Laminar Shear Stress–Induced GRO mRNA and Protein Expression in Endothelial Cells
Background—The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress.
Methods and Results—The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-β. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-α and/or -β protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress–induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti–GRO-α antibody.
Conclusions—The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.
The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing the endothelial functions. There is evidence that the expression of inducers of atherosclerosis, such as vascular cell adhesion molecule-1 (VCAM-1), on endothelium,1 macrophage accumulation on endothelium,2 and the enhancement of endothelial permeability and lipid deposition3 4 preferentially occur at turbulent low mean shear stress regions of the arteries. It was also reported that thick glycocalyx5 and an increase of zonular-type tight junctions between endothelial cells (ECs),6 which may function as antiatherosclerotic factors, are preferentially recognized at laminar high shear stress regions of the arteries. The morphologies of the ECs in these 2 regions are also different, which may reflect different functional activity of the ECs. In vitro studies in our laboratory revealed that laminar high shear stress promoted glycosaminoglycan synthesis,7 tight junction formation, and the expression of junction-related proteins in ECs,6 8 which may function as selective permeability barriers to large molecules. We and other investigators have shown that the mechanical signal of laminar shear stress was transmitted to cellular nuclei, in which it upregulated the expression of various genes and reacted to the shear stress–responsive element in the promoters, such as platelet-derived growth factor (PDGF), binding with transcription factors such as egr-1.9 The inhibition of gene expression by shear stress has also been reported (for a review, see Reference 1010 ). To obtain the appropriate probes for elucidating the molecular mechanisms of the antagonistic effect of shear stress on endothelial functions, we prepared an EC cDNA library to select specific clones expressed in response to laminar shear stress. Here we present evidence that laminar shear stress induces the expression of melanoma growth-stimulatory activity/growth-regulated gene (GRO) (a member of the chemokine family) in ECs (bovine aortic ECs [BAECs] and human umbilical vein ECs [HUVECs]) and that this expression functions as an adhesion factor of monocytes on ECs subjected to shear stress.
The Oligotex-dT30, cDNA Synthesis Kit, DNA Ligation Kit, Random Primer DNA Labeling Kit Version 2.0, and Taq Cycle Sequencing Core Kit were from Takara Shuzo Co. The λ EXlox EcoRI/HindIII arms kit, λ EXlox vector arms, PhageMaker In Vitro Lambda Packaging System, and host cells ER1647 and BM25.8 were obtained from Novagen Inc. The plasmid midikits were from Qiagen. All procedures were done according to the manufacturer’s technical manuals.
The goat polyclonal (AB-275-PB) and mouse monoclonal (MAB275) antibodies to human GRO-α and the mouse monoclonal antibody to GRO-β (MAB276) were from R&D Systems. The monoclonal antibody to human CD34 (QBEND 10) was from Cosmo Bio Co.
BAECs were scraped from bovine thoracic aortas with a surgical blade and grown on plastic culture plates (Becton-Dickinson) in DMEM containing 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. After the cells were removed from the dishes, cells at passages 7 to 12 were plated on a polyester sheet (54×89 mm, Plastic Suppliers) or on a plastic slide (36×74 mm) that was prepared from the bottom of tissue culture dishes, at a seeding density of 1×106 to 1.5×106 per sheet or 5×105 to 7×105 cells per slide. The cells were cultured until they reached confluence (2 to 3 days after seeding).
HUVECs were harvested from human umbilical vein with the use of 0.05% trypsin with 0.02% EDTA and plated on 0.1% gelatin-coated dishes and incubated in DMEM containing 20% FCS, 10 ng/mL basic fibroblast growth factor, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Confluent HUVECs at passages 6 to 9 on gelatin-coated polyester sheets were used for the experiments.
Human peripheral blood monocytes (THP-1) obtained from the American Type Culture Collection were maintained in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Cells at passages 8 to 10 were used for the experiments.
Exposure to Shear Stress
The flow experiments were performed according to a method similar to that described previously.11 Briefly, a confluent monolayer of ECs on a polyester sheet or plastic slide was placed in a parallel-plate flow chamber and subjected to steady laminar shear stress. The flow loop with reservoirs and the flow chamber were filled with DMEM containing 10% FCS. Control cells were grown on the same polyester sheets or plastic slides in the same medium as sheared cells until the cells reached confluence and were transferred into fresh medium before being maintained in the incubator.
After the BAECs on the polyester sheets were exposed to 30 dyne/cm2 shear stress for 4 hours, total RNA was extracted from the cells by the guanidinium isothiocyanate and cesium chloride gradient procedure of Chirgwin et al.12 Messenger RNA was isolated by Oligotex-dt30, and cDNA was synthesized with an oligo(dT)18 primer and a cDNA synthesis kit. After methylation by methylase EcoRI and HindIII, 5 μg cDNA in 5 μL of Tris-EDTA buffer was ligated to 4 μg dephosphorylated directional EcoRI/HindIII linkers (GCTTGAATTCAAGC) and digested with EcoRI and Hind III with a DNA ligation kit and λ EXlox EcoRI/HindIII arms kit. Linker cDNA (5 μg) was ligated to 0.5 μg λ EXlox vector arms, and then the in vitro packaging of vector to λ EXlox phage was performed with the In Vitro Lambda Packaging System.
Library Screening by Differential Hybridization
Specific cDNA clones expressed in response to the laminar shear stress were selected by screening the library by the differential plaque hybridization method. After amplification of the phage library, 250-μL phage libraries (2.27×104 pfu/mL) in λ EXlox vectors were incubated with an equal amount of Escherichia coli (strain ER1647, 5×108 cells/mL) suspension for 20 minutes at 37°C to allow the phage to adsorb to the host; top agar was added, and then 5700 phages per plate in top agar were plated. After incubation of the plates for 9 hours, plaques on each plate were transferred to 2 positively charged nylon membranes (Hybond-N+, Amersham International PLC). The nucleic acid probes that were labeled with [α-32P]dCTP (110 TBq per mmol/L) by a Random Primer DNA Labeling Kit Version 2.0 were synthesized from cDNA libraries of control BAECs, and cDNA on the membranes was hybridized with the probes. Shear stress–specific clones were identified as plaques that did not hybridize with the probes.
After isolation and amplification, phages with shear stress–specific clones (positive clones) were converted to plasmid clones automatically by infection to the host strain BM25.5. Positive plasmid clones were amplified by polymerase chain reaction (PCR) and screened twice (2nd and 3rd screening) by dot blotting hybridization using probes from the cDNA library of both sheared and static control BAECs, which were equally labeled by [α-32P]dCTP. The absorbency of dot images was evaluated quantitatively by the Bio Image Analyzer (BAS-2000 II, Fujifilm), and positive clones whose absorbencies were >2 times as large as that of the control were selected.
Northern Blot Analysis
Northern blotting was performed to confirm the mRNA expression of selected positive genes and to examine the time course of mRNA expression of these clones in ECs produced by shear stress according to a method similar to that described previously.11 Equal amounts of total RNA (20 μg per lane) were electrophoresed on 1% agarose gels and transferred to nylon membranes (Hybond-N+, Amersham). Blots were hybridized at 65°C overnight with selected positive clones and GAPDH13 labeled with [α-32P]dCTP by random priming. After the membranes had been washed, blots were visualized by autoradiography and quantified by the Bio Image Analyzer. The data are presented as relative values (target gene/GAPDH) and plotted against time.
Sequence Analysis and Homology Search
After amplification of the clones by the PCR method, the partial sequence of each clone was detected by the Taq Cycle Sequencing Core Kit (Takara Shuzo Co) and a fluorescein automatic sequencer (SQ3000/32, Hitachi Co) according to the manufacturer’s protocol. The homology of nucleotide and amino acid sequences was searched in the GenBank and SWISSPROT databases, respectively, accessed with Sequence Interpretation Tools (GenomeNet Japan).
Western Blot Analysis
HUVECs that had been subjected to shear stress were harvested by scraping, and the cells were lysed in 10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Triton X-100, 1 mmol/L PMSF, 10 μg/mL leupeptin, and 10 μg/mL pepstatin. Insoluble material was pelleted, and lysate proteins were separated on a 10% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was placed in a solution containing a monoclonal antibody to GRO-α or -β (1/500 dilution), and blots were developed with biotinylated secondary antibody (rabbit anti-mouse IgG) and peroxidase-conjugated streptavidin. Bound antigens were visualized with 0.02% 3,3′-diaminobenzidine tetrahydrochloride (DAB). Visualized blots were analyzed by the public domain NIH image program (developed at the US National Institutes of Health).
Human aorta at the bifurcation of the intercostal artery or at the bifurcation of the inferior mesenteric artery, obtained from 3 cadavers of people who died at 50 to 64 years old, were fixed with 4% paraformaldehyde and embedded in paraffin. BAECs and HUVECs that had been subjected to 30 dyne/cm2 shear stress for 4 and 12 hours, respectively, were fixed with 95% ethanol for 30 minutes. A monoclonal antibody to GRO-β (1/200 dilution), a polyclonal antibody to GRO-α (1/200 dilution), and a monoclonal antibody to CD34 (1/100 dilution) were used for staining human arteries. A monoclonal antibody to GRO-β was used at 1/50 and 1/100 dilution for staining the cultured cells, and bound antigens were visualized with biotin-labeled rabbit anti-mouse IgG or anti-goat IgG in combination with peroxidase-conjugated streptavidin and 0.02% DAB.
Monocyte Binding Assay
ECs on plastic slides were exposed to 30 dyne/cm2 laminar shear stress for 4 hours. Immediately after exposure to shear stress, the static and sheared cells were rinsed with DMEM containing 10% FCS at 37°C and then incubated with THP-1 (1.4×106 cells per slide) for 20 minutes at 37°C in the same medium. The cells were washed 3 times to remove unattached monocytes and then fixed in methanol containing 2% formaldehyde at room temperature for 5 minutes. After staining with Diff-Quik (Kokusai Shiyaku Co), a translucent plastic sheet with dots 5 mm apart was attached beneath the slide, and a grid was inserted into the microscope eyepiece. A field on the grid coinciding with the dots was selected to count the number of monocytes under the microscope. The number of attached monocytes on ECs in ≥60 fields was counted.
To study the expression of GRO protein on ECs subjected to shear stress, cells were treated by the antibody against GRO protein. After exposure to 30 dyne/cm2 laminar shear stress for 4 hours, the sheared and static cells on each slide were washed, and the area of each slide was divided into 3 equal parts. After the cells on the center part were stripped off with a rubber policeman, the polyclonal antibody for GRO-α (50 μg/mL protein) and normal goat serum (1:200 dilutions) were each applied to the cells on one of the remaining parts. The cells were then incubated for 15 minutes at 37°C. The cells were washed and then incubated with monocytes (2×106 cells per part) for 20 minutes at 37°C. After fixing and staining, the number of monocytes on ECs in at least 20 fields was counted under the microscope as described above.
The changes in monocyte number on 3 slides were averaged in each experiment, expressed as mean±SD, and analyzed by ANOVA with Scheffé’s test for multiple-group comparisons and by unpaired t test for 2 groups. Significance was defined as P<0.01.
Shear Stress–Specific Clones and Homology
A total of 7356 plaques containing phage cDNA library were screened, and 9 clones whose absorbency ratios of mRNA to GAPDH were >2 times higher than that of the static control were selected. The mRNA of these selected clones was either expressed transiently or increased up to 24 hours by the shear stress (30 dyne/cm2), and the absorbency ratio to GAPDH of clone 443 was 8.8-fold higher than that of the control at 24 hours (Figure 1⇓).
The partial base sequences of the 7 clones were analyzed. By subsequent homology searches, 65% to 94% homology to previously reported genes was detected in these clones. However, the functions of all of these clones except 1 are unknown. This clone, ≈1 kb in size (No. 539), was found to be 94% and 83% homologous to the GRO homolog and human gro-β mRNA, respectively. The resultant amino acid sequence of this clone was found to be 79% homologous to the human GRO-β protein.
GRO mRNA Expression
One main band was observed for the mRNA of GRO, which was ≈1 kb in size, by Northern blotting. Besides the main band, low levels of a smaller mRNA were detected occasionally, as described by Wen et al.14 The induction of expression was quite early and quick. Expression was first evident and became maximal at 1 hour after the exposure of the BAECs to the shear stress (30 dyne/cm2) and was maintained at a high level until 4 hours, with a nearly 12-fold increase over that of the control at this point; it decreased gradually to the control level by 12 hours (Figure 2⇓). When BAECs were subjected to shear stress for 4 hours, 5, 20, 30, and 70 dyne/cm2 shear stress induced GRO mRNA expression, although the level of expression by 70 dyne/cm2 was usually low (Figure 3⇓). Five dyne/cm2 shear stress induced a time course of expression similar to that induced by 30 dyne/cm2 in BAECs (Figure 4⇓). HUVECs also expressed GRO mRNA after 2- and 4-hour exposure to 5 and 30 dyne/cm2 shear stress (Figure 5⇓).
GRO Protein Expression in HUVECs, BAECs, and Human Aorta
HUVECs expressed both GRO-α and -β proteins after being subjected to shear stress for 4 to 24 hours, as analyzed with Western blotting (Figures 6⇓ and 7⇓). Immunohistochemistry revealed that both HUVECs and BAECs reacted to GRO-β monoclonal antibody after being subjected to shear stress for 12 and 4 hours, respectively (Figure 8⇓). The endothelium of human aorta at bifurcation also expressed both GRO-β and -α, even though not all of the cells reacted (Figure 9⇓).
Anti-GRO Antibody Inhibits Monocyte Adhesion to Sheared ECs
The shear stress (30 dyne/cm2 for 4 hours) significantly enhanced the monocyte adhesion on the ECs, to a level 2- to 2.5-fold that of the control cells (Figures 10⇓ and 11⇓). For the detection of the induction of the GRO protein (which has been shown to function as an adhesion factor of monocytes on the surface of ECs15 ) by shear stress, ECs were exposed to steady laminar shear stress (30 dyne/cm2) for 4 hours and then treated with the polyclonal antibody for the GRO protein before coculture with monocytes. The number of monocytes adhered on the surface of the static ECs treated with the antibody for GRO-α was similar to that of the static cells treated with control goat serum (3823.3±258.7 and 3697.0±238.9, respectively). However, this antibody inhibited the increased monocyte adhesion induced by shear stress significantly, by ≈53% (5282.3±734.8 and 7068.0±316.2 in sheared ECs with and without treatment by antibody, respectively, Figure 11⇓).
Nine clones were selected from the cDNA library of BAECs subjected to 30 dyne/cm2 shear stress for 4 hours by the differential plaque hybridization method. The Northern blotting analysis demonstrated an expression of mRNA of these clones in the ECs subjected to shear stress 2 to 12 times higher than that in the control cells. The sequence analysis of 7 clones revealed that the functions of these clones are unknown, except for 1 clone.
A number of important biological changes induced by shear stress in ECs have been observed within a short period of shear stress exposure. For example, the opening of a K+-selective channel16 and increases in intracellular calcium17 and IP318 occurred immediately after shear stress exposure; protein kinase-ε and MAP kinase20 were activated within 10 minutes (for a review, see References 10 and 2110 21 ). We also have shown that a stress fiber formation was observed within 30 minutes and a decrease of DNA synthesis produced by shear stress in ECs occurred within 4 hours.22 We therefore started to prepare cDNA libraries from ECs subjected to shear stress for 4 hours to select shear stress–specific clones. However, the hybridization of these selected genes disclosed the mRNA expression not only at 4 hours but also at 1 to 24 hours after the start of exposure to shear stress. As Topper et al23 demonstrated, the full-length cDNAs of these genes and antibodies for the proteins encoded in these genes might be useful tools for the elucidation of mechanisms of shear stress–dependent functional changes of vascular cells in atherogenesis.
Our selected clones had no homology to any known genes in the data banks examined, such as c-jun, intercellular adhesion molecule (ICAM)-1, PDGF-A and -B, transforming growth factor-β, endothelin-1, or heparin-binding epidermal growth factor-like growth factor, which had been shown to be expressed at 4 hours of exposure to shear stress. One of the possible reasons for this result might be the differences in the arteries from which the ECs were separated (human versus bovine, vein versus artery, and fetal vein versus adult aorta). The quality of the cDNA library and a loss of the low-expression genes during the selection might also be involved.
Recent findings indicate that factors related to inflammation, such as interleukin-1, tumor necrosis factor, lipopolysaccharide, and thrombin, induced GRO gene and protein in ECs14 and that GRO protein induced by oxidized LDL, which may cause fatty streak formation, bound to monocytes on the surface of ECs.15 The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein, in the absence of inflammatory or oxidized lipid stimulation. Because all of the above stimulants induced a maximal level of expression of GRO mRNA between 1 and 4 hours after the treatment and almost all of the expressions subsequently decreased to a basal level, it is possible that a similar signal transduction system is involved with the different stimulants.
GRO-β protein was expressed in the sheared HUVECs even after the mRNA expression was diminished. This could be due to an accumulation of GRO protein as a consequence of the low degradation rate.
Besides GRO, the expression of other endothelial adhesion molecules for monocytes (ie, ICAM-1,10 VCAM-1,10 monocyte chemotactic protein [MCP]-1,24 and E-selectin25 ) regulated by shear stress has been studied. Although ICAM-1 expression increased at 4 hours of shear stress, VCAM-1 and E-selectin expression were either unchanged or decreased to lower than the control level, and the MCP-1 mRNA expression induced by shear stress declined toward the control level at 4 hours. Our present results show that the increased monocyte adhesion induced by shear stress was inhibited by 53% by the antibody for GRO-α. Thus, the changes of GRO-α alone are probably not sufficient to account for the enhancement of monocyte adhesion in ECs under shear stress. These data indicate that the expression of multiple adhesion molecules at different times or at the same time upregulate or downregulate the monocyte adhesion on the ECs under the flow.
In addition to the adhesion function, a recent report indicated that GRO-α and -β can inhibit the growth factor–stimulated proliferation of ECs.26 We found that the DNA synthesis of ECs stimulated by serum was significantly inhibited by shear stress within 4 hours.22 These data indicate that GRO synthesized by ECs might be involved in shear stress–dependent mechanical signals for the inhibition of DNA synthesis in the ECs themselves.
Further investigations of the functions and participation in atherogenesis of our selected clones may clarify the significance of shear stress in atherogenesis.
This study was supported in part by a grant-in-aid from the Ministry of Science and Technology and by a grant-in-aid from the Ministry of Health and Welfare of Japan.
- Received June 18, 1998.
- Revision received July 13, 1998.
- Accepted July 21, 1998.
- Copyright © 1998 by American Heart Association
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Cao Y, Chen C, Weatherbee JA, Tsang M, Folkman J. gro-beta, a -C-X-C- chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice. J Exp Med. 1995;182:2069–2077.Nine clones whose expression was specifically enhanced by shear stress were selected from a library of endothelial cells (ECs) subjected to steady laminar shear stress (30 dyne/cm2) by a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA. The GRO mRNA and protein expression by bovine aortic ECs and human umbilical vein ECs were increased after these cells were subjected to the shear stress. The shear stress enhanced monocyte adhesion to ECs, and this enhancement was inhibited by anti–GRO-α antibody. These data indicate that shear stress enhanced the monocyte adhesion on ECs via GRO protein expression.