Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling - PubMed (original) (raw)

Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling

Peter J Mack et al. J Biol Chem. 2009.

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.

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Figures

FIGURE 1.

FIGURE 1.

Numerical simulation of human coronary collateral hemodynamics. A, schematic of a bridging type collateral vessel bypassing a site of coronary stenosis. B, a lumped parameter model of the coronary circulation, inclusive of coronary collaterals in parallel with a site of coronary stenosis, was used to simulate the shear stress waveforms present in collateral vessels. The model assigns electrical circuit equivalents to each vascular segment (i.e. vascular resistance (R) and compliance (C)) and uses the appropriate pressure (P(t)) boundary conditions to solve for the pressure difference across each segment, which is then used to obtain the time-varying wall shear stress acting along each segment. Both an NCC waveform, where the resistance through the stenosis (_R_st) equaled the resistance through the main coronary artery (_R_mca)(C), and an ACC waveform, where the resistance through the stenosis (_R_st) was equivalent to a 60% radius reduction of the main coronary artery (D), were generated with the model. The two waveforms are pulsatile over one cardiac cycle, with the NCC waveform having a mean shear stress value of 0.7 dyne/cm2 and the ACC waveform having a mean shear stress value of 12.4 dyne/cm2. Time-varying pressure tracing boundary conditions for the aorta (_P_aorta), right atrium (_P_ra), and left ventricle (_P_lv), which was used to calculate an external pressure, _P_ext = 0.75 ×_P_lv, were obtained from Heldt et al. (28).

FIGURE 2.

FIGURE 2.

Collateral flow regulates vascular cell molecular phenotypes. A, application of the simulated coronary collateral waveforms to cultured human EC resulted in genome-wide differences in endothelial transcriptional profiles. Significant differences across three independent samples were determined statistically for p < 0.001, using a_z_-statistic method of pooled variances (32). Normalizing ACC gene expression to NCC yielded 267 significantly up-regulated genes (red) and 414 significantly down-regulated genes (blue). B, real time TaqMan PCR validation of select differentially regulated genes identified by the transcriptional profiles as having a potential role in adaptive remodeling. EC conditioned medium from the simulated ACC compared with the NCC waveform and a no endothelium control increased SMC gene expression of_MYOCD_ (C) and decreased the expression of MMP-3 (D), as determined by real time TaqMan PCR. E, ACC flow-mediated EC conditioned medium delivered to SMC also increased the expression of myocardin-dependent SMC genes, including CNN1 and α_SMA_, compared with NCC flow-mediated EC conditioned medium. Data represent the mean of four independent experiments. All error bars show S.E.; *, p < 0.05; **, p < 0.01.

FIGURE 3.

FIGURE 3.

EC expressing KLF2 inhibit EC-directed SMC migration. A and_B_, a three-dimensional μFD assay was designed in order to investigate SMC migration in response to endothelium-derived factors. The device has two independent flow channels with each channel separated by a three-dimensional region filled with a hydrogel scaffold. C, SMC were cultured in the three-dimensional μFD in co-culture with EC. A type I collagen scaffold was injected into the three-dimensional region, and cells were introduced into their respective channels (smooth muscle channel and endothelial channel). Factors generated by EC were transported across the scaffold by passive diffusion. The image is shown at 4× magnification and rotated 90° relative to schematics shown in A and B. The cumulative effect of KLF2-dependent EC factors act to inhibit EC-directed three-dimensional SMC migration. Shown are 10× magnification fluorescent images of no endothelial control (NoEC) (D), endothelial cells expressing GFP only (EC-GFP)(E), and endothelial cells expressing KLF2 (EC-KLF2)(F). Blue, nuclei (DAPI); red, SMC (PKH-26 membrane stain). G, histogram quantification of the SMC migration distance into the three-dimensional region for the three culture conditions revealed that EC co-culture increases SMC migration compared with no endothelial control and that this effect is abolished by KLF2 induction in EC, as shown by quantifying the total number of SMC invading the three-dimensional area after 48 h (G, inset). Data are reported as the mean of 10 (EC-GFP and EC-KLF2) and nine (NoEC) independent experiments, with error bars representing S.E.. *, p < 0.05 across all conditions; ‡, p < 0.05 pairwise comparison between NoEC and EC-GFP only.

FIGURE 4.

FIGURE 4.

CTGF expression is reduced by KLF2 and induces SMC migration. Adenovirus-mediated endothelial KLF2 expression (EC-KLF2) reduces endogenous CTGF gene expression compared with control adenovirus-infected EC (EC-GFP), as determined by real time TaqMan PCR (A) and CTGF transcriptional activity compared with EC-GFP and negative control EC (EC-Only), as determined by a luciferase activity assay using an ∼2.0-kb fragment of the_CTGF_ proximal promoter (B). The binding of KLF2 to the_CTGF_ promoter was documented using a gel shift assay, with GST-KLF2 binding observed in only one (–1730 to –1706) of the five (25-bp) probes tested from the CTGF proximal promoter (C). All of the probes contained either one or two canonical CACCC KLF binding sites.D, 10× magnification fluorescent images of control (right) and 10 μg/ml CTGF (left) three-dimensional SMC migration. Blue, nuclei (DAPI); red, SMC (PKH-26 membrane stain). E, quantification of the total number of SMC invading the three-dimensional region after 48 h showed that CTGF induced more SMC migration compared with control. F, the addition of 10 μg/ml CTGF to the EC channel of the EC-KLF2 co-culture with SMC restored SMC migration to EC-GFP levels, as shown by normalizing the number of SMC migrating in each test condition to the number migrating in the EC-GFP co-culture condition. Data are reported as the mean of three (A), four (E), or five (B and F) experiments with error bars representing S.E.; *, p < 0.05. NS, not significant.

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