Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 46
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Incontrast, the unbranched, tubular portions of the arteries that carry uniform laminar flow are relatively protected from the atherogenesis. Ultrasound and magneticresonance imaging techniques demonstrated a close correlation between flow disturbances and arterial susceptibility to the development of atherosclerosis. Experiments using an in vitro flow chamber to simulate regions susceptible to atherosclerosis (flow disturbance) and resistant to atherosclerosis (undisturbed flow),also underscore the spatial relationships between hemodynamic shear stressforces and endothelial monolayer [12].Endothelial cells exposure to biological stimuli (cytokines, growth factors, hormones, metabolic products) produced by or in conjunction with pulsatile flow,generates a complex interplay of three distinct types of fluid mechanical forces:wall shear stresses, cyclic strain and hydrostatic pressures [13].
The impact ofthese hemodynamic factors on the structure and function of the cells that comprise the wall of the distributive vascular network is incompletely understood. Exposure of vascular endothelium to various fluid mechanical forces generated bypulsatile blood flow results in alterations in cell morphology [14], metabolic andsynthetic activities [12, 15], and gene regulation [16]. Single gene analyses have revealed that a broad spectrum of pathophysiologically relevant genes (PDGF-A andPDGF-B; TGF-b), and tissue plasminogen activators (tPA); ICAM, VCAM-1, etc.),are modulated by shear stress in cultured endothelial cells [16, 17].
This regulation is likely accomplished via the binding of transcription factors such as NF-jBand the immediate-early response gene, Egr-1, to shear-stress response elements15715810 Gene Expression Profiling and Vascular Cells10.3 Analysis of Gene Expression Profiling in Vascular Cellsthat are present in the promoters of biomechanically inducible genes [17, 18].Transcription profiles of the gap junctional connexin 43 gene in individual endothelial cells isolated from both disturbed and undisturbed flow regions exhibitedchronically elevated expression in disturbed than in undisturbed flow [12].
Bothconnexin 43-mediated channel assembly and cell-cell communication were impaired in the disturbed region compared with the undisturbed region confirmingregional detectable differences in the levels of gene expression, protein expressionand functional communication.With the relatively recent emergence of high throughput genomic technologies,novel mechanistic insights into the pathobiology of this tissue have been established. The application of high throughput microarray analysis of individual cellsof the vascular wall enables a more complete appreciation of the extent and biologic significance of vascular cell activation by biomechanical stimuli, particularlyin the context of pathophysiologically relevant phenotypic changes.
Available arrayand SAGE analysis of gene expression of specific vascular wall cellular components will be described.10.3.1Endothelial CellsEndothelial cells reside at the precise interface of the vessel wall and the circulation to rapidly sense changes in blood flow and respond by secreting or metabolizing vasoactive substances to maintain pressure/flow homeostasis. Furthermore, diverse pathophysiologic factors including biomechanical and humoral stimuli allserve to modulate the functional phenotype of the vascular endothelium [12, 19].Increasing evidence suggests that biomechanical stimulation plays a key role in3Fig.
10.1 Schematic of the serial analysis ofgene expression (SAGE) method. In the firststep, cDNA is synthesized from poly (A)+RNAusing the biotin-oligo-dT followed by cleavageof the cDNA (Step 2) by the anchoring enzyme (e.g. NlaIII), a frequent-cutting (fourbase-pair recognition sequence) enzyme andexpected to cleave most transcripts at leastonce. The 3'-portion of the cDNA is capturedby the streptavidin-coated magnetic beads.Next, two different linkers containing a fivebase-pair recognition site for a type II restriction enzyme called tagging enzyme such asBsmFI are ligated to two aliquots of the captured cDNA, respectively (Step 3). Short tagsplus linkers are then released from the cDNAby tagging enzyme digestion (Step 4), followed by blunting of the ends of the releasedshort tags with the DNA polymerase I, large(Klenow) fragment.
In step 6, the linker-tagmolecules are ligated tail to tail to form ditags which are amplified by PCR. The ditagsare released from linkers by the anchoring enzyme digestion and concatenated by ligase(Step 7) and the concatemers are cloned intoa sequencing vector and are subjected toDNA sequencing (Step 8). Finally, in step 9,sequencing of concatemer clones reveals theidentity and abundances of each tag. Relativeabundance can be calculated by dividing theobserved abundance of any tag by the totalnumber of tags analyzed to generate quantitative result. The digital data format of transcripts in both normal and diseased samplescan be easily analyzed by SAGE software toidentify the genes of interest in specific physiologic or disease states. This figure was modified from Ye et al.
[9].15916010 Gene Expression Profiling and Vascular Cellsthe maintenance of vascular integrity as well as in the development of vasculardisease [16, 20]. Other analyses of signal cascades, transcriptional factor systems,and the identification of shear-stress-response elements in the promoters of several genes (PDGF-A, VCAM-1, etc.) relevant to the atherosclerotic process [17, 18],have provided insight into the cellular mechanisms linking shear stress stimuliand genetic regulatory events [12, 15]. García-Cardeña G. et al. [21] applied transcriptional profiling via cDNA arrays to assess the gene expression in cultured human umbilical vein endothelial cells (HUVEC) exposed to either steady laminarshear stress (10 dynes/cm2) or turbulent or non-laminar shear stress (spatiallyand temporally averaged shear stress of 10 dynes/cm2) for 24 h. Whereas a greater number of genes were downregulated by either laminar or turbulent shearstress than were up-regulated, direct comparison of the two forms of shear stressrevealed 100 genes which were differentially regulated (68 up-regulated; 32 downregulated, turbulent vs.
laminar). The application of laminar shear stress to staticmonolayers resulted in greater than two-fold changes in 205 genes (from a totalof 11,397 unique genes) whereas only 86 genes increased greater than two-foldwith turbulent shear stress. These studies clearly indicate that cultured endothelial cells can discriminate between these distinct types of fluid mechanical stimulation.Similar studies in human aortic endothelial cells exposed to laminar shearstress at 12 dynes/cm2 for 24 hours [22] revealed 125 genes (from 1,185 gene arrays), which exhibited significant difference between the sheared and static samples.
Genes related to endothelial cell inflammation including MYD-88 (an adaptor protein essential for signaling via the IL-1 receptor), CD30 ligand (a memberof the TNF superfamily and an inducer for T-cell proliferation) and platelet basicprotein (a chemokine for neutrophils and monocytes) were down-regulated in response to shear stress as detected by microarray analysis and confirmed by Northern blotting. Since exposure of endothelial cells to proinflammatory cytokinessuch as IL-1 and TNF results in the modulation of “prothrombotic-proinflammatory” program, shear stress may antagonize inflammatory responses through genemodulation to prevent the induction of the atherogenic “prothrombotic-proinflammatory” program.
In contrast, genes such as Tie2 (angiopoeitin-1 and -2 receptor),Flk-1 (VEGF receptor) and MMP-1 (matrix metalloproteinase 1) were significantlyupregulated, supporting the concept that shear stress promotes endothelial cellsurvival, angiogenesis [23], migration and wound healing [24].Additional studies in human umbilical vein endothelial cells exposed to higherlevels of shear stress (25 dynes/cm2 for 6 or 24 h) also appear to provide evidenceto support the concept that physiological levels of shear stress are protective to endothelium [25].
A total of 52 genes were differentially regulated genes (32 up-, 20down-regulated out of 4,000 genes) in response to shear stress results which werefurther confirmed by Northern blot analysis. For example, expression of membersof the cytochrome P450 (CYP) family of genes (including 1A1 and 1B1) whosegene products are associated with cellular detoxification mechanisms, were dramatically increased. Consistent with this concept, the connective tissue growth factor(CTGF) gene whose product has been postulated to play a role in the develop-10.3 Analysis of Gene Expression Profiling in Vascular Cellsment of fibrotic disease [26], and is highly expressed in vascular cells in atherosclerotic lesion, was significantly downregulated [27]. These observations are consistent with the hypothesis that physiological arterial shear stress is protective andretards fibrotic and atherosclerotic disease processes.Nitric oxide (NO) is a key bioregulatory molecule with expression of endothelialnitric oxide synthase (NOS) regulated by shear stress.
Microarray studies have provided mechanistic information as to how shear stress regulates the synthesis ofNO in endothelial cells [25] with marked increases in arginiosuccinate synthetasegene expression (2.5 to 3 fold) suggesting that shear stress-induced increase in NOsynthesis may dependent on an increase in L-arginine synthesis from L-citrulline.The vasoactive intestinal polypeptide (VIP) receptor precursor gene (1.5 to 2 fold)is also upregulated by shear stress indicating that increased availability of VIP receptors potentially increase VIP-mediated NO production. Similarly, aside from increased NOS expression via shear stress promoter elements, increased elastingene expression (2 to 3 folds) by shear stress may increase the binding of elastinpeptides to the elastin-laminin receptor, leading to an increase in intracellularCa2+ and activation of eNOS with subsequent NO release.In contrast to shear stress, there is a paucity of studies in which the effect of cyclic strain on endothelial cell gene expression relevant to normal physiologic andpathophysiologic processes has been examined.
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