This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted 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 Khachigian, L. M. Articles by Collins, T. Search for Related Content PubMed PubMed Citation Articles by Khachigian, L. M. Articles by Collins, T. Pubmed/NCBI databases Substance via MeSH

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2280-2286.)
© 1997 American Heart Association, Inc.

Articles

Egr-1 is Activated in Endothelial Cells Exposed to Fluid Shear Stress and Interacts With a Novel Shear-Stress-Response Element in the PDGF A-Chain Promoter

Levon M. Khachigian; Keith R. Anderson; Nancy J. Halnon; Michael A. Gimbrone, Jr.,; Nitzan Resnick; ; Tucker Collins

From the Vascular Research Division, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston Mass.

Correspondence to Tucker Collins, MD, PhD, Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, 221 Longwood Avenue, Boston MA 02115.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Exposure of vascular endothelial cells to fluid mechanical forces can modulate the expression of many genes involved in vascular physiology and pathophysiology. Here, we report that platelet-derived growth factor (PDGF) A-chain gene expression is induced at the level of transcription in cultured bovine aortic endothelial cells exposed to a physiologic level of steady laminar shear stress (10 dyn/cm²). 5' Deletion analysis of the human PDGF-A promoter revealed that a GC-rich region near the TATA box was required for shear-inducible reporter gene expression. This element conferred shear inducibility onto a heterologous promoter-reporter construct that was otherwise unresponsive to shear stress. The induction of PDGF-A expression by shear was preceded by rapid and transient induction in the expression of the immediate-early gene, egr-1, which binds to GC-rich sequences. Gel shift studies indicated that shear-induced Egr-1 bound to the proximal PDGF-A promoter in a specific and time-dependent manner, displacing Sp1 from their overlapping recognition elements. Overlapping consensus binding sites for Egr-1 and Sp1 also appear in the proximal promoters of several other endothelial genes, including transforming growth factor-ß₁ and tissue factor, whose expression is modulated by shear stress. These findings define the Egr-1 binding site in the proximal PDGF-A promoter as a shear-stress-responsive element and suggest that shear-stimulated Egr-1 gene expression may be a unifying theme in the induction of various other endothelial genes exposed to biomechanical forces.

Key Words: platelet-derived growth factor A-chain • Egr-1 • fluid shear stress • vascular endothelial cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Biomechanical forces generated by circulating blood act on endothelial cells lining the vasculature and can modulate the expression of physiologically and pathophysiologically important genes at the level of transcription. One of these hemodynamic forces, fluid shear stress, can alter vessel structure and function, in part, via the elaboration of various growth-regulatory molecules. The promoter regions of several of these shear-inducible genes contain a "shear-stress-response element" (SSRE), first characterized in the platelet-derived growth factor (PDGF) B-chain promoter.¹ Recently, the PDGF-B SSRE was found to interact with the pluripotent transcription factor, nuclear factor-B,² which is activated in vascular endothelial cells by physiologic levels of fluid shear stress.^{2 3} The identification of this cis-acting element was an important first step in assigning a functional role to a particular promoter element in an endothelial gene responding to a defined biomechanical force. However, certain other endothelial genes, such as monocyte chemotactic factor-1,⁴ in which this SSRE does not appear to be functional,^{5 6} or the PDGF A-chain,^{7 8} in which this SSRE is absent in the promoter region, also are modulated by fluid shear stress. These findings therefore imply the existence of other promoter elements mediating shear-induced gene expression in vascular endothelial cells.

In this study, using the PDGF-A gene as a model, we have defined another SSRE as well as a positive regulatory nuclear factor that interacts with this DNA sequence during shear-induced endothelial gene expression. The immediate-early gene, egr-1 (krox-24; NGF-IA, zif268, TIS8), which encodes a nuclear transcription factor of the zinc-finger class,^{9 10} is rapidly and transiently induced in endothelial cells exposed to fluid shear stress. This increase precedes the induction of PDGF-A transcript levels by shear stress. 5' Deletion and transient transfection analysis revealed that the GC-rich region in the proximal PDGF-A promoter is crucial for shear-inducible reporter gene expression. Electrophoretic mobility shift assays revealed that shear-induced Egr-1 binds to this promoter element in a time-dependent and specific manner. Interestingly, the increase in PDGF-A gene expression by shear involves the displacement of Sp1 by Egr-1 from overlapping recognition elements. Overlapping consensus binding sites for Egr-1 and Sp1 are also present in the proximal promoters of several other endothelial genes whose expression is modulated by shear stress. Thus, Egr-1 activation may be a unifying theme in the induction of various genes in endothelial cells exposed to biomechanical forces.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Endothelial Culture and Shear Stress Conditions
Bovine aortic endothelial cells (BAEC) were grown in Dulbecco's modified Eagles' medium (Gibco BRL, Grand Island, NY) containing 10% calf serum (BioWhittaker, Walkersville, Md), 2 mmol/L of L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin in a humidified atmosphere of 5% CO₂/air at 37°C. Steady laminar shear stress (10 dyn/cm²) was applied to confluent monolayers of low passage (<6) BAEC for the times indicated using a cone-and-plate flow apparatus.^{1 11}

Nuclear Run-off Assays
Endothelial cells that were exposed to shear stress in 17-cm plates,¹ or their unstimulated counterparts, were assessed for rates of transcription by the protocol utilizing [³²P]-UTP (New England Nuclear) described previously.¹²

Transient Transfection Analysis
BAEC were transfected with 5' deletion constructs of the human PDGF-A promoter or SV40-based heterologous PDGF-A promoter-reporter constructs as previously described.¹³ Transfections were carried out in 100-mm Petri dishes using 25 µg of construct and the modified calcium phosphate precipitation protocol.¹⁴ The cells were washed twice with Hanks' balanced salts solution and allowed to recover in Dulbecco's modified Eagles' medium containing 10% calf serum for 5 hours. The cells were trypsinized and resuspended in Dulbecco's modified Eagles' medium containing 3% calf serum and allowed to attach to 1-cm coverslips or 17-cm plates.¹ The confluent monolayers were subjected to shear stress at 10 dyn/cm² for the times indicated. Endothelial cell extracts were prepared 4 hours after the completion of the shear stress stimulus. CAT activity in the lysates was assessed by the two-phase fluor-diffusion technique¹³ and normalized to protein concentrations of the lysate. When 5' deletion constructs were used in experiments, the cells were cotransfected with an SV40-based luciferase reporter construct, and the data were incorporated for normalization.

Northern Blot Analysis
Total RNA was prepared using RNAzol(TM) reagent (Tel-Test, Inc) and samples (15 µg) resolved on 1% formaldehyde/agarose gels. RNA was transferred to a Hybond nylon membrane (Amersham) and hybridized with cDNA that had been labeled with [³²P]-dCTP (New England Nuclear). Blots were washed with 0.2xSSC, 0.1% sodium dodecyl sulfate at 65°C, and exposed at -80°C using Kodak X-OMAT-AR film.

Oligonucleotide Synthesis and Radiolabeling
Oligonucleotides were synthesized using a 392 DNA synthesizer (Applied Biosystems) and purified by gel electrophoresis. For gel shift assays, oligonucleotides were radiolabeled with [³²P]-ATP (New England Nuclear) using T4 polynucleotide kinase (New England Biolabs).

EMSA
Nuclear extracts from cells subjected to shear stress in 17-cm plates were prepared as previously described² and incubated with 50 000 to 100 000 cpm of [³²P]-labeled Oligo A in 10 mmol/L of Tris-HCl, pH 8, 50 mmol/L of MgCl₂, 1 mmol/L of EDTA, 1 mmol/L of DTT, 5% glycerol, 5% sucrose, 1 mmol/L of PMSF containing 1 µg of poly(dI.dC)-poly(dI.dC), and 1 µg of salmon sperm DNA for 30 minutes at 22°C. In antibody inhibition studies, 1 µL of affinity purified rabbit antipeptide antibody (Santa Cruz) was incubated with the binding mixture for 10 minutes at 22°C before the addition of the probe. When recombinant proteins were used, binding reactions using [³²P]-Oligo A (50 000 to 100 000 cpm) were carried out in 10 mmol/L of Tris-HCl, pH 8, 50 mmol/L of NaCl, 1 mmol/L of EDTA, 5% glycerol, 2 mmol/L of DTT, 0.5% NP-40, and 1 µg/µL of bovine serum albumin in a total volume of 20 µL. Bound complexes were separated from the free probe by nondenaturing polyacrylamide gel electrophoresis using 1xTBE running buffer. The gels were run at 200 V (constant voltage) for approximately 2 hours and then dried under vacuum and autoradiographed overnight.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Fluid Shear Stress Induces PDGF-A Gene Transcription in Vascular Endothelial Cells
Nuclear run-off experiments were used to determine whether the PDGF A-chain gene could be induced at the transcriptional level in endothelial cells exposed to a physiologic level of fluid shear stress typical of large arterial geometries (10 dyn/cm²) using a well-characterized in vitro fluid mechanical system.¹¹ The rate of PDGF-A gene transcription was dramatically induced when BAEC were subjected to 2 hours of shear-stress stimulation (Fig 1). Enhanced PDGF-A transcription was also observed when human umbilical vein endothelial cells were used in place of BAEC in these experiments (L.M. Khachigian and T. Collins, unpublished data). In contrast, the rate of transcription of the ß-tubulin gene was unaffected by shear stress, and E-selectin was not induced (Fig 1), consistent with the inability to detect E-selectin mRNA expression in endothelial cells exposed to shear.¹⁵ Thus, the selective induction of PDGF-A expression in endothelial cells exposed to shear stress is mediated, at least in part, at the level of transcription.

View larger version (28K): [in this window] [in a new window]   Figure 1. Nuclear run-off analysis using nuclei from unstimulated vascular endothelial cells or cells exposed to physiologic levels of fluid shear stress. Nuclei were isolated from BAEC exposed to 10 dyn/cm2 of laminar shear stress for 2 hours, and the rate of newly transcribed [32P-UTP]-labeled RNA that hybridized with 5 µg of the appropriate cDNA was assessed. The data are representative of two independent determinations.

Identification of a Functional SSRE in the Human PDGF-A Promoter
5' Deletion analysis was used to determine the region in the PDGF-A promoter that could mediate increased promoter-dependent expression in endothelial cells exposed to shear stress. BAEC transfected with PDGF-A promoter-reporter constructs Sac, Xhol, and e33, which extend 630, 260, and 110 bp upstream of the transcriptional start site, respectively, responded to the biomechanical force by increased CAT expression in the order of two- to threefold (Fig 2). Shear-inducible reporter gene expression was abolished, however, in cells transfected with construct f36 (-55) (Fig 2), implicating a functional role for the promoter region between bp -110 and -55 in cells exposed to shear stress. This region contains repeating consensus overlapping recognition elements for the zinc-finger nuclear transcription factors Egr-1 and Sp1.

View larger version (22K): [in this window] [in a new window]   Figure 2. 5'-Deletion analysis of the human PDGF-A promoter using chloramphenicol acetyltransferase reporter constructs and cultured endothelial cells in a transient transfection setting. Confluent BAEC monolayers transfected with the appropriate reporter construct were grown on 1-cm coverslips and subjected to 10 dyn/cm2 shear stress for 4 hours. CAT activity in the cell lysate was assessed by the two-phase fluor-diffusion technique and normalized to luciferase activity and the protein concentration in the lysate. The percentage of chloramphenicol acetylation typically obtained was 5 to 20%. P<.001 for Xhol and e33, and P<.02 for Sac. pACCAT (n=9), f36 (n=12), e33 (n=4), Xhol (n=5), and Sac (n=4).

Transient Induction of Egr-1 mRNA Precedes the Induction of PDGF-A Gene Expression by Shear
The preceding findings led us to determine the effect of shear stress on levels of endothelial Egr-1 and Sp1 mRNA. Northern blot analysis revealed that exposure to shear stress dramatically increased steady-state Egr-1 transcripts in BAEC within 30 minutes (Fig 3). Egr-1 mRNA levels remained elevated for 1 hour and returned to basal levels by 2 hours (Fig 3). In contrast, Sp1 transcripts were constitutively expressed, and levels were only modestly affected by shear stress (Fig 3). The transient induction of Egr-1 mRNA preceded the induction of PDGF-A gene expression (Fig 3). This suggested that Egr-1 may play a functional role in the induced expression of the PDGF-A gene in endothelial cells exposed to shear stress.

View larger version (50K): [in this window] [in a new window]   Figure 3. Northern blot analysis using total RNA from unstimulated cultured endothelial cells or cells exposed to physiologic levels of fluid shear stress. Total RNA (15 µg) from BAEC exposed to 0, 0.5, 1, 2, and 4 hours was resolved on 1% formaldehyde/agarose gels and transferred to nylon membranes before hybridization with the appropriate [32P]-labeled cDNA. The blots were washed and exposed as described in Materials and Methods.

Shear-Induced Endothelial Nuclear Proteins Interact With the Proximal PDGF-A Promoter
When nuclear extracts from BAEC exposed to shear stress for 1 hour were incubated with an oligonucleotide ([³²P]-Oligo A) whose sequence spans the Egr-1/Sp1 binding site in the proximal PDGF-A promoter, a distinct nucleoprotein complex (A3) was obtained (Fig 4A). The ability of a 50-fold molar excess of the unlabeled oligonucleotide (Fig 4A)—but not of an irrelevant oligonucleotide (Fig 4A)—to compete demonstrated the specificity of this interaction. Although complexes A1, A2, and A5 also were found to be specific, these were not modulated by shear stress (Fig 4A).

View larger version (67K): [in this window] [in a new window]   Figure 4. Gel shift analysis using nuclear extracts from unstimulated cultured endothelial cells or cells exposed to 10 dyn/cm2 laminar shear stress. EMSA was performed with [32P]-labeled Oligo A (oligonucleotide A), which bears the nucleotide sequence between bp -76 and -47 in the proximal human PDGF-A promoter and nuclear extracts from BAEC exposed to 10 dyn/cm2 for the times indicated. A, competition studies using excess unlabeled oligonucleotides. The molar excess of the oligonucleotide is indicated. B, time course of appearance of the shear-induced complex (A3). C and SS denote nuclear extracts from static BAEC and cells exposed to fluid shear stress, respectively. Complexes A1 to A5, designated in a previous report,13 are indicated by arrows. The nucleotide sequences of oligonucleotide Oligo A and PEA-3 are 5'-GGGGGGGGCGGGGGCGGGGGCGGGGGAGGG-3' and 5'-GATCTCGAGCAGGAAGTTCGACTAG-3', respectively.

Time course experiments determined the transient appearance of complex A3. This complex was observed as early as 30 minutes after the application of shear stress. After 2.5 hours, however, the complex could no longer be detected (Fig 4B). The striking temporal similarity between the appearance of complex A3 (Fig 4B) and steady-state Egr-1 transcript levels (Fig 3) provided additional evidence that Egr-1 may indeed play a role in shear-induced PDGF-A gene expression.

Shear-Induced Egr-1 Attenuates the Ability of Sp1 to Interact With Proximal PDGF-A Promoter
To determine the identity of the nuclear proteins that bound to [³²P]-Oligo A, we utilized polyclonal antipeptide antibodies directed toward particular transcription factors in EMSA. In static cultures, antibodies to Sp1 significantly attenuated the appearance of complex A1. After 1 hour of shear stress, however, Sp1 (complex A1) was no longer detectable, and complex A3 was eliminated by antibodies to Egr-1 (Fig 5A) indicating that induced Egr-1 could displace prebound Sp1 from the promoter. This suggests that Egr-1 might displace Sp1 from the PDGF-A promoter in the nuclei of activated cells. Recombinant proteins were used to model these cellular events. Increasing concentrations of Egr-1 displaced prebound Sp1 from the proximal PDGF-A promoter in a dose-dependent manner (Fig 5B). Similarly, when the concentration of Egr-1 was decreased in the presence of a fixed amount of Sp1, the latter factor reoccupied the promoter (Fig 5B). These findings thus provide evidence for the interplay of Egr-1 and Sp1 at the proximal PDGF-A promoter in endothelial cells exposed to shear stress.

View larger version (51K): [in this window] [in a new window]   Figure 5. Interplay of Sp1 and Egr-1 at the proximal PDGF-A promoter. A, gel and antibody inhibition assays were performed with [32P]-Oligo A (20 000 to 25 000 cpm) using nuclear extracts from unstimulated BAEC or cells exposed to physiologic levels of fluid shear stress (10 dyn/cm2) for 1 hour. The binding reaction contained 1 µL of the antibody where indicated. C and SS denote nuclear extracts from static or shear-stressed BAEC, respectively. A3 is the shear-induced nucleoprotein complex. The apparent difference in the intensity of A1 and A2 between control and sheared samples is the result of a reduced amount of radiolabeled oligonucleotide in the reaction to allow those proteins in stoichiometric excess and/or those possessing greater affinity to bind the probe. B, EMSA using [32P]-Oligo A with recombinant Egr-1 and/or Sp1. Increasing amounts of Egr-1 were added to a solution containing Sp1 that was prebound to the probe (left panel). Alternatively, decreasing amounts of Egr-1 were incubated with the probe in the presence of a fixed amount of Sp1 (right panel). Binding and electrophoresis were carried out as described in "Methods." S denotes a supershift; F, free probe.

Shear-Induced PDGF-A Promoter-Dependent Expression Requires an Intact Egr-1 Binding Site
To determine whether the Egr-1 binding site in the PDGF-A promoter could serve as a shear-stress-responsive element without flanking PDGF-A promoter sequences, BAEC were transfected with heterologous promoter-reporter constructs bearing either the wild type or mutated sequence and exposed to 10 dyn/cm² of shear stress for 4 hours. The wild type element conferred shear responsiveness onto the heterologous construct (Fig 6), whereas subtle mutation of the sequence actually abolished the response to shear (Fig 6). Previous studies determined that the wild type sequence—but not the mutant sequence—could also mediate increased gene expression when Egr-1 is overexpressed or when the cells are exposed to phorbol 12-myristate 13-acetate in the context of both heterologous or native promoter-reporter constructs.¹³ These findings thus define the Egr-1 binding site in the proximal PDGF-A promoter as a portable shear-stress-responsive element.

View larger version (12K): [in this window] [in a new window]   Figure 6. Transient transfection analysis in cultured endothelial cells using heterologous PDGF-A promoter-reporter constructs. Confluent BAEC monolayers transfected with the appropriate reporter construct were grown on 1-cm coverslips and subjected to 10 dyn/cm2 shear stress for 4 hours. CAT activity in the cell lysate was assessed by the two-phase fluor-diffusion technique and normalized to the protein concentration in the lysate. The percentage of chloramphenicol acetylation typically obtained was 5 to 20. SV40-CAT (n=4), A.SV40-CAT (n=4), and Am.SV40-CAT (n=4) (P<.01). These constructs were comparably expressed in endothelial cells under static conditions. Previous studies indicate that recombinant Egr-1 is unable to interact with the mutant sequence in Am.SV40-CAT.13

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this paper, we report that the induction of PDGF A-chain gene expression in vascular endothelial cells exposed to a physiologic level of steady laminar shear stress involves a functional interaction of Egr-1 with the proximal PDGF-A promoter. Nuclear run-off analysis indicates that shear-inducible PDGF-A expression is, at least in part, the consequence of new transcription. 5' Deletion and transient transfection analysis of the promoter determined that a GC-rich region, bearing consensus binding sites for the zinc-finger transcription factors Egr-1 and Sp1, was necessary for shear-inducible gene expression. Northern blot analysis indicated that shear-induced PDGF-A mRNA expression was preceded by the dramatic induction of Egr-1 transcript levels, unlike Sp1 levels. Gel shift studies using nuclear extracts revealed that Sp1 occupied this region in static cells but was displaced from the promoter by Egr-1 in sheared cells. This element conferred shear inducibility onto a promoter-reporter construct that was otherwise unresponsive to shear stress, indicating the portability of the element. These findings thus define the GC-rich element in the proximal PDGF-A promoter as a functional cis-acting shear-stress-response element. Moreover, that fluid biomechanical forces (present study) and a phorbol ester¹³ can modulate PDGF A-chain gene expression through the same displacement mechanism demonstrates that certain transcription factors can integrate diverse signaling pathways with the inducible expression of pathophysiologically relevant genes.

Endothelial cells lining blood vessels are continually exposed to hemodynamic forces because of their intimate proximity to flowing blood. The nonrandom distribution of early atherosclerotic lesions¹⁶ and the positive correlation of plaque location with altered shear stress^{17 18} provide suggestive evidence for a link between fluid biomechanical forces and endothelial dysfunction. The induction of endothelial PDGF-A gene expression by shear stress has implications for the initiation and progression of vascular disease.^{19 20} PDGF-A may influence the migration and replication of smooth muscle cells underlying the endothelium in a paracrine manner.^{19 21} In addition, PDGF-A induced by shear may stimulate the production of inflammatory mediators and other growth-regulatory molecules in smooth muscle cells, which may then act in an autocrine fashion.¹⁹ Since PDGF is also a potent vasoconstrictor,²² it may play a role in the modulation of local blood flow and thus in the autoregulation of wall shear stress. These findings support the hypothesis that shear-dependent remodeling in vascular grafts may be mediated by the induction of PDGF-A gene expression.²³ A recent paper²⁴ indicates that changes in fluid shear stress, using a prosthetic graft model in baboons, can induce PDGF-A expression in vascular endothelium. Interestingly, elevated levels of Egr-1 and PDGF-A mRNA have been detected in cultured smooth muscle cells exposed to cyclic mechanical strain,²⁵ indicating that Egr-1-induced PDGF-A expression may not be restricted to endothelial cells or confined to one type of biomechanical force.

There is accumulating evidence for the involvement of the MAP kinase cascade as a mechanism for the activation of Egr-1 by shear stress. Recent studies indicate that MAP kinase and protein kinase C are activated in cultured BAEC within minutes of exposure to physiologic levels of fluid shear stress.^{26 27 28} MAP kinases can phosphorylate ternary complex factors, which interact synergistically with the serum response factor at the serum response element.^{29 30} Serum response factor is found in endothelial nuclei and interacts with the Egr-1 serum response element (L.M. Khachigian and T. Collins, unpublished data). Recent studies indicate that ERK 1/2 is activated in endothelial cells exposed to shear stress.^{28 31 32} Shear-stimulated MAP kinase activity²⁷ in endothelial cells is protein kinase C dependent.^{27 28} Protein kinase C has been previously suggested to mediate the induction of PDGF-A gene expression in endothelial cells exposed to fluid shear stress.³³ Accordingly, the signal transduction pathway(s) that integrates fluid biomechanical forces with the activation of Egr-1 and the subsequent induction of PDGF-A may involve MAP kinase and these phosphorylation events.

Egr-1 is one of a number of transcription factors activated in endothelial cells exposed to shear stress. For example, shear induces the rapid expression of c-fos and c-jun in human umbilical vein endothelial cells,³⁴ consistent with increased AP1 DNA binding activity.³ Similarly, shear induces the rapid translocation of nuclear factor-B from the cytoplasm to the nucleus where it can activate gene expression.^{2 3 35} Several transcription factors, including AP1 and nuclear factor-B, have been found to functionally cooperate over distinct promoter elements.³⁶ Egr-1 has not yet been reported to interact synergistically with other positive regulatory factors. Whether Egr-1 activation alone is sufficient for shear-induced PDGF-A gene expression in intact endothelial cells is presently an unresolved question. The role of Sp1 phosphorylation as a regulator of inducible PDGF-A expression is also unclear.

The induction of Egr-1 mRNA and DNA binding activity in endothelial cells by shear stress suggests a role for this factor in the regulation of a number of other pathophysiologically relevant genes. Overlapping binding sites for Egr-1 and Sp1 appear in the proximal promoters of transforming growth factor-ß₁,³⁷ basic fibroblast growth factor,³⁸ tissue factor,^{39 40} as well as cell surface adhesion molecules like intercellular adhesion molecule-1⁴¹ and CD44.⁴² Expression of the genes for transforming growth factor-ß₁, tissue factor, and intercellular adhesion molecule-1 is modulated by shear stress in endothelial cells.^{15 43 44} Recent reports indicate that both basic fibroblast growth factor and intercellular adhesion molecule-1 are under the transcriptional control of Egr-1 in other cell types.^{38 41} Gel shift studies indicate that both recombinant and shear-induced nuclear Egr-1 can interact with the tissue factor proximal promoter in a specific manner⁴⁵ (L.M. Khachigian and T. Collins, unpublished data) and that Egr-1 can displace prebound Sp1 from both the tissue factor and transforming growth factor-ß₁ proximal promoters.⁴⁵ Interestingly, a recent report has shown that shear-induced tissue factor gene expression in endothelial cells is also mediated by Sp1/Egr-1 interplay in the proximal promoter.⁴⁶ Thus, Egr-1 activation may be a unifying theme in the induction of various pathophysiologically relevant endothelial genes in response to biomechanical stimuli and provide a key link between hemodynamics and atherogenesis.

	Selected Abbreviations and Acronyms

 

BAEC = bovine aortic endothelial cells CAT = chloramphenicol acetyltransferase EMSA = electrophoretic mobility shift assay MAP kinase = mitogen-activated protein kinase PDGF = platelet-derived growth factor SSRE = shear-stress-response element

	Acknowledgments

 
We are grateful to Dr. Frank J. Rauscher, III (Wistar institute of Anatomy and Biology, Philadelphia, Penn) for recombinant Egr-1. This work was supported by research grants from the National Institutes of Health to M.A.G. (PO1 HL-36028, HL-51150) and T.C. (HL 35716, HL 45462) and an unrestricted grant for cardiovascular research from the Bristol-Meyers Squibb Research Institute to M.A.G. L.M.K. was supported by a C.J. Martin postdoctoral research fellowship from the National Health and Medical Research Council of Australia and a J. William Fulbright postdoctoral research award. T.C. is an established investigator of the American Heart Association. These data were presented in abstract form at the June 1996 Experimental Biology meeting in New Orleans, La.

Received December 10, 1996; accepted February 24, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF, Jr, Gimbrone MA, Jr. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear stress response element. Proc Natl Acad Sci U S A.. 1993;90:4591-4595.[Abstract/Free Full Text]

2. Khachigian LM, Resnick N, Gimbrone MA, Jr, Collins T. Nuclear factor-kappaB interacts functionally with the platelet-derived growth factor B-chain shear-stress-response-element in vascular endothelial cells exposed to fluid shear stress. J Clin Invest.. 1995;96:1169-1175.

3. Lan Q, Mercurius KO, Davies PF. Stimulation of transcription factors NF-kappaB and AP1 in endothelial cells subjected to shear stress. Biochem Biophys Res Commun.. 1994;201:950-956.[Medline] [Order article via Infotrieve]

4. Shyy Y-J, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein-1 gene expression. J Clin Invest.. 1994;94:885-891.

5. Shyy JYJ, Lin MC, Han JH, Lu YJ, Petrime M, Chien S. The cis-acting phorbol ester 12-O-tetradecanoylphorbol-13-acetate-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A.. 1995;92:8069-8073.[Abstract/Free Full Text]

6. Shyy JYJ, Li YS, Lin MC, Chen W, Yuan SL, Usami S, Chien S. Multiple cis-elements mediate shear stress-induced gene expression. J Biomech.. 1995;28:1451-1457.[Medline] [Order article via Infotrieve]

7. Hsieh H-J, Li N-Q, Frangos JA. Shear stress increases endothelial platelet-derived growth factor messenger RNA levels. Am J Physiol.. 1991;260:H642-H646.[Abstract/Free Full Text]

8. Halnon NJ, Collins T, Gimbrone MA, Jr, Resnick N. Regulation of the endothelial PDGF-A gene by shear stress. Circulation. Abstract. 1994;90:I-88.

9. Sukhatme VP, Cao X, Chang LL, Tsai-Morris C-H, Stamenkovich D, Ferreira PCP, Cohen DR, Edwards SA, Shows TB, Curran T, Le Beau MM, Adamson ED. A zinc-finger encoding gene corregulated with c-Fos during growth and differentiation and after depolarization. Cell.. 1988;53:37-43.[Medline] [Order article via Infotrieve]

10. Gashler A, Sukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol.. 1995;50:191-224.[Medline] [Order article via Infotrieve]

11. Dewey CF, Jr, Bussolari SR, Gimbrone MA, Jr, Davies PF. The dynamic response of endothelial cells to fluid shear stress. J Biomech Eng.. 1981;103:177-185.[Medline] [Order article via Infotrieve]

12. Neish AS, Williams AJ, Palmer HJ, Whitley MZ, Collins T. Functional analysis of the human vascular cell adhesion molecule-1 promoter. J Exp Med.. 1992;176:1583-1588.[Abstract/Free Full Text]

13. Khachigian LM, Williams AJ, Collins T. Interplay of Sp1 and Egr-1 in the proximal PDGF-A promoter in cultured vascular endothelial cells. J Biol Chem.. 1995;270:27679-27686.[Abstract/Free Full Text]

14. Khachigian LM, Fries JWU, Benz MW, Bonthron DT, Collins T. Novel cis-acting elements in the human platelet-derived growth factor B-chain core promoter that mediate gene expression in cultured vascular endothelial cells. J Biol Chem.. 1994;269:22647-22656.[Abstract/Free Full Text]

15. Nagel T, Resnick N, Atkinson WJ, Dewey CF, Gimbrone MA, Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest.. 1994;94:885-891.

16. Asakura T, Karino T. Flow patterns and spatial distribution atherosclerotic lesions in human coronary arteries. Circ Res.. 1990;66:1045-1066.[Abstract/Free Full Text]

17. Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma and arterial wall shear: observation, correlation and proposal of a shear dependent mass transfer mechanism for atherogenesis. Proc R Soc Lond (Biol.). 1971;177:109-159.[Medline] [Order article via Infotrieve]

18. Ku DN, Giddens DP, Zarins CK, Glasgov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis.. 1985;5:293-302.[Abstract/Free Full Text]

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

20. Gimbrone MA Jr. Vascular endothelium in health and disease. In: Haber E, ed. Molecular Cardiovascular Medicine. New York, NY: Scientific American Medicine, 1995.

21. Murry CE, Bartosek T, Giachelli CM, Alpers CE, Schwartz SM. Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas. Circulation.. 1996;93:1095-1106.[Abstract/Free Full Text]

22. Berk BC, Alexander RW, Brock TA, Gimbrone MA, Jr, Webb RC. Vasoconstriction: a new activity for platelet-derived growth factor. Science.. 1986;232:87-90.[Abstract/Free Full Text]

23. Geary RL, Kohler TR, Vergel S, Kirkman TR, Clowes AW. Time course of flow-induced smooth muscle cell proliferation and intimal thickening in endothelialized baboon vascular grafts. Circ Res.. 1994;74:14-23.[Abstract/Free Full Text]

24. Kraiss LW, Geary RL, Mattsson EJR, Vergel S, Au YPT, Clowes AW. Acute reductions in blood flow and shear stress induce platelet-derived growth factor-A expression in baboon prosthetic grafts. Circ Res.. 1996;79:45-53.[Abstract/Free Full Text]

25. Wilson E, Vives G, Collins T, Sukhatme V, Ives HE. Mechanical strain enhances expression of the PDGF-A and EGR-1 genes in vascular smooth muscle cells. FASEB J.. 1995;9:1200.

26. 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]

27. Tseng H, Peterson TE, Berk BC. Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. Circ Res.. 1995;75:869-878.

28. Berk BC, Corson MA, Peterson TE, Tseng H. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells—a hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech.. 1995;28:1439-1450.[Medline] [Order article via Infotrieve]

29. Whitmarsh AJ, Shore P, Sharrocks AD, Davis RJ. Integration of MAP kinase signal transduction pathways at the serum response element. Science.. 1995;269:403-407.[Abstract/Free Full Text]

30. Gille H, Sharrocks AD, Shaw PE. Phosphorylation of transcription factor p62^TCF by MAP kinase stimulates ternary complex formation at the c-fos promoter. Nature.. 1992;358:414-417.[Medline] [Order article via Infotrieve]

31. Hu Y-L, Chien S. Immunofluorescence studies on effects of shear stress on protein kinase C in endothelial cells. FASEB J.. 1995;9:A618.

32. Pearce MJ, McIntyre TM, Prescott SM, Zimmerman GA, Whatley RE. Shear stress activates cytosolic phospholipase A2 (cPLA2) and MAP kinase in human endothelial cells. Biochem Biophys Res Commun.. 1996;218:500-504.[Medline] [Order article via Infotrieve]

33. Hsieh H-J, Li N-Q, 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]

34. Hsieh H-J, Li N-Q, 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]

35. Resnick MA, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J.. 1995;9:874-882.[Abstract]

36. Tjian R, Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell.. 1994;77:5-8.[Medline] [Order article via Infotrieve]

37. Kim S-J, Glick A, Sporn MB, Roberts AB. Characterization of the promoter region of the transforming growth factor-beta1 gene. J Biol Chem.. 1989;264:402-408.[Abstract/Free Full Text]

38. Biesiada E, Razandi M, Levin ER. Egr-1 activates basic fibroblast growth factor transcription. J Biol Chem.. 1996;271:18576-18581.[Abstract/Free Full Text]

39. Mackman N, Morrissey JH, Fowler B, Edgington TS. Complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation cascade. Biochemistry.. 1989;28:1755-1762.[Medline] [Order article via Infotrieve]

40. Cui M-Z, Parry GCN, Oeth P, Larson KH, Smith M, Huang RP, Adamson ED, Mackman N. Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1. J Biol Chem.. 1996;271:2731-2739.[Abstract/Free Full Text]

41. Maltzman JS, Carman JA, Monroe JG. Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes. J Exp Med.. 1996;183:1747-1759.[Abstract/Free Full Text]

42. Maltzman JS, Carman JA, Monroe JG. Role of Egr1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol Cell Biol.. 1996;16:2283-2294.[Abstract]

43. Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production: modulation by potassium channel blockade. J Clin Invest.. 1995;95:1363-1369.

44. Lin M-C, F. Almus F, Shyy Y-J, Chien S. Fluid shear stress induces TF gene expression in vascular endothelium. FASEB J.. 1995;9:A412.

45. Khachigian LM, Lindner V, Williams AJ, Collins T. Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science.. 1996;271:1427-1431.[Abstract]

46. Braddock M, Houston P, Dickson MC, McVey J, Campbell CJ. Fluid shear stress activates the tissue factor promoter in vascular endothelial cells via upregulation of the cellular transcription factor, EGR-1. J Vasc Res. Abstract. 1995;33(suppl. 1):11.

This article has been cited by other articles:

				Z. Chen and E. Tzima PECAM-1 Is Necessary for Flow-Induced Vascular Remodeling Arterioscler Thromb Vasc Biol, July 1, 2009; 29(7): 1067 - 1073. [Abstract] [Full Text] [PDF]

				H. Beck, M. Semisch, C. Culmsee, N. Plesnila, and A. K. Hatzopoulos Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke Am. J. Pathol., July 1, 2008; 173(1): 77 - 92. [Abstract] [Full Text] [PDF]

				E. Sanchez-Guerrero, V. C. Midgley, and L. M. Khachigian Angiotensin II induction of PDGF-C expression is mediated by AT1 receptor-dependent Egr-1 transactivation Nucleic Acids Res., April 1, 2008; 36(6): 1941 - 1951. [Abstract] [Full Text] [PDF]

				H. Nakatsuka, T. Sokabe, K. Yamamoto, Y. Sato, K. Hatakeyama, A. Kamiya, and J. Ando Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G26 - G34. [Abstract] [Full Text] [PDF]

				L. M. Khachigian Early Growth Response-1 in Cardiovascular Pathobiology Circ. Res., February 3, 2006; 98(2): 186 - 191. [Abstract] [Full Text] [PDF]

				A. M. de Mestre, S. Rao, J. R. Hornby, T. Soe-Htwe, L. M. Khachigian, and M. D. Hulett Early Growth Response Gene 1 (EGR1) Regulates Heparanase Gene Transcription in Tumor Cells J. Biol. Chem., October 21, 2005; 280(42): 35136 - 35147. [Abstract] [Full Text] [PDF]

				J. Malakooti, R. Sandoval, V. C. Memark, P. K. Dudeja, and K. Ramaswamy Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G653 - G663. [Abstract] [Full Text] [PDF]

				D. Scholz and W. Schaper Preconditioning of arteriogenesis Cardiovasc Res, February 1, 2005; 65(2): 513 - 523. [Abstract] [Full Text] [PDF]

				T. Sokabe, K. Yamamoto, N. Ohura, H. Nakatsuka, K. Qin, S. Obi, A. Kamiya, and J. Ando Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2027 - H2034. [Abstract] [Full Text] [PDF]

				V. C. Midgley and L. M. Khachigian Fibroblast Growth Factor-2 Induction of Platelet-derived Growth Factor-C Chain Transcription in Vascular Smooth Muscle Cells Is ERK-dependent but Not JNK-dependent and Mediated by Egr-1 J. Biol. Chem., September 24, 2004; 279(39): 40289 - 40295. [Abstract] [Full Text] [PDF]

				F. S. Santiago and L. M. Khachigian Ets-1 Stimulates Platelet-Derived Growth Factor A-Chain Gene Transcription and Vascular Smooth Muscle Cell Growth via Cooperative Interactions With Sp1 Circ. Res., September 3, 2004; 95(5): 479 - 487. [Abstract] [Full Text] [PDF]

				M. Osawa, S. Itoh, S. Ohta, Q. Huang, B. C. Berk, N.-L. Marmarosh, W. Che, B. Ding, C. Yan, and J.-i. Abe ERK1/2 Associates with the c-Met-binding Domain of Growth Factor Receptor-bound Protein 2 (Grb2)-associated Binder-1 (Gab1): ROLE IN ERK1/2 AND EARLY GROWTH RESPONSE FACTOR-1 (Egr-1) NUCLEAR ACCUMULATION J. Biol. Chem., July 9, 2004; 279(28): 29691 - 29699. [Abstract] [Full Text] [PDF]

				A. A. Miyakawa, M. de Lourdes Junqueira, and J. E. Krieger Identification of two novel shear stress responsive elements in rat angiotensin I converting enzyme promoter Physiol Genomics, April 13, 2004; 17(2): 107 - 113. [Abstract] [Full Text] [PDF]

				E. Tzima, J. S. Reader, M. Irani-Tehrani, K. L. Ewalt, M. A. Schwartz, and P. Schimmel Biologically active fragment of a human tRNA synthetase inhibits fluid shear stress-activated responses of endothelial cells PNAS, December 9, 2003; 100(25): 14903 - 14907. [Abstract] [Full Text] [PDF]

				K. Boengler, F. Pipp, B. Fernandez, T. Ziegelhoeffer, W. Schaper, and E. Deindl Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp) Cardiovasc Res, September 1, 2003; 59(3): 573 - 581. [Abstract] [Full Text] [PDF]

				W. Schaper and D. Scholz Factors Regulating Arteriogenesis Arterioscler Thromb Vasc Biol, July 1, 2003; 23(7): 1143 - 1151. [Abstract] [Full Text] [PDF]

				J.-J. Chiu, L.-J. Chen, P.-L. Lee, C.-I Lee, L.-W. Lo, S. Usami, and S. Chien Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells Blood, April 1, 2003; 101(7): 2667 - 2674. [Abstract] [Full Text] [PDF]

				X.-L. Chen, S. E. Varner, A. S. Rao, J. Y. Grey, S. Thomas, C. K. Cook, M. A. Wasserman, R. M. Medford, A. K. Jaiswal, and C. Kunsch Laminar Flow Induction of Antioxidant Response Element-mediated Genes in Endothelial Cells. A NOVEL ANTI-INFLAMMATORY MECHANISM J. Biol. Chem., January 3, 2003; 278(2): 703 - 711. [Abstract] [Full Text] [PDF]

				Y. Zhao, B. P. C. Chen, H. Miao, S. Yuan, Y.-S. Li, Y. Hu, D. M. Rocke, and S. Chien Improved significance test for DNA microarray data: temporal effects of shear stress on endothelial genes Physiol Genomics, December 26, 2002; 12(1): 1 - 11. [Abstract] [Full Text] [PDF]

				D. G. Peters, X.-C. Zhang, P. V. Benos, E. Heidrich-O'Hare, and R. E. Ferrell Genomic analysis of immediate/early response to shear stress in human coronary artery endothelial cells Physiol Genomics, December 26, 2002; 12(1): 25 - 33. [Abstract] [Full Text] [PDF]

				E. V. Gerasimovskaya, S. Ahmad, C. W. White, P. L. Jones, T. C. Carpenter, and K. R. Stenmark Extracellular ATP Is an Autocrine/Paracrine Regulator of Hypoxia-induced Adventitial Fibroblast Growth. SIGNALING THROUGH EXTRACELLULAR SIGNAL-REGULATED KINASE-1/2 AND THE Egr-1 TRANSCRIPTION FACTOR J. Biol. Chem., November 15, 2002; 277(47): 44638 - 44650. [Abstract] [Full Text] [PDF]

				A. Zakrzewicz, T. W. Secomb, and A. R. Pries Angioadaptation: Keeping the Vascular System in Shape Physiology, October 1, 2002; 17(5): 197 - 201. [Abstract] [Full Text] [PDF]

				M. Fu, J. Zhang, Y. Lin, X. Zhu, M. U. Ehrengruber, and Y. E. Chen Early Growth Response Factor-1 Is a Critical Transcriptional Mediator of Peroxisome Proliferator-activated Receptor-gamma 1 Gene Expression in Human Aortic Smooth Muscle Cells J. Biol. Chem., July 19, 2002; 277(30): 26808 - 26814. [Abstract] [Full Text] [PDF]

				A. Shay-Salit, M. Shushy, E. Wolfovitz, H. Yahav, F. Breviario, E. Dejana, and N. Resnick VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells PNAS, July 9, 2002; 99(14): 9462 - 9467. [Abstract] [Full Text] [PDF]

				T. Abumiya, T. Sasaguri, Y. Taba, Y. Miwa, and M. Miyagi Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 907 - 913. [Abstract] [Full Text] [PDF]

				L. J. Landesberg, R. Ramalingam, K. Lee, T. K. Rosengart, and R. G. Crystal Upregulation of transcription factors in lung in the early phase of postpneumonectomy lung growth Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1138 - L1149. [Abstract] [Full Text] [PDF]

				M. Morimoto, N. Kume, S. Miyamoto, Y. Ueno, H. Kataoka, M. Minami, K. Hayashida, N. Hashimoto, and T. Kita Lysophosphatidylcholine Induces Early Growth Response Factor-1 Expression and Activates the Core Promoter of PDGF-A Chain in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 771 - 776. [Abstract] [Full Text] [PDF]

				R. Korenaga, K. Yamamoto, N. Ohura, T. Sokabe, A. Kamiya, and J. Ando Sp1-mediated downregulation of P2X4 receptor gene transcription in endothelial cells exposed to shear stress Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2214 - H2221. [Abstract] [Full Text] [PDF]

				H. J. Salacinski, S. Goldner, A. Giudiceandrea, G. Hamilton, A. M. Seifalian, A. Edwards, and R. J. Carson The Mechanical Behavior of Vascular Grafts: A Review J Biomater Appl, January 1, 2001; 15(3): 241 - 278. [Abstract] [PDF]

				A. D. Westmuckett, C. Lupu, S. Roquefeuil, T. Krausz, V. V. Kakkar, and F. Lupu Fluid Flow Induces Upregulation of Synthesis and Release of Tissue Factor Pathway Inhibitor In Vitro Arterioscler Thromb Vasc Biol, November 1, 2000; 20(11): 2474 - 2482. [Abstract] [Full Text] [PDF]

				X. Bao, C. B. Clark, and J. A. Frangos Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43 Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1598 - H1605. [Abstract] [Full Text] [PDF]

				W. Gosgnach, M. Challah, F. Coulet, J.-B. Michel, and T. Battle Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF Cardiovasc Res, January 14, 2000; 45(2): 486 - 492. [Abstract] [Full Text] [PDF]

				S. Ling, A. Dai, Y.-H. Ma, E. Wilson, K. Chatterjee, H. E. Ives, and K. Sudhir Matrix-Dependent Gene Expression of Egr-1 and PDGF A Regulate Angiotensin II-Induced Proliferation in Human Vascular Smooth Muscle Cells Hypertension, November 1, 1999; 34(5): 1141 - 1146. [Abstract] [Full Text] [PDF]

				K. Kawai-Kowase, M. Kurabayashi, Y. Hoshino, Y. Ohyama, and R. Nagai Transcriptional Activation of the Zinc Finger Transcription Factor BTEB2 Gene by Egr-1 Through Mitogen-Activated Protein Kinase Pathways in Vascular Smooth Muscle Cells Circ. Res., October 29, 1999; 85(9): 787 - 795. [Abstract] [Full Text] [PDF]

				R. C. Tam, C. J. Lin, C. Lim, B. Pai, and V. Stoisavljevic Inhibition of CD28 Expression by Oligonucleotide Decoys to the Regulatory Element in Exon 1 of the CD28 Gene J. Immunol., October 15, 1999; 163(8): 4292 - 4299. [Abstract] [Full Text] [PDF]

				C.-H. Heldin and B. Westermark Mechanism of Action and In Vivo Role of Platelet-Derived Growth Factor Physiol Rev, October 1, 1999; 79(4): 1283 - 1316. [Abstract] [Full Text] [PDF]

				E. S. Silverman, L. M. Khachigian, F. S. Santiago, A. J. Williams, V. Lindner, and T. Collins Vascular Smooth Muscle Cells Express the Transcriptional Corepressor NAB2 in Response to Injury Am. J. Pathol., October 1, 1999; 155(4): 1311 - 1317. [Abstract] [Full Text] [PDF]

				F. S. Santiago, D. G. Atkins, and L. M. Khachigian Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are Egr-1/NGFI-A-Dependent Am. J. Pathol., September 1, 1999; 155(3): 897 - 905. [Abstract] [Full Text] [PDF]

				J. J. Chiu, B. S. Wung, H. J. Hsieh, L. W. Lo, and D. L. Wang Nitric Oxide Regulates Shear Stress–Induced Early Growth Response-1 : Expression via the Extracellular Signal–Regulated Kinase Pathway in Endothelial Cells Circ. Res., August 6, 1999; 85(3): 238 - 246. [Abstract] [Full Text] [PDF]

				T. Nagel, N. Resnick, C. F. Dewey Jr, and M. A. Gimbrone Jr Vascular Endothelial Cells Respond to Spatial Gradients in Fluid Shear Stress by Enhanced Activation of Transcription Factors Arterioscler Thromb Vasc Biol, August 1, 1999; 19(8): 1825 - 1834. [Abstract] [Full Text] [PDF]

				L. M. Khachigian, F. S. Santiago, L. A. Rafty, O. L.-W. Chan, G. J. Delbridge, A. Bobik, T. Collins, and A. C. Johnson GC Factor 2 Represses Platelet-Derived Growth Factor A-Chain Gene Transcription and Is Itself Induced by Arterial Injury Circ. Res., June 11, 1999; 84(11): 1258 - 1267. [Abstract] [Full Text] [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]

				H. Morawietz, Y.-H. Ma, F. Vives, E. Wilson, V. P. Sukhatme, J. Holtz, and H. E. Ives Rapid Induction and Translocation of Egr-1 in Response to Mechanical Strain in Vascular Smooth Muscle Cells Circ. Res., April 2, 1999; 84(6): 678 - 687. [Abstract] [Full Text] [PDF]

				X. Bao, C. Lu, and J. A. Frangos Temporal Gradient in Shear But Not Steady Shear Stress Induces PDGF-A and MCP-1 Expression in Endothelial Cells : Role of NO, NF{kappa}B, and egr-1 Arterioscler Thromb Vasc Biol, April 1, 1999; 19(4): 996 - 1003. [Abstract] [Full Text] [PDF]

				J. E. Bishop and G. Lindahl Regulation of cardiovascular collagen synthesis by mechanical load Cardiovasc Res, April 1, 1999; 42(1): 27 - 44. [Full Text] [PDF]

				E. S. Silverman and T. Collins Pathways of Egr-1-Mediated Gene Transcription in Vascular Biology Am. J. Pathol., March 1, 1999; 154(3): 665 - 670. [Full Text] [PDF]

				F. S. Santiago, H. C. Lowe, F. L. Day, C. N. Chesterman, and L. M. Khachigian Early Growth Response Factor-1 Induction by Injury Is Triggered by Release and Paracrine Activation by Fibroblast Growth Factor-2 Am. J. Pathol., March 1, 1999; 154(3): 937 - 944. [Abstract] [Full Text] [PDF]

				B. K. Dieckgraefe and D. M. Weems Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK Am J Physiol Gastrointest Liver Physiol, February 1, 1999; 276(2): G322 - G330. [Abstract] [Full Text] [PDF]

				P. Houston, M. C. Dickson, V. Ludbrook, B. White, J.-L. Schwachtgen, J. H. McVey, N. Mackman, J. M. Reese, D. G. Gorman, C. Campbell, et al. Fluid Shear Stress Induction of the Tissue Factor Promoter In Vitro and In Vivo Is Mediated by Egr-1 Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 281 - 289. [Abstract] [Full Text] [PDF]

				M. Nagase, J. Abe, K. Takahashi, J. Ando, S. Hirose, and T. Fujita Genomic Organization and Regulation of Expression of the Lectin-like Oxidized Low-density Lipoprotein Receptor (LOX-1) Gene J. Biol. Chem., December 11, 1998; 273(50): 33702 - 33707. [Abstract] [Full Text] [PDF]

				H. Hagiwara, M. Mitsumata, T. Yamane, X. Jin, and Y. Yoshida Laminar Shear Stress–Induced GRO mRNA and Protein Expression in Endothelial Cells Circulation, December 8, 1998; 98(23): 2584 - 2590. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted 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 Khachigian, L. M. Articles by Collins, T. Search for Related Content PubMed PubMed Citation Articles by Khachigian, L. M. Articles by Collins, T. Pubmed/NCBI databases Substance via MeSH