RESUMO
Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.
Assuntos
Genômica , Mitocôndrias/patologia , Voo Espacial , Estresse Fisiológico , Animais , Ritmo Circadiano , Matriz Extracelular/metabolismo , Humanos , Imunidade Inata , Metabolismo dos Lipídeos , Análise do Fluxo Metabólico , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Músculos/imunologia , Especificidade de Órgãos , Olfato/fisiologiaRESUMO
The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.
Assuntos
Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/terapia , Imunocompetência , Células-Tronco Pluripotentes Induzidas/imunologia , Pneumopatias/imunologia , Pneumopatias/terapia , Transplante de Células-Tronco , Animais , Células Endoteliais/transplante , Insuficiência Cardíaca/terapia , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Isquemia/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/transplante , Transplante Homólogo , alfa 1-Antitripsina/metabolismoRESUMO
Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.
Assuntos
Aorta/lesões , Artérias/lesões , Constrição Patológica/prevenção & controle , Ácido Dicloroacético/farmacologia , Ácido Dicloroacético/uso terapêutico , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Angioplastia com Balão/efeitos adversos , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Apoptose/efeitos dos fármacos , Artérias/efeitos dos fármacos , Artérias/patologia , Proliferação de Células/efeitos dos fármacos , Constrição Patológica/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/lesões , Vasos Coronários/patologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Hiperplasia/tratamento farmacológico , Hiperplasia/patologia , Artéria Ilíaca/efeitos dos fármacos , Artéria Ilíaca/lesões , Artéria Ilíaca/patologia , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/lesões , Artéria Torácica Interna/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Coelhos , Ratos , Prevenção Secundária , Stents/efeitos adversos , Suínos , Túnica Íntima/lesõesRESUMO
BACKGROUND: In acute respiratory distress syndrome (ARDS), pulmonary perfusion failure increases physiologic dead space ventilation (VD/VT), leading to a decline of the alveolar CO2 concentration [CO2]iA. Although it has been shown that alveolar hypocapnia contributes to formation of atelectasis and surfactant depletion, a typical complication in ARDS, the underlying mechanism has not been elucidated so far. METHODS: In isolated perfused rat lungs, cytosolic or mitochondrial Ca2+ concentrations ([Ca2+]cyt or [Ca2+]mito, respectively) of alveolar epithelial cells (AECs), surfactant secretion and the projected area of alveoli were quantified by real-time fluorescence or bright-field imaging (n=3-7 per group). In ventilated White New Zealand rabbits, the left pulmonary artery was ligated and the size of subpleural alveoli was measured by intravital microscopy (n=4 per group). Surfactant secretion was determined in the bronchoalveolar lavage (BAL) by western blot. RESULTS: Low [CO2]iA decreased [Ca2+]cyt and increased [Ca2+]mito in AECs, leading to reduction of Ca2+-dependent surfactant secretion, and alveolar ventilation in situ. Mitochondrial inhibition by ruthenium red or rotenone blocked these responses indicating that mitochondria are key players in CO2 sensing. Furthermore, ligature of the pulmonary artery of rabbits decreased alveolar ventilation, surfactant secretion and lung compliance in vivo. Addition of 5% CO2 to the inspiratory gas inhibited these responses. CONCLUSIONS: Accordingly, we provide evidence that alveolar hypocapnia leads to a Ca2+ shift from the cytosol into mitochondria. The subsequent decline of [Ca2+]cyt reduces surfactant secretion and thus regional ventilation in lung regions with high VD/VT. Additionally, the regional hypoventilation provoked by perfusion failure can be inhibited by inspiratory CO2 application.
Assuntos
Hipocapnia/etiologia , Mitocôndrias/fisiologia , Surfactantes Pulmonares/metabolismo , Síndrome do Desconforto Respiratório/etiologia , Volume de Ventilação Pulmonar/fisiologia , Animais , Modelos Animais de Doenças , Alvéolos Pulmonares/irrigação sanguínea , Ratos , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/fisiopatologiaRESUMO
OBJECTIVE: The main objective of this study was to define a role of sphingosine-1-phosphate receptor 1 (S1PR1) in the arterial injury response of a human artery. The hypotheses were tested that injury induces an expansion of S1PR1-positive cells and that these cells accumulate toward the lumen because they follow the sphingosine-1-phosphate gradient from arterial wall tissue (low) to plasma (high). METHODS: A humanized rat model was used in which denuded human internal mammary artery (IMA) was implanted into the position of the abdominal aorta of immunosuppressed Rowett nude rats. This injury model is characterized by medial as well as intimal hyperplasia, whereby intimal cells are of human origin. At 7, 14, and 28 days after implantation, grafts were harvested and processed for fluorescent immunostaining for S1PR1 and smooth muscle α-actin. Nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride. Using digitally reconstructed, complete cross sections of grafts, intimal and medial areas were measured, whereby the medial area had virtually been divided into an outer (toward adventitia) and inner (toward lumen) layer. The fraction of S1PR1-positive cells was determined in each layer by counting S1PR1-positive and S1PR1-negative cells. RESULTS: The fraction of S1PR1-postive cells in naive IMA is 58.9% ± 6.0% (mean ± standard deviation). At day 28 after implantation, 81.6% ± 4.4% of medial cells were scored S1PR1 positive (P < .01). At day 14, the ratio between S1PR1-positive and S1PR1-negative cells was significantly higher in the lumen-oriented inner layer (9.3 ± 2.1 vs 6.0 ± 1.0; P < .01). Cells appearing in the intima at day 7 and day 14 were almost all S1PR1 positive. At day 28, however, about one-third of intimal cells were scored S1PR1 negative. CONCLUSIONS: From these data, we conclude that denudation of IMA specifically induces the expansion of S1PR1-positive cells. Based on the nonrandom distribution of S1PR1-positive cells, we consider the possibility that much like lymphocytes, S1PR1-positive smooth muscle cells also use S1PR1 to recognize the sphingosine-1-phosphate gradient from tissue (low) to plasma (high) and so migrate out of the media toward the intima of the injured IMA.
Assuntos
Aorta Abdominal/cirurgia , Oclusão de Enxerto Vascular/metabolismo , Artéria Torácica Interna/transplante , Músculo Liso Vascular/transplante , Miócitos de Músculo Liso/transplante , Neointima , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/patologia , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Artéria Torácica Interna/metabolismo , Artéria Torácica Interna/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ratos Nus , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Fatores de TempoRESUMO
Cardiac allograft vasculopathy (CAV) affects approximately 30% of cardiac transplant patients at 5 years post-transplantation. To date, there are few CAV treatment or prevention options, none of which are highly effective. The aim of the study was to investigate the effect of thalidomide on the development of CAV. The effect of thalidomide treatment on chronic rejection was assessed in rat orthotopic aortic transplants in allogeneic F344 or syngeneic Lew rats (n = 6 per group). Animals were left untreated or received thalidomide for 30 days post-transplant, and evidence of graft CAV was determined by histology (trichrome and immunohistochemistry) and intragraft cytokine measurements. Animals that received thalidomide treatment post-transplant showed markedly reduced luminal obliteration, with concomitant rescue of smooth muscle cells (SMCs) in the aortic media of grafts. Thalidomide counteracted neointimal hyperplasia by preventing dedifferentiation of vascular SMCs. Measurement of intragraft cytokine levels after thalidomide treatment revealed downregulation of matrix metalloproteinase 8 and monocyte chemotactic protein 1, cytokines involved in tissue remodelling and inflammation, respectively. Importantly, no negative side effects of thalidomide were observed. Thalidomide treatment prevents CAV development in a rodent model and is therefore potentially useful in clinical applications to prevent post-transplant heart rejection.
Assuntos
Aorta Torácica/transplante , Doença da Artéria Coronariana/prevenção & controle , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Talidomida/uso terapêutico , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Doença Crônica , Doença da Artéria Coronariana/etiologia , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Rejeição de Enxerto/complicações , Imunossupressores/farmacologia , Linfócitos/efeitos dos fármacos , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Talidomida/farmacologia , Túnica Média/efeitos dos fármacosRESUMO
OBJECTIVE: Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication. APPROACH AND RESULTS: We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed. CONCLUSION: This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.
Assuntos
Anticorpos Antinucleares/farmacologia , Materiais Revestidos Biocompatíveis , Reestenose Coronária/prevenção & controle , Regulação da Expressão Gênica , Oclusão de Enxerto Vascular/prevenção & controle , MicroRNAs/genética , Animais , Proliferação de Células/efeitos dos fármacos , Reestenose Coronária/genética , Reestenose Coronária/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/ultraestrutura , Modelos Animais de Doenças , Stents Farmacológicos , Feminino , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/imunologia , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , Neointima/metabolismo , Neointima/patologia , Desenho de Prótese , Ratos , Ratos NusRESUMO
Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.
Assuntos
Brônquios/transplante , Bronquiolite Obliterante/etiologia , Traqueia/transplante , Transplante Heterólogo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Complexo CD3/metabolismo , Modelos Animais de Doenças , ELISPOT , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Humanos , Sistema Imunitário , Transplante de Fígado , Período Pós-Operatório , Distribuição Aleatória , Ratos , Ratos Endogâmicos BN , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Resultado do TratamentoRESUMO
Human parvovirus B19 (B19V) is a common pathogen in microvascular disease and cardiomyopathy, owing to infection of endothelial cells. B19V replication, however, is almost restricted to erythroid progenitor cells (ErPCs). Endothelial regeneration attributable to bone marrow-derived circulating angiogenic cells (CACs) is a prerequisite for organ function. Because of many similarities of ErPCs and CACs, we hypothesized that B19V is a perpetrator of impaired endogenous endothelial regeneration. B19V DNA and messenger RNA from endomyocardial biopsy specimens, bone marrow specimens, and circulating progenitor cells were quantified by polymerase chain reaction analysis. The highest B19V DNA concentrations were found in CD34(+)KDR(+) cells from 17 patients with chronic B19V-associated cardiomyopathy. B19V replication intermediates could be detected in nearly half of the patients. Furthermore, chronic B19V infection was associated with impaired endothelial regenerative capacity. B19V infection of CACs in vitro resulted in expression of transcripts encoding B19V proteins. The capsid protein VP1 was identified as a novel inducer of apoptosis, as were nonstructural proteins. Inhibition studies identified so-called death receptor signaling with activation of caspase-8 and caspase-10 to be responsible for apoptosis induction. B19V causally impaired endothelial regeneration with spreading of B19V in CACs in an animal model in vivo. We thus conclude that B19V infection and damage to CACs result in dysfunctional endogenous vascular repair, supporting the emergence of primary bone marrow disease with secondary end-organ damage.
Assuntos
Apoptose , Cardiomiopatias/complicações , Eritema Infeccioso/virologia , Células Precursoras Eritroides/virologia , Parvovirus B19 Humano/fisiologia , Adulto , Idoso , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Estudos de Casos e Controles , Caspase 10/genética , Caspase 10/metabolismo , Linhagem Celular , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Células Precursoras Eritroides/fisiologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Parvovirus B19 Humano/genética , Regeneração , Transdução de Sinais , Replicação ViralRESUMO
Mechanisms of restenosis in type 2 diabetes mellitus (T2DM) are incompletely elucidated, but advanced glycation end-product (AGE)-induced vascular remodeling likely contributes. We tested the hypothesis that AGE-related collagen cross-linking (ARCC) leads to increased downstream vascular resistance and altered in-stent hemodynamics, thereby promoting neointimal hyperplasia (NH) in T2DM. We proposed that decreasing ARCC with ALT-711 (Alagebrium) would mitigate this response. Abdominal aortic stents were implanted in Zucker lean (ZL), obese (ZO), and diabetic (ZD) rats. Blood flow, vessel diameter, and wall shear stress (WSS) were calculated after 21 days, and NH was quantified. Arterial segments (aorta, carotid, iliac, femoral, and arterioles) were harvested to detect ARCC and protein expression, including transforming growth factor-ß (TGF-ß) and receptor for AGEs (RAGE). Downstream resistance was elevated (60%), whereas flow and WSS were significantly decreased (44% and 56%) in ZD vs. ZL rats. NH was increased in ZO but not ZD rats. ALT-711 reduced ARCC and resistance (46%) in ZD rats while decreasing NH and producing similar in-stent WSS across groups. No consistent differences in RAGE or TGF-ß expression were observed in arterial segments. ALT-711 modified lectin-type oxidized LDL receptor 1 but not RAGE expression by cells on decellularized matrices. In conclusion, ALT-711 decreased ARCC, increased in-stent flow rate, and reduced NH in ZO and ZD rats through RAGE-independent pathways. The study supports an important role for AGE-induced remodeling within and downstream of stent implantation to promote enhanced NH in T2DM.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Diabetes Mellitus/metabolismo , Oclusão de Enxerto Vascular/metabolismo , Neointima/metabolismo , Obesidade/metabolismo , Stents , Estresse Mecânico , Tiazóis/farmacologia , Resistência Vascular/efeitos dos fármacos , Animais , Aorta Abdominal/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Masculino , Neointima/prevenção & controle , Ratos , Ratos Zucker , Receptor para Produtos Finais de Glicação Avançada/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Resistência ao Cisalhamento , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
RATIONALE: CD34+ transplantation in dilated cardiomyopathy was associated with short-term improvement in left ventricular ejection fraction and exercise tolerance. OBJECTIVE: We investigated long-term effects of intracoronary CD34+ cell transplantation in dilated cardiomyopathy and the relationship between intramyocardial cell homing and clinical response. METHODS AND RESULTS: Of 110 dilated cardiomyopathy patients, 55 were randomized to receive CD34+ stem cell transplantation (SC group) and 55 received no cell therapy (controls). In the SC group, CD34+ cells were mobilized by granulocyte colony-stimulating factor and collected via apheresis. Patients underwent myocardial scintigraphy and cells were injected in the artery supplying segments with the greatest perfusion defect. At baseline, 2 groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal B-type natriuretic peptide levels. At 5 years, stem cell therapy was associated with increased left ventricular ejection fraction (from 24.3 ± 6.5% to 30.0 ± 5.1%; P=0.02), increased 6-minute walk distance (from 344 ± 90 m to 477 ± 130 m; P<0.001), and decreased N-terminal B-type natriuretic peptide (from 2322 ± 1234 pg/mL to 1011 ± 893 pg/mL; P<0.01). Left ventricular ejection fraction improvement was more significant in patients with higher myocardial homing of injected cells. During follow-up, 27 (25%) patients died and 9 (8%) underwent heart transplantation. Of the 27 deaths, 13 were attributed to pump failure and 14 were attributed to sudden cardiac death. Total mortality was lower in the SC group (14%) than in controls (35%; P=0.01). The same was true of pump failure (5% vs. 18%; P=0.03), but not of sudden cardiac death (9% vs. 16%; P=0.39). CONCLUSIONS: Intracoronary stem cell transplantation may be associated with improved ventricular function, exercise tolerance, and long-term survival in patients with dilated cardiomyopathy. Higher intramyocardial homing is associated with better stem cell therapy response.
Assuntos
Antígenos CD34/metabolismo , Cardiomiopatia Dilatada/cirurgia , Miocárdio/patologia , Transplante de Células-Tronco , Células-Tronco/imunologia , Função Ventricular Esquerda , Biomarcadores/metabolismo , California , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/imunologia , Cardiomiopatia Dilatada/mortalidade , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Causas de Morte , Movimento Celular , Rastreamento de Células , Distribuição de Qui-Quadrado , Circulação Coronária , Ecocardiografia , Teste de Esforço , Tolerância ao Exercício , Feminino , Seguimentos , Humanos , Injeções Intra-Arteriais , Interleucina-6/sangue , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Imagem de Perfusão do Miocárdio , Miocárdio/imunologia , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Modelos de Riscos Proporcionais , Recuperação de Função Fisiológica , Eslovênia , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/mortalidade , Volume Sistólico , Texas , Fatores de Tempo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangueRESUMO
RATIONALE: The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1-positive (Islet-1(+)) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life. OBJECTIVE: We investigated the distribution and identity of Islet-1(+) cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction. METHODS AND RESULTS: Distribution of Islet-1(+) cells in adult heart was investigated using gene targeted mice with nuclear ß-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1(+) cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1(+) cells within the outflow tract expressed the cardiomyocyte marker α-actinin. Islet-1(+) cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1(+) cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts. CONCLUSIONS: Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1(+) cells to serve as cardiac progenitors.
Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Aorta/citologia , Aorta/metabolismo , Biomarcadores/metabolismo , Compostos Cromogênicos , Galactosídeos , Indóis , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Miocárdio/citologia , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , RNA Mensageiro/metabolismo , Nó Sinoatrial/citologiaRESUMO
BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in the development of CAV. METHODS: We used an established preclinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy nontransplant controls. RESULTS: We showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found nonsignificantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, nontransplant controls. CONCLUSIONS: This study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.
Assuntos
Biomarcadores , Transplante de Coração , Leucotrieno B4 , Animais , Transplante de Coração/efeitos adversos , Leucotrieno B4/sangue , Leucotrieno B4/metabolismo , Ratos , Masculino , Biomarcadores/metabolismo , Biomarcadores/sangue , Humanos , Modelos Animais de Doenças , Aloenxertos , Pessoa de Meia-Idade , Ratos Endogâmicos Lew , Feminino , Neointima/patologiaRESUMO
Allogeneic transplantation of pancreatic islets for patients with difficult-to-control diabetes mellitus is severely hampered by the requirement for continuous immunosuppression and its associated morbidity. We report that allogeneic transplantation of genetically engineered (B2M-/-, CIITA-/-, CD47+), primary, hypoimmune, pseudo-islets (p-islets) results in their engraftment into a fully immunocompetent, diabetic non-human primate wherein they provide stable endocrine function and enable insulin independence without inducing any detectable immune response in the absence of immunosuppression. Hypoimmune primary p-islets may provide a curative cell therapy for type 1 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Humanos , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/metabolismo , Primatas , Diabetes Mellitus Tipo 1/terapia , Transplante HomólogoRESUMO
Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M-/-CIITA-/-CD47+). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Transplante das Ilhotas Pancreáticas , Camundongos , Animais , Macaca mulatta , Antígeno CD47 , Rejeição de EnxertoRESUMO
Despite significant progress in the treatment of chronic lung allograft rejection, obliterative bronchiolitis (OB) remains the major limitation to long-term survival after lung transplantation. The use of animal models is critical to an understanding of the pathological mechanisms behind OB, and to develop therapeutic strategies for OB. For almost 20 years, the technique of heterotopic tracheal transplantation was the leading experimental model in OB research. To address the need for a more physiologic experimental setup, a variety of small animal models have been proposed during the past two decades, such as the orthotopic tracheal transplantation model or the intrapulmonary trachea implantation model. The recent introduction of the orthotopic lung transplantation model in the mouse fulfilled the criteria for a physiologic lung transplantation setup, and also presents the advantage of being genetically modifiable. Here we review the evolution of OB models and their applications, from their beginning to the rapidly emerging physiologic models of chronic lung allograft rejection.
Assuntos
Bronquiolite Obliterante/patologia , Transplante de Pulmão/efeitos adversos , Mucosa Respiratória/patologia , Animais , Bronquiolite Obliterante/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Transplante de Pulmão/imunologia , Transplante de Pulmão/métodos , Camundongos , Óxido Nítrico/metabolismo , Ratos , Mucosa Respiratória/imunologia , Traqueia/metabolismo , Traqueia/transplanteRESUMO
Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESC(KD)). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESC(KD) was 88%-99% reduced. T cell activation after hESC(KD) transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESC(KD) was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESC(KD) was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.
Assuntos
Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Rejeição de Enxerto/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Animais , Células-Tronco Embrionárias/citologia , Técnicas de Silenciamento de Genes/métodos , Rejeição de Enxerto/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Transplante HeterólogoRESUMO
Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M-/-CIITA-/- CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their Fc, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.
Assuntos
Células Endoteliais , Receptores de IgG , Humanos , Animais , Camundongos , Células Endoteliais/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Imunoglobulina G/genética , Citotoxicidade Celular Dependente de Anticorpos , Imunidade CelularRESUMO
Immune rejection of allogeneic cell therapeutics remains a major problem for immuno-oncology and regenerative medicine. Allogeneic cell products so far have inferior persistence and efficacy when compared with autologous alternatives. Engineering of hypoimmune cells may greatly improve their therapeutic benefit. We present a new class of agonistic immune checkpoint engagers that protect human leukocyte antigen (HLA)-depleted induced pluripotent stem cell-derived endothelial cells (iECs) from innate immune cells. Engagers with agonistic functionality to their inhibitory receptors TIM3 and SIRPα effectively protect engineered iECs from natural killer (NK) cell and macrophage killing. The SIRPα engager can be combined with truncated CD64 to generate fully immune evasive iECs capable of escaping allogeneic cellular and immunoglobulin G (IgG) antibody-mediated rejection. Synthetic immune checkpoint engagers have high target specificity and lack retrograde signaling in the engineered cells. This modular design allows for the exploitation of more inhibitory immune pathways for immune evasion and could contribute to the advancement of allogeneic cell therapeutics.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Endoteliais/metabolismo , Antígenos HLA , Células Matadoras Naturais , Imunidade InataRESUMO
Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.