Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 48
Текст из файла (страница 48)
As smooth muscle cells that reside in the artery wall are constantly exposed to a mechanically active environment with variable mechanical loads,smooth muscle cells play a pivotal role in the inappropriate growth or remodelingof the vascular system during arteriosclerosis and hypertension. While incomplete, these studies highlight the possibility of assessing the biological effects ofmechanical strain in the vessel wall via gene expression analysis with microarrays.DNA microarray analysis was utilized to explore the synthesis of proteoglycans,a major component of arterial extracellular matrix, by vascular smooth musclecells in response to precisely controlled mechanical strains [36].
Large aggregatesformed by proteoglycans also contribute to tissue mechanical properties, providing a hydrated sponge-like matrix that resists or cushions against deformation ofarterial structure. Since proteoglycans are matrix molecules that participate in tissue mechanics, mechanical deformation (4% cyclic stretch) was applied to arterialsmooth muscle cells grown on a thin and transparent membrane to generate anearly homogeneous biaxial strain profile. While this magnitude of strain doesnot lead to cell injury, microarray data revealed increased expression of versican(3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels [35] with protein expression correlatingwell with gene expression.
Low-level cyclic stretch did not alter the hydrodynamicsize of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycansand heparan sulfate proteoglycans [36]. These and other data demonstrate thatmechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, suggesting that arterial smooth muscle cells maymodify their biomechanical environment in a manner that limits potential biomechanical injury.10.3 Analysis of Gene Expression Profiling in Vascular Cells10.3.3FibroblastsHigh throughput microarray analyses have been utilized to explore the temporalprofile of gene expression in fibroblasts exposed to serum, a suitable model forgrowth control and cell cycle progression studies.
Iyer V. R. et al. [37] utilizedcDNA microarrays (*8,600 human genes) to assess the transcriptional programin primary cultured foreskin fibroblasts stimulated with serum. Clustering analysis of the temporal patterns of expression revealed, that transcriptional alterationswere related to the physiology of wound repair. These data suggest that fibroblastsplay a larger and richer role in this complex multi-cellular response.Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor(TF) on cell surfaces trigger the intracellular signaling events.
Pendurthi et al. [38]performed limited microarray studies in human fibroblasts treated with factor VIIa(90 min), and found five upregulated genes and one downregulated gene. Two of theupregulated genes (Cyr61 and CIGF) are known to affect cell adhesion, as well asmitogenesis and migration in some cell types. Thus, a potential signaling pathwayrelevant to the known effects of factor VIIa has emerged from these studies.10.3.4Monocytes/MacrophagesMonocytes play a pivotal role in various human infectious and inflammatory diseases and participate as a key effector in the formation of the atheroscleroticplaque.
SAGE analysis in lipopolysaccharide (LPS)-stimulated human monocytes[39] yielded total of 35,874 tags corresponding to more than 12,000 different transcripts. LPS-inducible gene products are involved in cell activation and migration,angiogenesis, tissue remodeling, metabolism, and inflammatory reactions (including IL-6, IL-1a, IL-1b, TNF-a, COX2, macrophage inflammatory protein (MIP)MMP-9, PAI-2 RANTES, growth-regulated oncogene (GRO)-a and IL-8) [40]. Exposure of the monocyte/macrophage cell line (THP-1) to Ox-LDL (30 min to 4 days)identified 268 genes (10,000 human gene cDNA microarray) containing knownand novel molecular components of the cellular response implicated in thegrowth, survival, migratory, inflammatory, and matrix remodeling activity of vesselwall macrophages [41].
The observed induction of the orphan receptor LXR-alphaand retinoid X receptor (RXR) and the previously reported induction of peroxisome-proliferator-activated receptor (PPAR)-gamma under these conditions pointsto the potential involvement of nuclear receptor in the macrophage response toox-LDL loading.Although macrophages are critically involved in both atherogenesis and plaqueinstability where macrophages are subjected to excess mechanical stress, themechanism in which biomechanical forces affect macrophage function remainsincompletely defined.
DNA microarray analysis (1,056 genes) was used to assessthe transcriptional profile of mechanically induced genes in a human acute monocytic leukemia cell line (THP-1) [42]. The mechanical deformation (1 Hz) was ap-16716810 Gene Expression Profiling and Vascular Cellsplied to a thin and transparent membrane on which THP-1 cells were cultured.Among the 1,056 well-characterized genes with putative functions, only threegenes were induced more than 2.5-fold at 3 and 6 h, and no genes were mechanically induced at 1 h.
The increased expression of prostate apoptosis response-4(PAR-4), interleukin-8 and the NF-jB inducible immediate-early response gene,IEX-1 in an amplitude-dependent induction pattern were confirmed by RT-PCR[42]. Induction of IEX-1 by mechanical deformation may participate in differentiation of monocytes/macrophages and promotion of atherogenesis. Although thenumber of genes on the array platform was limited, these findings still suggestthat mechanical stress in vivo, such as that associated with hypertension, mayhave an important role in atherogenesis and instability of coronary artery plaques.Finally, the pathogenesis of unruptured intracranial aneurysms, a common vascular abnormality with a strong genetic component, is poorly understood. Peterset al.
[43] used a global gene expression analysis approach (SAGE-Lite) in combination with a novel data-mining approach to perform a high-resolution transcriptanalysis of a single intracranial aneurysm obtained from a 3-year-old girl. Aneurysmal dilation results in a highly dynamic cellular environment in which extensive wound healing and tissue/extracellular matrix remodeling are taking place.Specifically, significant over-expression of genes encoding extracellular matrixcomponents (collagen and elastin), genes involved in extracellular matrix turnover(TIMP-3, OSF-2), cell adhesion and anti-adhesion (SPARC, hevin), cytokinesis(PNUTL2), and cell migration (tetraspanin-5) was observed.
Although these datarepresent the analysis of only one individual, the study highlights the potential forthis technique to provide unique insights into the molecular basis of aneurysmaldisease and to define numerous candidate markers for future biochemical, physiological, and genetic studies of intracranial aneurysm.10.4Future PerspectivesAnalyses of vascular cell gene expression patterns, particularly when adequatelyvalidated (RT-PCR, Northern and Western blot analysis), may result in direct improvement of disease diagnosis and therapy, and ultimately reduce morbidity andmortality for these devastating illnesses. Unfortunately, vascular cells are not areadily accessible population and represent a heterogenous population of cellswhose cell-specific gene expression may be lost when cellular mixtures are analyzed.
The increasingly utilization of laser-assisted micro-dissection to provide amore homogenous sample may ultimately lead to the generation of gene expression profiles in disease tissues, tissue- or pathologic stage-specific markers, identify treatment responders and prove useful in identifying new targets for therapeutic agents. Further advances in functional proteomics analysis is vascular components (2D gel electrophoresis coupled to mass spectrometry) will not only confirmarray results at the protein level, but together, provide basic information integralto biologic and clinical investigation for years to come.10.5 References10.5References1234567891011Bertelsen A.
H., Velculescu V. E. Highthroughput gene expression analysisusing SAGE. DDT. 1998, 3, 152–159.Hedrick S. M., Cohen D. I., NielsenE. A., Davis M. M. Isolation of cDNAclones encoding T cell-specific membrane-associated proteins.
Nature. 1984,308, 149–153.Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA bymeans of the polymerase chain reaction.Science. 1992, 257, 967–971.Velculescu V. E., Zhang L., VogelsteinB., Kinzler K. W. Serial analysis of geneexpression. Science. 1995, 270, 484–487.Lockhart D. J., Dong H., Byrne M. C.,Follettie M. T., Gallo M. V., Chee M. S.,Mittmann M., Wang C., Kobayashi M.,Horton H., Brown, E.
L. Expressionmonitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 1996, 14,1675–1680.Schena M., Shalon D., Davis R. W.,Brown P. O. Quantitative monitoring ofgene expression patterns with a complementary DNA microarray. Science. 1995,270, 467–470.Southern E. M. et al.
Arrays of complementary oligonucleotides for analysis thehybridization behavior of nucleotide acids.Nucleic Acids Res. 1994, 22, 1368–1373.Madden, S. L., Wang, C. J., Landes, G.Serial analysis of gene expression: fromgene discovery to target identification.DDT 2000, 5, 415–425.Ye S. Q., Zhang L.
Q., Zheng F., VirgilD., Kwiterovich P. O. miniSAGE: geneexpression profiling using serial analysisof gene expression from 1 microgram total RNA. Anal Biochem. 2000, 287 (1),144–152.Virchow R. Der Atermatose Prozess derArterien. Wien. Med. Wochenshr. 1856, 6,825–841.Ku D. N., Giddens D. P., Zarins C. K.,Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation.
Positive correlation between plaquelocation and low and oscillating shearstress. Arteriosclerosis. 1985, 5, 293–301.12131415161718192021Davies P. F. Flow-mediated endothelialmechanotransduction. Physiol. Rev. 1995,75, 519–560.Dewey C. F. Jr., Bussolari S. R., Gimbrone M.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
