RESUMO
A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a (Prkar1a(+/-)), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). Prkar1a(+/-) mice were crossed with mice that were heterozygous for catalytic subunit Calpha (Prkaca(+/-)), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a(+/-)Prkaca(+/-)). Unexpectedly, Prkar1a(+/-)Prkaca(+/-) mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-II) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic subunit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors.
Assuntos
Envelhecimento/patologia , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Animais , Osso e Ossos/diagnóstico por imagem , Calcificação Fisiológica , Domínio Catalítico , Heterozigoto , Mesoderma/metabolismo , Camundongos , Diester Fosfórico Hidrolases/metabolismo , Análise Espectral Raman , Células Estromais/enzimologia , Células Estromais/patologia , Tomografia Computadorizada por Raios XRESUMO
In animal models of acute lung injury (ALI), gene expression studies have focused on the acute phase of illness, with little emphasis on resolution. In this study, the acute phase of intratracheal lipopolysaccharide (IT LPS)-induced lung injury was similar in wild-type (WT) and recombinase-activating gene-1-deficient (Rag-1(-/-)) lymphocyte-deficient mice, but resolution was impaired and resolution-phase lung gene expression remained different from baseline only in Rag-1(-/-) mice. By focusing on groups of genes involved in similar biological processes (gene ontologies) pertinent to inflammation and the immune response, we identified 102 genes at days 4 and 10 after IT LPS with significantly different expression between WT and Rag-1(-/-) mice. After adoptive transfer of isolated CD4+CD25+Foxp3+ regulatory T cells (Tregs) to Rag-1(-/-) mice at the time of IT LPS, resolution was similar to that in WT mice. Of the 102 genes distinctly changed in either WT or Rag-1(-/-) mice from our 7 gene ontologies, 19 genes reverted from the Rag-1(-/-) to the WT pattern of expression after adoptive transfer of Tregs, implicating those 19 genes in Treg-mediated resolution of ALI.
Assuntos
Lesão Pulmonar Aguda/genética , Perfilação da Expressão Gênica , Pulmão/imunologia , Linfócitos T Reguladores/imunologia , Cicatrização/genética , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Transferência Adotiva , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes RAG-1 , Lipopolissacarídeos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/transplante , Fatores de TempoRESUMO
Alveolar enlargement, which is characteristic of bronchopulmonary dysplasia, congenital matrix disorders, and cigarette smoke-induced emphysema, is thought to result from enhanced inflammation and ensuing excessive matrix proteolysis. Although there is recent evidence that cell death and oxidative stress punctuate these diseases, the mechanistic link between abnormal lung extracellular matrix and alveolar enlargement is lacking. We hypothesized that the tight-skin (TSK) mouse, which harbors a spontaneous internal duplication in the microfibrillar glycoprotein fibrillin-1, might show whether matrix alterations are sufficient to promote oxidative stress and cell death, injury cascades central to the development of clinical emphysema. We observed no evidence of increased metalloprotease activation by histochemical and zymographic methods. We did find initial oxidative stress followed by increased apoptosis in the postnatal TSK lung. Both blunted antioxidant production and reduced extracellular superoxide dismutase activity were evident in the neonatal lung. High-dose antioxidant treatment with N-acetylcysteine improved airspace caliber and attenuated oxidative stress and apoptosis in neonatal and adult TSK mice. These data establish that an abnormal extracellular matrix without overt elastolysis is sufficient to confer susceptibility to postnatal normoxia, reminiscent of bronchopulmonary dysplasia. The resultant oxidative stress and apoptosis culminate in profound airspace enlargement. The TSK lung exemplifies the critical interplay between extracellular matrix, oxidative stress, and cell-death cascades that may contribute to genetic and acquired airspace enlargement.
Assuntos
Apoptose/fisiologia , Matriz Extracelular/patologia , Estresse Oxidativo/fisiologia , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Animais , Antioxidantes/metabolismo , Western Blotting , Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilinas , Perfilação da Expressão Gênica , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Mutantes , Proteínas dos Microfilamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Enfisema Pulmonar/genéticaRESUMO
OBJECTIVE: Scleromyxedema (SMX) is a rare systemic sclerosis mimic that often responds to intravenous immunoglobulin (IVIG) therapy, yet the resulting clinical and biochemical changes have not been well characterized. To better understand the pathogenesis of the disease and the efficacy of IVIG, we sought to explore whether IVIG would introduce a measurable biologic effect corresponding with clinical improvement. METHODS: Fifteen patients with SMX were recruited for the study. Clinical information and peripheral blood mononuclear cells for flow cytometry were obtained immediately before and again 1-2 weeks after patients received IVIG therapy. Ten patients also underwent skin biopsies for gene expression analysis both before and after IVIG therapy. Clinical data included measures of skin involvement (modification of the modified Rodnan skin thickness score [MMRSS] and percentage of body surface area) and several patient-reported outcome measures assessing patients' skin. RESULTS: Posttreatment, the average MMRSS score decreased from mean ± SD 13.6 ± 2.6 to 10.3 ± 1.9; P = 0.003. There were also significant improvements in skin flexibility (mean ± SD 5.4 ± 0.8 to 3.2 ± 0.7; P = 0.003) and softening (mean ± SD 4.9 ± 0.9 to 2.6 ± 0.6; P = 0.022). Baseline levels of Tc17 cells (CD8+CCR6+CXCR3+CCR4-) correlated with the extent of skin involvement as measured by MMRSS pretreatment (r = 0.69, P = 0.012) and decreased after IVIG therapy (mean ± SD 3.4% ± 3.2% to 1.3% ± 1.7%; P = 0.008). Posttreatment analysis of RNA in skin tissue revealed a decrease in gene expression of transforming growth factor ß (TGFß) cytokines as well as several interferon-inducible proteins. CONCLUSION: This open-label study further supports the evidence that patients with SMX respond both objectively and subjectively to IVIG therapy. Biologic studies suggest a role for T lymphocytes in the pathogenesis of the disease and reveal the potential significance of TGFß and interferon pathways.
Assuntos
Imunoglobulinas Intravenosas/uso terapêutico , Escleromixedema/imunologia , Adulto , Biomarcadores/metabolismo , Contagem de Linfócito CD4 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Escleromixedema/metabolismo , Escleromixedema/terapia , Pele/metabolismoRESUMO
In cancer cells, cyclic AMP-dependent protein kinase (PKA) is secreted into the conditioned medium. This PKA, designated as extracellular protein kinase A (ECPKA), is markedly up-regulated in the sera of patients with cancer. The currently available tumor markers are based on the antigen determination method and lack specificity and sensitivity. Here, we present an ECPKA autoantibody detection method for a universal biomarker that detects cancer of various cell types. We tested sera from 295 patients with cancers of various cell types, 155 normal controls, and 55 patients without cancer. The specificity and sensitivity of this autoantibody enzyme immunoassay method were compared with the conventional antigen determination method by receiver-operating characteristic plots. In the sera, the presence of autoantibody directed against ECPKA was highly correlated with cancer. High anti-ECPKA autoantibody titers (frequency, 90%; mean titer, 3.0) were found in the sera of patients with various cancers, whereas low or negative titers (frequency, 12%; mean titer, 1.0) were found in the control group. The receiver-operating characteristic plot showed that autoantibody enzyme immunoassay exhibited 90% sensitivity and 88% specificity, whereas the enzymatic assay exhibited 83% sensitivity and 80% specificity. These results show that the autoantibody method distinguished between patients with cancer and controls better than the antigen method could. Our results show that autoantibody ECPKA is a universal serum biomarker for cancers of various cell types.
Assuntos
Autoanticorpos/sangue , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Técnicas Imunoenzimáticas/métodos , Neoplasias/enzimologia , Neoplasias/imunologia , Anticorpos Anti-Idiotípicos/sangue , Humanos , Neoplasias/sangueRESUMO
In mice, the recreational drug (+/-)3,4-methylenedioxymethamphetamine [MDMA ("ecstasy")] produces a selective toxic effect on brain dopamine (DA) neurons. Using cDNA microarray technology in combination with an approach designed to facilitate recognition of relevant changes in gene expression, the present studies sought to identify genes potentially involved in murine MDMA-induced toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein (Mt)-1 and Mt2 emerged as candidate genes possibly involved in MDMA-induced toxicity to DA neurons. Northern blot analysis confirmed the microarray findings and revealed a dynamic upregulation of Mt1 and Mt2 mRNA in the ventral midbrain within 4-12 hr after MDMA treatment. Western blot analysis showed a similar increase in MT protein levels, with peak times occurring subsequent to increases in mRNA levels. Mt1-2 double knock-out mice were more vulnerable to MDMA-induced toxicity to DA neurons than corresponding wild-type mice. Stimulation of endogenous expression of MT protein with zinc acetate conferred complete protection against MDMA-induced toxicity to DA neurons, and administration of exogenous MT protein afforded partial protection. Collectively, these results indicate that MDMA-induced toxicity to DA neurons is associated with increased Mt1 and Mt2 gene transcription and translation, possibly as part of a neuroprotective mechanism. The present findings may have therapeutic implications for neuropathological conditions involving DA neurons.
Assuntos
3,4-Metilenodioxianfetamina/toxicidade , Encéfalo/efeitos dos fármacos , Cocaína/análogos & derivados , Drogas Ilícitas/toxicidade , Metalotioneína/biossíntese , Neurônios/efeitos dos fármacos , 3,4-Metilenodioxianfetamina/antagonistas & inibidores , Animais , Northern Blotting , Encéfalo/metabolismo , Cocaína/farmacologia , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Masculino , Metalotioneína/genética , Metalotioneína/farmacologia , Metanfetamina/toxicidade , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Fatores de TempoRESUMO
Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.
Assuntos
Proliferação de Células/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Apoptose , Medula Óssea/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Meios de Cultura/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-ß signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-ß signaling would protect against CS-induced lung injury. We first confirmed that TGF-ß signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-ß signaling in CS-exposed mice. Systemic administration of a TGF-ß-specific neutralizing antibody normalized TGF-ß signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-ß signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-ß signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-ß-targeted therapies for patients with COPD.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Fumar/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Losartan/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos AKR , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor Tipo 1 de Angiotensina/metabolismo , Mecânica Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoRESUMO
The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eµ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.
Assuntos
Biomassa , Genes myc , Animais , Imunoprecipitação da Cromatina , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology.
Assuntos
Envelhecimento/patologia , Homeostase , Pulmão/patologia , Pulmão/fisiopatologia , Animais , Linfócitos B/imunologia , Morte Celular , Imunoglobulinas/metabolismo , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Monócitos/imunologia , Estresse Oxidativo , FenótipoRESUMO
OBJECTIVE: To evaluate the impact of two different luteal phase support protocols on gene expression of extracellular matrix (ECM) proteins and adhesion molecules in the human endometrium. DESIGN: Eighty-four ECM protein and adhesion molecule genes were analyzed using array-based reverse-transcription polymerase chain reaction. SETTING: Academic hospital. PATIENT(S): Nine oocyte donors. INTERVENTION(S): Endometrial biopsies were obtained on the day of oocyte retrieval (group I) and 3-5 days later (group II) after randomization into 3 groups. Group IIa had no luteal phase support, group IIb had luteal support with micronized progesterone, and group IIc had luteal support with progesterone plus 17ß-estradiol. MAIN OUTCOME MEASURE(S): Gene expression profiles in relation to different types of luteal phase support protocols. RESULT(S): Compared with the day of retrieval (group I), 24 genes were significantly up-regulated (4 in group IIa, 14 in group IIb, 22 in group IIc) and 14 genes were down-regulated (5 in group IIa, 2 in group IIb, 10 in group IIc) on day 3-5 (P<.05). In the luteal support group, up- regulation occurred predominantly in genes encoding for matrix metallopeptidases (MMP10, MMP3, MMP9), integrins (ITGA3, ITGA5, ITGB3, ITGB4), and laminin (LAMB3). In contrast, the most significant suppression was documented in genes encoding for catenin-D2, collagen-11A1, and the laminins (LAMA1 and LAMA3). Significant changes between groups IIb and IIc were also observed in 9 genes. CONCLUSION(S): Luteal phase support following controlled ovarian stimulation has a profound impact on the ECM pathway targeted genes.
Assuntos
Moléculas de Adesão Celular/genética , Implantação do Embrião , Endométrio/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Fármacos para a Fertilidade Feminina/farmacologia , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Fase Luteal/efeitos dos fármacos , Adulto , Moléculas de Adesão Celular/metabolismo , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Endométrio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Humanos , Fase Luteal/genética , Fase Luteal/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Indução da Ovulação/métodos , Adulto JovemRESUMO
The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HV(T); 20 ml/kg) or low (LV(T); 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HV(T) induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HV(T) also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from HV(T)-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-beta(6)) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Água Extravascular Pulmonar/metabolismo , Glicoproteínas/metabolismo , Proteína C/metabolismo , Respiração Artificial/efeitos adversos , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/fisiopatologia , Animais , Líquido da Lavagem Broncoalveolar , Capilares/metabolismo , Permeabilidade Capilar/fisiologia , Receptor de Proteína C Endotelial , Expressão Gênica/fisiologia , Glicoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , Proteína C/genética , Circulação Pulmonar/fisiologia , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , Edema Pulmonar/fisiopatologia , Receptores de Superfície Celular , Estresse Mecânico , Quinases Associadas a rho/metabolismoRESUMO
We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n = 9), PAH-SSc (n = 10) patients, and healthy controls (n = 5) were generated using HG_U133A_2.0 GeneChips and were processed by the RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real-time polymerase chain reaction (PCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), which are known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology (GO) analysis revealed significant (Z(score) > 1.96) alterations in angiogenesis genes according to PAH severity: matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) were significantly upregulated in mild as compared with severe PAH and healthy controls, as confirmed by real-time PCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.
Assuntos
Predisposição Genética para Doença , Hipertensão Pulmonar/genética , Metaloproteinase 9 da Matriz/genética , Artéria Pulmonar , Escleroderma Sistêmico/genética , Fator A de Crescimento do Endotélio Vascular/genética , Idoso , Feminino , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Artéria Pulmonar/fisiopatologia , Escleroderma Sistêmico/fisiopatologia , Índice de Gravidade de Doença , Regulação para Cima/genéticaRESUMO
BACKGROUND: Myosin light chain kinase (MYLK) is a multifunctional protein involved in regulation of airway hyperreactivity and other activities relevant to asthma. OBJECTIVE: To determine the role of MYLK gene variants in asthma among African Caribbean and African American populations. METHODS: We performed association tests between single nucleotide polymorphisms (SNPs) in the MYLK gene and asthma susceptibility and total serum IgE concentrations in 2 independent, family-based populations of African descent. Previously we identified variants/haplotypes in MYLK that confer risk for sepsis and acute lung injury; we compared findings from our asthma populations to findings in the African American sepsis and acute lung injury groups. RESULTS: Significant associations between MYLK SNPs and asthma and total serum IgE concentrations were observed in the African Caribbean families: a promoter SNP (rs936170) in the smooth muscle form gave the strongest association (P = .009). A haplotype including rs936170 corresponding to the actin-binding activity of the nonmuscle and smooth muscle forms was negatively associated with asthma (eg, decreased risk) in both the American (P = .005) and Caribbean families (P = .004), and was the same haplotype that conferred risk for severe sepsis (P = .002). RNA expression studies on PBMCs and rs936170 suggested a significant decrease in MYLK expression among patients with asthma with this variant (P = .025). CONCLUSION: MYLK polymorphisms may function as a common genetic factor in clinically distinct diseases involving bronchial smooth muscle contraction and inflammation. CLINICAL IMPLICATIONS: Genetic variants in MYLK are significantly associated with both asthma and sepsis in populations of African ancestry.
Assuntos
Asma/genética , Predisposição Genética para Doença , Quinase de Cadeia Leve de Miosina/genética , Sepse/genética , Negro ou Afro-Americano/genética , População Negra/genética , Região do Caribe , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
BACKGROUND: The gene encoding acyloxyacyl hydroxylase (AOAH), an enzyme that hydrolyzes secondary fatty acyl chains of LPS, is localized on chromosome 7p14-p12, where evidence for linkage to total IgE (tIgE) concentrations and asthma has been previously reported. OBJECTIVE: We hypothesized that variants in AOAH are associated with asthma and related phenotypes. Because both AOAH and soluble CD14 respond to LPS, we tested for gene-gene interaction. METHODS: We investigated the association between 28 single nucleotide polymorphisms throughout the AOAH gene and asthma, concentrations of tIgE, the ratio of IL-13/IFN-gamma, and soluble CD14 levels among 125 African Caribbean, multiplex asthmatic pedigrees (n = 834). Real-time PCR was used to assess whether AOAH cDNA expression differed with AOAH genotype. RESULTS: Significant effects were observed for all 4 phenotypes and AOAH markers in 3 distinct regions (promoter, introns 1-6, and the intron 12/exon 13 boundary/intron 13 region) by means of single-marker and haplotype analyses, with the strongest evidence for a 2-single-nucleotide-polymorphism haplotype and log[tIgE] (P = .006). There was no difference in AOAH expression levels by AOAH genotype for any of the markers. Comparing genotypic distributions at both the AOAH marker rs2727831 and CD14(-260)C >T raises the possibility of gene-gene interaction (P = .006-.036). CONCLUSION: Our results indicate that polymorphisms in markers within the AOAH gene are associated with risk of asthma and associated quantitative traits (IgE and cytokine levels) among asthmatic subjects and their families in Barbados, and there is an interactive effect on tIgE and asthma concentrations between an AOAH marker and the functional CD14(-260)C >T polymorphism. CLINICAL IMPLICATIONS: AOAH is a novel innate immunity candidate gene associated with asthma and related phenotypes in an African ancestry population.