High Glucose Attenuates Shear-Induced Changes in Endothelial Hydraulic Conductivity by Degrading the Glycocalyx

Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques

The Interaction between Fluid Wall Shear Stress and Solid Circumferential Strain Affects Endothelial Gene Expression

<div><p>Endothelial cells lining the walls of blood vessels are exposed simultaneously to wall shear stress (WSS) and circumferential stress (CS) that can be characterized by the temporal phase angle between WSS and CS (stress phase angle – SPA). Regions of the circulation with highly asynchronous hemodynamics (SPA close to -180°) such as coronary arteries are associated with the development of pathological conditions such as atherosclerosis and intimal hyperplasia whereas more synchronous regions (SPA closer to 0°) are spared of disease. The present study evaluates endothelial cell gene expression of 42 atherosclerosis-related genes under asynchronous hemodynamics (SPA=-180 °) and synchronous hemodynamics (SPA=0 °). This study used a novel bioreactor to investigate the cellular response of bovine aortic endothelial cells (BAECS) exposed to a combination of pulsatile WSS and CS at SPA=0 or SPA=-180. Using a PCR array of 42 genes, we determined that BAECS exposed to non-reversing sinusoidal WSS (10±10 dyne/cm²) and CS (4 ± 4 %) over a 7 hour testing period displayed 17 genes that were up regulated by SPA = -180 °, most of them pro-atherogenic, including NFκB and other NFκB target genes. The up regulation of NFκB p50/p105 and p65 by SPA =-180° was confirmed by Western blots and immunofluorescence staining demonstrating the nuclear translocation of NFκB p50/p105 and p65. These data suggest that asynchronous hemodynamics (SPA=-180 °) can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA may be an important parameter characterizing arterial susceptibility to disease.</p></div

Hemodynamic influences on gene expression.

<p>Asynchronous hemodynamic conditions significantly increased the expression of the adhesion molecules CDH5 and VCAM-1 (B). Asynchronous hemodynamic conditions significantly increased EPCR and SCD1 mRNA levels (C). Asynchronous hemodynamic conditions significantly increased the mRNA levels of transcription regulators OLR-1, ADFP, and SCARB1 (D) **p < 0.05, * p<0.1 indicate significant differences for SPA 0° with respect to control or SPA -180° with respect to control. An overbar indicates pairwise significant difference between SPA 0° and SPA -180°. (n = 11)</p

Hemodynamic influences on gene expression.

<p>Asynchronous hemodynamic conditions significantly increased the levels of mRNA of the apoptosis factor BCL2. Synchronous hemodynamics significantly reduced BAX and FAS mRNA levels (A). Asynchronous hemodynamics significantly increased the mRNA levels of PPARG, CCL5 and NFκB relative to synchronous conditions (B). Synchronous hemodynamics significantly decreased the mRNA levels of SOD1 compared to controls and SPA = −180° (C). Asynchronous hemodynamics significantly increased the mRNA levels of BMP4, GPC1, TGFb1 and VEGF compared to SPA 0°. Synchronous hemodynamic conditions decreased the gene expression of ANGTP2 and PRDX2 compared to control (D). (n = 11).</p

SPA modulates localization of NFKB p105/p50, NFKB p65 and CDH5.

<p>BAEC were exposed to asynchronous or synchornous condition for 7h. Stainings for NFKB p105/p50 (B) and p65 (D) were localized in the citoplasm and the nucleos for EC exposed to SPA = -180. NFKB localization where entirely citoplasmatic for EC exposed to SPA = 0 (A, C). The distribution for CDH5 were continous around the entire periphery of the cells after 7 when cells where exposed to SPA = 0 (E). Exposure of EC to SPA = -180 for 7 hours resulted in an intermitted pattern of CDH5 (F). images showed here are representative resutl from 3 individual experiments.</p

SPA modulates the protein expression levels of NFkB p105/p50 and NFkB p65 but does not affect CDH5.

<p>BAEC monolayer were exposed to WSS and CS with either SPA = 0 or SPA = -180 during 7 hours. Cell lysates from different samples (n = 6 each condition) were separated in gradient SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. Nitrocellulose membranes were split into two parts for immunoblotting with NFKB p105/p50 or NFkB p65 using βactin as the endogenous control. Representative blots are shown in Fig 6. Samples were analysed by densitometry and normalized by the βactin control; then the relative protein expressions at SPA = -180 and SPA = 0 were compared. The bar graphs in (A) represent the quantification of 6 individual experiments (mean ± SEM). SPA = -180 increases the expression of NFκB p50 by 1.9 fold compared to SPA = 0 (p = 0.001) and the expression of NFκB p105 by 1.98 fold (p = 0.058). The bar in (B) suggest that SPA = -180 increase the expression of the transcriptional factor NFKB p65 (n = 8) by 1.98 fold (p = 0.002) (ANOVA, all p<0.05). SPA did not affect the CDH5 protein expression levels (n = 3 for each condition) (ANOVA p>0.05) as shown in panel B.</p

Endothelial cells seeded on the inner surface of silicone substrates.

<p>(A) Control conditions. ECs formed a confluent monolayer that has a cobblestone appearance. (B) After 7-h-exposure to WSS (10±10 dynes/cm2) and CS (4 ± 4%) at SPA = 0° and (C) SPA = -180°. ECs remained confluent but showed subtle morphological changes. Flow is from left to right; strain is perpendicular to flow.</p

Atherosclerosis Related Gene List (43 genes) for PCR Array.

<p>Atherosclerosis Related Gene List (43 genes) for PCR Array.</p

Simultaneous WSS and CS on EC are characterized by the SPA.

<p>Blood flow in the axial direction induces wall shear stress (WSS) and the changes in pressure during the cardiac cycle induce circumferential strain and circumferential stress (CS) on the EC lining the wall of the blood vessel. Due to impedance of the distal circulation, local inertial effects associated with flow in larger vessels and arterial geometry, there is a time lag between WSS and CS characterized by the stress phase angle—SPA = φ(CS-WSS)</p

High glucose attenuates shear-induced changes in endothelial hydraulic conductivity by degrading the glycocalyx.

Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques