RESUMO
RATIONALE: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension. OBJECTIVE: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. METHODS AND RESULTS: We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis. CONCLUSIONS: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Lesões das Artérias Carótidas/metabolismo , Comunicação Celular , Proliferação de Células , Células Endoteliais/metabolismo , Metabolismo Energético , Epigênese Genética , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Notch1/metabolismo , Adulto , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Montagem e Desmontagem da Cromatina , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Receptor Notch1/deficiência , Receptor Notch1/genética , Transdução de Sinais , Remodelação Vascular , Adulto JovemRESUMO
RATIONALE: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. OBJECTIVE: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. METHODS AND RESULTS: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. CONCLUSIONS: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Animais , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Transplante de Células-Tronco/métodosRESUMO
BACKGROUND: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. RESULTS: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to ß-adrenergic stimulation mediated via canonical ß1- and ß2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.
Assuntos
Células-Tronco Embrionárias/transplante , Insuficiência Cardíaca/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Miócitos Cardíacos/transplante , Engenharia Tecidual/métodos , Remodelação Ventricular/fisiologia , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Insuficiência Cardíaca/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Impressão Tridimensional , Ratos , Ratos NusRESUMO
The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
Assuntos
Biomarcadores Tumorais/sangue , Imageamento por Ressonância Magnética/métodos , Células-Tronco Pluripotentes/patologia , Teratoma/sangue , Teratoma/diagnóstico , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gadolínio , Coração/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Lentivirus/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/transplante , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Ratos Nus , Teratoma/irrigação sanguínea , Carga Tumoral/efeitos dos fármacosRESUMO
RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
Assuntos
Células-Tronco Embrionárias/transplante , Sobrevivência de Enxerto , Transplante de Coração/métodos , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/transplante , Músculos Papilares/transplante , Engenharia Tecidual/métodos , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Conexina 43/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/metabolismo , Transplante de Coração/efeitos adversos , Xenoenxertos , Humanos , Imunossupressores/farmacologia , Masculino , Contração Miocárdica , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Músculos Papilares/imunologia , Músculos Papilares/metabolismo , Músculos Papilares/patologia , Músculos Papilares/fisiopatologia , Ratos Nus , Ratos Sprague-Dawley , Volume Sistólico , Fatores de Tempo , TransfecçãoRESUMO
NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.
Assuntos
Citotoxicidade Imunológica/imunologia , Rejeição de Enxerto/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Sequência de Aminoácidos , Animais , Citotoxicidade Imunológica/genética , Rejeição de Enxerto/genética , Células Matadoras Naturais/imunologia , Lectinas Tipo C/metabolismo , Ligantes , Proteínas de Membrana/genética , Dados de Sequência Molecular , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Ratos , Receptores Imunológicos/metabolismo , Homologia de Sequência do Ácido Nucleico , Transplante HomólogoRESUMO
BACKGROUND: Although outcomes for small intestine transplantation (SIT) have improved in recent years, allograft rejection rates remain among the highest of solid organ grafts. The high load of commensal bacteria in the small intestine may contribute through activation of the toll-like receptor (TLR) pathway. In this study, we examine the participation of TLR4 in acute allograft rejection in an orthotopic mouse model of SIT. METHODS: Wild-type C57Bl/6 (H-2b) or TLR49(-/-) (H-2b) mice were transplanted with syngeneic (C57Bl/6), allogeneic (BALB/c; H-2d), or F1 (BALB/cxC57Bl/6; H-2d/b) vascularized, orthotopic small intestine grafts. Graft recipients were killed on days 2 to 6 posttransplant. Serum cytokines were measured by Luminex, and tissue was obtained for histology and quantitative real-time polymerase chain reaction. RESULTS: BALB/c grafts transplanted into C57Bl/6 recipients exhibited mixed inflammatory infiltrates, destruction of the mucosa, and significant apoptosis. TLR2 and TLR4 transcripts were modestly increased in syngeneic grafts on days 2 and 6 compared with native bowel, whereas TLR2 and TLR4 were significantly increased on days 2 and 6 in allogeneic grafts. Although fully mismatched and F1 grafts were rejected by C57Bl/6 recipients (mean survival time=8.2 and 9.3 days, respectively), graft survival was significantly prolonged in TLR4(-/-) recipients (mean survival time=10.6 and 14.3 days, respectively). Proinflammatory cytokines were markedly reduced in TLR4(-/-) graft recipients. CONCLUSIONS: Small intestine graft survival is prolonged in the absence of TLR4, suggesting that gut flora associated with the graft may augment alloimmune responses through TLR4. Thus, the TLR pathway may be a novel therapeutic target for improving SIT allograft survival.
Assuntos
Rejeição de Enxerto/imunologia , Intestino Delgado/transplante , Receptor 4 Toll-Like/imunologia , Animais , Citocinas/sangue , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/deficiência , Transplante Homólogo/imunologia , Transplante Homólogo/patologiaRESUMO
OBJECTIVE: Unloading of the rodent heart activates the fetal gene program, decreases peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARalpha-regulated gene expression (MCAD), and induces cardiomyocyte atrophy. NF-kappaB regulates the fetal gene program and PPARalpha-regulated gene expression during cardiac hypertrophy and induces atrophy in skeletal muscle. Our objective was to test the hypothesis that NF-kappaB is the regulator for activation of the fetal gene program, for downregulation of PPARalpha and PPARalpha-regulated gene expression, and for cardiomyocyte atrophy in the heart subjected to mechanical unloading. METHODS: Activation of the inhibitory kappa B kinase beta (IKKbeta)/NF-kappaB pathways were measured in the heterotopically transplanted rat heart using Western blotting of total and phospho-IKKbeta and using transcription factor ELISA's for the five members of the NF-kappaB family (p65 (Rel A), p105/p50, c-Rel, RelB, and p100/p52). In loss of function experiments, we transplanted hearts of p105/p50 knockout mice into wildtype mice and compared changes in gene expression and cardiomyocyte size with wildtype hearts transplanted into wildtype mice. RESULTS: Total and phospho-IKKbeta levels significantly increased in the transplanted heart seven days after surgery. The activation of IKKbeta was paralleled by increased DNA binding activity of p65 and p105/p50. Mechanical unloading induced myosin heavy chain beta expression and decreased cardiomyocyte size in hearts of both wildtype and p105/p050 knockout animals. In contrast, the downregulation of PPARalpha and MCAD was significantly attenuated or prevented in the hearts of p105/p50 knockout mice. CONCLUSIONS: The IKKbeta/p65/p50 pathway is activated in the unloaded rodent heart and a likely regulator for the downregulation of PPARalpha and PPARalpha-regulated gene expression.
Assuntos
Regulação para Baixo , Transplante de Coração , Miocárdio/metabolismo , NF-kappa B/genética , PPAR alfa/metabolismo , Animais , Fenômenos Biomecânicos , Tamanho Celular , DNA/metabolismo , Regulação da Expressão Gênica , Genes Controladores do Desenvolvimento , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Ligação Proteica , Ratos , Ratos WistarRESUMO
The mechanism for the decrease in cardiomyocyte size with mechanical unloading is unknown. The calpain system regulates cardiomyocyte atrophy. We obtained samples from failing human hearts at the time of implantation and explantation of a left ventricular assist device. For mechanical unloading, we also heterotopically transplanted rat or mouse hearts for 1 week. The effect of calpain inhibition on cardiac atrophy was assessed in transplanted hearts overexpressing calpastatin. We measured transcript levels of calpain 1 and 2 in the human and the rodent model, as well as calpain activity, a calpain-specific degradation product and cardiomyocyte size in the two rodent models. Mechanical unloading of the failing human heart significantly increased calpain 2 gene expression. Transcript levels of calpain 1 and 2, calpain activity and a calpain-specific degradation product all significantly increased in the unloaded rat heart. Unexpectedly, in hearts of animals overexpressing calpastatin, cardiomyocyte size also decreased. Mechanical unloading of the mammalian heart activates the calpain system, although other proteolytic systems may compensate for decreased calpain activity when calpastatin is overexpressed.
Assuntos
Calpaína/biossíntese , Insuficiência Cardíaca/metabolismo , Transplante de Coração , Coração Auxiliar , Miocárdio/metabolismo , Transplante Heterotópico , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Tamanho Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Fatores de TempoRESUMO
STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Fator de Transcrição STAT5/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Western Blotting , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Transcrição STAT5/deficiência , Transplante Homólogo/imunologiaRESUMO
JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-alpha-driven NF-kappaB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.
Assuntos
Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/farmacologia , Bases de Mannich/farmacologia , Animais , Linhagem Celular , Ciclosporina/farmacologia , Humanos , Interleucina-2/fisiologia , Janus Quinase 3 , Transplante de Rim , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Baço/citologia , Baço/transplante , Transplante HomólogoRESUMO
BACKGROUND: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity. METHODS/RESULTS: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. CONCLUSIONS: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.
Assuntos
Ciclosporina/toxicidade , Imunossupressores/toxicidade , Molécula 1 de Adesão Intercelular/genética , Rim/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Tionucleotídeos/farmacologia , Animais , Células Cultivadas , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Transplante HomólogoRESUMO
Binding of the P-, L-, and E-selectins to sialyl Lewis(x) (sLe(x)) retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. Whether the selectin inhibitor bimosiamose (BIMO; C(46)H(54)O(16) . 0.25 H(2)O [867.4 molecular weight]) inhibits the rejection process of kidney allografts in a rat model was examined. Rat recipients acutely rejected kidney allografts at a mean survival time of 8.8 +/- 0.75 d. An intravenous 7-d infusion by osmotic pump of 2.5, 5, 10, or 20 mg/kg BIMO extended kidney allograft survival to 11.5 +/- 2.2 d (P < 0.03), 25.4 +/- 11.4 d (P < 0.006), 37.4 +/- 13.6 d (P < 0.001), and 39.8 +/- 34.5 d (P < 0.01), respectively. Combination of BIMO with cyclosporine produced synergistic interactions, as documented by the combination index (CI) values of 0.34 to 0.43 (CI <1 is synergistic; CI = 1 is additive; and CI >1 is antagonistic). Similarly, BIMO interacted synergistically with sirolimus (CI = 0.64) and FTY720 (CI = 0.22). While the mechanism of immunosuppression was being analyzed, decreased infiltration of CD4(+), CD8(+), and macrophages on day 7 after grafting was observed. Multiple cytokines were also expressed, including IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-alpha, and IFN-gamma in kidney allografts on days 3, 5, and 7 after grafting, as measured by a ribonuclease protection assay. Furthermore, at similar time points, BIMO treatment reduced intragraft expression of P-selectin glycoprotein ligand-1, CX(3)CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines.
Assuntos
Quimiocinas/antagonistas & inibidores , Citocinas/antagonistas & inibidores , Sobrevivência de Enxerto/efeitos dos fármacos , Hexanos/farmacologia , Transplante de Rim , Rim/metabolismo , Manose/análogos & derivados , Manose/farmacologia , Selectinas/metabolismo , Animais , Ciclosporina/farmacologia , Sinergismo Farmacológico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/farmacologia , Rim/irrigação sanguínea , Rim/patologia , Masculino , Ratos , Ratos Endogâmicos , Traumatismo por Reperfusão/patologia , Sirolimo/farmacologiaRESUMO
BACKGROUND: Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1. An rat alloantigen. We examined the potential of alpha1h-RT1. An versus alpha2h-RT1. An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts. METHODS: The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1. An AAs. Allochimeric and wild-type (WT)-RT1. An proteins were generated in an Escherichia coli expression system. RESULTS: A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1. An protein; alpha2h148-179n-RT1.Ac protein was ineffective. However, subcutaneous injection of 100 mug WT-RT1. An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation. CONCLUSIONS: The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Antígenos de Histocompatibilidade/imunologia , Polimorfismo Genético , Doadores de Tecidos , Tolerância ao Transplante , Animais , Citocinas/biossíntese , Antígenos de Histocompatibilidade/genética , Masculino , Estrutura Terciária de Proteína , Ratos , Ratos Endogâmicos , Proteínas Recombinantes de Fusão/imunologia , Fatores de Tempo , Transplante HomólogoRESUMO
Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO3- and Cl-, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (+/+) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (+/+) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (+/+) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis.
Assuntos
Proteínas de Transporte/genética , Desoxicorticosterona/farmacologia , Rim/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Animais , Gasometria , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proteínas de Transporte/análise , Cloretos/sangue , Cloretos/urina , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Concentração de Íons de Hidrogênio , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Mineralocorticoides , Potássio/sangue , Potássio/urina , Sódio/sangue , Sódio/urina , Transportadores de SulfatoRESUMO
BACKGROUND: The various toxicities associated with the general immune suppression resulting from current clinical immunosuppressive therapies continue to plague transplant recipients as well as jeopardize allograft survival. METHODS: The present study utilized allochimeric class I MHC antigens (alpha1hu70-77-RT1.Aa) bearing only four donor RT1.Au polymorphic amino acids (a.a.; His70, Val73, Asn74, and Asn77) superimposed on the recipient RT1.Aa background to induce transplantation tolerance in the rat cardiac transplant model. RESULTS: Oral delivery of alpha1hu70-77-RT1.Aa protein alone (days 0-6) induced tolerance, as evidenced by inhibition of both acute and chronic rejection processes. Delivery of alpha1hu70-77-RT1.Aa with therapeutic doses of cyclosporine (CsA) also prevented chronic rejection, otherwise readily developed after treatment with CsA alone. A polymerase chain reaction (PCR)-based analysis showed that tolerant recipients had reduced numbers of interleukin (IL)-2/interferon (IFN)-gamma-producing T helper (Th)1 cells and elevated numbers of IL-4/IL-10-producing Th2 cells. Adoptive transfer experiments revealed that potent regulatory T cells mediated tolerance. The same T cells displayed diminished T cell receptor (TCR)-driven signaling via extracellular regulated kinase, AP-1, and NF-kappaB, as well as the common gamma-chain (gammac) cytokine-receptor-induced signaling by Janus kinase 3 (Jak3)/stimulators and activators of transcription Stat/5 pathways. Tolerance induction was prevented in vivo by inhibition of signal 2 by CTL4Ig or of signal 3 by either rapamycin, which disrupts the mammalian target of rapamycin, or AG490, which inhibits Jak3. Finally, partial or complete tyrosine phosphorylation of Zap70 was observed in alloantigen-specific T cell clones in response to tolerogenic versus immunogenic peptides, respectively. CONCLUSIONS: Tolerance induction by allochimeric proteins is achieved by partial TCR activation in the presence of signals 2 and 3, resulting in a skewed Th2 phenotype.
Assuntos
Antígenos de Diferenciação/imunologia , Citocinas/imunologia , Transplante de Coração/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade/imunologia , Imunoconjugados , Cadeias lambda de Imunoglobulina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Quimeras de Transplante , Abatacepte , Transferência Adotiva , Animais , Antígenos CD , Antígeno CTLA-4 , Quimioterapia Combinada , Terapia de Imunossupressão/métodos , Imunossupressores/uso terapêutico , Ativação Linfocitária , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos WF , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
Janus kinase 3 (Jak3) is a cytoplasmic tyrosine (Tyr) kinase associated with the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)) that is activated by multiple T-cell growth factors (TCGFs) such as IL-2, -4, and -7. Using human T cells, it was found that a recently discovered variant of the undecylprodigiosin family of antibiotics, PNU156804, previously shown to inhibit IL-2-induced cell proliferation, also blocks IL-2-mediated Jak3 auto-tyrosine phosphorylation, activation of Jak3 substrates signal transducers and activators of transcription (Stat) 5a and Stat5b, and extracellular regulated kinase 1 (Erk1) and Erk2 (p44/p42). Although PNU156804 displayed similar efficacy in blocking Jak3-dependent T-cell proliferation by IL-2, -4, -7, or -15, it was more than 2-fold less effective in blocking Jak2-mediated cell growth, its most homologous Jak family member. A 14-day alternate-day oral gavage with 40 to 120 mg/kg PNU156804 extended the survival of heart allografts in a dose-dependent fashion. In vivo, PNU156804 acted synergistically with the signal 1 inhibitor cyclosporine A (CsA) and additively with the signal 3 inhibitor rapamycin to block allograft rejection. It is concluded that inhibition of signal 3 alone by targeting Jak3 in combination with a signal 1 inhibitor provides a unique strategy to achieve potent immunosuppression.