Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 43
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Ideally, data from geneexpression experiments require a uniform format so that the results can also beused for meta-analyses. The enormous amount of data may be most sophisticallypublished through www interface. One potential alternative would be the establishment of a centralized public data bank having a similar organization as e.g.Fig. 9.1 The principle and applications of laser microdissection technology. In lasermicrodissection laser beam isused to dissect small cell populations from paraffin or frozen sections. Cells can thenbe used to ordinary DNA,RNA and protein analysis.145Tab.
9.1 Minimal criteria for an array analysis in cardiovascular field–––Findings have to be confirmed by other, independent methods such as RT-PCR, Northernblot, immunohistochemistry or in situ hybridisationFindings must be reproducible i.e. confirmed with repeated hybridisations (at least 3times) using multiple sources of RNAstatistical methods must be used to interpret the dataGenBank.
Comparative databases in www should include the possibility to 1)search for the data by name of the gene, 2) find the expression value corrected toa uniform format, 3) have an access to brief annotations of specific genes andlinks to known biochemical pathways, including interactions at the transcriptionallevel, 4) download data for further analyses and 5) generate reports [3].Atherosclerotic plaques can usually be divided to macrophage-rich shoulderareas, fibrous cap and atheromatous core. By manual dissection it is not possibleto adequately separate these parts, and one has to be satisfied with heterogenoussamples containing various parts of the lesion, media and adventitia.
Recently, anovel method named “laser microdissection” has been developed to dissect verysmall cell populations or even single cells. Thin (< 5 lm) frozen or paraffin sections are cut to special plastic slides, stained and dissected by laser beam undermicroscopic control [28]. RNA, DNA and proteins can be extracted from microdissected cells and used for PCR and DNA-array analyses (Fig. 9.1). Because theamounts of RNA that can be extracted are very low, T7-RNA-polymerase-based amplification is needed before any array analyses can be performed [29].Tab.
9.1 states the minimal criteria for an array analysis in cardiovascular field.Analyses have to be repeated at least three times and statistical methods need tobe used to interpret the data. Findings must be confirmed by complementarymethods such as RT-PCR or immunohistochemistry since DNA arrays may stillproduce false signals due to sequence homologies and repetitive elements presentin many genes.9.4DNA Arrays in The Research of AtherosclerosisWe have analyzed gene expression in normal arteries and in different types of immunohistologically characterized human atherosclerotic lesions using a nylon-filter-based DNA-array method.
Radioactively labelled probes were generated from1.0 lg of mRNA from normal arteries, fatty streaks and advanced lesions and hybridized to filters of 18,376 cDNA clones (Incyte Genomics). Intensities were analyzed as duplicates with pairs of filters comparing pooled normal samples vs. fattystreaks and advanced lesions. Scores were calculated as described in formula 1.The sensitivity of the filter arrays currently is at the level of one molecule in100,000, which together with limitations in phosphoimaging allows detection of1469 Genes Involved in Atherosclerosisdifferential expression in excess of 1.5–2 folds. Only those genes/ESTs detected inthree repeated pairs of arrays for each lesion type and showing ³ 1.5 fold increaseor decrease were processed further [30]. We first validated the array by detecting agroup of genes (n = 17) that were already known to be connected to atherogenesis.Tab.
9.2 Some examples of differentially expressed genes in advanced atherosclerotic lesionsGenea) Genes upregulated in advanced lesionsMelanoma adhesion molecule MCAMneuronal PAS domain proteinHS solute carrier family 31 (copper transport)member 2ESTProteasome (prosome, macropain) subunit,alpha type 2Oligopherin 1Platelet/ endothelial cell adhesion molecule-1(PECAM-1) CD31 antigenESTUbiquitin-conjugating enzyme E2D 1Lectin, mannose-binding, 1NADH-ubiquinone oxidoreductase, 51 kDasubunitHuman non-muscle myosin alkali light chainZinc finger protein 7Proteasome, chain 7b) Genes downregulated in advanced lesionstranscript asssociated with monocyte tomacrophage differentiationHuman cleavage and polyadenylation specifityfactor mRNALIM binding domain 2ESTDeoxiribonuclease 1-like 1growth differentiation factor 11, bonemorphogenetic protein 11ESTEST, FLJ1321Diphosphoinositol polyphosphatephosphohydrolaseEST, Weakly similar to reverse transcriptaseRecQ protein like-5ESTprotein phosphatase 2, regulatory subunit B,alpha isoform PPP2R2AEST, similar to mus musculus AT3 gene forantithrombinGB accessFunctionScorep-valueR79246R78870R68089SignalingExpressionTransport56.549.844.8p < 0.05p < 0.05p < 0.05R68091H12633UnknownExpression40.837.6p < 0.05p < 0.05R81942R33252UnclassifiedSignaling34.429.5p < 0.05p < 0.05R36114H12682R62532R67754UnknownMetabolismMetabolismMetabolism29.021.321.311.6p < 0.05p < 0.05p < 0.05p < 0.05R70035AA005168R80719Unclass ifiedExpressionExpression10.16.04.5p < 0.05p < 0.05p < 0.05R34270Signaling66.5p < 0.05R82814Expression63.6p < 0.05R36692H02191R32385T81804UnclassifiedUnknownMetabolismUnclassified61.857.356.752.4p < 0.05p < 0.05p < 0.05p < 0.05R80390R69260H13795UnknownUnknownMetabolism51.340.537.0p < 0.05p < 0.05p < 0.05H12636R31058R81144R65749UnclassifiedUnknownUnknownMetabolism36.734.431.130.7p < 0.05p < 0.05p < 0.05p < 0.05R77725Unknown26.9p < 0.059.4 DNA Arrays in The Research of AtherosclerosisThese genes included e.g.
apoE, CD68, and tissue inhibitor of metalloproteinase(TIMP). Next we detected 150 differentially expressed genes that were previouslynot connected to atherogenesis. Among these genes we found upregulation of 82genes, 63 of which were known genes and downregulation of 68 genes, 33 ofwhich were known genes [30].
Tab. 9.2 presents some examples of the genes upor downregulated in advanced lesions. In Fig. 9.2 the expression intensities of allarrayed genes in advanced lesion are blotted against the intensities in normal artery, showing that the expression level of the majority of the genes has not changed.It is evident that cellular composition of the analyzed lesions has a major impact on the pattern of gene expression. For that reason, arterial samples were immunostained for the presence of SMC, macrophages and T-cells. Samples analyzed using the DNA array were confirmed to represent typical atherosclerotic lesions with only a moderate infiltration of inflammatory cells. Thus, it is likely thatif extensive infiltration of macrophages or T-cells had been present the results fromExpression intensities in advanced lesions blotted against the expression intensities innormal artery.Fig.
9.21471489 Genes Involved in Atherosclerosisgene expression profiling could have identified activation of different genes. Sinceadvanced lesions frequently involve microdomains of complex pathology it is likelythat laser microdissection of lesions will improve the accuracy of the analysis. Results obtained from atherosclerotic lesions using DNA arrays should always be confirmed by in situ hybridization and/or RT-PCR analyses of the same lesions sincecurrent DNA arrays may still produce false results.
It is also important to obtain information about the localization of mRNA species in different cell types.Macrophage-rich shoulder areas of human atherosclerotic lesion were laser microdissected and gene expression profiles were compared to normal intima. Upregulation of several macrophage-specific genes e.g.
IL-4 and additionally, upregulation of many known e.g. pancreatic lipase and unknown genes were detected(Tuomisto et al 2002, unpublished results).Studies published so far regarding gene expression profiles in atherogenesishave mainly focused on cultured cells or animal models. Foam cell formation hasbeen mimicked by treating macrophages with oxidized low density lipoproteinand changes in gene expression have been studied using a microarray of 9,808genes in timepoints ranging from 30 min to 4 days. 268 of the genes showed 2fold differential expression for at least 1 time points e.g. adipophilin, heparinbinding growth factor like growth factor and thrombomodulin [5].
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