© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF
Unit 460; CHU X. Bichat; 16, rue H. Huchard; 75870, Paris Cedex 18, France
* Corresponding author. Tel.: +33-1-44856-160; fax: +33-1-44-856-157
Received 11 June 1999; accepted 16 August 1999
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Abstract |
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Objective: Hemodynamic stresses are considered to be important regulators of gene expression in vascular cells. In this study, we have investigated the role of shear stress on ACE expression in cultured rat vascular cells, and focused on the regulation of ACE expression in smooth muscle cells. Methods: Rat aortic endothelial cells, smooth muscle cells and fibroblasts isolated from Wistar rats were submitted to shear stress using a laminar shear flow parallel chamber. Results: A 10 dynes/cm2 shear rate for 24 h increased ACE activity in the three vascular cell types (x 2.14 in endothelial cells, x 2.9 in smooth muscle cells, x 3.33 in fibroblasts). This induction was blocked by a 24 h pre-incubation with a translation blocker (10–4M cycloheximide) showing the role of protein neosynthesis. Therefore the study was focused on smooth muscle cells and we demonstrated that the increase in ACE activity was due to an elevation in ACE mRNA level in response to a 10 dynes/cm2 shear stress for 24 h. This induction was dependent on the shear intensity (P<0.0001). Six hours of a 15 dynes/cm2 shear stress showed no effect on ACE activity or mRNA expression. In contrast, the same duration of shear significantly increased bFGF mRNA level (x3.7). Conversely, bFGF dose dependently increased ACE mRNA expression and activity in smooth muscle cells. This result suggests that bFGF could be one of the potential inductors of ACE expression in the stressed smooth muscle cells. Conclusions: Mechanical stress increases ACE expression in vascular cells. bFGF could be one of the potential factors involved in this activation. This phenomenon could participate in the role of ACE activity in vascular wall remodeling.
KEYWORDS Angioplasty; Angiotensin; Blood flow; Growth factors; Hemodynamics; Gene expression
This article is referred to in the Editorial by S.J.L. Bakker and R.O.B. Gans (pages 270–272) in this issue.
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1 Introduction |
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Blood flowing through a vessel generates a frictional drag due to the movement of the fluid phase on the vascular wall surface, creating a shear stress at the interface. Physiologically, the endothelial cells lining the inside of the blood vessel are exposed to the blood-induced shear stress. In contrast, physiologically, the smooth muscle cells are protected from shear stress by endothelial cell lining but, after endothelial injury, smooth muscle cells proliferate at the interface between the flowing blood and the vascular wall, therefore becoming exposed to shear stress in pathological conditions. It has been demonstrated that angiotensin converting enzyme (ACE) expression is induced in smooth muscle cells of the neointima after vascular injury in vivo [1]. Fishel et al. showed that fibroblast growth factor (FGF) stimulated ACE expression in vascular smooth muscle and that there appeared to be a close spatial association between ACE expression in the injured artery and FGF expression [2]. Of particular interest in this study was the existence of a gradient of ACE expression with greater immunoreactivity in cells close to the lumen. This in vivo gradient of ACE expression, decreasing from the lumen to the media, was associated with a similar gradient of FGF expression suggesting that shear stress and FGF could be concomitantly involved in the regulation of ACE expression in the neointima. In view of the different phenotypic expressions of proliferating smooth muscle cells in the neointima, the hypothesis underlying our study was that the exposure of SMC to shear stress forces due to the absence of the endothelial cell lining could modify ACE expression.
We therefore wanted to determine whether mechanical stress could induce ACE expression in vascular cell cultures, and could be one of the factors responsible for the observed increase in ACE expression in smooth muscle cells of injured arteries in vivo. For doing so, we used a laminar shear flow parallel chamber device. Our cell isolation technique allowed us to culture the three main cell types (endothelial cells, smooth muscle cells and fibroblasts) derived from the three layers of the rat aorta. Using activity and mRNA detection, we studied ACE and FGF overexpression induced by shear stress and focused this study on smooth muscle cell regulation of ACE expression.
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2 Materials and methods |
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2.1 Animals
Normotensive male Wistar rats (weighing 160–180 g) were obtained from Iffa Credo (Labresle, France). The procedure followed for the care and euthanasia of the study animals was in accordance with the European Community Standards on the care and use of laboratory animals (Ministère de l’Agriculture, France; authorisation No 00577).
2.2 Cell isolation and culture
The three cell types from the aorta (i.e.: endothelial cells from the intima, smooth muscle cells from the media and fibroblasts from the adventitia) were isolated and cultured as described previously [3].
The purity of the cultures was assessed using morphological and immunohistological criteria. Rat aortic endothelial cells (RAEC) were stained for Von Willebrand factor and by RECA-1, a monoclonal antibody specific of rat endothelial cells [4]. Smooth muscle cells (SMC) were characterised using antibodies raised against alpha actin [5]. Staining of fibroblasts proved to be negative for both antibodies.
2.3 The shear stress device
The three cell types were individually seeded on a rectangular plastic (cell-culture treated) plate previously coated with collagen (0.1% in hydroxychloride, SIGMA) and were allowed to reach confluence. The experimental surface of shear-subjected cells was of 18 cm2. Collagen was chosen as a substrate in order to increase the adherence forces of the cells submitted to shear stress. As a control, unstressed cells were seeded in the same conditions without insertion in the flow chamber device. Cells were used 2 to 4 days after having reached confluence (from 6 to 8 days after trypsinisation). All cells were used from passage 2 to passage 3. Each isolation gave from 18 to 24 shear stress plates. Each experiment included at least 2 isolations.
Confluent cells were then exposed to a fluid-imposed shear stress using a parallel-plate-channel-flow device derived from the one described by Levesque and Nerem [6]. The cell culture flow chamber was designed to provide steady, uniform laminar flow. It was positioned in a closed continuous flow loop. This latter consisted of an elevated reservoir (placed under a class II laminar flow hood) which provided the required pressure drop across the chamber and a roller pump to return the outflow from the collecting reservoir back to the feeding reservoir. The upper reservoir was filled with 350 ml of Dulbecco Modified Essential Medium (DMEM) at 37°C (equilibrated with 95% air, 5% CO2) supplemented with 10% heat-inactivated fetal calf serum, whose pH, temperature and flow rate were monitored continuously. The same culture medium was added on the control cells and they were placed in a dry incubator. The pH of the medium was 7.4 in the shear stress device and in control plates. It was verified that warming the medium for 1.5 h at 65°C completely inhibited endogenous ACE expression.
2.4 Shear stress values
The shear stress intensity ¥ (dynes/cm2) can be calculated using the equation [6]: ¥=6 µQ/ph2 where µ is the media viscosity (DMEM 0.0084±0.08 poise at 37°C), Q the flow rate (ml/s), p the flow path length (1.8 cm) and h the gap height over the cell layer (0.025 cm). The viscosity of the media and the flow chamber cross section are constant in the device. In order to change the intensity of shear stress, the induction flow rate had to be modified.
2.5 Shear stress protocol
First, the shear stress was fixed at 10 dynes/cm2 for 24 h, and the ACE activity was measured in the three vascular cell types. Then the relationship between shear intensity and ACE activity was studied on smooth muscle cells. Shear stress intensity was fixed at near physiological values (15–17 dynes/cm2) and the time dependence of shear-induced ACE activity and mRNA were studied on smooth muscle cells. We thereafter studied time dependence of shear-induced bFGF mRNA.
2.6 Post-shear cell treatment
After being submitted to shear, the cell plates were removed from the flow chambers under sterile conditions. To measure ACE activity, cells from each plate were scraped off into 1 ml of PBS and centrifuged at 1500xg for 15 min. The pellet was resuspended in 250 µl of Tris–HCl buffer with 8 mM CHAPS and sonicated for 2x15 s. Samples were frozen at –20°C until further investigations.
To determine ACE and bFGF mRNA expressions, cells were scraped from each plate into 1 ml of Trizol solution (Gibco-BRL). Total RNA was prepared using the manufacturer's instructions. Relative changes in ACE and bFGF mRNA levels were detected by RT-PCR.
2.7 Measurement of ACE activity
The ACE activity was measured in the cell lysate by measuring the hydrolysis of a radiolabeled synthetic substrate [glycine-1-14C] hippuryl-L-histidyl-L-leucine (3 mCi.mmol–1, Amersham), with or without 10–6 M enalaprilat as proposed by Cushman and Cheung [7]. Cell lysate was incubated for 1 h with [glycine-1-14C] hippuryl-L-histidyl-L-leucine in Tris–HCl 50 mM, pH 7.4, 1% NaCl. The reaction was stopped by heat inactivation at 80°C for 5 min after which 1,2-phtalicdicarboxaldehyde (PDA) and NaOH 0.28 M were added to the mix. After 10 min incubation, the reaction was stopped by adjunction of 2 M HCl. Results are expressed as pmol of synthetic substrate cleaved per mg of protein per min.
2.8 Quantification of ACE and bFGF mRNA expressions
2.8.1 Reverse transcription
One hundred and fifty ng of total mRNA were reverse transcribed (final volume 18 µl) with reverse transcriptase (8 U/µl, 1 h at 37°C) (Gibco-BRL) in the presence of 1 µg of oligo d(T).
2.8.2 PCR reaction
Part of the reverse transcription solution (3 µl) was mixed with the PCR mix (1x Taq-buffer containing 1.5 mM MgCl2, 1U Taq DNA polymerase (Gibco-BRL), 0.1 mM dNTP (Pharmacia), 8 pmoles of sense and antisense primers and 400 000 cpm of 33P-radiolabelled mix of both primers) and subjected to 4 min of initial denaturation at 94°C; 31 cycles of 30 s at 94°C, 30 s at 64°C and 1 min at 72°C; then 1 min at 62°C and 10 min at 72°C.
Primers for ACE include 5'-AGAAGGCCAAGGAGCTGTATG-3' (sense) and 5'-GACAAAGGCATGGAGGTTCAG-3' (antisense). The amplification generates a 478 bp product spanning bases 337 to 815 of rat ACE cDNA. ACE mRNA expression was calculated by normalising ACE mRNA to GAPDH mRNA. Primers for GAPDH include 5'-GTGAAGGTCGGAGTCAACG-3' (sense) and 5'-GGTGAAGACGCCAGTGGAC-3' (antisense) which amplify a 299 bp mRNA region. The annealing temperature for GAPDH primers was 55°C, and the PCR was performed for 27 cycles.
For bFGF mRNA amplification, 1 µg of total RNA was reverse transcribed in the same conditions as described previously. Primers for FGF included 5'-AAGCAGAAGAGAGAGGAGTTG-3' (sense) and 5'-TTAGCAGACATTGGAAGAAAC-3' (antisense) which amplify a 270 bp mRNA region. The annealing temperature for bFGF primers was 62°C, and the PCR was performed for 31 cycles. The primers were chosen to encompass several introns in order to avoid amplification of contaminating genomic cDNA. A negative control was used for each set of samples to check the reverse transcription and the PCR amplification reagents for any contamination. PCR amplification was verified to be exponential and the product proportional to the input.
The PCR products were separated on a 8% acrylamide/DHEBA (29/1) gel in 1x TBE buffer using miniprotean II cell apparatus (Biorad). N,N'-(1,2-dihydroxyethylene)-bis-acrylamide (DHEBA) was obtained from ICN biochemicals. After ethidium bromide staining, the bands were excised, dissolved for 2 h at 50°C in 750 µl of 25 mmol/l per-iodic acid and the radioactivity was counted in a β-scintillation counter. On the negative control lane, gel slices corresponding to the position of the bands were excised, counted and used as a background.
2.9 Statistical method
Results are expressed as means±s.e. mean. Significance was estimated by analysis of variance or student t test. A P value less than 0.05 was considered significant.
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3 Results |
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3.1 Shear stress induced ACE activity in the three vascular cell types
Results of basal and shear-induced ACE levels are shown in Fig. 1. Exposure of cells to a shear stress of 10 dynes/cm2 for a period of 24 h increased the angiotensin converting enzyme activity in all three cell types. Endothelial cell basal ACE activity was 0.41±0.07 fmol of substrate/mg prot/min in quiescent conditions. Shear stress increased this activity by 114%. Smooth muscle cell ACE activity was raised by 190% in stressed cells. Fibroblast ACE activity was increased by 233% under shear conditions. In order to determine the role of protein neosynthesis in this phenomenon, cells were pre-incubated for 24 h with 10–6M cycloheximide, a translation blocker. Cycloheximide abolished the response to shear stress in the three cell types (Fig. 1).
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3.2 Shear-induced ACE mRNA in smooth muscle cells
Rat aortic smooth muscle cells submitted to a 10 dynes/cm2 shear stress for 24 h also showed increased ACE mRNA expression in comparison with non-sheared cells. The ACE/GAPDH mRNA ratio rose from 1±0.02 to 32±20 (Fig. 2).
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3.3 Shear-intensity dependency of ACE expression
In smooth muscle cells, ACE activity correlated with shear stress intensity (r=0.99, P<0.0001). A level of 10 dynes/cm2 was necessary to induce a significant rise in ACE activity. A shear value between 15 dynes/cm2 and 20 dynes/cm2 appears to be required to induce the maximum response (Fig. 3).
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3.4 Time-dependence of shear stress-induced ACE expression
The induction of ACE activity and mRNA expression appeared to be time-dependent. After 6 h of 15 dyne/cm2 shear, no increase in ACE activity or mRNA expression was detected in aortic smooth muscle cells. The ACE/GAPDH mRNA ratio was 1±0.04 in control cells and 1.2±0.02 in stressed cells. ACE activity was 0.376±0.05 fmol substrate/mg prot/min in control cells and 0.316±0.05 in 6 h stressed cells. In contrast, 24 h of the same level of shear stress increased significantly both mRNA expression and ACE activity (Fig. 4).
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3.5 Time-dependence of shear-induced bFGF mRNA expression
Because shear stress-induced ACE expression was delayed, the hypothesis that shear-induced FGF expression preceded ACE expression has been tested. Basic FGF mRNA expression was measured in smooth muscle cells after 6 and 24 h of shear stress at 15 dynes/cm2. Basic bFGF/GAPDH mRNA ratio was low in the control cells. After 6 h, bFGF/GAPDH mRNA ratio was significantly increased and remained significantly increased after 24 h of shear stress (Fig. 5).
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3.6 Effect of basic FGF on ACE activity and mRNA expression in SMC
ACE activity was measured in smooth muscle cells incubated with increasing levels of bFGF for 48 h. bFGF significantly increased ACE activity in the smooth muscle cells. ACE activity was 0.1±0.02 fmol substrate/mg prot/min in quiescent smooth muscle cells whereas in treated cells ACE activity ranged from 0.35±0.03 to 0.45±0.07 fmol substrate/mg prot/min (Fig. 6). In view of the above results the effect of FGF accumulation on ACE expression has been tested on smooth muscle cells. Increasing levels of bFGF from 0.1 nM to 2.5 nM correspondingly increased ACE mRNA level in a dose-dependent manner in cultured smooth muscle cells (Fig. 7).
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4 Discussion |
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The present study shows that ACE expression can be up-regulated by shear stress in vascular cells. Nevertheless, in smooth muscle cells, this up-regulation is delayed, suggesting an indirect rather than a direct effect of shear stress on ACE expression. Shear stress also induced the expression of basic FGF in smooth muscle cells which preceded the expression of ACE. Because bFGF was able to induce, in a dose-dependent manner, the expression of ACE, these results suggest that shear stress-induced ACE expression could be mediated in part by FGF. As FGF has no signal sequence for secretion the possible respective roles of secreted and non-secreted FGF remain to be determined. This shear-dependency of ACE expression was not limited to smooth muscle cells, but was observed also in other vascular cell types including primary cultures of endothelial cells and fibroblasts. Therefore, in the three vascular cell types, (endothelial cells, smooth muscle cells and fibroblasts of adventitial origin), shear stress positively correlated with ACE expression.
Our results agree with the in vivo data reported by Rakugi et al. [1] demonstrating ACE overexpression in the post-ballooning, proliferating intima and with the data of Fishel et al. [2] showing that bFGF was able to induce ACE overexpression in the proliferating smooth muscle cells. Since smooth muscle cells migrate into the intima after vascular injury, they acquire a proliferative and secretory phenotype. Similarly, several passages of smooth muscle cells in culture changed their phenotypic expression from a contractile to a proliferative and secretory phenotype, mimicking what is observed in vivo. Our present in vitro data also correspond with our observations in vivo in experimental congestive heart failure [8]. In this early study, we observed in vivo that ACE expression decreased in the lung in proportion to the degree of heart failure. We suggested that the decrease in shear stress induced by the decrease in blood flow and the increase in radius of the pulmonary arterial vessels could account for the down-regulation of endothelial ACE expression. Similar results have been reported in pulmonary hypertension [9]. Conversely, the high tensile stress perceived by the fibrotic scar of myocardial infarction and by heart valves in physiological conditions could be responsible in part for ACE expression by fibroblasts in the myocardial fibrotic scar [10] and by mitral valve leaflets [11]. Therefore in vivo ACE expression correlated positively with hemodynamic stresses.
In contrast, our data disagrees with those of the study by Rieder and coworkers [12] showing that shear stress diminished ACE expression in bovine pulmonary artery endothelial cells in culture. This discrepancy could be due to the differences in the shear device and the cell types used, to the resistance of cultured cells to shear and to the complexity of the ACE-gene promoter. In their study, Rieder and coworkers used a luciferase reporter assay coupled to a 1.3 kb segment of the ACE promoter. In our own experiments, we have sequenced the 1.3 kb of the rat ACE promoter (data not shown) and have shown that this 5' sequence upstream to the coding sequence did not contain the element of the complex behaviour of the ACE promoter. For example, the mutated segment of the gene promoter responsible for the genetic determination of ACE expression in human [13] as in rats [14] could not be identified in the 1.3kb segment of the promoter.
Our data showed that all vascular cells shared shear stress-induced ACE activity and that this induction was blocked by cycloheximide suggesting that ACE overactivity was dependent on protein neosynthesis. The effect on protein expression correlated well with a similar effect on mRNA expression suggesting that ACE expression was regulated at the mRNA level by shear stress. Moreover, the increase in ACE activity was dependent on the shear stress intensity. Several potential sites of transactivation have been identified in the human ACE promoter: TRE at –5266 bp upstream to the TATA box, Egr-1 site at –59 bp and SP-1 sites at –131, –68 and –41bp [15] but no SSRE element have been identified. Moreover the delayed response of ACE expression to shear suggest an indirect rather than a direct relationship.
We were not able to detect any changes in ACE expression after 6 h of shear stress, whereas a reproducible induction of ACE activity and mRNA expression was detected at 24 h. FGF was able to dose-dependently induce ACE expression in smooth muscle cells [2]. It has been shown that shear stress was able to induce FGF expression in smooth muscle cells [16,17] and that the produced FGF was detectable in the conditioned medium [16] demonstrating the secretion of the growth factor. Nevertheless the mechanism by which cells secreted FGF in response to stress remains to be determined. We therefore tested the hypothesis that in our experimental system the overexpression of basic FGF could precede the expression of ACE. Our experimental results showed that bFGF mRNA was induced after a 6 h exposure to shear stress, i.e. earlier than ACE overexpression. Therefore these data suggested that shear-induced ACE overexpression in smooth muscle cells could, at least in part, be mediated indirectly by shear-induced basic FGF overexpression and accumulation. Nevertheless, shear and bFGF increased more mRNA level than ACE activity. This discrepancy could be due in part to a partial translation of mRNA in protein in such experimental conditions. Moreover ACE activity was measured only in cell lysate and soluble form present in the medium was not measured. Our results probably represent only a part of ACE activity.
The promoter region of FGF has been better characterised than the promoter of ACE gene. Several sites of transactivation have been identified in the promoter of FGF: AP-1, SP-1, TRE [18,19] and could be involved in its upregulation by shear. Shear is able to activate several, complex signal transduction pathways: calcium mobilisation [20,21], activation of protein phosphorylation cascades including protein kinase C, focal adhesion kinase [22], MAP kinases, ERK1/2 [23], and activation of several transactivator nuclear factors as Egr-1, NF kappa B, SP-1, TRE...(see [24] for review). FGF induced EGR-1 binds to two DNA elements in the FGF promoter inducing an auto-amplification of FGF expression [25]. Therefore Egr-1 could be involved in the FGF response to shear stress [26] and in the response of ACE to shear and FGF. Nevertheless demonstration of this last point requires further studies.
It is well known that shear stress increases the production of NO in vascular endothelial cells. Furthermore, Takemoto et al. recently demonstrated that NOS inhibition results in an up-regulation of ACE expression in various vascular and myocardial tissues [27]. In addition, Ackermann et al. showed that NO and NO-releasing compounds inhibit ACE activity in a concentration-dependent and a competitive way [28]. This suggest that particularly in endothelial cells, the ACE overexpression in response to shear stress might have been influenced by the simultaneous production of NO.
In conclusion, our data in vitro suggest that mechanical stress positively influenced ACE expression in the three vascular cell types and could have pathophysiological relevance in vivo. Modification of hemodynamic conditions could influence ACE expression in endothelial cells but also in smooth muscle cells and could participate in the observed shift of ACE expression from the endothelium, in physiological conditions, to other vascular cell types, (including smooth muscle cells and fibroblasts) in pathological conditions such as endothelial injury.
Time for primary review 24 days.
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Acknowledgements |
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This study was supported by the French Ministry of Research and Technology ACCSV9 contract no. 1A015A.
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  S. J.L Bakker and R. O.B Gans About the role of shear stress in atherogenesis Cardiovasc Res, January 14, 2000; 45(2): 270 - 272. [Full Text] [PDF]
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