Objective-Comparison of gene expression in stable versus unstable atherosclerotic plaque may be confounded by interpatient variability. The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. Methods and Results-Human carotid endarterectomy specimens were segmented and macroscopically classified using a morphological classification system. Two analytical methods, an intraplaque and an interplaque analysis, revealed 170 and 1916 differentially expressed genes, respectively using Affymetrix gene chip analysis. A total of 115 genes were identified from both analyses. The differential expression of 27 genes was also confirmed using quantitative-polymerase chain reaction on a larger panel of samples. Eighteen of these genes have not been associated previously with plaque instability, including the metalloproteinase, ADAMDEC1 (Ϸ37-fold), retinoic acid receptor responder-1 (Ϸ5-fold), and cysteine protease legumain (Ϸ3-fold). Matrix metalloproteinase-9 (MMP-9), cathepsin B, and a novel gene, legumain, a potential activator of MMPs and cathepsins, were also confirmed at the protein level. Key Words: atherosclerosis Ⅲ gene expression Ⅲ stroke Ⅲ affymetrix Ⅲ MMP-9 Ⅲ legumain Ⅲ plaque instability A therosclerosis is a chronic inflammatory disease that remains a major cause of morbidity in the Western world. The composition and vulnerability of the atherosclerotic plaque are considered to be important factors in the development of arterial thrombus and embolic complications. 1 However, the precise mechanisms by which plaque ruptures remain to be determined 2,3 Gene expression techniques such as microarrays and representational difference analysis are powerful tools that can be used to probe the complexities underlying atherosclerotic plaque initiation and progression. 4 -6 These techniques have already been used to show altered gene expression between normal and diseased arteries, 6,7 between different stages in disease progression 8,9 and differential expression in samples of atherosclerotic plaque classified according to patient symptomatology. 10 However, there are drawbacks to these types of comparisons. The differences in the cellular composition and morphology between plaque and normal arterial wall may lead to differences in gene expression that simply reflect this variation. In addition, the high degree of variability in plaque composition and gene expression in different patients may confound comparative analysis in studies that use pooled samples. [11][12][13] Features of unstable plaque such as surface ulceration and rupture occur in both symptomless and symptomatic patients, 14 and this can also confound studies that classify samples according to patient symptomatology. Conclusions-TheThe aim of this study was to use a whole transcriptome analysis to characterize the gene expression signature of unstable regions of carotid endarterectomy (CEA) specimens using a stable region of the same specimen as an internal control. ...

Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels. Here, we demonstrate that a biomimetic topography, consisting of microhemispheres arrayed over the cell culture substrate, promotes podocyte differentiation in vitro. We define new methods for fabricating microscale curvature on various substrates, including a thin porous membrane. By growing podocytes on our topographic substrates, we found that these biophysical cues augmented nephrin gene expression, supported full-size nephrin protein expression, encouraged structural arrangement of F-actin and nephrin within the cell, and promoted process formation and even interdigitation compared to the flat substrates. Furthermore, the topography facilitated nephrin localization on curved structures while nuclei lay in the valleys between them. The improved differentiation was also evidenced by tracking barrier function to albumin over time using our custom topomembranes. Overall, our work presents accessible methods for incorporating microcurvature on various common substrates, and demonstrates the importance of biophysical stimulation in supporting higher-fidelity podocyte cultivation in vitro.

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Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.

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