Primary Porcine Aortic Endothelial Cells Research Articles

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<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.

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.

Abstract 12268: Overexpression of Endothelial p25 Causes Vascular Remodeling by Inducing Premature Aging

Introduction: Cardiovascular diseases are the leading cause of disability and death worldwide. The cyclin-dependent kinase 5 (CDK5) signaling pathway has been implicated in the regulation of SIRT1, a mammalian longevity regulator involved in the cellular homeostasis and aging. However, the participation of p25, the regulatory subunit of CDK5 in cardiovascular diseases has not been fully understood. Hypothesis: Selective overexpression of p25 in the vascular endothelium leads to vascular remodeling by accelerating vascular aging. Methods: A transgenic mouse model with selective overexpression of p25 in endothelial cells (EC-p25) was established. Aortas were collected from 12- or 30-weeks old EC-p25 and control mice. Vascular tone response, vascular senescence, aortic stiffness and aortic wall hypertrophy were evaluated with wire myography, senescence-associated β-galactosidase (SA-β-gal), alizarin red and heamatoxylin-eosine staining respectively. Protein levels were measured by Western blotting. Primary porcine aortic endothelial cells (PAECs) were transfected with p25, Sirt1 and Sirt1 lacking deacetylase activity, cell death and senescence were evaluated. Sirt1 activity was measured in EC-p25 and Control mice. A p-value p<0.05 was considered as statistically significant. Results: Young EC-p25 mice exhibited increased vascular tone compared to the control mice. Alizarin red staining was more abundant in the aorta of 30 weeks old EC-p25 compared with the control mice, whereas SA-β-gal staining and aortic wall thickness were increased in EC-p25 mice. Overexpression of p25 in PAECs induced endothelial senescence and cell death. EC-p25 mice showed increased Sirt1 activity levels. Western blot analysis demonstrated that SIRT1 protein was fragmented in aorta of EC-p25 mice with increased levels of shorter fragments of Sirt1. PAECs transfected with Sirt1 mutant exhibited increased senescence. Conclusion: Selective overexpression of p25 in the vascular endothelium may regulate vascular remodeling by increasing vascular tone, vascular and endothelial senescence, aortic stiffness and aortic wall hypertrophy possible by modulating Sirt1 activity and integrity in the aorta of EC-p25 mice, accelerating premature vascular aging.

The Cell-specific Engagement of Notch and Wnt Pathways in Calcific Aortic Valve Disease

Objective: The Notch and Wnt pathways participate in calcific aortic valve disease (CAVD), but with conflicting findings to date. We hypothesize that Notch and Wnt pathway engagement during CAVD initiation are cell-type specific. Methods: Primary porcine aortic valvular endothelial (PAVEC) and/or interstitial (PAVIC) cells were cultured in a 3D spring mechanical constraint system or in 2D culture and challenged with osteogenic media (OGM) to induce calcification. DAPT and IMR-1 were used as Notch1 inhibitors, and lentiviral transduction was used to create constitutively-active b-catenin (B-cat+) PAVIC and doxycycline-inducible Notch1 intracellular domain (NICD+) PAVEC and PAVIC. Calcification was evaluated with alizarin red staining. Results: PAVIC treatment with Notch inhibitors DAPT and IMR-1 resulted in opposite calcific responses, and this correlated with active-form b-catenin signaling. Further interrogation using B-cat+ PAVIC showed increased calcification compared to wild-type (WT) cells. Treatment of B-cat+ PAVIC with DAPT, but not IMR-1, was able to reduce OGM-induced calcification to different extents in B-cat+ and WT cells. NICD overexpression was investigated in PAVIC and PAVEC. Culture of NICD+ PAVIC in 3D decreased calcification compared to controls, while NICD+ PAVIC and PAVEC co-culture increased calcification. Gene expression data in 2D NICD+ VEC found no difference in EndMT SNAI1/2 expression, and an increase in osteogenic genes Runx2, BMP2, SPARC, and SPP1 vs. WT controls. Conclusions: We demonstrate a divergence in Notch1 regulation of calcification with PAVIC DAPT and IMR-1 treatment with and without constitutively active b-catenin, and with NICD+ PAVIC monoculture vs. NICD+ PAVIC and PAVEC co-culture. Given our previous findings demonstrating early upregulation and later downregulation of Notch1 in VEC with TNFa-stimulus, our results are consistent with the hypothesis that Notch1 signaling has a dynamic and cell-specific influence over CAVD progression. Nuances in Notch1 and Wnt signaling could be vital to development of a CAVD therapeutic.

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the "natural" anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging.

BackgroundHendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.MethodsA range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy.ResultsSignificant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions.ConclusionThese findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.

Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.

Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non-Galα(1,3)Gal (non-Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized GalTKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non-Gal pig antigens using primary porcine aortic endothelial cells (pAECs) and cell culture-adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (GalTKO) swine. The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture-adapted GalTKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non-Gal antibodies in human and baboon serum. We have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non-Gal antibody responses.

RNA interference of GGTA1 physiological and immune functions in immortalized porcine aortic endothelial cells

BackgroundPig organs are commonly used in xenotransplantation, and α-1,3-galactose has been shown to be the main cause of hyperacute rejection. The development of transgenic pigs that lack α-1,3-galactosyltransferase (GGTA1) has overcome this problem to a certain extent, but transgenic pigs are difficult to maintain, making their usefulness in basic research limited. For this reason, we propose to establish a cell model to study hyperacute rejection. MethodsImmortalized primary porcine aortic endothelial cells were transfected with a short hairpin RNA targeted to GGTA1. Cell proliferation, apoptosis, complement C3 activation, and the binding of human immunoglobulins and components of the complement system, including IgM, IgG, C3, and C5b-9, were examined. ResultsAfter RNA interference, GGTA1 was found to be reduced at both the transcript and protein level as assessed by quantitative polymerase chain reaction and flow cytometry, respectively. When cultured in the presence of human serum, the proliferation rate of the transfected cells was higher than that of untransfected cells, and the apoptosis rate was lower. Additionally, activation of C3 and the binding of human immunoglobulins IgM and IgG and complement component C3 and C5b-9 to the transfected cells were lower than in the immortalized group but higher than in untransfected cells. ConclusionsRNA interference of GGTA1 in cultured porcine endothelial cells reduces the reaction of immunoglobulin and complement system with the cells. Therefore, this in vitro cell model could be useful for further study of xenotransplantation.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.

An Energy-Sensor Network Takes Center Stage During Endothelial Aging

See related article, pages 1384–1393 Blood vessels form a critical interface between the environment and the organism by supplying nutrients, oxygen and macromolecules to all tissues and cells in the body. Endothelial cells (ECs) line the inner surface of the hierarchically branched blood vessel network and integrate functionally into different organs to support tissue growth and function in development, physiology, and disease. In healthy postnatal tissues, ECs are mostly quiescent and rarely migrate or proliferate. However, ECs possess a remarkable phenotypic plasticity in terms of being able to proliferate rapidly in response to vascular injury, tissue ischemia, or other stress conditions. This phenotypic plasticity progressively declines when ECs age and/or are permanently exposed to metabolic (eg, hyperglycemia) and environmental stressors (eg, oxidative stress) and coincides with the development of endothelial senescence.1 Cellular senescence is a stress response that is characterized by a robust inhibition of cell proliferation, which often becomes irreversible and independent of the initial stress signal.2 Senescence can be observed in most mammalian cells after extended propagation in culture and is related to the attrition of telomeres after repetitive cycles of cell divisions.2 In addition to telomere shortening, other stressors such as DNA damage, genomic instability, and oxidative stress can induce a similar growth arrest and trigger senescence.2 Senescent cells display characteristic alterations in cell morphology and gene expression that may weaken cellular functions. In ECs, these changes cause a phenotype that is proinflammatory, prothrombotic, and proatherosclerotic, which also negatively affects the vasodilatory, angiogenic, and regenerative properties of ECs and, thus, accelerates the development of several cardiovascular diseases.1 Despite advances in understanding the molecular mechanisms of endothelial senescence and vascular aging, the signaling networks governing the development and progression of EC senescence remain less well defined. In this issue of Circulation Research …

Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

SIRT1 Promotes Proliferation and Prevents Senescence Through Targeting LKB1 in Primary Porcine Aortic Endothelial Cells

Endothelial senescence causes endothelial dysfunction, promotes atherogenesis and contributes to age-related vascular disorders. SIRT1 is a conserved NAD(+)-dependent deacetylase possessing beneficial effects against aging-related diseases, despite that the detailed functional mechanisms are largely uncharacterized. The present study is designed to evaluate the protective effects of SIRT1 on endothelial senescence and to elucidate the underlying mechanisms. An in vitro senescence model was established by prolonged culture of primary endothelial cells isolated from porcine aorta. The freshly isolated "young" cells gradually underwent senescence during 1 month of repetitive passages. Both mRNA and protein expressions of SIRT1 were progressively decreased. In contrast, the protein levels of LKB1, a serine/threonine kinase and tumor suppressor, and the phosphorylation of its downstream target AMPK(Thr172) were dramatically increased in senescent cells. Overexpression of LKB1 promoted cellular senescence and retarded endothelial proliferation, which could be blocked by increasing SIRT1 levels. Knocking down of SIRT1 induced senescence and elevated the protein levels of LKB1 and phosphorylated AMPK(Thr172). Regardless of the nutritional status, hyperactivation of AMPK was able to induce endothelial senescence. SIRT1 antagonized LKB1-dependent AMPK activation through promoting the deacetylation, ubiquitination and proteasome-mediated degradation of LKB1. The survival signaling of Akt was also found to be modulated by SIRT1 and LKB1, and could cross-regulate AMPK activity. SIRT1 and LKB1/AMPK are the 2 key sensor systems for regulating endothelial cell survival, proliferation and senescence. The protective activities of SIRT1 may be achieved at least in part by fine tuning the acetylation/deacetylation status and stabilities of LKB1 protein.

Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin

BackgroundHeparan sulfate proteoglycans (HSPGs) contain glycosaminoglycan (GAG) chains made primarily of heparan sulfate (HS). Hyperglycemia in diabetes leads to endothelial injury and nephropathy, retinopathy and atherosclerosis. Decreased HSPG may contribute to diabetic endothelial injury. Decreased tissue HS in diabetes has been reported, however, endothelial HS changes are poorly studied.ObjectiveTo determine total GAGs, including HS, in endothelium under hyperglycemic conditions and the protective effect of insulin and heparin.MethodsConfluent primary porcine aortic endothelial cells (PAECs) were divided into control, glucose (30 mM), insulin (0.01 unit/ml) and glucose plus insulin treatment groups for 24, 48 and 72 hours. Additionally, PAECs were treated with glucose, heparin (0.5 μg/ml) and glucose plus heparin for 72 hours. GAGs were isolated from cells and medium. GAG concentrations were determined by the carbazole assay and agarose gel electrophoresis.ResultsGAGs were significantly increased only in control and glucose plus insulin groups at 72 versus 24 hours. Glucose decreased cell GAGs and increased medium GAGs, and insulin alone decreased cell GAGs at all times compared to control. In the glucose plus insulin group, cell GAGs were less than control at 24 hours, and greater than glucose or insulin alone at 48 and 72 hours while GAGs in medium were greater than control at all times and glucose at 72 hours. Heparin increased GAGs in glucose treated cells and medium.ConclusionHigh glucose and insulin alone reduces endothelial GAGs. In hyperglycemic conditions, heparin or insulin preserves GAGs which may protect cells from injury. Insulin is an effective diabetic therapy since it not only lowers blood glucose, but also protects endothelium.

Characterization of porcine UL16‐binding protein 1 endothelial cell surface expression

Natural killer (NK) cells participate in the immune response against solid organ allo- and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL-16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell-surface expression on primary porcine aortic endothelial cells (PAEC). A monoclonal antibody (mAb; aE5-63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). Neither human nor porcine IFN-gamma stimulation changed pULBP1 expression, whereas both human and porcine TNF-alpha stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti-pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. In conclusion, (i) the novel anti-pULBP1 IgM mAb aE5-63 represents a useful tool to study pULBP1/hNKG2D-mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human-pig cross-species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.

Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels

The interactions between endothelial cells and the underlying extracellular matrix regulate adhesion and cellular responses to microenvironmental stimuli, including flow-induced shear stress. In this study, we investigated the adhesion properties of primary porcine aortic endothelial cells (PAECs) and valve endothelial cells (PAVECs) in a microfluidic network. Taking advantage of the parallel arrangement of the microchannels, we compared adhesion of PAECs and PAVECs to fibronectin and type I collagen, two prominent extracellular matrix proteins, over a broad range of concentrations. Cell spreading was measured morphologically, based on cytoplasmic staining with a vital dye, while adhesion strength was characterized by the number of cells attached after application of shear stresses of 11, 110, and 220 dyn cm(-2). Results showed that PAVECs were more well spread on fibronectin than on type I collagen (P < 0.0001), particularly for coating concentrations of 100, 200, and 500 microg mL(-1). PAVECs also withstood shear significantly better on fibronectin than on collagen for 500 microg mL(-1). PAECs were more well spread on collagen compared to PAVECs (P < 0.0001), but did not have significantly better adhesion strength. These results demonstrate that cell adhesion is both cell-type and matrix dependent. Furthermore, they reveal important phenotypic differences between vascular and valvular endothelium, with implications for endothelial mechanobiology and the design of microdevices and engineered tissues.

ATM/ATR-related checkpoint signals mediate arsenite-induced G2/M arrest in primary aortic endothelial cells

Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular disease. Our recent in vitro studies have linked this vascular damage to vascular endothelial dysfunction induced by arsenic exposure. However, cell-cycle arrest induced by arsenic and its involvement in vascular dysfunction remain to be clarified. In this study, we employed primary porcine aortic endothelial cells to investigate regulatory mechanisms of G2/M phase arrest induced by arsenite. Our study revealed that lower concentrations of arsenite (1 and 3 microM) increased cell proliferation, whereas higher concentrations of arsenite (10, 20, and 30 microM) inhibited cell proliferation together with correlated increases in G2/M phase arrest. We found that this arsenite-induced G2/M phase arrest was accompanied by accumulation and/or phosphorylation of checkpoint-related molecules, including p53, Cdc25B, Cdc25C, and securin. Inhibition of activations of these checkpoint-related molecules by caffeine significantly attenuated the 30-microM arsenite-induced G2/M phase arrest by 93%. Our data suggest that the DNA damage responsive kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) play critical roles in arsenite-induced G2/M phase arrest in aortic endothelial cells possibly via regulation of checkpoint-related signaling molecules including p53, Cdc25B, Cdc25C, and securin.

Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12

Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques. Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis. Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.

Endothelial Cells Derived from Pigs Lacking Gal??(1,3)Gal: No Reduction of Human Leukocyte Adhesion and Natural Killer Cell Cytotoxicity

The expression of galactose-alpha(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal-/- pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal-/- and Gal+/+ pigs, and (2) the susceptibility of such Gal-/- porcine endothelial cells to human natural killer (NK) cell cytotoxicity. Primary porcine aortic endothelial cells (PAEC) were isolated from Gal-/- (PAEC-Gal-/-) and Gal (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal-/- cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal-/- and PAEC-Gal+/+ was detected. Tumor-necrosis factor-alpha stimulation of PAEC-Gal-/- and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal-/- expression on PAEC-Gal cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2-activated NK cells. Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

Cell-mediated cytotoxicity to porcine aortic endothelial cells is not dependent on galactosyl residues when baboon peripheral blood lymphocytes are previously primed with pig xenoantigens.

In the pig-to-baboon model, acute vascular rejection remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knockout pigs could solve concomitantly the problem of hyperacute and acute vascular rejection. This work studies in vitro the cell-mediated cytotoxicity of natural killer (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. PBLs from naive and primed baboons were used as effectors on primary porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated or galactosidase-digested PAECs were used to evidence the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T+NK cells (LO-CD2b) or NK cells alone (LO-CD94). When using PBLs from naive animals, spontaneous lysis occurred and was inhibited by both LOCD-2b and LO-CD94. In comparison, lysis of PAECs was significantly higher when baboon PBLs were first primed in vivo with pig xenoantigens. In this case, cytotoxicity was completely inhibited by LO-CD2b but only partially by LO-CD94. Reduction of galactosyl residues by galactosidase digestion showed that PAEC lysis almost completely disappeared with naive baboon PBLs but not with primed baboon PBLs, thereby indicating that anti-pig T-cell response is not dependent on galactosyl residues. Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.

Novel employment of lactate dehydrogenase release from porcine aortic endothelial cells (PAEC) as a quantitative marker of cytotoxic activity in thermophilic Campylobacter spp. from human faecal isolates, poultry and environmental sources.

The aim of this study was to employ a novel cytotoxicity assay based on primary porcine aortic endothelial cells in combination with a lactate dehydrogenase release assay to quantitatively determine differences in cytotoxin production between Campylobacter jejuni, C. coli, C. lari and urease-positive thermophilic campylobacters (UPTC), isolated from human faeces, animals and environmental sources. Campylobacter isolates totalling 34 and comprising of C. jejuni (n = 24) C. coli (n = 5) and UPTC (n = 4) and C. lari (n = 1) were analysed. The cytotoxic response ranged from 32.15 to 64.47% and 33.08 to 59.41%, for C. jejuni from chicken and human isolates, respectively and there was no statistically significant difference (P > 0.05) in cytotoxic response between C. jejuni isolated from humans and chicken isolates (50.78% versus 50.55% cytotoxicity, respectively). However, there was a difference in response between C. jejuni and C. coli isolated from chickens (50.78% versus 33.22% cytotoxicity, respectively). The greatest cytotoxic response was obtained with the UPTC group of organisms examined (n = 4 isolates) (mean cytotoxic response = 57.11% cytotoxicity. Employment of this cytotoxin assay may help identify virulent strains in poultry that could potentially proceed to cause clinical problems for humans and thus intervention measures targeted at the reduction or elimination of such specific strains, may be sought.