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1.
Purinergic Signal ; 12(3): 575-81, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27030122

RESUMO

Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers γH2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.


Assuntos
Trifosfato de Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Dano ao DNA/fisiologia , Células Endoteliais/metabolismo , Animais , Western Blotting , Células Endoteliais/patologia , Espaço Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley
2.
Am J Physiol Lung Cell Mol Physiol ; 308(10): L1046-57, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25820525

RESUMO

Endothelial cell (EC) dysfunction plays a role in the pathobiology of occlusive vasculopathy in pulmonary arterial hypertension (PAH). Purinergic signaling pathways, which consist of extracellular nucleotide and nucleoside-mediated cell signaling through specific receptors, are known to be important regulators of vascular tone and remodeling. Therefore, we hypothesized that abnormalities in the vascular purinergic microenvironment are associated with PAH. Enzymatic clearance is crucial to terminate unnecessary cell activation; one of the most abundantly expressed enzymes on the EC surface is E-NTPDase1/CD39, which hydrolyzes ATP and ADP to AMP. we used histological samples from patients and healthy donors, radioisotope-labeled substrates to measure ectoenzyme activity, and a variety of in vitro approaches to study the role of CD39 in PAH. Immunohistochemistry on human idiopathic PAH (IPAH) patients' lungs demonstrated that CD39 was significantly downregulated in the endothelium of diseased small arteries. Similarly, CD39 expression and activity were decreased in cultured pulmonary ECs from IPAH patients. Suppression of CD39 in vitro resulted in EC phenotypic switch that gave rise to apoptosis-resistant pulmonary arterial endothelial cells and promoted a microenvironment that induced vascular smooth muscle cell migration. we also identified that the ATP receptor P2Y11 is essential for ATP-mediated EC survival. Furthermore, we report that apelin, a known regulator of pulmonary vascular homeostasis, can potentiate the activity of CD39 both in vitro and in vivo. we conclude that sustained attenuation of CD39 activity through ATP accumulation is tightly linked to vascular dysfunction and remodeling in PAH and could represent a novel target for therapy.


Assuntos
Antígenos CD/biossíntese , Apirase/biossíntese , Hipertensão Pulmonar/enzimologia , Artéria Pulmonar/enzimologia , Remodelação Vascular , Trifosfato de Adenosina/metabolismo , Adulto , Apelina , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Hipertensão Pulmonar/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Artéria Pulmonar/patologia , Receptores Purinérgicos P2/metabolismo
3.
Am J Respir Cell Mol Biol ; 50(6): 1118-28, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24433082

RESUMO

Occlusive vasculopathy with intimal hyperplasia and plexogenic arteriopathy are severe histopathological changes characteristic of pulmonary arterial hypertension (PAH). Although a phenotypic switch in pulmonary endothelial cells (ECs) has been suggested to play a critical role in the formation of occlusive lesions, the pathobiology of this process is poorly understood. The goal of this study was to identify novel molecular mechanisms associated with EC dysfunction and PAH-associated bone morphogenetic protein receptor 2 (BMPR2) deficiency during PAH pathogenesis. A bioinfomatics approach, patient samples, and in vitro experiments were used. By combining a metaanalysis of human idiopathic PAH (iPAH)-associated gene-expression microarrays and a unique gene expression-profiling technique in rat endothelium, our bioinformatics approach revealed a PAH-associated dysregulation of genes involving chromatin organization, DNA metabolism, and repair. Our hypothesis that altered DNA repair and loss of genomic stability play a role in PAH was supported by in vitro assays where pulmonary ECs from patients with iPAH and BMPR2-deficient ECs were highly susceptible to DNA damage. Furthermore, we showed that BMPR2 expression is tightly linked to DNA damage control because excessive DNA damage leads to rapid down-regulation of BMPR2 expression. Moreover, we identified breast cancer 1 (BRCA1) as a novel target for BMPR2 signaling and a novel modulator of pulmonary EC homeostasis. We show here that BMPR2 signaling plays a critical role in the regulation of genomic integrity in pulmonary ECs via genes such as BRCA1. We propose that iPAH-associated EC dysfunction and genomic instability are mediated through BMPR2 deficiency-associated loss of DNA damage control.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Reparo do DNA , Hipertensão Pulmonar/genética , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Regulação para Baixo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hipertensão Pulmonar Primária Familiar , Expressão Gênica , Predisposição Genética para Doença , Humanos , Hipertensão Pulmonar/metabolismo , Pulmão/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Transdução de Sinais
4.
Mol Genet Genomic Med ; 3(4): 354-62, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26247051

RESUMO

The genetic basis of pulmonary arterial hypertension (PAH) among Finnish PAH patients is poorly understood. We adopted a novel-targeted next-generation sequencing (NGS) approach called Oligonucleotide-Selective Sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions, insertions, and deletions in seven genes associated with PAH (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3) from Finnish PAH patients. This study represents the first clinical study with OS-Seq technology on patients suffering from a rare genetic disorder. We analyzed DNA samples from 21 Finnish PAH patients, whose BMPR2 and ACVRL1 mutation status had been previously studied using Sanger sequencing. Our sequencing panel covered 100% of the targeted base pairs with >15× sequencing depth. Pathogenic base substitutions were identified in the BMPR2 gene in 29% of the Finnish PAH cases. Two of the pathogenic variant-positive patients had been previously tested negative using Sanger sequencing. No clinically significant variants were identified in the six other PAH genes. Our study validates the use of targeted OS-Seq for genetic diagnostics of PAH and revealed pathogenic variants that had been previously missed using Sanger sequencing.

5.
J Oral Microbiol ; 6: 25835, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25412607

RESUMO

BACKGROUND: We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. OBJECTIVE: To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic-anatomic findings linked to cardiovascular diseases. METHODS: Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009-2010. Of the autopsies, 10 (45.5%) were free of coronary artery disease (CAD), 7 (31.8%) had mild and 5 (22.7%) had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra) and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes). RESULTS: Of 22 cases, 14 (63.6%) were positive for endodontic and 8 (36.4%) for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035). CONCLUSIONS: Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

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