Endothelial cells express a unique transcriptional profile under very high wall shear stress known to induce expansive arterial remodeling

Abstract

Chronic high flow can induce arterial remodeling, and this effect is mediated by endothelial cells (ECs) responding to wall shear stress (WSS). To assess how WSS above physiological normal levels affects ECs, we used DNA microarrays to profile EC gene expression under various flow conditions. Cultured bovine aortic ECs were exposed to no-flow (0 Pa), normal WSS (2 Pa), and very high WSS (10 Pa) for 24 h. Very high WSS induced a distinct expression profile compared with both no-flow and normal WSS. Gene ontology and biological pathway analysis revealed that high WSS modulated gene expression in ways that promote an anti-coagulant, anti-inflammatory, proliferative, and promatrix remodeling phenotype. A subset of characteristic genes was validated using quantitative polymerase chain reaction: very high WSS upregulated ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motif-1), PLAU (urokinase plasminogen activator), PLAT (tissue plasminogen activator), and TIMP3, all of which are involved in extracellular matrix processing, with PLAT and PLAU also contributing to fibrinolysis. Downregulated genes included CXCL5 and IL-8 and the adhesive glycoprotein THBS1 (thrombospondin-1). Expressions of ADAMTS1 and uPA proteins were assessed by immunhistochemistry in rabbit basilar arteries experiencing increased flow after bilateral carotid artery ligation. Both proteins were significantly increased when WSS was elevated compared with sham control animals. Our results indicate that very high WSS elicits a unique transcriptional profile in ECs that favors particular cell functions and pathways that are important in vessel homeostasis under increased flow. In addition, we identify specific molecular targets that are likely to contribute to adaptive remodeling under elevated flow conditions.

Fig. 1.

Schematic of the flow chamber. Flow enters a parallel section with uniform wall shear stress (WSS) of 2 Pa and then tapers to a second parallel section with uniform WSS of 10 Pa.

Fig. 2.

Venn diagrams of the number of genes that are differentially regulated by various flow conditions. A: genes that are differentially expressed when endothelial cells (ECs) under high WSS (10 Pa) or normal WSS (2 Pa) are compared with static cultures. The overlap of the two circles represents genes whose expression is altered when static ECs encounter flow, regardless of whether WSS is normal or high. B: genes that are expressed when ECs under high WSS or maintained in static culture are compared with normal WSS. The overlap represents genes that are affected when static ECs encounter normal WSS, but also change further, or in the opposite direction, when normal WSS is increased to 10 Pa.

Fig. 3.

Quantitative PCR validation of microarray results. A: the mRNA concentration of tissue plasminogen activator (PLAT), urokinase plasminogen activator (PLAU), a disintegrin and metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1), interleukin-8 (IL-8), tissue inhibitor of matrix metalloproteinase 3 (TIMP3), chemokine (C-X-C motif) ligand 5 (CXCL5), and thrombospondin 1 (THBS1) relative to the housekeeping gene 18SrRNA. B: the mRNA expression ratio (log2 fold change) of IL-8, THBS1, and PLAU under normal vs. static conditions and high vs. normal WSS. Error bars are means ± SE; n = 3. *Statistically significant difference for delta cycle threshold values (P < 0.05, paired t-test).

Fig. 4.

Stimulation of urokinase plasminogen activator (uPA) and ADAMTS1 expression by WSS in the basilar artery. The basilar artery of either sham rabbits (A and D) or rabbits 5 days after carotid artery ligation (B and E) were stained for uPA (A and B) and ADAMTS1 (D and E) by indirect immunofluorescence. In sham animals, both uPA (A) and ADAMTS1 (D) staining was weak in ECs and the other cells of the basilar artery. uPA staining was intense in 5-day animals and strongly localized to the endothelium (B; open arrow). ADAMTS1 staining increased not only in ECs (E; open arrows), but also in other cells in the 5-day ligated animals. Staining intensity specifically within the ECs was measured as described in materials and methods for uPA (C) and ADAMTS1 (F). Bars represent the average mean intensity ± SE, (n = 5 for both ligated groups and for the uPA sham group; n = 4 for the sham control group for ADAMTS1). *Statistically significant difference between sham and ligated groups (unpaired t-test, P < 0.03).