ABSTRACT
Little is known about the altered lipid metabolism-related transcriptional events occuring in sebaceous glands of patients with acne vulgaris. Peroxisome proliferator-activated receptor (PPAR)γ, a lipid-activated transcription factor, is implicated in differentiation and lipid metabolism of sebocytes. We have observed that PPARγ and its target genes, ADRP (adipose differentiation related protein) and PGAR (PPARγ angioprotein related protein) are expressed at lower levels in sebocytes from patients with acne than in those from healthy controls (HCs) Furthermore, endogenous PPARγ activator lipids such as arachidonic acid-derived keto-metabolites (e.g. 5KETE, 12KETE) are increased in acne-involved and nonacne-involved skin of patients with acne, compared with skin from healthy individuals. Our findings highlight the possible anti-inflammatory role of endogenous ligand-activated PPARγ signaling in human sebocyte biology, and suggest that modulating PPARγ- expression and thereby signaling might be a promising strategy for the clinical management of acne vulgaris.
Subject(s)
Acne Vulgaris/metabolism , PPAR gamma/metabolism , Sebaceous Glands/metabolism , Signal Transduction/physiology , Adult , Analysis of Variance , Angiopoietin-Like Protein 4 , Angiopoietins/metabolism , Case-Control Studies , Eicosanoids/metabolism , Female , Gene Expression Regulation , Humans , Immunohistochemistry , Male , Perilipin-2/metabolism , RNA, Messenger/metabolismABSTRACT
Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies.
Subject(s)
Cysteine Endopeptidases/genetics , Intracellular Signaling Peptides and Proteins/genetics , Liver/metabolism , Animals , Apoptosis , Cell Proliferation , Cyclin A/metabolism , Cyclin D1/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cysteine Endopeptidases/deficiency , Cysteine Endopeptidases/metabolism , Hepatectomy , Hepatocytes/cytology , Hepatocytes/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/metabolism , Lipid Metabolism , Liver/surgery , Liver Regeneration , Mice , Mice, Knockout , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Up-RegulationABSTRACT
Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.
Subject(s)
Carbon Monoxide/pharmacology , Cell Differentiation/drug effects , Heme Oxygenase-1/metabolism , Macrophages/drug effects , Membrane Proteins/metabolism , Myeloid Progenitor Cells/drug effects , Animals , Bone Marrow Transplantation , Carbon Monoxide/metabolism , Cell Lineage , Cell Proliferation , Chemokine CCL2/blood , Gases , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Heme Oxygenase-1/genetics , Humans , Interleukin-1alpha/blood , Lipopolysaccharide Receptors/metabolism , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/enzymology , Macrophages/immunology , Macrophages/transplantation , Membrane Proteins/genetics , Mice , Mice, Knockout , Myeloid Progenitor Cells/enzymology , Myeloid Progenitor Cells/immunology , Myeloid Progenitor Cells/transplantation , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Signal Transduction , Time Factors , U937 CellsABSTRACT
Ischemia/reperfusion injury and delayed graft function (DGF) following organ transplantation adversely affect graft function and survival. A large animal model has not been characterized. We developed a pig kidney allograft model of DGF and evaluated the cytoprotective effects of inhaled carbon monoxide (CO). We demonstrate that donor warm ischemia time is a critical determinant of DGF as evidenced by a transient (4-6 days) increase in serum creatinine and blood urea nitrogen following transplantation before returning to baseline. CO administered to recipients intraoperatively for 1 h restored kidney function more rapidly versus air-treated controls. CO reduced acute tubular necrosis, apoptosis, tissue factor expression and P-selectin expression and enhanced proliferative repair as measured by phosphorylation of retinol binding protein and histone H3. Gene microarray analyses with confirmatory PCR of biopsy specimens showed that CO blocked proinflammatory gene expression of MCP-1 and heat shock proteins. In vitro in pig renal epithelial cells, CO blocks anoxia-reoxygenation-induced cell death while promoting proliferation. This large animal model of DGF can be utilized for testing therapeutic strategies to reduce or prevent DGF in humans. The efficacy of CO on improving graft function posttransplant validates the model and offers a potentially important therapeutic strategy to improve transplant outcomes.
Subject(s)
Carbon Monoxide/therapeutic use , Delayed Graft Function/drug therapy , Kidney Transplantation/physiology , Animals , Carbon Monoxide/pharmacokinetics , Cell Death/drug effects , Cell Proliferation/drug effects , Female , Gene Expression Profiling , Graft Rejection/prevention & control , Kidney/metabolism , Kidney Tubular Necrosis, Acute/etiology , Kidney Tubular Necrosis, Acute/immunology , Reperfusion Injury/prevention & control , Swine , Tacrolimus/pharmacokineticsABSTRACT
Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.
Subject(s)
Peripheral Nervous System Diseases/chemically induced , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Ubiquitin/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Blotting, Western , Boronic Acids/pharmacology , Bortezomib , Cell Proliferation , Cell Survival , Dose-Response Relationship, Drug , Immunohistochemistry , Inhibitory Concentration 50 , Mice , Mice, Inbred BALB C , Oligopeptides/pharmacology , PC12 Cells , Peripheral Nervous System Diseases/enzymology , Peripheral Nervous System Diseases/metabolism , Proteasome Endopeptidase Complex/metabolism , Pyrazines/pharmacology , Rats , Ubiquitin/antagonists & inhibitorsABSTRACT
Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.
Subject(s)
Adenosine Triphosphatases/metabolism , Antigens, CD/metabolism , Apyrase/metabolism , Ovary/enzymology , Pyrophosphatases/metabolism , Testis/enzymology , Animals , Cell Membrane/enzymology , Endothelial Cells/enzymology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Smooth Muscle/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.
Subject(s)
Epithelial Cells/microbiology , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , Plasminogen Activator Inhibitor 1/biosynthesis , Up-Regulation , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Gastric Mucosa/pathology , Gene Deletion , Gene Expression Profiling , Humans , Male , Middle AgedABSTRACT
BACKGROUND: Growing evidence implicates the involvement of extracellular nucleotides in the regulation of platelet, leukocyte, endothelial cell (EC) and vascular smooth muscle cell (VSMC) phenotype and function. Within the quiescent vasculature, extracellular nucleotides are rapidly hydrolyzed by CD39, the dominant endothelial nucleoside triphosphate diphosphohydrolase (NTPDase-1). However, vascular CD39/NTPDase-1 activity is lost in EC activated by oxidative stress or proinflammatory mediators, and upon denudation of the endothelium following balloon injury. The consequent increase in extracellular nucleotide concentrations triggers signaling events leading to prothrombotic responses and increased VSMC proliferation. OBJECTIVES: To investigate the effect of overexpressed CD39/NTPDase-1 in injured aorta. METHODS: Using adenoviral-mediated gene transfer we expressed CD39/NTPDase-1 in mechanically denudated rat aortas. We measured intima formation by morphometry and VSMC proliferation by the [(3)H]-thymidine incorporation assay. RESULTS: Targeted expression of CD39 in injured vessels increased NTPDase activity (from 2.91 +/- 0.31 to 22.07 +/- 6.7 nmols Pi mg(-1) protein, 4 days after exposure to the adenovirus) and prevented the formation of neointima. The thickness of the intimal layer in injured aortas exposed to Ad-CD39 was 26.2 +/- 3.9 microm vs. 51.8 +/- 6.1 microm and 64.4 +/- 22.2 microm (P < 0.001) in vessels treated with Ad-beta-gal and saline, respectively. Moreover, targeted expression of CD39/NTPDase-1 caused a 70% (P < 0.01) decrease in proliferation of VSMC isolated from transduced rat aortas as compared with VSMC derived from control vessels. CONCLUSIONS: The presented data suggest that increasing CD39/NTPDase-1 activity in VSMC could represent a novel therapeutic approach for the prevention of stenosis associated with angioplasty and other occlusive vascular diseases.
Subject(s)
Angioplasty/adverse effects , Antigens, CD/physiology , Apyrase/physiology , Cell Proliferation , Myocytes, Smooth Muscle/cytology , Tunica Intima/cytology , Animals , Antigens, CD/genetics , Apyrase/genetics , Gene Expression Regulation , Humans , Male , Mice , Mice, Transgenic , Muscle, Smooth, Vascular/cytology , Rats , Rats, Inbred F344ABSTRACT
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.
Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins/therapeutic use , Endothelium, Vascular/physiology , Muscle, Smooth, Vascular/physiology , NF-kappa B/antagonists & inhibitors , Transplantation, Homologous/immunology , Animals , Anti-Inflammatory Agents/pharmacology , Carotid Arteries/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , NG-Nitroarginine Methyl Ester/pharmacology , Rats , Tumor Necrosis Factor alpha-Induced Protein 3ABSTRACT
Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.
Subject(s)
Adenosine Triphosphatases/metabolism , Antigens, CD/metabolism , Caveolae/enzymology , Placenta/enzymology , Receptors, Purinergic P2/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/ultrastructure , Antigens, CD/genetics , Antigens, CD/ultrastructure , Apyrase , Caveolae/ultrastructure , Caveolin 1 , Caveolins/genetics , Caveolins/metabolism , Caveolins/ultrastructure , Female , Gene Expression Regulation , Humans , Immunohistochemistry , Microscopy, Electron, Scanning/methods , Placenta/ultrastructure , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/ultrastructure , Receptors, Purinergic P2Y1 , Receptors, Purinergic P2Y2ABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/physiology , Antigens, CD/physiology , Cell Movement/physiology , Neovascularization, Physiologic/physiology , Acid Anhydride Hydrolases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/pharmacology , Animals , Antigens/analysis , Antigens, CD/analysis , Antigens, CD/genetics , Apyrase , Blood Vessels/chemistry , Blood Vessels/growth & development , Chemokine CCL2/pharmacology , Drug Synergism , Female , Genotype , Immunohistochemistry , Integrin beta3 , Macrophages/cytology , Macrophages/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Monocytes/cytology , Monocytes/drug effects , Mutation , Nucleoside-Triphosphatase , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Platelet Membrane Glycoproteins/analysis , Proteoglycans/analysis , Receptor Protein-Tyrosine Kinases/analysis , Receptor, Platelet-Derived Growth Factor beta/analysis , Receptors, Growth Factor/analysis , Receptors, Vascular Endothelial Growth Factor , Serotonin/pharmacologyABSTRACT
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.
Subject(s)
Hematopoietic Stem Cell Transplantation/adverse effects , Thrombosis/etiology , Thrombosis/physiopathology , Transplantation, Heterologous/adverse effects , Animals , Antibodies, Monoclonal/therapeutic use , Eptifibatide , Hematopoietic Stem Cell Transplantation/mortality , L-Lactate Dehydrogenase/blood , P-Selectin/immunology , Papio , Peptides/therapeutic use , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Swine , Thrombocytopenia/etiology , Transplantation, Heterologous/mortalityABSTRACT
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.
Subject(s)
Carbon Monoxide/metabolism , Graft Rejection/metabolism , Graft Rejection/prevention & control , Heart Transplantation/immunology , Heme Oxygenase (Decyclizing)/metabolism , Transplantation, Heterologous/immunology , Acute Disease , Animals , Apoptosis/immunology , Carbon Monoxide/administration & dosage , Carbon Monoxide/physiology , Cell Line , Cell Movement/immunology , Endothelium, Vascular/enzymology , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Environmental Exposure , Enzyme Activation/immunology , Graft Rejection/enzymology , Graft Rejection/pathology , Graft Survival/drug effects , Heart Transplantation/pathology , Heme Oxygenase (Decyclizing)/biosynthesis , Heme Oxygenase (Decyclizing)/physiology , Heme Oxygenase-1 , Macrophages/pathology , Male , Membrane Proteins , Mice , Mice, Inbred BALB C , Monocytes/pathology , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/immunology , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Platelet Aggregation/immunology , Rats , Rats, Inbred Lew , Thrombosis/pathology , Thrombosis/prevention & control , Transplantation, Heterologous/pathology , Up-Regulation/immunologySubject(s)
Apoptosis/immunology , Graft Survival/immunology , Heart Transplantation/immunology , Immunosuppression Therapy/methods , Transplantation, Heterologous/immunology , Acute Disease , Animals , Cricetinae , Cytotoxicity, Immunologic , Graft Survival/physiology , Heart Transplantation/physiology , Time Factors , Transplantation, Heterologous/physiologySubject(s)
Heart Transplantation/immunology , Membrane Proteins , Proteins/immunology , Th2 Cells/physiology , Transplantation, Heterologous/immunology , Animals , Cricetinae , Cyclosporine/therapeutic use , Immunity, Cellular , Immunosuppressive Agents/therapeutic use , Interleukin-10/metabolism , Interleukin-4/metabolism , Rats , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
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.
Subject(s)
Adenosine Triphosphatases , Adenoviridae/genetics , Antigens, CD/genetics , Genetic Vectors/immunology , Heart Transplantation/immunology , Transplantation, Heterologous/immunology , Animals , Apyrase/metabolism , Blotting, Western , Gene Transfer Techniques , Graft Survival/genetics , Guinea Pigs , Humans , Kinetics , Male , Rats , Transplantation, Heterologous/pathologyABSTRACT
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.
Subject(s)
Adenosine Triphosphatases , Adenosine Triphosphate/analogs & derivatives , Antigens, CD/metabolism , Apoptosis , Apyrase/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Adenosine Triphosphate/pharmacology , Adenoviridae/genetics , Adenoviridae/metabolism , Antigens, CD/genetics , Apyrase/genetics , Cell Nucleus/metabolism , Cells, Cultured , Culture Media, Conditioned , Culture Media, Serum-Free , E-Selectin/biosynthesis , E-Selectin/genetics , Endothelium, Vascular/drug effects , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Transfer Techniques , Hemostatics/pharmacology , Humans , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thrombin/pharmacology , Umbilical Veins , Weibel-Palade Bodies/metabolism , von Willebrand Factor/metabolismABSTRACT
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.
Subject(s)
Graft Enhancement, Immunologic , Graft Survival/immunology , Heart Transplantation/immunology , Immunization , Transplantation, Heterologous , Animals , Antibodies, Heterophile/biosynthesis , Antibody-Dependent Cell Cytotoxicity , Complement Activation , Cricetinae , Cyclosporine/pharmacology , Cytotoxicity, Immunologic , Exchange Transfusion, Whole Blood , Graft Enhancement, Immunologic/methods , Graft Rejection/immunology , Graft Rejection/pathology , Graft Survival/drug effects , Heart Transplantation/pathology , Immunization/methods , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Immunoglobulin G/classification , Immunohistochemistry , Immunosuppression Therapy/methods , Killer Cells, Natural/immunology , Macrophages/immunology , Mesocricetus , Monocytes/immunology , Rats , Rats, Inbred Lew , Transplantation, Heterologous/methodsABSTRACT
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.
Subject(s)
Adenosine Triphosphatases , Apyrase/chemistry , Bile Canaliculi/enzymology , Liver/enzymology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Antigens, CD/metabolism , Apyrase/isolation & purification , Apyrase/metabolism , Calcium/pharmacology , Chromatography, Agarose , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Kidney/enzymology , Kinetics , Microsomes/metabolism , Sodium Azide/pharmacology , Substrate Specificity , Swine , Time Factors , Tissue DistributionABSTRACT
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.