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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. IntroductionAtherosclerosis is an inflammatory disease 1,2 preferentially occurring in arterial regions exposed to disturbed flow characterized by low and oscillatory shear stress, whereas straight arterial regions exposed to table flow are protected from atherosclerosis. 3,4 Despite the close association between the 2, in vivo evidence directly linking disturbed flow conditions to atherosclerosis has been scarce.The differential mechanisms by which disturbed and stable flow promotes and inhibits atherogenesis, respectively, have been a subject of intense study, mostly using cultured endothelial cells. [5][6][7][8] To define molecular mechanisms responsible for these changes, investigators have carried out DNA microarray studies using endothelial cells [9][10][11][12][13][14][15][16][17] and have subsequently identified numerous shear-sensitive genes, such as kruppel-like factor 2 and 4 (Klf2, Klf4), endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), bone morphogenic protein 4 (BMP-4), cathepsins, and angiopoietin-2 (Angpt2). 11,14,[18][19][20][21][22][23][24][25][26][27][28][29] Functional studies based on these shear-sensitive genes and their protein products have revealed the critical roles that they play in regulation of inflammation, thrombosis, vascular remodeling, angiogenesis, and arteriogenesis. 11,[19][20][21][22][26][27][28][29][30] Although these in vitro studies have provided critical insights regarding shear-sensitive mechanisms in cultured endothelial cells using modeled flow conditions, it cannot be assumed that identical mechanosensitive genes and pathways are involved in vivo regulating flow-dependent vascular responses and diseases. Therefore, it is critical to study how arterial endothelium responds to different flow conditions in vivo. However, the adequate pathophysiologic animal models enabling acute and reproducible modulation of flow conditions that rapidly lead to atherosclerosis have been lacking.Recently, we have shown that partial ligation of mouse carotid artery causes disturbed flow with characteristic low and oscillatory wall shear stress, which in turn rapidly induces atherosclerosis, directly demonstrating the causal relationship between disturbed flow and atherosclerosis. 31 In this model, disturbed flow induces e...

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.

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 due in part to 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. ApoE−/− mice underwent left carotid artery (LCA) partial ligation and fed high-fat diet for 4 to 28 days. Monocyte/macrophages, dendritic cells, granulocytes, NK cells, and CD4 T-cells significantly infiltrated the LCA as early as 4d. Monocyte/macrophages and dendritic cells decreased between 7d and 14d, while T-cell numbers remained steady. Leukocyte numbers peaked at 7d, preceding atheroma formation at 14d. B-cells entered LCA by 14d. Control right carotid and sham-ligated LCAs showed no significant infiltrates. PCR and ELISA arrays showed that expression of pro-inflammatory cytokines and chemokines peaked at 7 and 14-days post-ligation, respectively. Conclusions 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.

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.

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.

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.

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