Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 47
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We have evaluated the effect ofFig. 10.2 Scatter Plot on Human Lung Endo-thelium Gene Expression Exposed to CyclicStretch. Expression profiling of HPAEC geneexpression after 18% cyclic stretch for 48 hwas analyzed using the Affymetrix MicroarraySuite software (MAS, ver 5.0). The X axis represents the signal intensity of gene expression in cells exposed to static condition. TheY axis represents the signal intensity of geneexpression in cells exposed 18% cyclic stretchfor 48 h.
Colored dots represent the level ofexpression whereas parallel lines representfold changes (2, 5, 10 and 30 folds). Expression levels high to low correlate with colorchange from red to yellow.16116210 Gene Expression Profiling and Vascular CellsFig. 10.3 K-mean Clustering Analysis of GeneExpression in Human Lung Endothelium exposed to 18% cyclic stretch. Expression profiling of HPAEC gene expression after 18% cyclic stretch for varied time intervals was analyzed using GeneSpring (ver.
4.2, Silicon Genetics). The representative clusters representing 1,232 genes are displayed. X axis lists thetime points and Y axis represent the normalized intensity. Illustrated clusters identify aunique class of genes that are upregulated at48 h.
Selection of a particular gene (such asCASP 9 (487_g_ at, white line), an apoptosisregulator gene (listed in Tab. 10.1) illustratesthe expression behavior of the individual genein a time dependent manner.human pulmonary artery endothelial cell exposure to 18% cyclic stretch (CS) ongene expression profiling (Birukov et al. manuscript submitted). Extended cyclicstretch (simulates pathophysiological lung expansion) exposure (48 hr) enhancedthrombin-induced paracellular gap formation and decreased transcellular electricalresistance in conjunction with significant alterations in gene expression detectedby Affymetrix human Genechip oligonucleotide arrays. Our preliminary results indicate approximately 60 genes (of *12,000 genes) were upregulated with *140genes down-regulated (more than 2-fold) following cyclic stretch preconditioning(Fig.
10.2). Fig. 10.3 illustrates the expression profiling of human lung endothelium exposed to 18% cyclic stretch for varied time intervals with a representativeK-mean cluster (1232 genes) depicted by K-mean analysis which identifies aunique class of genes that are upregulated at 48 h. To assess gene-gene interaction within the cluster, a tree view (dendrogram) analysis was applied and illustrated in Fig. 10.4. Through dendrogram analysis, the related genes within thesame cluster, which shows the similar expression pattern, can be identified. Our10.3 Analysis of Gene Expression Profiling in Vascular Cellsdata reveal that apoptosis-related genes such as DED caspase, DAD-1, CED-3, ICEetc. were among the most up-regulated genes (Tab.
10.1). While experiments utilizing RT-PCR, real-time RT-PCR, Northern and Western blots are currently inprogress to validate these data, these data suggest that excessive mechanical cyclicstrain promotes programmed cell death and impairs vascular functions.To identify the repertoire of genes differentially expressed after stimuli associated with the atherosclerotic process, de Waard et al. [28] used serial analysis ofgene expression (SAGE) to evaluate quiescent human arterial endothelial cellsFig.
10.4 A Tree View (Dendrogram) Gene Ex-pression in Human Lung Endothelium Exposed to Cyclic Stretch. Panel A: Expressionprofiling of HPAEC gene expression after 18%cyclic stretch treatment for 0, 6, 24 and 48 hwas analyzed using GeneSpring (ver. 4.2, Silicon Genetics). Approximately 1,232 genes aredisplayed which allow us to identify the related genes within the same cluster whichshows the similar expression pattern. Panel B:Display the adjacent genes of 487_g_ at(Caspase 9) identified in K-mean cluster. Thecolor depicts the raw gene expression levelsafter CS where the brighter the more expressed.
Gray rectangle represents absent ofgene. Within the vicinity of CASP9, at least 3genes (Tissue specific extinguisher, Microtube-associated protein 1A/1B light chain 3,and Quinone oxidoreductase homolog) wereknown to be involved in apoptosis. Inversin,syntaxin 7 and integrin a V may be implicatedto have functions related to apoptosis. Thehypothetical protein 37 884_f_ at) may be indicated to have a functional role in the apoptosis.16316410 Gene Expression Profiling and Vascular CellsFig. 10.4 BTab.
10.1 HPAEC apoptosis regulatory genes showing upregulation after 18% CS for 48 hProbe IDGene name48 h32 746_ at33 774_ at34 892_ at35 662_ at38 413_ at39 436_ at487_g_ at34 449_ at33 419_ atDED caspaseDeath triggerDR4 homologNew memberDAD-1BNIP 3aCASP 9ICEPutative1.661.531.361.281.501.561.661.302.88treated with oxidized LDL, a strong atherogenic stimulus (6 hrs). Among the12,000 tags analyzed, *600 tags (*5%) were differentially expressed representing56 differentially expressed genes (42 known genes), including the hallmark endothelial cell activation markers such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), plasminogen10.3 Analysis of Gene Expression Profiling in Vascular CellsTab. 10.2 List of genes within the same cluster identified by tree view analysis shows similarexpression patterns after 18% CS for 48 hGene orderProbe IDGene nameFold ofexpression12337 132_ at37 884_ f_ at39 370_ at1.591.581.5745678487_ g_ at226_ at36 079_ at38 744_ at39 071_ atInversinHypothetical proteinMicrotube-associated protein 1A/1B,light chain 3Caspase 9 (CASP9)CAMP dependent protein kinaseQuinone oxidoreductase homologSyntaxin 7Integrin alpha V1.561.481.451.411.38activator inhibitor 1 (PAI-1), Gro-a, Gro-b and E-selectin.
Differential transcriptionof a selection of the upregulated genes was confirmed by Northern blot analysis.An important application of the genomic information obtained by microarrayanalysis is the potential for novel disease-specific therapeutic approaches. To explore aspects of tumor angiogenesis [29, 30], St. Croix et al.
[31] compared geneexpression patterns of endothelial cells derived from blood vessels of normal andmalignant colorectal tissues. Of the 170 transcripts predominantly expressed inendothelium, 46 were specifically elevated in tumor-associated endothelium including extracellular matrix proteins (such as several collagens: type IV, a2; typeVI, a1 and a2; CD146), while many genes remain with an as yet unknown function. These studies provide molecular information on tumor biological processes,which may have significant implications for the development of anti-angiogenictherapies. In fact, data obtained from St. Croix’s group have been further examined by Novatachkova et al.
[32] concluded that up-regulated transcripts in angiogenesis are involved in extracellular matrix remodeling, cellular migration, adhesion, cell-cell communication rather than in angiogenesis initiation or integrativecontrol. These studies strongly suggest potential application of microarray in therapeutic approaches.10.3.2Smooth Muscle CellsIn the artery wall, vascular smooth muscle cells participate in regulating vasculartone and extracellular matrix synthesis and degradation. Under pathophysiologicalconditions, smooth muscle cells respond to growth factors and cytokines secretedby endothelial cells and surrounding macrophages and de-differentiate into a synthetic, proliferative phenotype and migrate from the media into the intima to produce large amounts of extracellular matrix constituents.
Cumulative data (http://www.ncbi.nlm.nih.gov/UniGene) identified genes (ICAM-1, GM-CSF, IL-8, NF-jB,16516610 Gene Expression Profiling and Vascular CellsFGF-5 etc.) that are specifically induced in cultured smooth muscle cells upon exposure to atherosclerotic stimuli. The overall alterations in smooth muscle geneexpression are largely unknown.In addition to growth factors and cytokines, the mechanical environment is amajor stimulus to alter smooth muscle cell function. Cheng et al. [33] have shownthat large deformations in vitro (10% deformation) lead to transient cell injury, release of FGF-2) and cell proliferation. Similar observations were reported by Lindner et al. [34] using an in vivo balloon injury model and suggest that mechanicalforces can directly regulate vascular smooth muscle cell function.
To explore therelationship between mechanical forces and vascular diseases, Feng et al. [35] utilized a biaxial cyclic device that generates uniform deformation on vascularsmooth muscle cell and monitored gene expression (*5,000 gene microarrays) incells exposed to 12 and 24 hours of continuous uniform strain. Only three genes(cyclooxygenase-1, tenascin-C and plasminogen activator inhibitor-1) were upregulated and 13 genes such as matrix metalloproteinase-1 and thrombomodulin weredownregulated by strain, with the results verified by Northern and Western blotanalyses.
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