Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

Abstract

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli-a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.

Figure 1

Global patterns of biomechanical activation of human endothelial genes revealed by transcriptional profiling. Scatterplots (log-log) of normalized intensities of each array element (13,325 unique clones) under the various experimental conditions examined: static 1 and static 2 (two separate no-flow experiments), TSS, and LSS. Differential expression of a given clone is reflected by deviation from the black diagonal line. Red diagonal defines ≥2-fold up-regulation; blue diagonal, ≥2-fold down-regulation. Gray box includes genes with levels of expression less than 1,500 (see Materials and Methods).

Figure 2

Hierarchical clustering analysis of genes responding to biomechanical stimulation in cultured endothelial cells. Named genes whose mRNA levels showed significant up- or down-regulation (≥2-fold) and whose level of expression was ≥1,500 under laminar vs. static or turbulent vs. static conditions were selected. These 143 genes were clustered hierarchically into groups on the basis of the patterning of their expression profiles, by the procedure of Eisen et. al. (17). Each column represents a single experiment, and each row represents a single gene. For each gene, the ratio of mRNA levels for LSS/static conditions and TSS/static conditions, is denoted by a color code. Blue squares represent lower than static levels of gene expression in the LSS or TSS samples (ratios less than 1); white squares represent genes equally expressed (ratios near 1); red squares represent higher than static levels of gene expression (ratios greater than 1). Color saturation reflects the magnitude of the log/ratio (see color scale). Full cluster diagram with complete names and accession numbers is available at http://vessels.bwh.harvard.edu/papers/PNAS2001.

Figure 3

Changes in cytoskeletal elements of endothelial cells exposed to LSS. (a) Cytoskeleton-related genes showing significant up- or down-regulation in response to LSS stimulation. (b) Fluorescence confocal micrographs of the apical (supranuclear) region of HUVEC stained green for F-actin (Top), myosin heavy chain (Middle), and plectin (Bottom). Cells were counterstained with SYTOX (red) to identify nuclei. (Left) HUVEC under static (no flow) conditions; (Right), HUVEC exposed to LSS (10 dyn/cm² for 24 h).

Figure 4

Cell cycle dynamics in biomechanically stimulated endothelial cells. (a) Cell cycle-related genes regulated by LSS or TSS compared with static conditions. (b) Relative mRNA levels of cyclin B1 and cyclin D1 as measured by TaqMan assay. β2-microglobulin, a gene not regulated by these stimuli, was used for normalization. Measurements were done in duplicate by using mRNA samples from three independent experiments. Bars represent means ± SE. (c) (Upper) Representative propidium iodide DNA-staining profile of HUVEC under static (no flow), LSS, or TSS (10 dyn/cm² for 24 h). (Lower) Quantitative analysis of FACS data for indicated phases of the cell cycle. Percentages represent means from three independent experiments (SE in parentheses).