This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, D. L. Articles by Chien, S. Search for Related Content PubMed PubMed Citation Articles by Wang, D. L. Articles by Chien, S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL

(Circulation Research. 1995;77:294-302.)
© 1995 American Heart Association, Inc.

Articles

Mechanical Strain Induces Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells

Effects of Mechanical Strain on Monocyte Adhesion to Endothelial Cells

Danny Ling Wang, Being-Sun Wung, Yeun-Jund Shyy, Cheng-Fu Lin, Yuh-Jen Chao, Shunichi Usami, Shu Chien

From the Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Monocyte chemotactic protein-1 (MCP-1), a potent monocyte chemoattractant secreted by endothelial cells (ECs), is believed to play a key role in the early events of atherogenesis. Since vascular ECs are constantly subjected to mechanical stresses, we examined how cyclic strain affects the expression of the MCP-1 gene in human ECs grown on a flexible membrane base deformed by sinusoidal negative pressure (peak level, -16 kPa at 60 cycles per minute). Northern blot analysis demonstrated that the MCP-1 mRNA levels in ECs subjected to strain for 1, 5, or 24 hours were double those in control ECs (P<.05). This strain-induced increase was mainly serum independent, and MCP-1 mRNA level returned to its control basal level 3 hours after release of strain. Culture media from strained ECs contained approximately twice the MCP-1 concentration and more than twice the monocyte chemotactic activity of media from control ECs (P<.05). Pretreatment of collected media with anti–MCP-1 antibody suppressed such activity. Monocyte adhesion to ECs subjected to strain for 12 hours was 1.8-fold greater than adhesion to unstrained control ECs (P<.05). A protein kinase C inhibitor, calphostin C, abolished the strain-induced MCP-1 gene expression. In addition, cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 and KT5823, respectively) partially inhibited such expression. Pretreatment with EGTA or the intracellular Ca²⁺ chelator BAPTA/AM strongly suppressed the strain-induced MCP-1 mRNA. Verapamil, a Ca²⁺ channel blocker, greatly reduced MCP-1 mRNA levels in both strained and unstrained ECs. These results indicate that mechanical strain can stimulate monocyte chemotaxis and adhesion by increasing MCP-1 gene expression in ECs. This increased gene expression is predominantly mediated via the protein kinase C pathway and requires Ca²⁺ influx. Such strain-induced MCP-1 expression might contribute to the trapping of monocytes in the subendothelial space. Strain-induced gene expression might provide a molecular mechanism for the role of hypertension in atherogenesis.

Key Words: atherosclerosis • endothelial cells • gene regulation • mechanical strain • monocyte chemotactic protein-1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The involvement of macrophages in atherogenesis is well known. Many atherosclerotic risk factors enhance the transendothelial movement of monocytes, their accumulation in the subendothelial space where they transform into macrophages, and their subsequent conversion to foam cells by taking up oxidized LDL.^{1 2} However, the molecular mechanisms involved in the recruitment and accumulation of monocytes within the arterial wall are not yet fully understood. Although growth factors and/or cytokines that promote the expression of adhesive molecules on the surfaces of monocytes and ECs are known to be involved,^{1 3 4} recent studies suggest that MCP-1, a potent chemoattractant for monocyte recruitment, may play an important role in the early events of atherogenesis.^{5 6 7 8} MCP-1 is a 14-kD glycoprotein secreted by many cells,^{9 10 11 12 13} including vascular smooth muscle cells and ECs.⁹ The MCP-1 gene, known as the JE gene in the murine system, belongs to a family of small, early-response, and serum-inducible genes.^{10 11 14 15} The MCP-1 gene can be induced by cytokines such as tumor necrosis factor-,¹⁶ interleukin-1 and interleukin-4,^{17 18} and IgG.¹⁹ Because of its potent monocyte chemotactic activity and its secretion by vascular endothelium, MCP-1 may play an essential role in the recruitment and accumulation of monocytes/macrophages in the subendothelial space of the arterial wall during atherogenesis. The participation of MCP-1 in the progression of atherosclerotic plaque has been demonstrated by reports that MCP-1 is expressed in macrophage-rich regions of human and rabbit atherosclerotic lesions⁵ and that it is induced by modified LDL.^{6 7} Furthermore, Shyy et al⁸ have recently demonstrated that monocyte colony–stimulating factor stimulates MCP-1 gene expression in human ECs and that this increases the adhesion of monocytes to ECs. Although the activation of MCP-1 gene expression has been suggested to be mediated through multiple signaling pathways,²⁰ the detailed signaling mechanism for its induction in ECs remains unclear.

Vascular ECs, which synthesize and release substances important for maintaining normal vascular homeostasis, are constantly exposed to mechanical forces, including blood flow–induced shear stress and pressure-induced strain. A large body of evidence shows that physiological shear stress can regulate gene expression and the production of many types of substances, including prostacyclin²¹ and TPA,²² by ECs. However, modulation of gene expression in the endothelium in response to cyclic strain, which results from the oscillatory deformation of the vascular wall caused by pulsatile intravascular pressures associated with systole and diastole, is relatively uncharacterized. Previous studies from this laboratory have demonstrated that cyclic strain enhances endothelin-1 mRNA levels in human ECs and thus increases the secretion of endothelin-1.^{23 24} In the present study, we demonstrate further that mechanical strain can stimulate the chemotaxis and adhesion of monocytes to ECs by increasing MCP-1 levels in ECs. Evidence is also provided that this enhancement is mediated predominantly through the PKC pathway and appears to require extracellular Ca²⁺. The increased gene expression of MCP-1 and its release from ECs suggest that mechanical strain might modulate the migration of monocytes across the arterial wall, thus providing a molecular mechanism to link hypertension to atherogenesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Calphostin C, KT 5720, and KT 5823 were purchased from Kamiya Biomedical Co. A 0.6-kb fragment of MCP-1 cDNA isolated from a human aortic endothelium cDNA library^{8 20} was used as a probe in Northern blot analysis. Recombinant human MCP-1 was obtained from Pepro Tech Inc. Rabbit anti-human MCP-1 antibody was obtained from Genzyme. Monoclonal anti-human MCP-1 antibody (IgG₁) was purchased from R&D Systems. Goat anti-mouse IgG–alkaline phosphatase conjugate and phosphatase substrate 1 were purchased from Sigma Chemical Co. BAPTA/AM, a membrane-permeating form of BAPTA, was purchased from Calbiochem. Goat anti-mouse IgG–alkaline phosphatase conjugate, phosphatase substrate 1, verapamil, and other chemicals of reagent grade were obtained from Sigma Chemical Co. Nunc-Immuno IF ELISA plates were supplied by GIBCO BRL.

EC Cultures
HUVECs were isolated from human umbilical cord according to the procedures described previously.²⁵ After 3 days of growth in medium 199 (GIBCO BRL) containing 20% fetal calf serum, ECs (2.0x10⁵ cells per well) were seeded on the flexible membrane base of a culture well (Flex 1, Flexcell Co) and grown for 3 more days until the monolayer became confluent. The medium for the cultured ECs was then changed to the same medium containing only 2% fetal calf serum, and the cells were incubated overnight before the experiment.

In Vitro Cyclic Strain on Cultured ECs
The strain unit Flexcell FX-2000 (Flexcell), which has been described previously,^{23 24} consists of a vacuum unit linked to a valve controlled by a computer program. ECs cultured on the flexible membrane base were subjected to cyclic strain produced by this computer-controlled application of sinusoidal negative pressure. The flexible membranes supporting the cultured cells were deformed by a sinusoidal negative pressure with a peak level of 16 kPa at a frequency of 1 Hz (60 cycles per minute) for various periods of time. After the strain experiment, the conditioned supernatant and the total RNA from the strained cells were collected for Boyden chamber study and Northern blot analysis, respectively. In experiments involving signaling inhibitors, HUVECs were pretreated with either calphostin C (2.5 µmol/L), a specific PKC inhibitor; KT 5720 (0.5 µmol/L, K_i=56 nmol/L), a cAMP-dependent protein kinase inhibitor; KT 5823 (1 µmol/L, K_i=0.234 µmol/L), a cGMP-dependent protein kinase inhibitor; verapamil (10 µmol/L); or BAPTA/AM (2.5 µmol/L) for 30 minutes before the strain treatment. The specificity of calphostin C was shown by the IC₅₀ values, which were 50 nmol/L for PKC, >50 µmol/L for cAMP-dependent protein kinase, and >25 µmol/L for cGMP-dependent protein kinase. In the EGTA experiments, cells were preincubated with EGTA (4 mmol/L) for 5 minutes before strain application. After incubation with the inhibitors or other agents, HUVECs remained intact on the flexible membrane, as revealed by trypan blue staining and by the quantities and qualities of total RNA collected.

ELISA for MCP-1
Human MCP-1 concentration was measured by ELISA, with recombinant human MCP-1 used as a standard. Briefly, 200 µL per well of anti–MCP-1 polyclonal antibody in bicarbonate buffer (pH 9.6) was coated onto Nunc-Immuno plates at a concentration of 5 µg protein/mL overnight. After washing with phosphate buffer containing EDTA and Tween 20 (buffer A), the plates were incubated with phosphate buffer (pH 7.2) containing 0.2% Triton X-100 and 1% bovine serum albumin for 2 hours at room temperature. Those plates were washed with phosphate buffer, and then 200 µL of serially diluted culture medium from each sample was added. A series of MCP-1 dilutions (0.15 to 15 ng per well) was used as the standard. After incubation for 2 hours with shaking, media were removed, and wells were rinsed with buffer A. Anti–MCP-1 monoclonal antibody (200 µL per well of 5 µg/mL) was then added and incubated for 2 hours at room temperature. After washing with buffer A, the wells were incubated with goat anti-mouse IgG–alkine phosphatase conjugate for 2 hours. Phosphatase substrate (1 mg/mL) was then added. The reaction products were measured at 405 nm by a microplate reader (Molecular Device). The standard concentration curve for MCP-1 measured by this ELISA was linear from 0.15 to 15 ng/mL.

Monocyte Chemotaxis Assays
Blood was drawn by venipuncture from healthy adults into anticoagulant-dextrose buffer. Monocytes were isolated by the Ficoll-Paque (Pharmacia) density centrifugation method as previously described.²⁶ In brief, a standard solution of Ficoll-Hypaque was prepared, and crystalline NaCl was added to achieve a final density of 1.078. Separation of monocytes was achieved by layering 3 vol of leukocyte-rich plasma on 1 vol of the hyperosmotic separation medium and centrifuging at 600g for 15 minutes at room temperature. The monocytes were harvested at the interface between the Ficoll-Hypaque cushion and the plasma. The collected monocytes were washed twice and resuspended in RPMI 1640 (GIBCO BRL) containing 25 mmol/L HEPES and 30% autologous serum at pH 7.2 to a final concentration of 3x10⁶ cells/mL. This preparation method routinely harvested >95% of monocytes and maintained >95% viability as assessed by trypan blue exclusion. The chemotaxis assay was performed in a Boyden chamber containing two wells separated by a filter membrane (pore size, 5 µm).^{27 28} In brief, monocytes (3.0x10⁵ cells) were added to the upper well, and the test medium was added to the lower well. After 1 hour of incubating the chamber at 37°C, the nonadherent monocytes were rinsed off. The filters were then removed, fixed in paraformaldehyde, and stained with Giemsa. The number of monocytes that had migrated to the lower surface of the filter was determined microscopically from 10 random areas at a magnification of x1000. Monocytes were easily recognized by their characteristic horseshoe-shaped nuclei and blue cytoplasm on Giemsa staining. Fresh medium containing FMLP (0.1 µmol/L) and the culture medium from ECs grown without mechanical strain were used as positive and negative controls, respectively. The number of attached monocytes was counted in each of 10 microscopic fields from a set of three separate experiments. Approximately 16 cells per x1000 magnification field (2.5x10⁴ µm²) were seen in negative controls, and 82 cells per field were seen in positive controls. In some experiments, the collected medium was preincubated with an anti–MCP-1 antibody (1 µg/mL) for 1 hour before its introduction into the Boyden chamber.

Monocyte Adhesion to ECs
Freshly isolated monocytes were radiolabeled by incubation with ⁵¹Cr (500 µCi/2x10⁷ cells) for 30 minutes. The labeled monocytes were then added to HUVECs, which had been strained for 4 or 12 hours (3x10⁵ monocytes per well) and incubated for 30 minutes. The monocytes that had adhered to ECs were then washed twice with phosphate-buffered saline and lysed with 0.5N NaOH, and the total radioactivity was counted in a gamma counter. In some experiments, the media from the strained ECs were removed and replaced with fresh medium 199. The conditioned media collected were then used to replace the medium of control unstrained ECs before the introduction of monocytes in a crossover experiment.

RNA Isolation and Northern Hybridization
Total cellular RNA was obtained as described previously.²⁹ RNA was transferred onto a membrane by a vacuum blotting system (VacuGene XL, Pharmacia). After hybridizing with the ³²P-labeled MCP-1 cDNA, the membrane was washed with 1x standard saline citrate containing 1% SDS at room temperature for 15 minutes and then exposed to x-ray film (Kodak X-Omat-AR) at -70°C. Autoradiographic results were scanned and analyzed by using a densitometer (Computing Densitometer 300S, Molecular Dynamics).

Statistical Analysis
Statistical analyses were performed by using Student's t test for experiments consisting of two groups only and by ANOVA for experiments consisting of more than two groups. Data are expressed as mean±SEM. Statistical significance was defined as P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Strain on MCP-1 mRNA Levels
The application of cyclic strain to ECs cultured on flexible membrane bases for 1, 5, or 24 hours caused 2.2-, 2.3-, and 2.2-fold increases in MCP-1 mRNA levels, respectively, compared with the level in control unstrained cells (Fig 1A). To elucidate whether this strain-induced MCP-1 gene expression was serum independent, we preincubated the ECs overnight with medium containing only 0.5% bovine serum albumin, and those cells were then subjected to strain treatment for 5 hours. Although the basal MCP-1 expression in these control unstrained ECs was lower than that in ECs maintained in medium containing 2% serum, the MCP-1 level in ECs under serum-free conditions could still be increased by stretch (data not shown). To further demonstrate that this gene induction was strain dependent, ECs were strained for 2 hours and then allowed to rest. As shown in Fig 1B, ECs strained for 2 hours showed an 2-fold MCP-1 gene expression. When these strained ECs were allowed to rest, their MCP-1 mRNA level gradually decreased, returning to near the control basal level in 3 hours. By 4 hours after the onset of resting, the MCP-1 mRNA level decreased to a level lower than the control level. These results indicate that mechanical strain induced increases in MCP-1 mRNA levels in ECs.

View larger version (22K): [in this window] [in a new window]   Figure 1. Cyclic strain increases the MCP-1 gene expression in HUVECs. A, Confluent cells grown on the flexible membrane bases of culture wells were mechanically strained for various durations as indicated, and the total RNAs were isolated for Northern blot analysis with a 32P-labeled 0.6-kb MCP-1 cDNA as the probe. The ethidium bromide staining of 18S rRNA indicates that equal amounts of RNA were loaded. Autoradiographic results from three experiments were analyzed by densitometry, and the data are presented as mean±SEM from three to four separate experiments. *P<.05 vs unstrained controls. B, Cultured HUVECs were strained (S) for 2 hours. Those culture wells containing strained cells were then removed from the strain unit and rested in an incubator for 1, 2, 3, or 4 hours (R1, R2, R3, and R4, respectively) before RNA extraction and Northern blot analysis. C indicates control. Densitometric results from the autoradiographic pictures are presented under each lane.

Effect of Strain on Monocyte Adhesion to ECs
Since mechanical strain can induce MCP-1 mRNA levels that may result in increased release of MCP-1 protein into the culture medium of ECs, we measured the MCP-1 concentration by ELISA and tested the monocyte chemotactic activity of these collected media by using a Boyden chamber. MCP-1 concentration in culture medium from ECs after strain for 7 hours was 0.5 nmol/L compared with 0.3 nmol/L from control unstrained cells. MCP-1 concentration increased further from 0.5 nmol/L in control cells to 0.9 nmol/L in ECs after strain for 12 hours. Fresh medium containing FMLP (0.1 µmol/L), which served as a positive control, induced an increase in monocyte migration to nearly five times the control value (data not shown). The degree of increase in monocyte chemotactic activity was reduced progressively when the collected medium from 7-hour–strained ECs was diluted 2-, 4-, or 8-fold with medium 199 (Fig 2A). Fig 2B shows that the media collected from ECs strained for 7 or 12 hours contained sufficient chemotactic activity to induce monocyte migration to 2.4 and 2.5 times that of the control media, respectively. Preincubation of the cultured media collected from ECs strained for 7 or 12 hours with an antibody to MCP-1 decreased their monocyte chemotactic activity (Fig 2B). These results indicate that the MCP-1 released from strained ECs causes an increase in monocyte chemotactic activity. Since previous studies^{7 8} have shown that elevated MCP-1 gene expression leads to increases in EC adhesion and transendothelial migration of monocytes, we tested whether the induced MCP-1 release in culture medium can lead to an increase in monocyte adhesion to strained ECs. Isolated radiolabeled monocytes were incubated either with ECs that had been strained for 4 or 12 hours or with unstrained control ECs. As shown in Fig 2C, monocyte adhesion to 12-hour–strained ECs increased by 80%, but no increase occurred in the 4-hour–strained ECs. To confirm that the MCP-1 released into the culture medium contributed to the monocyte adhesion, we replaced the medium from 12-hour–strained ECs with the control medium; the increased monocyte adhesion was reduced to 35%. Furthermore, when we replaced the medium from control ECs with medium from the 12-hour–strained ECs, this resulted in a 60% increase in monocyte adhesion. However, when medium from these 12-hour–strained ECs was preincubated with anti–MCP-1 before its introduction into control ECs, the increased monocyte adhesion was less. All these results indicate that mechanical strain increases the adhesion of monocytes to strained ECs by stimulating MCP-1 production in ECs.

View larger version (15K): [in this window] [in a new window]   Figure 2. Cyclic strain increases monocyte chemotactic activity and monocyte adhesion to ECs. A, Monocyte chemotactic activity in original (no dilution [no dil]) and diluted culture medium from cells strained for 7 hours is shown. The MCP-1 concentration analyzed by ELISA was 0.3 and 0.5 nmol/L from unstrained control and strained ECs, respectively. Note the decrease in chemotactic activity with progressive dilution. MCP-1 activity was tested in Boyden chambers as described in the text. *P<.05 vs unstrained controls. B, Monocyte chemotactic activity of the culture medium from cells strained for 7 hours (S7) or 12 hours (S12) was suppressed by preincubation of the medium with MCP-1 antibody (Ab, 1 µg/mL) for 1 hour. MCP-1 concentrations in culture medium from unstrained control ECs and ECs strained for 12 hours were 0.5 and 0.9 nmol/L, respectively. Monocytes were counted from 10 different microscopic fields (original magnification x1000) from each of three experiments. The results are shown as mean±SEM. *P<.05 vs unstrained controls. #P<.05 vs the respective strained ECs not treated with Ab. C, 51Cr-labeled monocytes were added to cultured ECs that had been strained for 4 hours (S4) or 12 hours (S12), and the cells were coincubated for 1 hour then used in adhesion assays. Bars on the right represent experiments in which the medium from strained ECs was replaced with fresh medium (S12+control medium) and vice versa (control medium [C]+S12 medium) before monocyte addition. Some experiments were carried out by preincubating the S12 medium with anti–MCP-1 Ab (1 µg/mL) for 1 hour before its introduction into control unstrained ECs (C+S12 medium+Ab). Results are shown as mean±SEM from three or four independent experiments. *P<.05 vs unstrained controls. #P<.05 vs strained ECs untreated with Ab. +P<.05 vs C+S12 medium.

Roles of PKC-, cAMP-, and cGMP-Dependent Protein Kinases in MCP-1 Gene Expression
To study the intracellular signal pathways involved in the strain-enhanced MCP-1 gene expression, we pretreated ECs with calphostin C (2.5 µmol/L), a specific PKC inhibitor, for 30 minutes before strain treatment. As indicated in Fig 3A, calphostin C treatment strongly reduced the MCP-1 gene expression in strained and unstrained ECs to 39% and 15% of unstrained control levels, respectively. Pretreatment with cAMP- or cGMP- dependent protein kinase inhibitors (KT 5720 or KT 5823, respectively) also inhibited the increase in MCP-1 gene expression in strained ECs, but to lesser extents (Fig 3B). These inhibitors did not significantly inhibit the MCP-1 mRNA levels in unstrained cells (data not shown). These results suggest that the PKC pathway plays a significant role in mediating the strain-modulated MCP-1 gene expression. However, the contributions of the cAMP and cGMP pathways to the induced MCP-1 gene expression cannot be excluded. These results indicate that mechanical strain can modulate the MCP-1 gene expression in ECs via the PKC pathway.

View larger version (20K): [in this window] [in a new window]   Figure 3. Roles of PKC-, cAMP-, and cGMP-dependent protein kinases in strain-induced MCP-1 gene expression. A, Cells grown on flexible membranes were pretreated with calphostin C (2.5 µmol/L) for 30 minutes before to the 2-hour strain treatment. Total cellular RNA was extracted and subjected to Northern blot analysis with MCP-1 cDNA as the probe. The ethidium bromide staining of 18S rRNA indicates that equal amounts of RNA were loaded. The results are shown as mean±SEM from three experiments. *P<.05 vs respective unstrained or strained controls. B, ECs were pretreated with the cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 [0.5 µmol/L] or KT5823 [1 µmol/L], respectively) for 30 minutes before strain treatment for 2 hours. *P<.05 vs respective unstrained control or strained untreated ECs.

Role of Ca²⁺ in Strain-Induced MCP-1 Gene Expression
Since it has been reported that mechanical perturbation of ECs causes rapid increases in intracellular Ca²⁺,³⁰ we investigated whether Ca²⁺ influx or intracellularly released Ca²⁺ was involved in strain-induced MCP-1 gene expression. ECs were pretreated with EGTA (4 mmol/L) to chelate the extracellular Ca²⁺ or BAPTA/AM (2.5 µmol/L) to chelate the intracellular Ca²⁺ before strain treatment. The densitometric results in Fig 4A show that the EGTA and BAPTA/AM treatments caused the same degree of inhibition of MCP-1 gene expression in both control and strained ECs. To study further whether Ca²⁺ channels are involved in this strain-induced MCP-1 expression, ECs were pretreated with verapamil, a Ca²⁺ channel blocker, for 30 minutes before a 2-hour strain. As shown in Fig 4B, verapamil reduced MCP-1 mRNA levels in the control and strained groups to 50% and 58% of the unstrained control levels, respectively. Collectively, the above results indicate that the strain-induced MCP-1 gene expression and MCP-1 secretion from ECs are predominantly mediated via the PKC pathway and that this gene induction requires Ca²⁺.

View larger version (21K): [in this window] [in a new window]   Figure 4. Role of Ca2+ in strain-induced MCP-1 gene expression. A, Cultured ECs were pretreated with EGTA (4 mmol/L) for 5 minutes or BAPTA/AM (2.5 µmol/L) for 30 minutes before strain for 2 hours. For BAPTA/AM experiments, ECs in culture medium containing BAPTA/AM were washed, and the medium was replaced with fresh medium before strain. Total RNAs were collected and analyzed by Northern blot analysis with MCP-1 cDNA probe. C indicates control; S, strain experiments. B, Cultured ECs were pretreated with verapamil (10 µmol/L) for 30 minutes before strain for 2 hours. The results are shown as mean±SEM from three experiments. *P<.05 vs respective unstrained or strained ECs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
ECs, after treatment with modified LDL, show increased monocyte chemotactic activity and monocyte binding or migration.^{7 31} Our recent studies have also demonstrated that monocyte colony–stimulating factor can induce MCP-1 gene expression and increase the adhesion of monocytes to endothelial monolayers.⁸ The increased monocyte adhesion/migration can be inhibited by an antibody to MCP-1.^{7 8} In addition to increased release of vasoactive substances, such as prostacyclin²¹ and endothelin-1,³² a biphasic response of MCP-1 gene expression has recently been demonstrated in ECs under shear stress treatment.³³

The effect of strain on cells has only recently been investigated. Pressure overload of the heart and stretching of cardiac myocytes activate PKC and induce the expression of proto-oncogenes.^{34 35} We have demonstrated that ECs under strain can induce endothelin-1 gene expression,^{23 24} which may contribute to cardiac growth and hypertrophy.²⁹ In the present study, we show that mechanical strain can also induce the gene expression and secretion of MCP-1. This gene induction is strain dependent, since the induced MCP-1 mRNA level returned to the control basal level after strain was released. Mechanical strain increased the production and subsequent release of MCP-1 into the culture medium, which would contribute to the increased monocyte adhesion to ECs. Evidence of increased MCP-1 protein production also includes the elevated monocyte chemotaxis mediated by culture medium from strained cells and increased monocyte adhesion to strained ECs, both of which were blocked by the addition of anti–MCP-1 antibody.

Our previous studies have demonstrated that increased MCP-1 gene expression in ECs is sustained after treatment with monocyte colony–stimulating factor⁸ but is transient after the continued application of shear stress³³ or TPA treatment.³⁶ Another previous study of ours has shown that mechanical strain also causes a sustained increase in endothelin-1 gene expression.^{23 24} Thus, the signal transduction pathway for strain-induced gene expression in ECs may differ from that for transient expression of MCP-1 after shear stress or TPA treatment. The reason for the discrepancy between these two mechanical effects on MCP-1 gene induction is unclear but may be due to the nature of these mechanical forces. Steady laminar flow–induced shear stress produces a constant stress to ECs, whereas our strain system provides cyclic changes of pressure to cells. ECs may be sensitive to these cyclic changes. Shear and strain may produce different responses in terms of PKC activation, Ca²⁺ mobilization, and other intracellular signals. The effects of shear stress have been linked to PKC activation.^{37 38 39} However, the PKC activation in strain-treated ECs is sustained.⁴⁰ Whereas fluid shear stress causes either little effect^{41 42} or a transient increase in intracellular Ca²⁺,⁴³ stretch appears to stimulate intracellular Ca²⁺ release and Ca²⁺ flux. The stretch-activated Ca²⁺ influx may help to stimulate PKC activity. Stretch has been demonstrated to cause a sustained increase in cellular content of diacylglycerol in cardiac myocytes⁴⁴ and increases in IP₃ and DAG in ECs after initiation of cyclic strain.⁴⁵ It has been suggested that phosphatidylcholine hydrolysis by phospholipase D can contribute to the sustained DAG formation in ECs subjected to cyclic strain.⁴⁶ An increase in DAG is consistent with reports that stretching increases PKC activity.⁴⁰ Since stretch and phorbol esters induce proto-oncogene c-fos gene expression via a common PKC pathway, it has therefore been suggested that mechanical strain might directly stimulate PKC activity via phospholipase C and/or phospholipase D activation to induce early genes such as c-fos and c-myc.^{35 46} We have previously shown that PKC is involved in strain-induced endothelin-1 gene expression in ECs.^{23 24} Therefore, we conclude that mechanical strain–induced MCP-1 gene expression is predominantly mediated through the PKC pathway. The cAMP- or cGMP-dependent protein kinase pathway plays a minor role and may be involved partially or indirectly through the PKC pathway in regulating this strain-induced MCP-1 gene expression. This is in agreement with a previous report that cyclic strain does not significantly increase intracellular cAMP in HUVECs.⁴⁷

In addition to the presence of elevated DAG in strained cells, an increase in IP₃ might trigger the release of intracellularly stored Ca²⁺, which would contribute to a rapid increase in [Ca²⁺]_i.³⁰ The intracellular Ca²⁺ chelator BAPTA/AM and EGTA exert the same effects in inhibiting the strain-induced MCP-1 gene expression. This finding suggests that both intracellular Ca²⁺ release and Ca²⁺ influx play important roles in such gene expression. A recent report suggests that Ca²⁺ mobilization in stretch-treated ECs involves stretch-activated cation channels,^{48 49} and there is some evidence suggesting the presence of voltage-sensitive Ca²⁺ channels in ECs.^{50 51} Morphological changes induced by TPA and free radical injury of ECs can be prevented by pretreating the cells with the voltage-sensitive Ca²⁺ channel blocker verapamil.^{52 53} The inhibition of MCP-1 gene expression by EGTA and verapamil indicates that Ca²⁺ influx through Ca²⁺ channels during strain is crucial for strain-induced MCP-1 gene expression.

The mechanical strain–induced MCP-1 gene expression in ECs is low compared with that induced in cells by cytokines.^{54 55} However, this could be because our strain device did not provide uniform stretch. Our recent studies of stretch effects have indicated that cells at the periphery of wells show higher MCP-1 induction (data not shown). The results we show in the present study represent a mixture of high- and low-stretched cells, and this may contribute to the lower MCP-1 induction we observed. Unfortunately, there is no in vitro model system that can completely mimic the in vivo situation. In vivo, the local MCP-1 induction in vessels subjected to strain might be higher. Nevertheless, this small induction attracted monocytes, as demonstrated by its monocyte chemotactic activity, which is potent when compared with that of FMLP. Our results clearly demonstrate that MCP-1 production can be induced by mechanical strain in a force-dependent manner. Because increasing gene expression is not usually reflected by functional activity of the same magnitude and because our finding of a 1.8-fold increase in monocyte adhesion to strained ECs is approximately the same magnitude as that for cytokine-treated ECs previously reported,⁵⁶ we believe that mechanical strain plays an important role in increasing adhesion of monocytes to strained ECs. Whereas steady shear stress produces a biphasic response, cyclic strain induces a sustained increase in MCP-1 production. Thus, the in vivo MCP-1 expression in vascular ECs would be useful to know. In ECs, c-fos induction was found to be higher under pulsatile flow than under steady flow treatment.⁵⁷ The in vivo concentration of MCP-1 in normal human plasma has never been determined. Whether vascular ECs under strain release most of their MCP-1 into the luminal side of the vessel wall is not known, but the secretion of endothelin-1 by ECs predominantly into abdominal sites has been demonstrated.⁵⁸

The increased MCP-1 gene expression and secretion induced by strain may be relevant to pathological states of the cardiovascular system, including atherosclerosis and hypertension. First, it is well known that atherosclerotic lesions occur preferentially at coronary bifurcations and arterial branch sites. Recent analysis of stress concentration and strains in the arterial walls has indicated that strains are significantly higher at branch sites than in the straight segments.⁵⁹ Those branch regions subjected to higher strain may constantly express higher levels of certain growth factors and/or cytokines, such as endothelin-1 and MCP-1. Hypertension, in general, is associated with an increase in both the extent and severity of atherosclerosis.^{60 61} Hypertension lesions can be found in some locations usually spared of disease.^{62 63} There is evidence that a reduction in blood pressure may retard lesion progression if previous hypercholesterolemia is eliminated,⁶⁴ but the basic mechanism of the role of hypertension in atherosclerosis is complicated and remains unclear. The present study suggests that MCP-1 gene expression in the vessel wall, especially at branch sites where the strain is large, may be elevated in hypertensive patients. Increased adherence of circulating white blood cells to the endothelial surface and their recruitment into the subendothelial space in the aortas in hypertensive rats^{65 66} supports this hypothesis. Finally, MCP-1 is likely to be an important protein released from ECs under mechanical deformation during cardiovascular interventions such as balloon angioplasty or angioscopic procedures. After these cardiovascular interventions, activation of vascular cells and leukocytes in the aorta has been suggested to contribute to restenosis.⁶⁷ Balloon injury can induce the expression in the vessel wall of early response genes, including JE (ie, the MCP-1 gene).⁶⁸ Our present study indicates that balloon-induced stretch itself may further induce MCP-1 gene expression in ECs around lesion areas. This stretch-induced MCP-1 release may directly participate in the local inflammatory activation of vascular cells and thus may contribute, at least initially, to the intimal hyperplasia response after balloon injury.

In summary, the present study shows that mechanical strain is an important factor in regulating MCP-1 production in the endothelium. This strain-induced MCP-1 production may contribute to the trapping of monocytes in the subendothelial space during atherogenesis and thus may provide a molecular mechanism for the role of hypertension in atherogenesis. The mechanism(s) by which mechanical deformation leads to increased gene expression and protein release remains an important question that deserves further investigation.

	Selected Abbreviations and Acronyms

 

DAG = diacylglycerol EC = endothelial cell ELISA = enzyme-linked immunosorbent assay FMLP = formyl-Met-Leu-Phe-OH HUVEC = human umbilical vein endothelial cell IP3 = inositol tris-phosphate LDL = low-density lipoprotein MCP-1 = monocyte chemotactic protein-1 PKC = protein kinase C TPA = tissue-type plasminogen activator

	Acknowledgments

 
This study was supported by the National Science Council, Taiwan, ROC. We thank J.F. Cheng for his preparation of cultured cells and Dr Cathy Fletcher for her reading of the manuscript.

	Footnotes

 
Reprint requests to Dr Danny Ling Wang, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.

Received September 28, 1994; accepted May 1, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]

2. Gerrity RG, Naito HK, Richardson ME, Schwartz CJ. Dietary induced atherogenesis in swine: morphology of the intima in prelesion stages. Am J Pathol. 1979;95:775-792. [Medline] [Order article via Infotrieve]

3. Bevilacqua MP, Pobers JS, Wheeler ME, Cotran RS, Gimbrone MA. Interleukin I acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest. 1985;76:2003-2011.

4. Prieto J, Beatty PG, Clark EA, Patarroyo M. Molecules mediating adhesion of T-and B-cells, monocytes and granulocytes to vascular endothelial cells. Immunology. 1988;63:631-637. [Medline] [Order article via Infotrieve]

5. Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Lenonard E, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A. 1991;88:5252-5256. [Abstract/Free Full Text]

6. Valente AJ, Rozek MM, Spraque EA, Schwartz CJ. Mechanism in intimal monocyte-macrophage recruitment: special role for monocyte chemotactic protein-1. Circulation. 1992;86(suppl III):III-20-III-25.

7. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H, et al. Monocyte transmigration induced by modified low density lipoprotein in coculture of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991;88:2039-2046.

8. Shyy YJ, Wichham LL, Hagan JP, Hsieh HJ, Hu YL, Telian SH, Valente AJ, Sung KLP, Chien S. Human monocyte colony-stimulating factor stimulates the gene expression of monocyte chemotactic protein-1 and increases the adhesion of monocytes to endothelial monolayers. J Clin Invest. 1993;92:1745-1751.

9. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ, Fogelman AM. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990;87:5134-5138. [Abstract/Free Full Text]

10. Yoshimura T, Yuhki N, Moore SK, Appella E, Lerman MI, Leonard EJ. Human monocyte chemoattractant protein-1 (MCP-1): full length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence. FEBS Lett. 1989;244:487-493. [Medline] [Order article via Infotrieve]

11. Rollings BJ, Stier P, Ernst T, Wong GG. The human monology of the JE gene encodes monocyte secretory protein. Mol Cell Biol. 1989;9:4687-4695. [Abstract/Free Full Text]

12. Colotta F, Borre A, Wang JM, Tattanelli M, Maddalena F, Polentarutti N, Peri G, Mantovani A. Expression of a monocyte chemotactic cytokine by human mononuclear phagocytes. J Immunol. 1992;148:760-765. [Abstract]

13. Hall DJ, Brownlee C, Stiles CD. Interleukin-1 is a potent regulator of JE and KC gene expression in quiescent BALB/c fibroblasts. J Cell Physiol. 1992;141:154-159.

14. Yoshimura T, Robinson EA, Tanaka S, Appella E, Leonard EJ. Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes. J Immunol. 1962;142:1956-1962. [Abstract]

15. Rollings BJ, Morrison ED, Stiles CD. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci U S A. 1988;85:3738-3742. [Abstract/Free Full Text]

16. Hanazawa S, Takeshita A, Amano S, Semba T, Nirazuka T, Katoh H, Kitano S. Tumor necrosis factor-alpha induces expression of monocyte chemoattractant JE via fos and jun genes in clonal osteoblastic MC3T3-E1 cells. J Biol Chem. 1993;268:9526-9532. [Abstract/Free Full Text]

17. Strieter RM, Wiggins R, Phan SH, Wharaam BL, Showell DG, Chensue SW, Kunkel SL. Monocyte chemotactic protein gene expression by cytokine-treated human fibroblasts and endothelial cells. Biochem Biophys Res Commun. 1989;162:694-700. [Medline] [Order article via Infotrieve]

18. Brach MA, Gruss HJ, Riedel D, Asano Y, De Vos S, Herrmann F. Effect of antiinflammatory agents on synthesis of MCP-1/JE transcripts by human blood monocytes. Mol Pharmacol. 1992;42:63-68. [Abstract]

19. Satriano JA, Shuldiner M, Hora K, Xing Y, Shan Z, Schlondorff D. Oxygen radicals as second messenger for expression of the monocyte chemoattractant protein, JE/MCP-1 and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor- and immunoglobulin G. J Clin Invest. 1993;92:1564-1571.

20. Shyy YJ, Li YS, Kolattukudy PE. Activation of MCP-1 gene expression is mediated through multiple signaling pathways. Biochem Biophys Res Commun. 1993;192:693-699. [Medline] [Order article via Infotrieve]

21. Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science. 1985;227:1477-1479. [Abstract/Free Full Text]

22. Diamond S, Eskin S, McIntire L. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science. 1989;243:1483-1485. [Abstract/Free Full Text]

23. Wang DL, Tang CC, Wung BS, Chen HH, Hung MS, Wang JJ. Cyclical strain increases endothelin-1 secretion and gene expression in human endothelial cells. Biochem Biophys Res Commun. 1993;195:1050-1056. [Medline] [Order article via Infotrieve]

24. Wang DL, Wung BS, Peng YC, Wang JJ. Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. J Cell Physiol. 1995;163:400-406. [Medline] [Order article via Infotrieve]

25. Gimbrone MA. Culture of Vascular Endothelium. In: Spaet TH, ed. New York, NY: Grune & Stratton; 1976;3:1-28.

26. Recalde HR. A simple method of obtaining monocytes in suspension. J Immunol Methods. 1984;69:71-77. [Medline] [Order article via Infotrieve]

27. Snyderman R, Altman LC, Hausman MS, Morgenhagen SE. Human mononuclear leukocyte chemotaxis: a quantitative assay for humoral and cellular chemotactic factors. J Immunol. 1972;108:857-860. [Abstract/Free Full Text]

28. Berliner JA, Territo M, Almada L, Carter A, Shafonsky E, Fogelman AM. Monocyte chemotactic factor produced by large vessel endothelial cells in vitro. Arteriosclerosis. 1986;6:254-258. [Abstract/Free Full Text]

29. Wang DL, Chen JJ, Shih NL, Kao YC, Hsu KH, Huang WY, Liew CC. Endothelin stimulates cardiac - and ß-myosin heavy chain gene expression. Biochem Biophys Res Commun. 1992;183:1260-1265. [Medline] [Order article via Infotrieve]

30. Sigurdson WJ, Sachs F, Diamond SL. Mechanical perturbation of cultured human endothelial cells causes rapid increases of intracellular calcium. Am J Physiol. 1993;264:H1745-H1752. [Abstract/Free Full Text]

31. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260-1266.

32. Morita T, Kurihara H, Maemura K, Yoshizumi M, Yazaki Y. Disruption of cytoskeletal structure mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells. J Clin Invest. 1993;92:1706-1712.

33. Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci U S A. 1994;91:4678-4682. [Abstract/Free Full Text]

34. Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. J Biol Chem. 1992;267:10551-10560. [Abstract/Free Full Text]

35. Komuro I, Kotoh Y, Kaida T, Shibazaki Y, Kurabayashi EH, Takaku E, Yazaki Y. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. J Biol Chem. 1991;266:1265-1268. [Abstract/Free Full Text]

36. Shyy YJ, Li YS, Kolattukudy PE. Structure of human monocyte chemotactic protein gene and its regulation by TPA. Biochem Biophys Res Commun. 1990;169:346-351. [Medline] [Order article via Infotrieve]

37. Hsieh HJ, Li NQ, Frangos JA. Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J Cell Physiol. 1992;150:552-558. [Medline] [Order article via Infotrieve]

38. Kuchan MJ, Frangos JA. Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol. 1993;264:H150-H156. [Abstract/Free Full Text]

39. Morita T, Kurihara H, Maemura K, Yoshizumi M, Nagai R, Yazaki Y. Role of Ca²⁺ and protein kinase C in shear stress–induced actin depolymerization and endothelin 1 gene expression. Circ Res. 1994;75:630-636. [Abstract/Free Full Text]

40. Rosales OS, Sumpio BE. Protein kinase C is a mediator of the adaptation of vascular endothelial cells to cyclic strain in vitro. Surgery. 1992;112:459-466. [Medline] [Order article via Infotrieve]

41. Dull RO, Davies PF. Flow modulation of agonist (ATP)-response (Ca²⁺) coupling in vascular endothelial cells. Am J Physiol. 1991;261:H149-H154. [Abstract/Free Full Text]

42. Mo M, Eskin S, Schilling WP. Flow-induced changes in Ca²⁺ signaling in vascular endothelial cells: effect of shear stress and ATP. Am J Physiol. 1991;260:H1698-H1707. [Abstract/Free Full Text]

43. Shen J, Luscinskas FW, Connolly A, Dewey CF, Gimbrone MA. Fluid shear stress modulates cytosolic free calcium in vascular endothelial cells. Am J Physiol. 1992;262:C384-C390. [Abstract/Free Full Text]

44. Sadoshima JI, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 1993;12:1681-1692. [Medline] [Order article via Infotrieve]

45. Rosales OR, Sumpio BE. Changes in cyclic strain increase inositol triphosphate and diacylglycerol in endothelial cells. Am J Physiol. 1992;262:C956-C962. [Abstract/Free Full Text]

46. Evans LV, Brophy C, Sumpio BE. Transient activation of phospholipase C and sustained activation of phospholipase D in endothelial cells subjected to cyclical strain. FASEB J. 1994;8:A35. Abstract.

47. Manolopoulos VG, Lelkes PI. Cyclic strain and forskolin differentially induce cAMP production in phenotypically diverse endothelial cells. Biochem Biophys Res Commun. 1993;191:1379-1385. [Medline] [Order article via Infotrieve]

48. Lansman JB, Hallan TJ, Ring TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature. 1987;325:811-813. [Medline] [Order article via Infotrieve]

49. Naruse K, Sokabe M. Involvement of stretch-activated ion channels in Ca²⁺ mobilization to mechanical stretch in endothelial cells. Am J Physiol. 1993;264:C1037-C1044. [Abstract/Free Full Text]

50. Crutchley DJ, Ryan JW, Rya US, Fisher GH. Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Biochem Biophys Acta. 1983;751:99-107. [Medline] [Order article via Infotrieve]

51. Rubanyi GM, Schwartz A, Vanhoutte PM. The calcium agonists BAY K8644 and (+)202,891 stimulate the release of endothelial relaxing factor from canine femoral arteries. Eur J Pharmacol. 1985;117:143-144.[Medline] [Order article via Infotrieve]

52. Northover AM. Inhibition by calcium antagonists of shape changes in vitro by proinflammatory mediators in venous endothelial cells. Agents Actions. 1992;36:237-242. [Medline] [Order article via Infotrieve]

53. Mak IT, Boehme P, Weglicki WB. Antioxidant effects of calcium channel blockers against free radical injury in endothelial cells: correlation of protection with preservation of glutathione levels. Circ Res. 1992;70:1099-1103. [Abstract/Free Full Text]

54. Rollins BJ, Yoshimura T, Leonard EJ, Pober JS. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol. 1990;136:1229-1233. [Abstract]

55. Rollins BJ, Pober JS. Interleukin-4 induces the synthesis and secretion of MCP-1/JE by human endothelial cells. Am J Pathol. 1991;138:1315-1319. [Abstract]

56. Beekhuizen H, Van Furth R. Monocyte adherence to human vascular endothelium. J Leukocyte Biol. 1993;54:363-378. [Abstract]

57. Hsieh HJ, Li NQ, Frangos JA. Pulsatile and steady flow induces c-fos expression in human endothelial cells. J Cell Physiol. 1993;154:143-151. [Medline] [Order article via Infotrieve]

58. Wagner OS, Christ G, Wojta J, Vierhapper H, Parzer S, Nowotny PJ, Schneider B, Waldhausl W, Binder BR. Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem. 1992;267:16066-16068. [Abstract/Free Full Text]

59. Thubrikar MJ, Roskelley SK, Eppink RT. Study of stress concentration in the walls of the bovine coronary arterial branch. J Biomech. 1990;23:15-26. [Medline] [Order article via Infotrieve]

60. Breterton KN, Day AJ, Skinner SL. Hypertension-accelerated atherogenesis in cholesterol-fed rabbits. Atherosclerosis. 1977;27:79-87. [Medline] [Order article via Infotrieve]

61. Chobanian AV. The influence of hypertension and other hemodynamic factors in atherogenesis. Cardiovasc Dis. 1983;26:177-196.

62. Glagov S, Ozoa AK. Significance of the relatively low incidence of atherosclerosis in the pulmonary, renal and mesenteric arteries. Ann N Y Acad Sci. 1968;149:940-955. [Medline] [Order article via Infotrieve]

63. Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis. Arch Pathol Lab Med. 1988;112:1018-1031. [Medline] [Order article via Infotrieve]

64. Zarins CK, Bomberger RA, Taylor KE, Glagov S. Artery stenosis inhibits regression of diet-induced atherosclerosis. Surgery. 1980;88:86-92. [Medline] [Order article via Infotrieve]

65. Haudenschild CC, Prescott MF, Chobanian AV. Aortic endothelial and subendothelial cells in experimental hypertension and aging. Hypertension. 1981;3(suppl I):I-148-I-153.

66. Haudenschild CC, Prescott MF, Chobanian AV. Effects of hypertension and its reversal on aortic intimal lesions of the rat. Hypertension. 1980;2:33-44. [Abstract/Free Full Text]

67. Tanaka H, Sukhova GK, Swanson SJ, Clinton SK, Ganz P, Cybulsky MI, Libby P. Sustained activation of vascular cells and leukocytes in the rabbit aorta after balloon injury. Circulation. 1993;88:1788-1803. [Abstract/Free Full Text]

68. Taubman MB, Rollings BJ, Poon M, Marmur J, Green RS, Berk BC, Nadal-Ginard B. JE mRNA accumulates rapidly in aortic injury and in platelet-derived growth factor–stimulated vascular smooth muscle cells. Circ Res. 1992;70:314-325.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. C. van Oostrom, O. van Oostrom, P. H. A. Quax, M. C. Verhaar, and I. E. Hoefer Insights into mechanisms behind arteriogenesis: what does the future hold? J. Leukoc. Biol., December 1, 2008; 84(6): 1379 - 1391. [Abstract] [Full Text] [PDF]

				R. Rossi, A. Nuzzo, G. Origliani, and M. G. Modena Metabolic Syndrome Affects Cardiovascular Risk Profile and Response to Treatment in Hypertensive Postmenopausal Women Hypertension, November 1, 2008; 52(5): 865 - 872. [Abstract] [Full Text] [PDF]

				T.-D. Liao, X.-P. Yang, Y.-H. Liu, E. G. Shesely, M. A. Cavasin, W. A. Kuziel, P. J. Pagano, and O. A. Carretero Role of Inflammation in the Development of Renal Damage and Dysfunction in Angiotensin II-Induced Hypertension Hypertension, August 1, 2008; 52(2): 256 - 263. [Abstract] [Full Text] [PDF]

				A. Stempien-Otero, A. Plawman, J. Meznarich, T. Dyamenahalli, G. Otsuka, and D. A. Dichek Mechanisms of Cardiac Fibrosis Induced by Urokinase Plasminogen Activator J. Biol. Chem., June 2, 2006; 281(22): 15345 - 15351. [Abstract] [Full Text] [PDF]

				L. Pascarella, A. Penn, and G. W. Schmid-Schonbein Venous Hypertension and the Inflammatory Cascade: Major Manifestations and Trigger Mechanisms Angiology, November 1, 2005; 56(6_suppl): S3 - S10. [Abstract] [PDF]

				M. Voskuil, I. E Hoefer, N. van Royen, J. Hua, S. de Graaf, C. Bode, I. R Buschmann, and J. J Piek Abnormal monocyte recruitment and collateral artery formation in monocyte chemoattractant protein-1 deficient mice Vascular Medicine, November 1, 2004; 9(4): 287 - 292. [Abstract] [PDF]

				G. J. Blake, N. Rifai, J. E. Buring, and P. M Ridker Blood Pressure, C-Reactive Protein, and Risk of Future Cardiovascular Events Circulation, December 16, 2003; 108(24): 2993 - 2999. [Abstract] [Full Text] [PDF]

				N. C. Weber, S. B. Blumenthal, T. Hartung, A. M. Vollmar, and A. K. Kiemer ANP inhibits TNF-{alpha}-induced endothelial MCP-1 expression--involvement of p38 MAPK and MKP-1 J. Leukoc. Biol., November 1, 2003; 74(5): 932 - 941. [Abstract] [Full Text] [PDF]

				S. Takase, L. Lerond, J. J. Bergan, and G. W. Schmid-Schonbein Enhancement of reperfusion injury by elevation of microvascular pressures Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1387 - H1394. [Abstract] [Full Text] [PDF]

				B.S. Wung, J.J. Cheng, S.-K. Shyue, and D.L. Wang NO Modulates Monocyte Chemotactic Protein-1 Expression in Endothelial Cells Under Cyclic Strain Arterioscler. Thromb. Vasc. Biol., December 1, 2001; 21(12): 1941 - 1947. [Abstract] [Full Text] [PDF]

				Y.-L. Chang, J.-J. Shen, B.-S. Wung, J.-J. Cheng, and D. L. Wang Chinese Herbal Remedy Wogonin Inhibits Monocyte Chemotactic Protein-1 Gene Expression in Human Endothelial Cells Mol. Pharmacol., September 1, 2001; 60(3): 507 - 513. [Abstract] [Full Text] [PDF]

				S. P Schwarzacher, P. S Tsao, M. Ward, M. Hayase, J. Niebauer, J. P Cooke, and A. C Yeung Effects of stenting on adjacent vascular distensibility and neointima formation: role of nitric oxide Vascular Medicine, August 1, 2001; 6(3): 139 - 144. [Abstract] [PDF]

				D. Weiss, J. J. Kools, and W. R. Taylor Angiotensin II-Induced Hypertension Accelerates the Development of Atherosclerosis in ApoE-Deficient Mice Circulation, January 23, 2001; 103(3): 448 - 454. [Abstract] [Full Text] [PDF]

				I.-W. Park, J.-F. Wang, and J. E. Groopman HIV-1 Tat promotes monocyte chemoattractant protein-1 secretion followed by transmigration of monocytes Blood, January 15, 2001; 97(2): 352 - 358. [Abstract] [Full Text] [PDF]

				M. P. Gupta, M. D. Ober, C. Patterson, M. Al-Hassani, V. Natarajan, and C. M. Hart Nitric oxide attenuates H2O2-induced endothelial barrier dysfunction: mechanisms of protection Am J Physiol Lung Cell Mol Physiol, January 1, 2001; 280(1): L116 - L126. [Abstract] [Full Text] [PDF]

				J. C. Parker Inhibitors of myosin light chain kinase and phosphodiesterase reduce ventilator-induced lung injury J Appl Physiol, December 1, 2000; 89(6): 2241 - 2248. [Abstract] [Full Text] [PDF]

				H. Kito, E. L. Chen, X. Wang, M. Ikeda, N. Azuma, N. Nakajima, V. Gahtan, and B. E. Sumpio Role of mitogen-activated protein kinases in pulmonary endothelial cells exposed to cyclic strain J Appl Physiol, December 1, 2000; 89(6): 2391 - 2400. [Abstract] [Full Text] [PDF]

				E. Bush, N. Maeda, W. A. Kuziel, T. C. Dawson, J. N. Wilcox, H. DeLeon, and W. R. Taylor CC Chemokine Receptor 2 Is Required for Macrophage Infiltration and Vascular Hypertrophy in Angiotensin II-Induced Hypertension Hypertension, September 1, 2000; 36(3): 360 - 363. [Abstract] [Full Text] [PDF]

				Y Shafrir, D ben-Avraham, and G Forgacs Trafficking and signaling through the cytoskeleton: a specific mechanism J. Cell Sci., January 8, 2000; 113(15): 2747 - 2757. [Abstract] [PDF]

				B. S. Wung, J. J. Cheng, Y. J. Chao, H. J. Hsieh, and D. L. Wang Modulation of Ras/Raf/Extracellular Signal–Regulated Kinase Pathway by Reactive Oxygen Species Is Involved in Cyclic Strain–Induced Early Growth Response-1 Gene Expression in Endothelial Cells Circ. Res., April 16, 1999; 84(7): 804 - 812. [Abstract] [Full Text] [PDF]

				J. Pugin, I. Dunn, P. Jolliet, D. Tassaux, J.-L. Magnenat, L. P. Nicod, and J.-C. Chevrolet Activation of human macrophages by mechanical ventilation in vitro Am J Physiol Lung Cell Mol Physiol, December 1, 1998; 275(6): L1040 - L1050. [Abstract] [Full Text] [PDF]

				C. Schmidt, H. Pommerenke, F. Durr, B. Nebe, and J. Rychly Mechanical Stressing of Integrin Receptors Induces Enhanced Tyrosine Phosphorylation of Cytoskeletally Anchored Proteins J. Biol. Chem., February 27, 1998; 273(9): 5081 - 5085. [Abstract] [Full Text] [PDF]

				J.-J. Cheng, B.-S. Wung, Y.-J. Chao, and D. L. Wang Cyclic Strain-Induced Reactive Oxygen Species Involved in ICAM-1 Gene Induction in Endothelial Cells Hypertension, January 1, 1998; 31(1): 125 - 130. [Abstract] [Full Text] [PDF]

				S. Chien, S. Li, and J. Y-J. Shyy Effects of Mechanical Forces on Signal Transduction and Gene Expression in Endothelial Cells Hypertension, January 1, 1998; 31(1): 162 - 169. [Abstract] [Full Text] [PDF]

				B. S Lewis, M. Y Flugelman, A. Weisz, I. Keren-Tal, and W. Schaper Angiogenesis by gene therapy: a new horizon for myocardial revascularization? Cardiovasc Res, September 1, 1997; 35(3): 490 - 497. [Abstract] [Full Text] [PDF]

				B. S. Wung, J. J. Cheng, H. J. Hsieh, Y. J. Shyy, and D. L. Wang Cyclic Strain–Induced Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Involves Reactive Oxygen Species Activation of Activator Protein 1 Circ. Res., July 19, 1997; 81(1): 1 - 7. [Abstract] [Full Text]

				W. D. Ito, M. Arras, B. Winkler, D. Scholz, J. Schaper, and W. Schaper Monocyte Chemotactic Protein-1 Increases Collateral and Peripheral Conductance After Femoral Artery Occlusion Circ. Res., June 19, 1997; 80(6): 829 - 837. [Abstract] [Full Text]

				J.-J. Cheng, B.-S. Wung, Y.-J. Chao, and D. L. Wang Cyclic Strain Enhances Adhesion of Monocytes to Endothelial Cells by Increasing Intercellular Adhesion Molecule-1 Expression Hypertension, September 1, 1996; 28(3): 386 - 391. [Abstract] [Full Text]

				J.-J. Cheng, B.-S. Wung, Y.-J. Chao, and D. L. Wang Sequential Activation of Protein Kinase C (PKC)-alpha and PKC-epsilon Contributes to Sustained Raf/ERK1/2 Activation in Endothelial Cells under Mechanical Strain J. Biol. Chem., August 10, 2001; 276(33): 31368 - 31375. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, D. L. Articles by Chien, S. Search for Related Content PubMed PubMed Citation Articles by Wang, D. L. Articles by Chien, S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL