The authors have declared that no competing interests exist.
Conceived and designed the experiments: RAA JMT. Performed the experiments: RAA. Analyzed the data: RAA. Contributed reagents/materials/analysis tools: RAA AP. Wrote the paper: RAA.
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.
The fluid wall shear stress (WSS) driven by pulsatile blood flow and the solid circumferential stress (CS) driven by pulsatile blood pressure and associated strain, act simultaneously on endothelial cells (EC) lining blood vessels modulating their biological activity. Due to distal impedance at a vascular site of interest (global effect) and the inertial effects of blood flow at the site (local effect), a time lag arises between CS and WSS that is referred to as the “Stress phase angle”—SPA [
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)
Most studies of fluid mechanics and atherosclerosis have used the time-average WSS and oscillatory shear index (OSI—an index of direction changes in WSS; it measures the extent to which the shear stress reverses its direction over the flow cycle, as the parameters that correlate the relationship between hemodynamics and atherosclerosis since the work of Ku et al. [
To further support the hypothesis that SPA is an important parameter, we have used a novel hemodynamic simulator to compare gene expression profiles (39 target genes and 3 housekeeping genes) of BAECs under highly asynchronous, atheroprone hemodynamics (SPA = −180°) with atheroprotective, synchronous hemodynamics (SPA = 0°). BAECs were chosen since they have been utilized extensively as a model system for the study of mechanical effects on EC. A limited set of genes was investigated rather than a large array because the SPA is still not widely accepted as an important mechanical factor in EC mechano-stimulation. We observed that the expression of 17 out of 39 genes differed significantly after exposure to identical WSS and CS waveforms that differed only by the SPA (0° or -180°).
Cell culture components obtained from Sigma (St. Louis, MO) include: bovine serum albumin (BSA, 30% solution), trypsin, penicillin streptomycin, MEM (phenol red free), sodium bicarbonate, fibronectin, fetal bovine serum (FBS), L-Glutamine. Dulbecco's PBS (1x without Ca2+ and Mg2+) from Fisher Scientific (Houston, TX).
BAECs were purchased from VEC Technologies (Rensselaer, NY) and grown in T-75 flasks with 10% FBS-MEM. Silicone sheets (Down Corning Corporation, MI) were used as the substrates for cell culture. BAECs were seeded onto the upper surface of silicone membranes of 0.020" thickness. First the membranes were laser cut to the geometrical specifications of the bioreactor, and then silicone substrates were washed for 20min with a gentle detergent and autoclaved for 30 minutes. A region delimited by an area of 1.5 x 4.5 cm on each substrate was coated using bovine plasma fibronectin (30μg/ml in MEM) for 1 hour at room temperature. The BAECs between passages 3 and 7 were plated at a density of 1.0x105 cells/cm2 onto the treated silicone substrates. ECs were grown using with 10% FBS until confluency in a controlled environment (37C and 5% CO2 air). The EC monolayer reached confluence within four days.
At the end of the stretch and shear experiments, RNA was isolated using TRIzol reagent (Invitrogen) following the manufacture’s protocol. RNA was purified using the RNeasy kit (Invitrogen), and then purified RNA was converted into cDNA by reverse transcription (RT). For analyzing gene expression, quantitative real-time PCR (RT-qPCR) was performed for 42 genes on the ABI PRISM 7000 sequence detection system (Applied Biosystems). Using the geNorm algorithm, βactin, B2M, and HPRT were determined to be the best performing housekeeping genes (HKG) and the geometric mean quantities of the HKG were used as the normalization factor [
eNOS, EDN1(ET-1), PTGS2(COX-2) |
THBD (Thrombomodulin), endothelial protein C receptor (EPCR), CD36, syndecan-1(SCD-1) |
ABCA1 (ABC-1), APOE, OLR1 (oxLDL or LOX-1), ADFP (adipophilin), SCARB1 (SR BI) |
SOD1, SOD2. |
BAX, BCL2, TNFAIP3 (A20), TNFRSF6 (Fas) |
ANGPT-2, PRDX2, KLF2, BMP4, GPC1, TGFB1, VEGF |
GAPDH, 18S, HPRT, B2M, βactin |
At the end of the experiments silicone membranes with confluent monolayers were stained with specific antibodies for NFKB p50/p105 (Santacruz, California), NFKB p65 (Cell Signaling, Massachusetts) and CDH5 and visualized using fluorescence microscopy. Silicone membranes were washed twice with PBS and fixed in 1% PFA and 4% PFA for NFKB p65 for 10 min, cut into sections, and permeabilized with Triton X-100 in PBS for 10 min. After permeabilization samples were blocked in 10% BSA and 0.1% Triton X-100 in PBS (NFKB and CDH5) for 1h. After washing with 0.1% Triton X-100 in PBS, the silicone sheets were incubated with polyclonal rabbit anti- NFKB, and CDH5 primary antibodies overnight at 4°C followed by washing with 0.1% Triton X-100 in PBS. Samples were subjected to secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:500 to 1:200; Invitrogen) secondary antibody for 1.5 h for CDH5 and with secondary antibody Alexa Fluor 488 donkey anti-goat (1:100 to 400; Santa Cruz) for 1.5h for NFKB. Samples were washed again with 0.1% Triton X-100 in PBS and mounted with vectashield mounting media with DAPI on glass slides with cover slips in contact with cells. These slides were imaged using a Nikon Eclipse TE2000-E inverted fluorescence microscope with a Photometrics Cascade 650 camera (Roper Scientific) and MetaVue 6.2r2 imaging software (Universal Imaging).
The monolayers were washed once with ice-cold PBS and scraped from the silicone membranes with a plastic scraper in the presence of RIPA extraction buffer (1 mM NaHCO3, 2 mM PMSF, 1 mM Na3VO4, 5 mM EDTA, 10% protease and phosphatase inhibitor cocktail tablet and 1% Triton-X) followed by 30 s sonication on ice. Protein concentration was determined with the Protein determination kit from Cayman chemical (Ann Harbor, Mi) using the spectrophotometer Synergy HT from Biotek. Western blotting was carried out by standard techniques, loading 30 μg of protein into gradient precast-gels from Biorad, (Berkeley, Ca) and incubating overnight with antibodies to NFκB (p50/P105 dilution 1:800), NFKB (p65 dilution 1:1000) and CDH5 and the constitutively expressed protein β-actin from Cell Signalling Technologies (Beverly, MA), followed by specific secondary HRP conjugated anti-rabbit, anti-mouse and anti-goat IgG from Cell Signalling Technologies (Beverly, MA). The blots were scanned with the Biorad western blot scanner and quantified with Image J software.
The hemodynamic simulator is described in detail in (
To explore how endothelial cells acquire different phenotypes in response to vascular pulsatile WSS and CS at SPA characteristic of athero-protective and athero-prone regions, we used the hemodynamic simulator to reproduce the same sinusoidal WSS and CS waveforms, but at different phase angles. Changes in the expression of 39 genes, after 7 hours of mechanical stimulation, were studied. This time point was selected for comparison because EC are considered nearly flow-adapted with respect to gene expression after this time exposure. Details of the imposed WSS and CS conditions are given below.
Oscillatory flow with 10±10 (mean ± amplitude) dyn/cm2 WSS, 4 ± 4% CS, frequency = 1 Hz, and SPA of -180° (atheroprone).
Oscillatory flow with 10±10 dyn/cm2 WSS, 4 ± 4% CS, frequency = 1 Hz, and SPA = 0° (atheroprotective).
No hemodynamic forces were applied on the silicon substrate. No flow (WSS = 0 dyn/cm2), and no strain (CS = 0%).
Note that in each dynamic case the mean WSS is typical of arterial flow in a non-separated flow zone and that there is no flow reversal (OSI = 0). The CS is in a normal physiological range. The mean and amplitude of WSS and CSS do not vary between conditions, only the SPA.
Results are presented as mean ± SEM obtained from at least eleven independent experiments for gene expression and independent experiments for Western blots (n = 6 for NFκB, and n = 3 for CDH5). Samples were obtained from monolayers that did not show damage or desquamation. Statistical analysis was performed by one-way analysis of variance (ANOVA) with either the least significant difference (LSD) test or Tamhane’s T2 test (depending on Levene’s statistic for homogeneity of variance) using SPSS 20.0 software tool. Difference in means were considered significant if P<0.1.
BAECs were exposed to WSS (10±10 dyn/cm2) and CS (4 ± 4) for 7 h in the hemodynamic simulator. The cells remained viable, with no signs of injury or desquamation, throughout the experiments at SPA = -180° and SPA = 0°. Staining with PI showed no increase in dead cells after exposure to shear stress and stretch (Data not shown). The cells showed morphological changes in response to WSS and CS, becoming partially elongated and aligned parallel to the direction of flow and perpendicular to the CS as shown in
(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.
We analyzed the regulation of mRNA levels for vasoactive genes endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1) and cyclooxygenase-2 (COX-2). WSS and CS induced 2.3 and 3.3 fold increase in eNOS mRNA levels for SPA = 0° and SPA = -180°, respectively, compared to static control, indicating that BAECs were responsive to WSS and CS (
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)
We analyzed tight junction proteins OCLN and ZO-1 and adhesion molecules SELE, CDH-5, ICAM-1, VCAM-1 (
We analyzed the regulation of mRNA levels for the blood coagulation factors Thrombomodulin (THBD/TM), endothelial protein c-receptor (EPCR) and syndecan-1 (SCD1), and the cell surface receptor cluster of differentiation (CD36) as shown in
We analyzed the regulation of mRNA levels for the lipid transporter ABCA-1, Apolipoprotein E (apoE), oxidized LDL receptor-1 (ORL1), Adipose Differentiation-Related Protein / Adipophilin (ADFP), and Scavenger receptor class B type 1 (SCARB-1 / SR-B1) as shown in
We analyzed the gene expression of apoptosis regulating proteins: the BCL2-associated X (BAX), B-cell lymphoma 2 (BCL2), Tumor Necrosis Factor-Alpha-Induced Protein-3 (TNFAIP-3 / A20), and Apoptosis Stimulating Factor (Fas) as shown in
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).
We analyzed the regulation of Nuclear Receptor Sub-Family 1, Group H, Member 3 (NR1H3), Peroxisome proliferator-activated receptor G (PPARG) and the inflammatory response genes Chemokine (C-C Motif) Ligand-2 (CCL-2), Chemokine (C-C Motif) Ligand-5 / RANTES (CCL-5), interleukins (IL6, IL8), and cluster of differentiation CD40, and the transcriptional factor Nuclear factor-kappa B (NFκB-1/ NF-κB) as shown in
We analyzed the regulation of Superoxide Dismutase-1 / Cu,Zn Superoxide Dismutase (SOD-1) and Superoxide dismutase-2 (SOD2) (
We analyzed the regulation of Angiopoietin-2 (ANGPT-2 /Ang-2), peroxiredoxin-2 (PRDX-2), Kruppel-like factors 2 (KLF2), bone morphogenetic protein-4 (BMP4), glypican-1 (GPC-1), transforming growth factor beta-1 (TGFb1) and vascular endothelial growth factor (VEGF) (
NFκB gene expression was significantly up-regulated by SPA = −180° compared to SPA = 0° (
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.
CDH5 (VE-cadherin) gene expression was significantly up-regulated by SPA = −180 compared to SPA = 0 (
Asynchronous conditions increased the level of protein expression for NFκB p105, p50 and p65 compared to synchronous hemodynamics (
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.
Most in vitro studies of the role of hemodynamics in atherosclerosis have emphasized the analysis of isolated forces, mainly fluid shear stress, but also cyclic stress (strain). Different hemodynamic conditions such as low mean wall shear stress, disturbed flow, and reversal flow (high OSI) have been associated with the localization and development of atherosclerosis. The coronary arteries, the locations most prone to atherosclerosis, exhibit highly asynchronous hemodynamic conditions (SPA close to -180°); approximately 50% of the total coronary blood flow occurs in early diastole, 25% in late diastole, and only 25% in systole [
Asynchronous and synchronous hemodynamic waveforms differentially regulate endothelial gene expression. We have shown that identical WSS and CS waveforms elicit different gene expression profiles depending only on differences in the stress phase angle. Statistical analysis based on duplicate sets of cDNA template experiments using eleven different biological samples from each experimental condition (SPA = 0° and SPA = -180) revealed that 17 out of 38 genes were up-regulated by asynchronous hemodynamics relative to synchronous hemodynamics and classified as atheroprone, atheroprotective or of undetermined role in the disease (
Gene Bank N° | Gene name | Atheroprone/atheroprotective ratio | Characteristic | p-Value |
---|---|---|---|---|
BC151459.1 | Vascular Cell Adhesion Molecule-1 (VCAM-1) | 2.3 | Proatherogenic | 0.073 |
M32976 | Vascular Endothelial Growth Factor (VEGF) | 2.5 | Proatherogenic | 0.099 |
XM_002694894 | Cadherin-5 (CDH-5 / VE-Cadherin) | 2.1 | Proatherogenic | 0.013 |
NM_174132.2 | Oxidized Low Density Lipoprotein Receptor-1 (OLR-1) | 2.7 | Proatherogenic | 0.047 |
BC102211 | Adipose Differentiation-Related Protein / Adipophilin (ADFP) | 2.8 | Proatherogenic | 0.01 |
BC102064 | Chemokine (C-C Motif) Ligand-5 / RANTES (CCL-5) | 2.8 | Proatherogenic | 0.034 |
DQ464067 | Nuclear Factor of Kappa(NFKB-1 / NF-kB) | 3.0 | Proatherogenic | 0.04 |
BC105344 | Bone Morphogenetic Protein-4 (BMP-4) | 2.0 | Proatherogenic | 0.066 |
M_001166068 | Transforming growth factor b1 (TGFB1) | 2.0 | Proatherogenic | 0.001 |
NM_174662 | Apoptosis Stimulating Factor (Fas) | 2.3 | Proatherogenic | 0.097 |
BC134513 | Scavenger Receptor, Class B, Type 1 (SCARB-1 / SR-B1) | 2.5 | Antiatherogenic | 0.033 |
BC102432 | Superoxide Dismutase-1 (SOD-1) | 2.4 | Antiatherogenic | 0.023 |
BC116074 | Endothelial Protein C Receptor (EPCR) | 2.1 | Antiatherogenic | 0.013 |
NM_001075924 | Syndecan-1 (SDC-1) | 2.5 | Indeterminate | 0.028 |
NM_181024 | Peroxisome Proliferator-Activated Receptor-g (PPAR-G) | 2.0 | Indeterminate | 0.043 |
NM_001166486 | B-Cell CLL/Lymphoma-2 (BCL-2) | 4.0 | Indeterminate | 0.04 |
BC105496 | Glypican-1 (GPC-1) | 2.3 | Indeterminate | 0.002 |
Asynchronous hemodynamic conditions elicited an up-regulation of genes across all the different gene groups. Direct comparison of SPA = 0° and SPA = -180° conditions illustrated the differences in gene profile pattern. Most significantly, we showed that asynchronous hemodynamics (SPA = -180°) evoke mostly athero-prone genes relative to synchronous hemodynamics (SPA = 0°).
Asynchronous hemodynamics induced the expression of the transcription factor NFκB p50/p105 and p65 which plays a central role in the regulation of inflammation, adhesion and proliferation genes- all precursors to the onset of atherosclerosis [
In contrast with the above results, asynchronous hemodynamic seems to activate an atheroprotective mechanism as well. Asynchronous hemodynamics increased the mRNA levels of three atheroprotective genes: SCARB-1, which is a cell-surface HDL receptor that mediates HDL cholesterol efflux reducing atherosclerosis progression [
Asynchronous hemodynamics also increased the expression of genes having both pro- and anti-atherogenic features (indeterminate): Glycocalyx core proteins SDC-1, which is involved in events of cell migration, proliferation, early inflammatory response and matrix remodeling [
Athero-prone waveforms alter the endothelial apoptosis state. Asynchronous hemodynamics increases the gene expression of FAS (pro-apoptotic) and BCL-2 (anti-apoptotic). The overall effects of these genes in the development of the disease are undetermined. A previous study showed that asynchronous hemodynamics increases apoptosis rate [
Our results did not show any significant difference in mRNA levels for the tight junction protein genes OCLN-1 and ZO-1. A previous study showed that both synchronous and asynchronous hemodynamics had no effect on either OCLN-1 or ZO-1 protein expression after 12 hours [
Our results showed that ET-1 mRNA levels were not significantly altered from the basal level under any hemodynamic condition. These results agree with previous studies where it was observed that ET-1 gene expression was not significantly affected at SPA = 0° or -180° after 5 hours [
Both asynchronous and synchronous hemodynamics significantly increased the expression of three atheroprotective genes relative to static controls: ENOS, COX2 and KLF2 (Figs
We also examined the expression and distribution of proteins whose genes were up-regulated by asynchronous relative to asynchronous conditions (Figs
On the other hand we found that SPA = -180° increased the protein expression level for NFκB p105/p50 and NFkB p65 compared to SPA = 0° (
In conclusion, we have reported the results of novel experiments in which BAECs were exposed to a well-defined combination of WSS and CS, accurately simulating synchronous (athero-protective), and asynchronous (athero-prone) hemodynamics. We have shown that asynchronous hemodynamics up-regulate many atheroprone genes including those that modulate pro-inflammatory signal transduction or produce components that enhance the binding of lipoproteins. Of special note, we showed that endothelial cells exposed to asynchronous hemodynamics acquire a pro-inflammatory phenotype with enhanced expression of the important chemokine CCL5 and the activation of NFκB as well as the activation of adhesion molecules such as VCAM-1 and several growth factors including VEGF and TGFβ1. We also reported post translational modifications of CDH5 and NFκB induced by asynchronous SPA that are consistent with an atheroprone environment. Based on these results, the SPA appears to be an important parameter characterizing the hemodynamic environment.
Analysis of the flow dynamics in the novel device and strain characterization of silicone substrate sheets. Table A in S1 File. PCR primer sequences.
(DOCX)
The bioreactor combines a customized pulsatile flow valve mechanically linked to a membrane stretching mechanism and a parallel flow chamber. Different SPA values can be generated by changing the configuration of the phase link station.
(TIF)
Displacement over time of reference markings recorded by video, showing a sinusoidal displacement when the reference markings were tracked over 8 complete cycles for a CS = 4 ± 4%. The strain characterization of the silicone substrate using computational software ABAQUS determined a uniform strain distribution in the center of the flow channel at a maximum strain of 10%. The cells were plated in the uniform strain region.
(TIF)
The upper panel of the LabVIEW screen shows a typical flow waveform and the lower panel shows the associated FFT indicating very little contribution from the second or higher harmonics.
(TIF)
We like to thank Pablo Saez for his help with the strain characterization model of cell culture substrate.
This work was supported by NIH Grant HL 086543.