Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47(phox)-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

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...

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signalregulated kinase (ERK) activation, Egr-1 expression, and Erg DNAbinding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-␣. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxiainduced Egr-1 mRNA expression was ERK-dependent, and COmediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with L-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP͞protein kinase A-and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.