Analysis of Gene Expression Profiling in Vascular Cells

As early as 1856, Virchow et al. [10] recognized that vascular endothelium might participate as a central component in the atherosclerotic disease process. Ku et al. [11] reported that atherosclerotic lesions often originate near branches, bifurcations and curvatures of arteries, where the laminar flow pattern is disturbed. In contrast, the unbranched, tubular portions of the arteries that carry uniform laminar flow are relatively protected from the atherogenesis. Ultrasound and magnetic resonance 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 stress forces 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 of these 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 by pulsatile blood flow results in alterations in cell morphology [14], metabolic and synthetic activities [12, 15], and gene regulation [16]. Single gene analyses have revealed that a broad spectrum of pathophysiological^ relevant genes (PDGF-A and PDGF-B; TGF-/3), 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-kB and the immediate-early response gene, Egr-1, to shear-stress response elements


2. Anchoring enzyme (e.g., NlaHI) digestion Streptoavidin beads( <i—|) binding i-r


4. Tagging enzyme(e.g., BsrnFI) digestion

5. Blunting ends

6. Ligation and PCR amplification

7. Anchoring enzyme digestion Concatenation by ligase

8. Cloning and DNA sequencing

Gene Products

9. Data analysis

Gene Products that 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 exhibited chronically elevated expression in disturbed than in undisturbed flow [12]. Both connexin 43-mediated channel assembly and cell-cell communication were impaired in the disturbed region compared with the undisturbed region confirming regional detectable differences in the levels of gene expression, protein expression and 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 cells of the vascular wall enables a more complete appreciation of the extent and biologic significance of vascular cell activation by biomechanical stimuli, particularly in the context of pathophysiologically relevant phenotypic changes. Available array and SAGE analysis of gene expression of specific vascular wall cellular components will be described.

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