© 1996 American Heart Association, Inc.
Articles |
Pressure and Angiotensin II Synergistically Induce Aortic Fibronectin Expression in Organ Culture Model of Rabbit Aorta
Evidence for a Pressure-Induced Tissue Renin-Angiotensin System
the Institut National de la Sante et de la Recherche Medicale, Unite 141 (N.B., R.M., J.B., A.T.) and Unite 127 (J.-L.S.), IFR Circulation, Hopital Lariboisiere, Paris, France.
Correspondence to Dr Alain Tedgui, PhD, INSERM Unite 141, 41, Blvd de la Chapelle, 75475 Paris Cedex 10, France.
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Abstract |
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Aortic fibronectin (FN) expression is augmented in hypertension. Increasing evidence suggests that both angiotensin II (Ang II) and mechanical factors may induce vascular remodeling in response to hypertension. We have previously shown that, in vitro, increased transmural pressure enhances FN expression in rabbit aortic media. To investigate the existence of a link between the effects of pressure and Ang II and to explore the mechanisms underlying such a relationship, we quantified the effect of Ang II and Ang II inhibitors on the pressure-dependent FN expression in a 3-day organ culture model of rabbit aorta using immunolabeling analysis and detected FN mRNAs by in situ hybridization. A dose-dependent effect of Ang II on FN expression was observed at both 80 and 150 mm Hg but not at 0 mm Hg (relaxed vessels). One µmol/L Ang II increased the media cross-sectional surface, showing FN expression from 7.9±0.7% (n=9) to 18.9±1.1% (n=4) at 80 mm Hg (P<.01) and from 17.4±1.8% (n=9) to 56.6%±3.6 (n=4) at 150 mm Hg (P<.001). In situ hybridization revealed that Ang II and pressure upregulated FN mRNA expression. Losartan, an AT1 antagonist, not only blocked the Ang II effect but also inhibited the transmural pressure effect. Angiotensin-converting enzyme inhibition abolished the pressure-dependent FN expression and significantly diminished the effect of pressure in the presence of Ang II. The effect of renin-angiotensin system inhibitors was specific for FN, since neither bFGF nor laminin expression was affected by these agents. Taken together, the results demonstrate that (1) the effect of transmural pressure is mediated by the stimulation of a local renin-angiotensin system, resulting in a net Ang II production in the culture medium, (2) transmural pressure and Ang II act synergistically to enhance vascular FN expression, (3) AT1 receptors mediate both the effects of pressure and of exogenous Ang II, and (4) the effect of Ang II on FN expression is regulated at a pretranslational level.
Key Words: hypertension • vascular remodeling • mechanical factors • extracellular matrix • smooth muscle cells
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Introduction |
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Hemodynamic forces, including pressure and wall shear stress, are among the most important environmental factors implicated in the physiology and pathobiology of the vascular wall. Arterial hypertension is associated with structural and functional changes of the vascular wall.1 2 Modifications of the extracellular matrix, including FN, have been previously reported in vessels of hypertensive animals.3 4 5 Such changes in the composition of the extracellular matrix participate in vascular wall remodeling and could affect cell-to-cell interactions.
Vascular remodeling in hypertension might be an adaptive response to increased mechanical stresses.6 7 8 9 Pressure, per se, seems to play a direct role in remodeling of the vessel wall, since stretch is capable of enhancing protein synthesis by cultured smooth muscle cells.10 However, neuronal or humoral factors might be critical in hypertension-induced vascular hypertrophy. Several in vivo studies have suggested that Ang II might play a direct role in vascular remodeling in hypertension. Treatment of hypertensive animals with ACE inhibitors appears to be more effective in causing regression of vascular hypertrophy than other treatments that lead to an equivalent decrease in blood pressure.11 12 In rats made hypertensive by in vivo infusion of Ang II, FN biosynthesis increased in the aorta, and normalization of blood pressure by direct vasodilators did not prevent Ang II–induced FN mRNA increase, whereas treatment with an ACE inhibitor at doses that did not lower arterial blood pressure was able to markedly reduce the increase in FN mRNA.13 On the other hand, in animals made hypertensive by coarctation of the abdominal aorta between the two renal arteries, vascular hypertrophy was only seen in the upstream part of the aorta subjected to elevated blood pressure, whereas the downstream part, maintained at normal pressure, did not show hypertrophy,14 even though it is known that Ang II is dramatically increased in this model.15 Moreover, in a model of infusion of pressive and subpressive doses of Ang II, an increase in aortic FN mRNA was reported in both hypertensive and normotensive animals,16 but the Ang II–induced increase in FN mRNA was clearly more important in hypertensive than in normotensive animals in regulating vascular metabolism. Therefore, a critical link between mechanical factors and Ang II might exist, as has been shown recently in cardiac myocytes.17 A single preliminary qualitative observation in our own laboratory suggested that such a relationship could also be present in blood vessels.18 To establish whether or not such a link was indeed demonstrable in a definitive series of quantitative observations and to explore the mechanisms underlying such a relationship, we studied vascular FN expression in an organ culture system in which rabbit thoracic aortas were perfused at a constant flow and subjected to varying levels of transmural pressure for 3 days. This organ culture model has been previously characterized in regard to cellular viability.19
We report in the present study a highly significant synergy between transmural pressure and Ang II in enhancing c-FN expression in vascular tissue. In situ hybridization revealed that this effect is pretranslationally regulated. Finally, we provide evidence that the increase in c-FN caused by elevated transmural pressure is due to the stimulation of a local RAS.
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Materials and Methods |
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Artery Preparation
Pressurized segments of rabbit aortas were excised and cultured using a procedure described in detail previously.19 Briefly, male New Zealand White rabbits (2 to 2.5 kg) anesthetized with sodium pentobarbital (30 mg·kg-1 IV) were intubated and mechanically ventilated. The entire length of the descending thoracic aorta was exposed under sterile conditions, and the intercostal arteries were cauterized. The proximal and distal portions of the thoracic aorta were cannulated and excised while held at in vivo length. Endothelial damage was minimized during the whole in situ preparation by maintaining physiological pressure within the vessel and avoiding shortening of the arterial segment. We have shown that the procedure used for artery excision preserves the structural and metabolic integrity of the vessel wall.20 21
Excised aortic segments were immersed into an organ culture bath placed in an incubator and filled with DMEM (GIBCO) containing antibiotics (100 IU·L-1 penicillin, 100 mg·L-1 streptomycin, and 10 µg·L-1 amphotericin B), supplemented with 1.5% albumin (fraction V, tested for cell culture, Sigma Chemical Co). In order to vary the transmural pressure and the flow independently, each arterial segment was connected to a perfusion circuit consisting of a custom-designed and -constructed three-port glass reservoir, a peristaltic pump (Masterflex 60648, Cole-Palmer Instrument Co), and a pressure chamber.19 The upper port of the glass reservoir was connected to the pressure chamber, which permitted the application of a controlled hydrostatic pressure to the intraluminal compartment, and the two lateral ports of the glass reservoir were used for the input and the output of the circulating intraluminal medium, which was the same as the extraluminal medium described above. The peristaltic pump was connected between the distal end of the aortic segment and the upper lateral port of the glass reservoir in order to maintain a constant flow.
Vessels were kept for 3 days at 37°C in an incubator under humidified air gassed with 5% CO2.
Experimental Protocols
In order to investigate the effect of transmural pressure, arteries were pressurized at 80 mm Hg (n=9) or 150 mm Hg (n=9) and perfused at a constant flow of 40 mL/min. In an additional series of experiments, vessels were cultured under relaxed conditions (zero flow and zero transmural pressure) (n=4).
To study the effect of exogenous Ang II, vessels maintained at 0 mm Hg (n=4), 80 mm Hg (n=9), or 150 mm Hg (n=9) were cultured in medium containing extraluminal and intraluminal Ang II (Sigma) at a concentration of 1 µmol/L.
A dose-response effect of exogenous Ang II was evaluated in arterial segments maintained at both 80 mm Hg (n=4) and 150 mm Hg (n=4) at each Ang II concentration. Ang II was added to the intraluminal and extraluminal solutions to achieve final concentrations of 0.01, 0.1, or 1 µmol/L.
To verify the specificity of the effect of Ang II, saralasin ([Sar1,Thr8]Ang II, 3 µmol/L, Sigma Chemical Co), a competitive inhibitor of Ang II, was added to culture medium containing Ang II (1 µmol/L) in experiments conducted at 80 mm Hg (n=4) and 150 mm Hg (n=4).
To assess the role of AT1 receptors of Ang II, losartan (3 µmol/L, Merck), a specific antagonist of these receptors, was used in the presence of Ang II (1 µmol/L) in experiments performed at 80 mm Hg (n=6) and 150 mm Hg (n=6).
The possible role of the tissue RAS in pressure-induced FN synthesis was explored in a series of experiments in which vessels pressurized at 150 mm Hg without addition of exogenous Ang II in culture medium were studied in the presence of losartan (3 µmol/L) (n=4) or lisinopril (10 µmol/L, Zeneca), an ACE inhibitor (n=4). In order to verify the specific effect of the ACE inhibitor on the blockade of the vascular RAS, vessels pressurized at 150 mm Hg were incubated in the presence of both exogenous Ang II (1 µmol/L) and lisinopril (10 µmol/L) (n=4).
Immunofluorescence Method
At the end of the 3-day culture, arterial segments were processed for immunostaining analysis. Rings were mounted in embedding medium (Miles Inc), frozen in isopentane (Rhone-Poulenc Ltd), cooled with liquid nitrogen, and stored at -80°C.
Serial frozen transverse cross sections (5 µm) were incubated for either 1 hour at room temperature or overnight at 4°C in specific antibodies. Sections were then postincubated for 30 minutes at 37°C in either fluorescein- or biotin-conjugated anti–species-specific antibodies (Amersham International PLC), which were amplified by the streptavidin–Texas red system (Amersham International PLC). Autofluorescence of elastic lamina was avoided by using a specific filter for Texas red detection. Fluorescence staining was visualized using a Leitz microscope equipped with an epifluorescence system (Leica).
For detection of c-FN expression, mouse anti-human c-FN monoclonal antibody (Sigma) was used at the dilution of 1:200. These antibodies have been shown to recognize only cellular and not plasma FN.22
For detection of bFGF and laminin expression, respectively, rabbit anti-bovine bFGF polyclonal antibody (Sigma) and rabbit anti-rat laminin polyclonal antibody (Chemicon International Inc) were used at a dilution of 1:100.
The specificity of the antibodies used was verified in control studies performed using nonimmune rabbit or mouse serum (anti–species-specific antibodies) instead of specific antibodies. Another series of experiments was performed using only the first antibodies (anti-protein antibodies) to control the absence of self-fluorescence.
Quantitative Analysis
Quantitative analyses of thoracic aorta labelings with antibodies against c-FN, b-FGF, and laminin were performed using a video imaging microscopy technique. Video images from a low-light-level camera (C-2400, Hamamatsu) were transmitted to a microcomputer (Macintosh IIfx) equipped with an image analyzer program (Optilab, Graphtek). This software allowed us to store several images from the same cross section at the same time in digitally calibrated formats. All slides labeled on the same day were analyzed under identical microscope lighting and camera settings. Each slide was given a coded number to allow unbiased blind analysis, and data were stored on 44-megabyte disk cartridges. The image analyzer software automatically divided the slide image into pixels, and a value of 100% was designated to represent total aortic surface area. The percentage of pixels with positive labeling was then evaluated by using a threshold method, ie, by selecting pixels whose intensity level was greater than a predetermined threshold value (background). All quantitative analyses were performed blinded, and all images were quantified using the same settings for analysis. For each aortic fragment, a minimum of four sections were quantified to give one mean value. Data represent the mean of the independent experiments performed under each experimental condition. Quantitative analysis was expressed as percent total aortic surface occupied by c-FN, bFGF, or laminin.
Reproducibility of the method was assessed by analysis of selected slides performed by at least two operators who obtained similar values.
In Situ Hybridization Analysis
The vector used in this study was pSPT19 (Boehringer Mannheim). Subcloning and in vitro transcriptions have been described elsewhere.23 The SnaBI–Sty I cDNA fragment encoding the signal peptide, the propeptide, and the first FN-I segment excised from a full-length human FN cDNA expression vector was subcloned in pSPT19. Single-strand antisense RNA probes were synthesized in the presence of [
-35S]UTP (Amersham International PLC) and SP6 RNA polymerases from a commercially available transcription kit (Boehringer Mannheim), as previously described.23 The RNA probes were separated from the free ribonucleotide triphosphates by repeated ethanol precipitation and used without prior alkaline treatment. The probes were diluted to a final concentration of 
60 000 cpm/µL in 50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl (pH 8), 5 mmol/L EDTA, 10 mmol/L sodium phosphate (pH 8), 10% dextran sulfate, 1x Denhardt's solution, 0.5 mg/mL yeast RNA, and 20 mmol/L dithiothreitol.
Aortic cryosections (5 µm thick) were transferred to chrome-alum-gelatin–coated slides, air-dried a few minutes at room temperature, then fixed in 4% paraformaldehyde for 5 minutes, washed in PBS (twice for 5 minutes), dehydrated in ethanol, and stored at -70°C with dessicant until used for in situ hybridization.
Hybridization procedures were those described by Wilkinson et al.24 Approximately 7 µL of hybridization mixture (60 000 cpm/µL) was applied onto each section, and the slides were incubated at 50°C overnight. After washing at 50°C in 5x SSC and 10 mmol/L dithiothreitol, the sections were subjected to stringent washing at 65°C in 50% formamide, 2x SSC, and 10 mmol/L dithiothreitol. They were then washed in Tris-EDTA buffer before treatment with 20 µg/mL RNase A for 30 minutes at 37°C. After washes in 2x SSC and 0.1x SSC at room temperature for 15 minutes, the sections were dehydrated, dipped in Kodak NTB2 nuclear track emulsion (Eastman Kodak), and exposed for 12 days in light-tight boxes with dessicant at 4°C. Sections were developed in Kodak D19, mounted, and analyzed using both light- and dark-field optics of a Leitz Dialux microscope.
The homology between rabbit and human was better than 80%, and the specificity of hybridization has been previously assessed.25 However, a sense RNA was synthesized from the FN-I plasmid, as described above using T7 polymerase, and used as a negative control probe generating background signal whatever the experimental conditions (not shown).
Ang II RIA
Culture media of experiments conducted at 0, 80 and 150 mm Hg were kept after 3 days of culture and stored at -30°C until Ang II RIA could be performed.
All samples were purified with an Amprep minicolumn containing phenylsilyl-silica (Amersham International PLC) as described previously.26 Cold culture medium was rapidly passed through the column, followed by a wash with 2 mL of distilled water. Adsorbed angiotensins were then eluted with 1 mL of methanol. Thereafter, the methanol was evaporated, and RIAs were performed on the extraction residues according to the instructions of the manufacturer (E.R.I.A. Diagnostics Pasteur).
Statistical Analysis
Results are expressed as mean±SEM. A two-way ANOVA was constructed with data of c-FN, bFGF, or laminin quantification to test the effect of pressure and Ang II. Comparisons were performed using Bonferroni's test. Differences were considered significant at P<.05.
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Results |
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Effect of Transmural Pressure
In order to investigate the effect of the transmural pressure on c-FN expression in the arterial wall, perfused aortic segments were maintained in culture for 3 days under different transmural pressure levels. As shown in Fig 1
, c-FN expression was increased at 150 mm Hg compared with 80 mm Hg or relaxed conditions (zero transmural pressure and zero flow). At 150 mm Hg, FN was expressed in the internal third of the media and occupied 17.4±1.8% of the aortic wall (Figs 1 and 2), whereas the protein was only observed on the luminal side of the vessel wall at 0 or 80 mm Hg and occupied 6.1±0.1% or 7.9±0.7% of the aortic wall, respectively.
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Effect of Exogenous Ang II
Ang II was determined by RIA in the culture medium supplemented with exogenous Ang II, used at a concentration of 1 µmol/L after 1, 2, and 3 days of incubation at 37°C. The levels of Ang II immunoreactivity remained unchanged during the 3-day culture period.
In the presence of Ang II (1 µmol/L), FN expression was increased in pressurized vessels, whereas no changes in c-FN were detected in relaxed vessels (no pressure and no flow) (Fig 1
). At 80 mm Hg, c-FN was detected in the inner third of the media and occupied 18.9±1.1% of the aortic wall (Figs 1 and 2). At high pressure, c-FN expression was markedly increased, being detected in the whole media (Fig 1) and occupying 56.6±3.6% of the aortic wall (Fig 2).
The effect of Ang II on c-FN expression was dose dependent at both low and high pressures (Fig 3). No modification of c-FN detection was observed when Ang II was used at 0.01 µmol/L. At low pressure, c-FN expression became detectable in the inner third of the media (not shown) when Ang II was used at both 0.1 and 1 µmol/L and occupied 17.3±0.1% and 18.9±1.1% of the aortic wall, respectively (Fig 3). In vessels pressurized at 150 mm Hg, the expression of FN was increased throughout the media and occupied 46.8±0.6% and 56.6±3.6% of the aortic wall in the presence of Ang II used at 0.1 and 1 µmol/L, respectively.
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To assess the possibility that increased FN mRNA synthesis was regulated at a pretranslational level, in situ hybridization analysis was performed. When the FN-I probe was hybridized to aortic sections cultured for 3 days, at 80 mm Hg, only endothelial cells were positive, whereas the signal increased in the inner part of the media at 150 mm Hg (Fig 4A and 4C). When Ang II was added to the culture medium for 3 days, FN mRNAs were detected in the inner media at 80 mm Hg and in the whole media at 150 mm Hg (Fig 4B and 4D). Therefore, a comparison of Figs 1 and 4 shows that the distributions of FN mRNA and protein throughout the media are superimposable, indicating that the effects of transmural pressure and Ang II regulated FN expression at a transcriptional level and that the protein is secreted locally.
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Evidence for a Vascular RAS
In order to verify the specificity of the Ang II effect on FN expression, arterial segments were incubated at 150 mm Hg for 3 days in the presence of Ang II (1 µmol/L) and saralasin (3 µmol/L), a competitive inhibitor of Ang II. Surprisingly, the addition of saralasin totally inhibited the Ang II–induced expression of c-FN, which was no longer detected in the media and persisted only at the luminal side of the vessel wall, occupying 7.5±0.6% of the aortic wall. Therefore, saralasin inhibited not only FN expression due to exogenous Ang II but also FN expression induced by increased pressure, suggesting that the pressure-induced increase in FN synthesis was associated with the activation of an RAS in the arterial wall.
The possible role of AT1 receptors in mediating the effect of exogenous Ang II was then examined. Arteries were incubated in the presence of Ang II (1 µmol/L) and losartan (3 µmol/L). The addition of this AT1 receptor antagonist in the culture medium containing Ang II markedly decreased FN expression, which remained detectable only on the luminal side of the vessel wall (8.5±0.6% of the aortic wall), showing that AT1 receptors mediated the effect of Ang II on FN expression and further supporting the involvement of pressure-stimulated vascular RAS in pressure-induced FN expression (Figs 5A and 6). Interestingly, when arterial segments were cultured at 150 mm Hg in medium containing Ang II (1 µmol/L) and an ACE inhibitor (lisinopril, 10 µmol/L), FN expression was significantly reduced (threefold, P<.001 versus Ang II) but not totally inhibited, as seen with saralasin or losartan. In the presence of both exogenous Ang II and lisinopril, c-FN expression was observed in the inner media (16.6±0.4% of the aortic wall) (Figs 5B and 6), as it was observed at 80 mm Hg in the presence of exogenous Ang II.
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P<.01, lisinopril+Ang II vs losartan+Ang II.
To confirm that the vascular RAS was stimulated by transmural pressure, arterial segments pressurized at 150 mm Hg were cultured in the presence of losartan (3 µmol/L) or lisinopril (10 µmol/L) for 3 days in medium without exogenous Ang II. In these culture conditions, both losartan and lisinopril inhibited the pressure-induced rise in FN expression in the media, with the protein being detected only in the subendothelium (Fig 5C and 5D) and occupying 9.3±0.5% and 6.03±0.6% of the aortic wall, respectively.
In order to ensure that the effect of lisinopril and losartan on c-FN expression was indeed specific, we evaluated the effect of these agents on the expression of bFGF and of another extracellular matrix protein, laminin. bFGF appeared to be expressed by both endothelial and smooth muscle cells (Fig 7), whereas laminin expression was observed at the level of basal lamina and around medial smooth muscle cells (Fig 8). Immunostaining was similar in arteries freshly removed from animals and in vessels cultured for 3 days at 0, 80, or 150 mm Hg. The addition of Ang II, saralasin, losartan, or lisinopril modified neither bFGF nor laminin expression in the arterial wall (Table).
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To further support the hypothesis of a local RAS stimulated by transmural pressure, Ang II was assayed in the culture medium of relaxed vessels and of perfused vessels pressurized at 80 and 150 mm Hg for 3 days (Fig 9). The amount of Ang II detected in the culture medium of vessels cultured at 0 and 80 mm Hg was 5.7±1.1 pg/mg wet tissue (n=5) and 10.9±2.5 pg/mg wet tissue (n=5), respectively, and markedly rose to 67.1±9.9 pg/mg wet tissue (n=7) in the culture medium of vessels maintained at 150 mm Hg (P<.001).
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Discussion |
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It is well known that in arterial hypertension, medial hypertrophy of large arteries develops as a result of increased smooth muscle cell mass and extracellular matrix content.11 27 However, it remains to be determined whether mechanical load is the primary mediator of the vascular trophic response and, if so, how it regulates this process. Several studies in vivo suggest that Ang II may be a critical factor in mediating vascular hypertrophy.28 29 30 The results of the present study, which used a novel organ culture model of arteries previously characterized regarding viability19 and which permitted us to separately control the mechanical and humoral environments of the vessel wall, demonstrate that (1) pressure and Ang II act synergistically to stimulate c-FN expression at a pretranslational level, (2) the effect of Ang II is mediated through AT1 receptors, and (3) the effector mechanism of the pressure-induced c-FN overexpression involves the local vascular RAS, as evidenced by the Ang II production by vessels cultured at high pressure.
Our finding that pressure, per se, induced an increase in FN mRNA and protein expression in cultured vessels confirms and extends our previous results.19 It is in agreement with earlier studies conducted in in vitro cell culture models to evaluate the effect of stretch. In fact, Leung and colleagues31 32 have demonstrated that cyclic mechanical stimulation of arterial smooth muscle cells induced an increase in both type I and type III collagen. Recently, stretch-induced myofiber hypertrophy has been reported in cultured avian smooth muscle cells, associated with short-term increase in insulin growth factor-1 secretion.33 Similar results have been obtained in neonatal cardiac myocytes, in which mechanical stretch caused hypertrophy.17 34 35 Several in vivo studies have shown that there is a close correlation between blood pressure levels and an increased content in extracellular matrix proteins.3 27 However, in vivo studies have also revealed that vascular hypertrophy may not be simply a response to increased blood pressure but that other humoral factors, such as Ang II, may be important in vascular remodeling.28 In vivo Ang II infusion using an osmotic minipump has been shown to induce an increase in rat aortic FN mRNA3 13 16 and to cause cardiac hypertrophy accompanied by fibrosis and FN accumulation in ventricles.36 In vitro experiments have also provided evidence that Ang II might be a mediator of vascular hypertrophy. Indeed, Ang II promotes hypertrophy in rat aortic cells in culture29 37 38 and increases protein synthesis in intact rat aortic rings.39 In agreement with these studies, we found that aortic c-FN mRNA and protein content were markedly increased in 3-day cultured pressurized vessels in the presence of Ang II. We further demonstrated that this effect was mediated through AT1 receptors, since incubation in the presence of losartan prevented the effect of exogenous Ang II. Interestingly, Kim et al40 have recently reported, in a model of intimal hyperplasia induced by balloon injury, that increased intimal FN expression was prevented by AT1 receptor blockade. The effect of Ang II via the AT1 receptor might be direct or indirect (by induction of TGF-ß). Kagami et al41 found that Ang II stimulation of extracellular matrix protein synthesis in mesangial cells was mediated by TGF-ß. However, in the study by Kim et al, Ang II–induced expression of FN and TGF-ß in the intima of injured arteries was clearly dissociated, since AT1 receptor blockade prevented FN accumulation but did not affect TGF-ß overexpression.
A major finding of the present study is the demonstration of a highly significant synergy between transmural pressure and exogenous Ang II on c-FN mRNA levels and protein expression (Figs 2, 3, and 4). We have now established quantitatively the existence of a link between the effects of transmural pressure and Ang II in blood vessels, which extends our preliminary qualitative observation.18 Recently, Noda et al42 reported that cyclic stretch and Ang II synergistically stimulated cultured rat aortic smooth muscle cells to induce a marked increase in the expression of parathyroid hormone–related peptide mRNA and protein. Such a link between mechanical overload and Ang II in causing a trophic response is also supported by recent in vivo studies. Kim et al16 showed that the increase in aortic FN mRNA levels was higher in animals receiving a hypertensive dose of Ang II than in animals treated with subpressive doses. We have previously found in an in vivo model of aortic coarctation between the two renal arteries, known to be associated with increased plasma renin activity,15 that medial hypertrophy occurred only in the hypertensive part of the aorta, upstream from the abdominal coarctation.14 Furthermore, treatment of hypertensive animals with ACE inhibitors has been reported to be more effective in reducing vascular hypertrophy than other treatments leading to a similar decrease in blood pressure.12
Our findings demonstrate for the first time, to the best of our knowledge, that the effector mechanism of pressure-induced trophic response involves the RAS in the vascular wall. Several lines of evidence have suggested that circulating RAS and local RAS are involved in the regulation of vascular remodeling, especially in intimal hyperplasia following endothelial denudation.40 43 44 45 The existence of RAS in the endothelial and smooth muscle cells has been shown using molecular biology and biochemical techniques.46 47 48 In the present work, when vessels were cultured at high pressure in the presence of an ACE inhibitor or an AT1 receptor antagonist in the absence of exogenous Ang II, we observed that the pressure-induced increase in c-FN expression was totally inhibited. Interestingly, in the presence of exogenous Ang II, losartan was able to totally prevent c-FN expression, whereas lisinopril markedly reduced, but did not totally inhibit, c-FN expression. The effect of losartan and lisinopril appeared to be specific for c-FN, since bFGF and laminin expression was not modified by these agents. Our results strongly support the hypothesis that transmural pressure stimulated the endogenous production of Ang II, which was prevented by ACE inhibitors and blocked by AT1 receptor antagonism. The present findings extend the results recently reported by Himeno et al,13 who showed that ACE inhibition was able to prevent in vivo the increase in aortic FN in hypertensive rats infused with Ang II without any significant effect on the blood pressure. The effect of ACE inhibition on FN expression might involve the bradykinin pathway, as suggested by Himeno et al, in an in vivo model of Ang II infusion. However, this was unlikely in the present study since ACE inhibition or AT1 receptor antagonism decreased pressure-induced FN expression to the same extent. Therefore, our results demonstrate that a local tissue RAS was activated by the increase of transmural pressure. In agreement with this finding, recent studies have demonstrated that mechanical stretch stimulates a tissue RAS in cultured cardiac myocytes.17 49 50 For example, Miyata et al49 demonstrated that both the ACE inhibitor captopril and the AT1 receptor antagonist losartan partially inhibited increased protein synthesis induced by stretching cardiac myocytes.
Strong additional evidence that a local RAS was indeed stimulated by transmural pressure comes from the finding of a marked increase (6- to 12-fold) in Ang II immunoreactivity in the culture medium of vessels pressurized at high level compared with relaxed or low-pressure cultured vessels. Therefore, we can speculate that the effect of transmural pressure was mediated locally via the proteolytic cleavage of angiotensin I into Ang II by ACE. Whether transmural pressure increased angiotensin I production and/or ACE levels in the vascular wall remains to be elucidated. It has been recently reported that smooth muscle cells are capable of expressing ACE in response to dexamethasone activation.51 However, in our organ culture model, we previously found that ACE was expressed in endothelial cells but was undetectable in medial cells, regardless of the pressure level.19 Therefore, it is unlikely that the effect of transmural pressure was mediated by enhanced ACE expression in the media of pressurized vessels. Further investigations are required to identify which parts of the RAS (angiotensinogen, renin, and/or AT1 receptors) are activated by high transmural pressure. Interestingly, Malhotra et al52 have recently reported that stretch in neonatal cardiac myocytes in culture upregulates the level of expression of the renin and angiotensinogen genes, as well as the AT1 receptor gene. If this is also the case in vascular smooth muscle cells, this latter effect could account for the observed synergy between exogenous Ang II and transmural pressure.
In conclusion, our results suggest that mechanical overload in the vascular wall can cause an autocrine/paracrine release of Ang II from vascular cells via a local RAS, which in turn activates the intracellular signal transduction pathways leading to c-FN expression. Mechanical and humoral factors have therefore associative and synergistic roles to play in the biosynthesis of extracellular matrix proteins in the arterial wall.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by an INSERM/MSD grant, and Nathalie Bardy holds a Glaxo Laboratories graduate studentship. We thank Dr Stephanie Lehoux for her help with the angiotensin II RIA.
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Footnotes |
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Previously presented in part in abstract form at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994 (Circulation. 1994;90[pt 2]:I-2771).
Received August 8, 1995; accepted March 19, 1996.
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References |
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