RESUMO
Fluorogenic sequencing is a sequencing-by-synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement- arily matched terminal phosphate-labeled fluorogenic nucleotides into the DNA template and release halogen-fluorescein as the reporter. This red-emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35-base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal-to-background performance.
Assuntos
Fluoresceína/química , Corantes Fluorescentes/química , Halogênios/química , Nucleotídeos/química , Análise de Sequência de DNA/métodos , DNA/química , DNA/genética , Cinética , Nucleotídeos/genética , Espectrometria de Fluorescência , Coloração e RotulagemRESUMO
OBJECTIVE: Inflammation plays a central role in atherosclerosis. However, the detailed changes in the composition and quantity of leukocytes in the arterial wall during atherogenesis are not fully understood in part because of the lack of suitable methods and animal models. METHODS AND RESULTS: We developed a 10-fluorochrome, 13-parameter flow cytometry method to quantitate 7 major leukocyte subsets in a single digested arterial wall sample. Apolipoprotein E-deficient mice underwent left carotid artery (LCA) partial ligation and were fed a high-fat diet for 4 to 28 days. Monocyte/macrophages, dendritic cells, granulocytes, natural killer cells, and CD4 T cells significantly infiltrated the LCA as early as 4 days. Monocyte/macrophages and dendritic cells decreased between 7 and 14 days, whereas T-cell numbers remained steady. Leukocyte numbers peaked at 7 days, preceding atheroma formation at 14 days. B cells entered LCA by 14 days. Control right carotid and sham-ligated LCAs showed no significant infiltrates. Polymerase chain reaction and ELISA arrays showed that expression of proinflammatory cytokines and chemokines peaked at 7 and 14 days postligation, respectively. CONCLUSION: This is the first quantitative description of leukocyte number and composition over the life span of murine atherosclerosis. These results show that disturbed flow induces rapid and dynamic leukocyte accumulation in the arterial wall during the initiation and progression of atherosclerosis.
Assuntos
Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/imunologia , Citometria de Fluxo , Imunofenotipagem/métodos , Leucócitos/imunologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Artérias Carótidas/fisiopatologia , Artérias Carótidas/cirurgia , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/fisiopatologia , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Imunidade Inata , Mediadores da Inflamação/metabolismo , Contagem de Leucócitos , Leucócitos/classificação , Ligadura , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Fluxo Sanguíneo Regional , Fatores de TempoRESUMO
Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.
Assuntos
Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/genética , Artéria Carótida Primitiva/fisiologia , Mecanorreceptores/fisiologia , Animais , Doenças das Artérias Carótidas/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Endotélio/fisiologia , Perfilação da Expressão Gênica , Hemorreologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.
Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Inflamação/fisiopatologia , MicroRNAs/metabolismo , Estresse Mecânico , Estresse Fisiológico/fisiologia , Regulação para Cima/fisiologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , Monócitos/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , Veias Umbilicais/fisiopatologiaRESUMO
A high-throughput microfluidic device is developed to handle liquid dispensation in nanoliter range. The dispenser system shows no cross-contamination between the microwells, indicating its great potential in large-scale screening experiments. An array of 115 nl PCR reactions, as well as the single channel addressable chip demonstrate the high flexibility and wide applications of this novel system.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/economia , Plasmídeos/genética , Reação em Cadeia da Polimerase/economia , Tamanho da AmostraRESUMO
In atherosclerosis, plaques preferentially develop in arterial regions of disturbed blood flow (d-flow), which alters endothelial gene expression and function. Here, we determined that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase-dependent (DNMT-dependent) manner. Induction of d-flow by partial carotid ligation surgery in a murine model induced DNMT1 in arterial endothelium. In cultured endothelial cells, DNMT1 was enhanced by oscillatory shear stress (OS), and reduction of DNMT with either the inhibitor 5-aza-2'-deoxycytidine (5Aza) or siRNA markedly reduced OS-induced endothelial inflammation. Moreover, administration of 5Aza reduced lesion formation in 2 mouse models of atherosclerosis. Using both reduced representation bisulfite sequencing (RRBS) and microarray, we determined that d-flow in the carotid artery resulted in hypermethylation within the promoters of 11 mechanosensitive genes and that 5Aza treatment restored normal methylation patterns. Of the identified genes, HoxA5 and Klf3 encode transcription factors that contain cAMP response elements, suggesting that the methylation status of these loci could serve as a mechanosensitive master switch in gene expression. Together, our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.
Assuntos
Aterosclerose/genética , Aterosclerose/metabolismo , Metilação de DNA , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/fisiopatologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Decitabina , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/fisiopatologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fluxo Sanguíneo Regional , Estresse Mecânico , Fatores de TranscriçãoRESUMO
Epigenetic regulation plays an important role in cell migration. Although many methods have been developed to measure the motility of mammalian cells, accurate quantitative assessments of the migration speed of individual cells remain a major challenge. It is difficult for conventional scratch assays to differentiate proliferation from migration during the so-called wound-healing processes because of the long experimental time required. In addition, it is also challenging to create identical conditions for evaluating cell migration by conventional methods. We developed a microfluidic device with precisely created blanks allowing for robust and reproducible cell migration inside accurately-controlled microenvironments to study the regulatory effect of the epigenetic regulator histone deacetylase 7 (HDAC7) on cell migration. Through analyzing time-lapse imaging of the cells migrating into individual blank regions, we can measure the migration speed parameter for human primary cells within a few hours, eliminating the confounding effect of cell proliferation. We also developed an automatic image analysis and a numeric model-based data fitting to set up an integrated cell migration analysis system at single-cell resolution. Using this system, we measured the motility of primary human umbilical vein endothelial cells (HUVECs) and the migration speed reduction due to the silencing of HDAC7 and various other genes. We showed that the migration behaviour of these human primary cells are clearly regulated by epigenetic mechanisms, demonstrating the great potential of this accurate and robust assay in the fields of quantitatively migration studies and high-throughput screening.