 and occludin rather than the adherens junction protein VE-cadherin in co-cultured porcine aortic endothelial cells (PAECs). Furthermore, we demonstrate that resistin regulated claudin-5 and occludin expression and monolayer PAEC permeability in an LKB1/AMPK/mTOR pathway-dependent manner. Additionally, we reveal that the outer membrane lipoprotein gene lppA in H. parasuis induced resistin expression in PAMs, as deleting lppA reduced resistin expression in H. parasuis-infected PAMs, causing a significant change in LKB1/AMPK/mTOR pathway activity in co-cultured PAECs, thereby restoring tight junction protein levels and endothelial monolayer permeability. Thus, we postulate that the H. parasuis lppA gene enhances resistin production in PAMs, disrupting tight junctions in PAECs and causing endothelial barrier dysfunction. These findings elucidate the pathogenic mechanism of exudative inflammation caused by H. parasuis for the first time and provide a more profound angle of acute exudative inflammation caused by bacteria.

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 μM 3a or 0.5 μM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Abstract 14587: Dysregulation of Vascular Autophagy by Overexpression of p25/CDK5 is Associated With Vascular Senescence

Introduction: Cardiovascular diseases are the main causes of death worldwide. These diseases are often associated with cardiovascular aging. Autophagy is a degradative cellular process that has been implicated in aging regulation. Cyclin-dependent kinase 5 (CDK5), which is normally activated by its regulatory subunit p25, has been implicated in several diseases. However, the role of p25/CDK5 signaling pathway in cardiovascular diseases and autophagy have not been fully understood. Hypothesis: Upregulation of vascular p25/CDK5 signaling pathway modifies autophagy induction and participates in vascular senescence. Methods: Selective overexpression of p25 (CDK5 activator) in the vascular endothelium was generated in a transgenic mouse model (EC-p25). Then, aortas from 30-weeks old EC-p25 and control mice were collected. Vascular senescence was evaluated with senescence-associated β-galactosidase (SA-β-gal) staining. Protein levels were measured by Western blotting. Primary porcine aortic endothelial cells (PAECs) were transfected with pcDNA, p25 and CDK5. Autophagy activity, autophagic flux, cell death, and senescence were evaluated. A p-value of p&lt;0.05 was considered as statistically significant. Results: EC-p25 mice exhibited increased SA-β-gal staining compared to the Control mice suggesting accelerated aging in the aorta. EC-p25 mice showed increased levels of autophagy compared to the Control mice. On the other hand, overexpression of CDK5 increased cell death, endothelial senescence, autophagy and proteins involved in autophagy induction in PAECs. Conclusion: Accelerated premature vascular aging induced by selective overexpression of the p25/CDK5 signaling pathway in the vascular endothelium may be associated with increased vascular senescence in the aorta by modifying autophagy activity and induction process.

Recovery from cold-induced mitochondrial fission in endothelial cells requires reconditioning temperatures of ≥ 25◦C

Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37◦C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4◦C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10◦ or 15◦C. At 21◦C some re-fusion was visible, which became more marked at 25◦C. Networks of tubular mitochondria similar to control cells only re-appeared at 37◦C. ATP content decreased at 4◦C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/106 cells and decreased even further when rewarming cells to 10◦ and 15◦C. Values after rewarming at 21◦C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4◦C incubation and recovered with increasing temperatures to 36 ± 10% at 25◦C and 78 ± 17% at 37◦C. Integrity of monolayers, largely disturbed at 4◦C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15◦C upwards, complete recovery at 37◦C. Endothelial repair processes (scratch assay) at 25◦C were clearly inferior to those at 37◦C. These data suggest that reconditioning temperatures below 21◦C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25◦C appear required, with 30◦C being superior and 37◦C yielding the best results.

Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease.

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.

Effects of TolC on the pathogenicity of porcine extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a well-known critical pathogenic zoonosis that causes extraintestinal infections in humans and animals by affecting their immune organs. Recently, research on the outer membrane protein of E. coli, tolerant colicin (TolC), a virulent protein in the formation of the ExPEC efflux pump, has been an attractive subject. However, the pathogenic mechanisms remain unclear. This study aimed to explore the role of TolC in the pathogenesis of the ExPEC strain PPECC42; a complementation strain (Cm-TolC) and an isogenic mutant (ΔTolC) were constructed. Loss of TolC drastically impaired the virulence of ExPEC in an experimental mouse model. ΔTolC showed a substantial decrease in the porcine aortic vascular endothelial cell (PAVEC) adherence, invasion, and pro-inflammatory response, in contrast to that of the wild type, with a reduced survival ratio in both the bacterial load and whole blood in mice. ΔTolC also showed decreased expression of necroptosis signals such as receptor-interacting protein kinase 1, phosphorylated mixed-lineage kinase domain-like protein, and mitochondrial proteins such as phosphoglycerate mutase family member 5. Our data suggest that TolC is closely associated with ExPEC pathogenesis. These results provide scientific grounds for exploring the potential of TolC as an effective drug target for controlling ExPEC infection, screening new inhibitors, and developing new drugs. This will allow for further prevention and control of ExPEC infection.

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.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

Development and Preliminary Testing of Porcine Blood-Derived Endothelial-like Cells for Vascular Tissue Engineering Applications: Protocol Optimisation and Seeding of Decellularised Human Saphenous Veins.

Functional endothelial cells (EC) are a critical interface between blood vessels and the thrombogenic flowing blood. Disruption of this layer can lead to early thrombosis, inflammation, vessel restenosis, and, following coronary (CABG) or peripheral (PABG) artery bypass graft surgery, vein graft failure. Blood-derived ECs have shown potential for vascular tissue engineering applications. Here, we show the development and preliminary testing of a method for deriving porcine endothelial-like cells from blood obtained under clinical conditions for use in translational research. The derived cells show cobblestone morphology and expression of EC markers, similar to those seen in isolated porcine aortic ECs (PAEC), and when exposed to increasing shear stress, they remain viable and show mRNA expression of EC markers similar to PAEC. In addition, we confirm the feasibility of seeding endothelial-like cells onto a decellularised human vein scaffold with approximately 90% lumen coverage at lower passages, and show that increasing cell passage results in reduced endothelial coverage.

Porcine cytomegalovirus: A very unwelcome stowaway.

The announcement of the first pig-to-human heart transplantation early this year was the culmination of a very long and arduous path. The groundbreaking first life-sustaining pig-to man heart transplantation by the team of University of Maryland at Baltimore under the lead of Professor Griffith provided the long-awaited proof that it can work–even if survival was cut short by a hitherto still to be defined process. In a fascinating webinar, Professor Griffith described in detail the course of the patient.1 One potential element in the deterioration was the increase of porcine cytomegalovirus (PCMV) DNAemia, detected in the blood of the patient. The exact role of PCMV in the sequence of events is unclear and currently analyzed in detail, and will undoubtedly increase our knowledge. As a young researcher in the laboratory of Jay Fishman, I had the privilege to play a small role in early pig-to-baboon xenotransplantation experiments done in the Transplantation Biology Research Center at MGH, headed by Professor David Sachs. Organ- and recipient survival was short in the early 2000s, and reasons were explored on many different angles. From the experience in human-to-human solid organ transplantation, one avenue was to look for activation or primary infection of latent viruses, primarily from the herpesvirus family. PCMV had been identified as a potential zoonotic agent before.2 Using quantitative PCR assays, we demonstrated activation of PCMV and baboon cytomegalovirus (BCMV) in a series of pig-to primate organ kidney xenotransplantations. High copy numbers of PCMV DNAemia were detected in recipient baboon tissue and particularly in the transplanted porcine graft tissues.3, 4 In a model of primary porcine aortic endothelial cells infected in vitro with PCMV, an increase in porcine tissue factor was observed, and we hypothesized that this was a potential cofactor in the development of an often fatal consumptive coagulopathy observed in pig-to baboon kidney xenotransplantation.5 Using PCMV-free pig donors, which was achieved by early weaning of piglets, consumptive coagulopathy was prevented in pig-to-baboon heart transplantation, resulting in an improved graft survival.6 Given the many modifications in the protocols over time, it was not possible to establish PCMV as the sole cause of this coagulopathy. Nevertheless, at this point elimination of PCMV was the clear goal given the harmful potential of this virus.7 Eradication protocols were developed and successfully tested.8 This was all the more important, as ganciclovir failed to control PCMV activation.9 BCMV activation in the recipient baboons, on the other hand, by then was controlled by prophylactic use of ganciclovir in the majority of recipients and had become part of all protocols. The most comprehensive study on the role of PCMV was recently published by Denner et al. in 2020. In a series of orthotopic pig-to-baboon heart transplantations, transmission of PCMV was observed from PCMV-positive donor pigs in some but not all baboon recipients, whereas no transmission events occurred for a number of other potentially harmful latent porcine viruses tested, including porcine lymphotropic herpesviruses, and porcine endogenous retroviruses. In baboons transplanted with a heart from a PCMV-positive donor pig, survival was reduced, inflammatory cytokines such as IL-6 and TNF-α were increased and tPA-PAI-1 complexes circulated in high concentrations in the recipients, pointing to substantial alterations of the coagulation system. This detailed report provided additional evidence for the deleterious effects of PCMV, and reiterated the need for elimination of PCMV from the pig donor.10 Why then are PCMV-positive donor pigs still an issue? Breeding pathogen-free pigs was shown to be feasible, but the effort to achieve this is considerable. In a publication on virus prevalence in different minipig breeds in 2021, PCMV was still detected in some but not all breeds.11 Recent work did primarily focus on porcine endogenous retroviruses, as elimination of these viruses is incomparably more challenging than that of traditional latent viruses. Testing for elimination requires not only the use of PCR for detection PCMV DNAemia, but also by serology, as PCR can be negative in swabs from a healthy swine. Validated serological assays for PCMV are not readily available. Efforts did understandably concentrate on the genetic modifications of donor pigs. Given the potential unfavorable role in the first human case of orthotopic heart transplantation, the focus must be redirected to the elimination of PCMV. It is a feasible and necessary step. The amazing initial clinical course of the patient is a huge incentive to keep the momentum going and continue on this path.

Common Variation in EDN1 Regulatory Regions Highlights the Role of PPARγ as a Key Regulator of Endothelin in vitro.

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied.