Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling

Peter J Mack,Yuzhi Zhang,Seok Chung,Vernella Vickerman,Roger D Kamm,Guillermo García-Cardenña

doi:10.1074/jbc.m804524200

Peter J Mack, Yuzhi Zhang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m804524200

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Abstract

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.

Highlights

A number of signaling molecules have previously been characterized for their role in controlling smooth muscle cell phenotypes in both blood vessel maturation and vascular remodeling
To address the question of how elevated collateral flow acting on the endothelium affects adaptive remodeling processes, we developed previously undefined numerical simulations of the shear stress waveforms present in coronary collaterals and used these waveforms to characterize the effect of collateral flow on endothelial cell (EC) gene expression and the subsequent flow-mediated endothelial paracrine effects on smooth muscle cell (SMC) molecular phenotype
Collateral Shear Stress Regulates Endothelial Gene Expression Patterns—The two simulated coronary collateral waveforms were applied to cultured human umbilical vein EC for 24 h, and differences in the resulting EC molecular phenotype were determined by genome-wide transcriptional profiling

Summary

EXPERIMENTAL PROCEDURES

Lumped Parameter Numerical Simulation of Collateral Vessel Shear Stress Waveforms—A lumped parameter hemodynamic model was created to numerically simulate shear stress waveforms acting along the no stenosis/normal coronary collateral (NCC) and partial stenotic/adaptive remodeling coronary collateral vessels (ACC). With boundary pressure tracings obtained from previously published data, fourth order Runga-Kutta numerical integration (Matlab; Mathworks) was used to solve for the individual time-varying nodal pressure tracings (see supplemental material) [28] These results were used to calculate the instantaneous pressure difference across each vessel segment, ⌬P, for both the NCC and ACC simulations, and subsequently the wall shear stress ␶ acting along the collateral vessel by the equation, r⌬P ␶ ϭ 2L (Eq 1). 2 h prior to SMC seeding, EGM-2 in the microfluidic device was exchanged for co-culture medium (Medium-199, supplemented with 2% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin plus 100 ␮g/ml streptomycin, and 0.03 mg/ml endothelial cell growth supplement) in order to allow the three-dimensional region to equilibrate with the co-culture medium. Statistical significance for the microarray data is described under “Transcriptional Profiling.”

RESULTS

Remodeling function

DISCUSSION