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E Csizmadia

Publications and source records attributed to E Csizmadia.

At least 19 recordsLinked to original sources
PubMed2006

The universal NF-kappaB inhibitor a20 protects from transplant vasculopathy by differentially affecting apoptosis in endothelial and smooth muscle cells.

Transplant vasculopathy (TV) is an accelerated form of atherosclerosis resulting in chronic rejection of vascularized allografts. The causes of TV are multifactorial and integrate at the level of the vascular wall, leading to a phenotypic switch of endothelial cells (ECs) and smooth muscle cells (SMCs). A20 is a NF-kappaB-dependent stress response gene in ECs and SMCs with potent anti-inflammatory effect in both cell types through blockade of NF-kappaB. A20 expression in ECs and SMCs correlates with the absence of TV in rat kidney allografts and long-term functioning human kidney allografts. We demonstrate that A20 protects ECs from tumor necrosis factor, Fas, and natural killer cell-mediated apoptosis by inhibiting proteolytic cleavage of caspase 8. A20 also safeguards ECs from complement-mediated necrosis. Hence, effectively shutting down cell death pathways initiated by inflammatory and immune offenders associated with TV. In contrast, A20 sensitizes SMCs to cytokine and Fas-mediated apoptosis through a novel nitric oxide (NO)-dependent mechanism. The unexpected proapoptotic effect of A20 in SMCs translates in vivo by the regression of established neointimal carotid lesions following balloon angioplasty in rats. Antedating apoptosis of SMCs, expression of the inducible NO synthase increases in A20-expressing neointimal SMCs, corroborating the involvement of NO in causing the proapoptotic effect of A20 in SMCs. Combined anti-inflammatory and anti- or proapoptotic functions of A20 in ECs and SMCs respectively qualify the positive effect of A20 upon vascular remodeling and healing. We propose that A20-based therapies may be effective in prevention and treatment of TV.

Animals↗
PubMed2001

Disordered cellular migration and angiogenesis in cd39-null mice.

BACKGROUND: Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 is the major ectonucleotidase of endothelial cells and monocytes and catalyzes phosphohydrolysis of extracellular nucleoside diphosphates (NDP) and triphosphates (NTP, eg, ATP and UTP). Deletion of cd39 causes perturbations in the hydrolysis of NTP and NDP in the vasculature. Activation of P2 receptors appears to influence endothelial cell chemotactic and mitogenic responses in vitro. Therefore, aberrant regulation of nucleotide P2 receptors may influence angiogenesis in cd39-null mice. Methods and Results- In control mice, implanted Matrigel plugs containing growth factors were rapidly populated by monocyte/macrophages, endothelial cells, and pericytes, with the development of new vessels over days. In cd39-null mice, migrating cells were completely confined to the tissue-Matrigel interface in a clearly stratified manner. Absolute failure of new vessel ingrowth was consistently observed in the mutant mice. Linked to these findings, chemotaxis of cd39-null monocyte/macrophages to nucleotides was impaired in vitro. This abnormality was associated with desensitization of nucleotide receptor P2Y-mediated signaling pathways. CONCLUSIONS: Our findings demonstrate a role for NTPDase1 and phosphohydrolysis of extracellular nucleotides in the regulation of the cellular infiltration and new vessel growth in a model of angiogenesis.

Acid Anhydride Hydrolases↗
PubMed2001

Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation.

INTRODUCTION: Attempts to induce tolerance though mixed hematopoietic chimerism in the discordant pig-to-baboon xenotransplantation model are sometimes complicated by a potentially fatal thrombotic microangiopathy in the recipient baboons. This state develops immediately after the infusion of porcine mobilized peripheral blood leukocytes, containing progenitor cells (PBPC). In our study, we examined the interaction of infused porcine PBPC with recipient platelets in vivo in baboons and investigated the underlying mechanisms using an in vitro model. METHODS: Two naïve baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. The interaction of porcine and baboon PBPC with baboon platelets was investigated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for potential to induce platelet aggregation. Immunohistochemical staining was performed on platelet-leukocyte aggregates and potential inhibition of aggregation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/IIIa receptor antagonist), was tested. RESULTS: All baboons that received porcine PBPC rapidly developed marked thrombocytopenia (<20,000/microl), elevated serum lactate dehydrogenase (>1,500U/liter), schistocytosis, and platelet aggregates on blood smear. Three baboons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvasculature of lungs and kidneys. In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistological examination of these aggregates confirmed the incorporation of porcine leukocytes. Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition of eptifibatide, and modulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recognize adhesion receptors on activated platelets. Purified fractions (granulocytes, CD2+, and CD- cells) of porcine PBPC did not initiate aggregation, whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells, and/or platelets) to the purified fractions exacerbated the aggregation response. CONCLUSIONS: These data indicate that porcine PBPC mediate aggregation of baboon platelets. This process likely contributes to the thrombotic microangiopathy observed after PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the progenitor cells from porcine PBPC and/or treatment of baboons with eptifibatide may be beneficial.

Animals↗
PubMed2001

Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants.

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.

Acute Disease↗
PubMed2000

Recombinant adenoviral mediated CD39 gene transfer prolongs cardiac xenograft survival.

BACKGROUND: Extracellular ATP and ADP may be important mediators of vascular inflammation and thrombosis. Nucleoside triphosphate diphosphohydrolase (NTPDase or CD39) is a vascular ectoenzyme that hydrolyses ATP and ADP; however, this activity is lost during reperfusion injury. We show that the supplementation of NTPDase activity within xenograft vasculature using CD39 recombinant adenoviruses (AdCD39) has protective effects in vivo. METHODS: Recombinant adenoviruses containing human CD39 or beta-galactosidase (Adbeta-gal) encoding genes were constructed. Hartley guinea pig coronary arteries were perfused ex vivo with University of Wisconsin solution containing 10(9) plaque-forming units of the recombinant adenovirus. Infected grafts were then implanted in the abdomen of complement depleted Lewis rats. RESULTS: NTPDase activities decreased in all grafts within the first 24 hr and subsequently recovered only in those hearts infected with AdCD39. Immunohistological examination of AdCD39-infected grafts confirmed successful CD39 gene transfer into the endocardium and macrovasculature. Expression of CD39 modestly prolonged graft survival (90.2+/-5.4 hr, mean+/-SD, n=5) when compared with Adbeta-gal-infected grafts (67.4+/-5.4 hr, P<0.005) and perfusion controls (66.4+/-5.2 hr; P<0.005). CONCLUSIONS: Recombinant adenoviral infection can induce expression of CD39 within cardiac xenografts and provide survival benefits in vivo. Our data show that ex vivo infection by recombinant adenovirus vectors can result in vascular expression of a potential therapeutic agent.

Adenosine Triphosphatases↗
PubMed2000

Long-term survival of hamster hearts in presensitized rats.

We transplanted hamster hearts into rats that had been sensitized to hamster cardiac grafts 5 days earlier as a model for discordant xenotransplantation. Sensitized rats had high serum levels of elicited anti-donor IgM and IgG that caused hyperacute rejection. Transient complement inhibition with cobra venom factor (CVF) plus daily and continuing cyclosporin A (CyA) prevented hyperacute rejection. However, grafts underwent delayed xenograft rejection (DXR). DXR involved IgG and associated Ab-dependent cell-mediated rejection, because depletion of IgG or Ab-dependent cell-mediated rejection-associated effector cells prolonged graft survival and the serum-mediated Ab-dependent cell-mediated cytotoxicity in vitro. Blood exchange in combination with CVF/CyA treatment dramatically decreased the level of preexisting Abs, but DXR still occurred in association with the return of Abs. Splenectomy and cyclophosphamide acted synergistically to delay Ab return, and when combined with blood exchange/CVF/CyA facilitated long-term survival of grafts. These grafts survived in the presence of anti-donor IgM, IgG, and complement that precipitated rejection of naive hearts, indicating that accommodation (survival in the presence of anti-graft Abs and complement) had occurred. We attribute the long-term survival to the removal of preexisting anti-donor Abs and therapy that attenuated the rate of Ab return. Under such conditions, the surviving hearts showed expression in endothelial cells and smooth muscle cells of protective genes and an intragraft Th2 immune response. Th2 responses and protective genes are associated with resistance to IgM- and IgG-mediated, complement-dependent and -independent forms of rejection.

Animals↗
PubMed2000

Identification and characterization of a novel hepatic canalicular ATP diphosphohydrolase.

We have identified and characterized a novel ATP diphosphohydrolase (ATPDase) with features of E-type ATPases from porcine liver. Immunoblotting with a specific monoclonal antibody to this ectoenzyme revealed high expression in liver with lesser amounts in kidney and duodenum. This ATPDase was localized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium. In contrast, ATPDase/cd39 was detected in vascular endothelium and smooth muscle in these organs. We purified the putative ATPDase from liver by immunoaffinity techniques and obtained a heavily glycosylated protein with a molecular mass estimated at 75 kDa. This enzyme hydrolyzed all tri- and diphosphonucleosides but not AMP or diadenosine polyphosphates. There was an absolute requirement for divalent cations (Ca(2+) > Mg(2+)). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases. Kinetic parameters derived from purified preparations of hepatic ATPDase indicated V(max) of 8.5 units/mg of protein with apparent K(m) of 100 microM for both ATP or ADP as substrates. NH(2)-terminal amino acid sequencing revealed near 50% identity with rat liver lysosomal (Ca(2+)-Mg(2+))-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions that are distinct from the vascular ATPDase/cd39.

Adenosine↗
PubMed2000

CD39 modulates endothelial cell activation and apoptosis.

BACKGROUND: CD39 is the dominant vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that exerts major effects on platelet reactivity by the regulated hydrolysis of extracellular adenine nucleotides. The effects of NTPDases on endothelial cell (EC) activation and apoptosis remain unexplored. MATERIAL AND METHODS: Recombinant replication-deficient adenoviruses were constructed with human CD39 cDNA (rAdCD39) or the bacterial beta-galactosidase (rAdbetagal). RESULTS: Intact human umbilical vein EC cultures infected with rAdCD39 had substantial and stable increases in NTPDase biochemical activity (14.50 +/- 3.50 Pi nmole/well/min), when contrasted with noninfected cells (0.95 +/- 0.002) and rAdbetagal infected cells (1.01 +/- 0.02; p<0.005). Increased NTPDase activity efficiently inhibited immediate type 2Y purinergic receptor (P2Y)-mediated EC activation responses viz. von Willebrand factor secretion in response to extracellular ATP. In addition, CD39 up-regulation blocked ATP-induced translocation of the transcription nuclear factor (NF)-kappaB to the cell nucleus, and abrogated transcription of mRNA encoding E-selectin, and consequent protein synthesis. CD39 also decreased the extent of apoptosis triggered by putative type-2X purinergic (P2X7) receptors in response to high concentrations of extracellular ATP in vitro. CONCLUSION: These properties of CD39 indicate primary vascular protective effects with potential therapeutic applications.

Adenosine Triphosphatases↗
PubMed1999

Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells.

Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities. Suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could predispose to thrombosis and inflammation. To study the effects of suppression of CD39 synthesis, stable 25-base antisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured human endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P(i) min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 protein expression was also observed by Western blotting. Decreases in ATP diphosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endothelial cells. Rates of initial hydrolysis of extracellular ATP released from purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5.0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense transfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascular endothelial cells.

Adenosine Diphosphate↗
PubMed1999

CD39 as a caveolar-associated ectonucleotidase.

CD39 is a human lymphoid cell activation antigen, (also referred to E-ATPDase or apyrase) that hydrolyzes extracellular ATP and ADP. Although it has been widely studied, its physiological role, however, still remains unclear. This ectonucleotidase generally is said to be evenly distributed in the membrane of the cells. However, we observed that in cell types which possess caveolae, specialised membrane invaginations involved in signalling, CD39 is preferentially targeted to these membrane microdomains. Since all molecules involved in signalling (eNOS, G-proteins, receptors) which are targeted to the caveolae undergo posttranslational modifications (e.g., palmitoylation) we hypothesize the same to be the case for CD39. Furthermore, its presence in the caveolae supports its participation in signalling events.

Adenosine Triphosphatases↗
PubMed1999

Accommodated xenografts survive in the presence of anti-donor antibodies and complement that precipitate rejection of naive xenografts.

Hamster hearts transplanted into transiently complement-depleted and continuously cyclosporin A (CyA)-immunosuppressed rats survive long-term despite deposition of anti-donor IgM Abs and complement on the graft vascular endothelium. This phenomenon is referred to as "accommodation." The hypothesis tested here is that accommodated xenografts are resistant to IgM Abs and complement that could result in rejection of naive xenografts. After first hamster hearts had been surviving in cobra venom factor (CVF) + CyA-treated rats for 10 days, a time when the anti-donor IgM Ab level was maximal and complement activity had returned to approximately 50% of pretreatment levels, naive hamster hearts or hamster hearts that had been accommodating in another rat for 14 days were transplanted into those rats carrying the surviving first graft. The naive hearts were all hyperacutely rejected. In contrast, a majority of regrafted accommodating hearts survived long-term. There was widespread Ab and activated complement deposition on the vascular endothelium of accommodating first hearts, second accommodating hearts, and rejected second naive hearts. However, only the rejected naive hearts showed extensive endothelial cell damage, myocardial necrosis, fibrin deposition, and other signs of inflammation. Accommodating first and second hearts but not rejected second naive hearts expressed high levels of the protective genes A20, heme oxygenase-1 (HO-1), bcl-2, and bcl-xL. These data demonstrate that accommodated xenografts become resistant to effects of anti-donor IgM Abs and complement that normally mediate rejection of xenografts. We hypothesize that this resistance involves expression by accommodated xenografts of protective genes.

Adoptive Transfer↗
PubMed1999

Rejection of cardiac xenografts by CD4+ or CD8+ T cells.

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 +/- 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 +/- 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 +/- 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 +/- 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 +/- 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 +/- 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.

Adoptive Transfer↗
PubMed1999

Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

Adenosine Triphosphatases↗
PubMed1999

Modulation of nucleoside [correction of nucleotide] triphosphate diphosphohydrolase-1 (NTPDase-1)cd39 in xenograft rejection.

BACKGROUND: There is increasing evidence showing that extracellular nucleosides [corrected] may be important mediators of vascular inflammation. Nucleoside [corrected] triphosphate diphosphohydrolase-1 (NTPDase-1, identical to CD39), the major vascular endothelial ectonucleotidase, is responsible for the hydrolysis of both extracellular ATP and ADP in the blood plasma to AMP. Studies were therefore conducted to evaluate the role of vascular NTPDase-1/cd39 in modulating platelet activation and vascular injury in cardiac xenografts. MATERIALS AND METHODS: Cardiac xenografts from both wild-type and cd39 knockout mice (C57BL/6 x 129 Svj) were transplanted into Lewis rats. Alterations in cd39 mRNA transcripts and NTPDase activity expression were evaluated in wild-type grafts in untreated rats and then following complement depletion and immunosuppression. Rejection responses were studied with both mutant and wild-type grafts in the following models: presensitization with or without complement depletion, complement depletion alone, and with chronic immunosuppression to induce long-term graft survival. RESULTS: NTPDase biochemical activity in wild-type xenografts rapidly decreased after transplantation but soon rebounded with graft survival. Elevated levels of cd39 mRNA with associated increases in NTPDase activity were observed in all long-term surviving wild-type grafts. Hyperacute xenograft rejection times were comparable in wild-type and mutant grafts but cd39-deficient grafts were subject to more rapid rejection and exhibited pronounced vascular injury in complement-depleted, presensitized rats. The cd39-deficient grafts in immunosuppressed recipients were subject to increased intravascular platelet sequestration and fibrin deposition; this resulted in focal myocardial infarction in long-term surviving mutant xenografts. CONCLUSIONS: Augmentation of NTPDase-1 activity may be an important adaptive response for graft survival. Our results suggest that NTPDase-1/cd39 influences pathways of vascular injury in cardiac xenografts.

Adenosine Triphosphatases↗
PubMed1998

Expression of heme oxygenase-1 can determine cardiac xenograft survival.

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

Animals↗