Elevated Perfusion Pressure Upregulates Endothelin-1 and Endothelin B Receptor Expression in the Rabbit Carotid Artery
Abstract—To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ETA-R) expression were pressure-insensitive, whereas that of the ETB-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ETB-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins β and δ were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ETB-R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling.
Pressure overload of the vessel wall has been implicated in hypertension-induced arterial remodeling, ultimately leading to manifest hypertension.1 In resistance vessels, remodeling is mainly associated with medial hyperplasia, whereas in conduit arteries it is characterized by medial hypertrophy and enhanced matrix protein synthesis. Among the various mediators implicated in these adaptive processes, endothelin-1 (ET-1) may play a pivotal role.2
Predominantly formed by endothelial cells (EC), this 21–amino acid peptide is not only a powerful vasoconstrictor but also a potent mitogen for vascular smooth muscle cells (SMC). It is derived from a 212–amino acid precursor, prepro-ET-1 (ppET-1), which is sequentially processed to big ET-1 and ET-1 by a furinlike protease and an endothelin-converting enzyme (ECE-1).2 3 ET-1 exerts its biological effects mainly through activation of 2 types of G-protein–coupled receptors, ETA-R and ETB-R.4 Although arterial SMC express both types of receptors, ETA-R activation primarily modulates their tone and growth, whereas the functional significance of the ETB-R in these cells remains to be properly defined.2 3 4
Cyclic strain has been reported to enhance ET-1 peptide synthesis and ppET-1 mRNA expression in cultured EC.5 6 In the vessel wall in situ, EC are not normally exposed to this hemodynamic force because the bulk of the physiological increase in transmural pressure is transformed into a circumferential tensile strain that almost exclusively affects the SMC.7 However, in situations in which the pressure-induced distention of the vessel wall is more pronounced and/or chronically elevated, as in arterial hypertension, EC may also be deformed to a significant extent. To delineate as to whether a supraphysiological increase in (blood) pressure affects ppET-1 gene expression also in the vessel wall in situ, we have developed an experimental model in which isolated segments of the rabbit carotid artery are perfused at different levels of intraluminal pressure.
Male New Zealand White rabbits (2.1±0.1 kg body wt, n=53) were anesthetized with pentobarbitone sodium (60 mg/kg IV; Sigma-Aldrich) and exsanguinated. The left and right common carotid arteries were dissected, cleansed of adventitial adipose and connective tissue, and cut in half. The 4 segments were mounted into a specially designed 4-position perfusion chamber, where they were stretched back to their in situ length (20.6±0.3 mm, n=152) with the aid of moveable cannulas. Their diameter was continuously monitored by videomicroscopy (Visitron Instruments). The lumen of the segments and the surrounding tissue baths were individually perfused (lumen: 1 mL/min; bath: 0.5 mL/min) with warmed (37°C), oxygenated (lumen: 75% N2, 20% O2, 5% CO2; Po2=140 mm Hg, Pco2=15 to 20 mm Hg, pH 7.4; bath: 95% CO2, 5% O2; Po2 >300 mm Hg, Pco2=18 to 38 mm Hg, pH 7.4) Tyrode solution of the following composition (in mmol/L): Na+ 144.3, K+ 4.0, Cl− 138.6, Ca2+ 1.7, Mg2+ 1.0, HPO42− 0.4, HCO3− 19.9, d-glucose 10.0. An IPC roller pump (Ismatec) was used for perfusion, pumping at a frequency of 1.33 Hz. After a 30-minute equilibration period, the segments were perfused at 2±1, 90±3, or 160±4 mm Hg for 3 to 12 hours with the aid of an adjustable afterload device system (Hugo Sachs Elektronik). Perfusion pressure without the afterload device attached was 2 mm Hg, as determined with a pressure transducer (Gould) connected to a side arm of the outflow tubing.
In experiments with drug or vehicle pretreatment, the segments were perfused with defined drug doses at a reduced flow rate (0.5 mL/min) for up to 1 hour directly after the resting phase. In another series of experiments, the segments were mechanically denuded by gentle abrasion with a roughened stainless steel cannula (2.0 mm OD). At the end of the perfusion, the segments were snap-frozen in liquid nitrogen and stored at −80°C.
Superfusion bioassay experiments with 3- to 4-mm-wide ring segments of the rabbit carotid artery were performed essentially as described8 except that passive tension was adjusted to 2 g for experiments with ET-1 or sarafotoxin 6c (Alexis).
Staining of Nuclear DNA With Hoechst 33342
After perfusion of the segments with Tyrode solution for 6 hours at 2 or 160 mm Hg, they were incubated in Waymouth/Ham F12 medium 1:1 (vol/vol) containing 20% fetal bovine serum (Gibco BRL via Life Technologies) and standard additives for 18 hours. Thereafter, the segments were fixed with 5% (vol/vol) formaldehyde in 145 mmol/L sodium chloride, 10 mmol/L HEPES for 12 hours at 4°C followed by the addition of the DNA-binding fluorescent dye Hoechst 333429 (10 μg/mL; Calbiochem) for 72 hours. The fixed dye-treated segments were then embedded in paraffin blocks, of which 3-μm-thick sections were cut that were examined in a blinded fashion by 2 independent investigators with the aid of a videoimaging system (Visitron Instruments) and the MetaMorph 3.51 software package (Universal Imaging). The media of 2 sections from each segment were evaluated at 800-fold magnification, and only those nuclei were judged apoptotic that showed definite chromatin condensation or fragmentation. On average, a total of 682 nuclei were counted per section.
Reverse Transcription–Polymerase Chain Reaction Analysis
The frozen segments were minced under liquid nitrogen with the aid of a mortar and pestle. Total RNA was isolated with the Qiagen RNeasy kit (Qiagen) followed by cDNA synthesis with a maximum of 3 μg RNA and 200 U Superscript II reverse transcriptase (Gibco, Life Technologies) in a total volume of 20 μL, according to the manufacturer’s instructions. For normalization of cDNA load, 5 μL (≈75 ng cDNA) of the resulting cDNA solution and 20 pmol of each primer (Gibco) were used for elongation factor 1 (EF-1) polymerase chain reaction (PCR) with 1 U Taq DNA polymerase (Gibco) in a total volume of 50 μL according to the manufacturer’s instructions. PCR products were electrophoretically separated on 1.5% agarose gels containing 0.1% ethidium bromide, and the intensity of the detected bands was determined densitometrically to adjust cDNA volumes for subsequent PCR analyses.10 All PCR reactions were performed individually for each primer pair in a Hybaid OmnE thermocycler (AWG) that was programmed as follows: A unique 2-minute period for complete denaturation at 94°C in the beginning followed by a primer-specific number of cycles of 30-second denaturation at 94°C, 30-second annealing at 53° to 60°C (see below), and 1-minute primer extension at 72°C, with an additional 5 minutes at 72°C for final extension in the end. To ensure that the PCR amplification was indeed semiquantitative, several PCR runs were performed on each set of samples to establish the adequate numbers of cycles that usually corresponded to those indicated. Individual PCR conditions were as follows: Prepro ET-1: product size 517 bp, 33 cycles, annealing temperature 53°C, (forward)5′-GCTCCTGCTCCTCGCTGAT-3′, (reverse) 5′-AAGAGCGAGTGAGAGAGTGA-3′ (corresponding to nucleotide sequences 270 to 289 and 786 to 767 of the rabbit prepro ET-1 gene; GenBank accession No. X59931); ECE-1: 309 bp, 29 cycles, 58°C, 5′-GCACCCTCAAGTGGATGGAC-3′, 5′-CCGGAAACACGATCT-CGTTC-3′(1425–1444, 1734–1715, human ECE-1, Z35307); ETA-R: 334 bp, 30 cycles, 58°C, 5′-CAGGGCATCCTTTTGGCTGGC ACTG-3′, 5′-GCGCGTTGGGGCCATTCCTCATAC-3′ (24–48, 358–335, human ETA-R, E07649); ETB-R: 446 bp, 33 cycles, 53°C, 5′-GTGCT-GGGGATCATCGGGAAC-3′, 5′-TGAACGGGATGAAGCAAGCAG-3′(570–590, 1015–995, human ETB-R, E07650) or 304 bp, 33 cycles, 53°C, 5′-TGTTGGCTTCCCCTTCATCT-3′, 5′-TGGAGCGGAAGTTGTCGTAT-3′(1203–1219, 1506–1487, rat ETB)-R, X57764); EF-1: 951 bp, 22 cycles, 58°C, 5′-TGCCGTCCTGATTGTTGCTGC-3′, 5′-ATCACGGACAGCGAAACGACC-3′ (346–366, 1297–1276, rabbit EF-1, X62245); CD31 (PECAM-1): 362 bp, 30 cycles, 56°C, 5′-AACTTCACCATCCAGAAGG-3′, 5′-CACTGGTATTCCACGTCTT-3′(1207–1225, 1568–1550, human CD31, M28526); iNOS (inducible nitric oxide synthase): 576 bp, 30 cycles, 60°C, 5′-CAG CTACTGGGTC-AAAGACAAGAGG- 3′, 5′-TGCTGAGAGTCATGGAGCCG-3′(543–567, 1118–1099, rabbit iNOS, U85094).
To verify the identity of the amplification products with the designed primer pairs, we cloned and sequenced the ECE-1, ETA-R, ETB-R, EF-1, CD31, and iNOS PCR products and found an 87% to 92% homology with the published sequences of the corresponding human and rat genes. The homology of the ppET-1 PCR product with the corresponding rabbit gene was 100%.
Measurements of ET-1 Tissue Concentrations
ET-1 was extracted from the weighted segments according to the methods of Hisaki et al11 and Moreau et al,12 with minor modifications. Overall recovery of ET-1 was 69.4%; interassay and intra-assay variabilities were 15.3% and 11.7%, respectively. The concentration of ET-1 in the tissue extracts was determined with the use of a commercially available ELISA kit (Amersham) according to the manufacturer’s instructions.
Electrophoretic Mobility Shift Analysis
Preparation of nuclear extracts from the minced segments, incubations with 10 μg nuclear protein and 10 to 20 000 cpm of the double-stranded consensus oligodeoxynucleotides (Santa Cruz Biotechnology), nondenaturing polyacrylamide gel (4%) electrophoresis, autoradiography, and supershift analyses were performed essentially as described.10 The single-strand sequences of the oligodeoxynucleotides were as follows (core sequences are italicized): Activator protein-1 (AP-1), 5′-CGCTTGATGACTCA-GCCGGAA-3′; CCAAT enhancer binding protein (C/EBP), 5′-TGCAGATTGCGCAATCTGCA-3′; nuclear factor-κB (NF-κB), 5′-AGTTGAGGGGACTTTCCCAGG-3′; CREB, 5′-AGAGATTGCC-TGACGTCAAGAAGCTAG-3′; GATA, 5′-CACTTGATAACAGA-AAGTGATAACTCT-3′; egr-1, 5′-GGATCCAGCGGGGGCGA-GCGGGGGCGA-3′; ets-1, 5′-GATCTCGAGCAGGAAGTTCGA-3′; Sp1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′.
Unless indicated otherwise, all data in the figures and text are expressed as mean±SEM of n observations. Statistical evaluation was performed by Student’s t test for unpaired data with the Instat for Windows statistics software package (GraphPad Software Inc), and a value of P<0.05 was considered statistically significant.
Raising the intraluminal pressure from 2 to 220 mm Hg resulted in a progressive increase in (outer) diameter of the perfused segments that reached a maximum at 160 mm Hg (Figure 1a⇓), with an average distention of 208±6% (n=76). Of note was that the segments were already distended close to the maximum at the physiological pressure of 90 mm Hg. Circumferential (tensile) strain (ε), defined as pressure-induced change in diameter (d−d0) as a fraction of the diameter at zero pressure (d0),13 14 was also maximal at 160 mm Hg and close to the maximum at 90 mm Hg (compare with Figure 1a⇓).
The mRNA level of the housekeeping reference gene, EF-1, was not altered by an increase in perfusion pressure under any of the experimental conditions described below (compare with Figure 1b⇑). Moreover, there was no apparent loss of EC from the endothelium-intact perfused segments, even after 12 hours of exposure to 160 mm Hg, as judged by the virtually constant expression of the EC-specific marker CD31 (Figure 1c⇑) and light microscopy analysis of paraffin-embedded hematoxylin and eosin–stained tissue sections as well as transmission electron microscopy (not shown). Even though the experiments were not performed under sterile conditions, there was no microscopically visible contamination at any time point and no expression of iNOS mRNA, an extremely sensitive marker for the presence of bacterial lipopolysaccharides (not shown).
Prepro ET-1 Expression and ET-1 Synthesis
In endothelium-intact segments, a distinct basal expression of ppET-1 was detectable that decreased by 61±13% (n=4, P<0.05) after mechanical denudation. Efficient EC removal was verified by a 93% loss of CD31 mRNA abundance in the denuded segments (Figure 1d⇑). Perfusion at either 2 or 90 mm Hg for up to 9 hours had no appreciable effect on ppET-1 mRNA abundance in endothelium-intact segments (Figure 2a⇓), which, on the other hand, was markedly elevated when the segments were perfused at 160 mm Hg for 3 to 12 hours (Figures 2a⇓ and 2b⇓). As shown in Figure 2b⇓, this pressure-induced increase in ppET-1 mRNA was confined to the EC. Moreover, a comparable pressure-induced increase in ppET-1 mRNA was observed when the segments were first equilibrated at 90 mm Hg for 3 hours and then exposed to 160 mm Hg for 6 hours (Figure 2a⇓). For reasons of simplicity, therefore, all other experiments were performed by comparing segments perfused at 2 mm Hg with those perfused at 160 mm Hg. In addition to the increase in ppET-1 mRNA (917%), the intravascular concentration of ET-1 peptide was elevated to a similar extent (931%) after 6-hour perfusion at 160 mm Hg (Figure 4a⇓).
ECE-1 and ETA-R Expression
In contrast to ppET-1, there was no pressure-induced increase in ECE-1 mRNA under any of the aforementioned experimental conditions (compare with Figure 2⇑, a and b). ETA-R mRNA expression was also largely unaffected by raising the intraluminal pressure to 160 mm Hg for 3 to 12 hours (compare with Figure 3b⇓ for the lack of effect after 6 hours), although in some experiments a small increase was noted (Figure 3a⇓).
ETB-R mRNA abundance, on the other hand, was significantly increased after 3 to 12 hours of exposure to 160 mm Hg (inserts in Figures 2a⇑, 3a⇑, and 3b⇑). This pressure-induced increase in ETB-R mRNA, however, was reproducibly detectable only in the rostral part, that is, next to the bifurcation of the internal and external carotid artery (Figure 3c⇑). Both basal and pressure-induced ETB-R expression appeared to be largely confined to the SMC (Figure 3b⇑ insert). Parallel analysis of 2 completely different PCR products, designed from the human and rat ETB-R sequence, corroborated this pressure-induced rise in ETB-R mRNA abundance (341±24% and 342±55% of 2 mm Hg, respectively, n=6). Raising the intraluminal pressure further (ie, to 250 mm Hg) finally resulted in an increase in ETB-R mRNA also in the caudal segments (218% as compared with 506% of control at 160 mm Hg for the rostral segment from the same animal). The different pressure sensitivity of ETB-R expression did not appear to be due to a greater dilatability of the rostral (ε=0.93, n=39) as compared with the caudal segments (ε=1.14, n=37). Unlike ETB-R expression, there was no difference in the pressure-induced rise in ppET-1 mRNA abundance between the 2 parts of the carotid artery (Figure 3c⇑).
Because both Western blot and [125I]-ET-1 receptor binding analyses failed for technical reasons to confirm that in addition to the pressure-induced increase in ETB-R mRNA there is a corresponding increase in ETB-R protein, the superfusion bioassay technique was used. However, despite being sensitive toward ET-1 (threshold 30 pmol), no constrictor response to the specific ETB-R agonist15 16 sarafotoxin 6c, up to a dose of 1 nmol (corresponding to a final concentration of 1 μmol/L), was observed (not shown). The ET-1–induced constriction, on the other hand, was markedly reduced (to 14±3% of control at a concentration of 0.1 μmol/L ET-1, n=3, P<0.05) by the selective ETA-R antagonist4 BQ 123 (1 μmol/L).
Therefore, another assay method had to be established that finally demonstrated a pressure-dependent increase in functional ETB-R protein. Preliminary findings from this laboratory had indicated that ET-1 through activation of the ETB-R exerts a proapoptotic effect on rat aortic cultured smooth muscle cells (M. Cattaruzza, C. Dimigen, and M. Hecker, unpublished data, 1999). In the media of endothelium-intact segments of the rabbit carotid artery (rostral part), an increased number of apoptotic nuclei, characterized by chromatin condensation and fragmentation, was detected 18 hours after the segments had been exposed to a perfusion pressure of 160 mm Hg for 6 hours. Moreover, this pressure-induced increase in apoptosis was completely prevented by the ETB-R–selective antagonist4 BQ 788 but not by BQ 123 (1 μmol/L each; Figure 4b⇓). This increase in chromatin condensation was preceded by a rise in caspase-3 activity in homogenates of the segments (not shown), which in combination justify the term apoptosis.
Transcriptional Control of Pressure-Induced Gene Expression, Role of Protein Kinases
Blockade of RNA synthesis with actinomycin D (1 μmol/L) abolished the pressure-induced increase in ppET-1 and ETB-R mRNA (Figure 5a⇓) as well as the increase in intravascular ET-1 (Figure 5a⇓, insert). Basal ppET-1 and ETB-R mRNA but not ET-1 peptide levels (Figure 5a⇓, insert) were also reduced by actinomycin D.
Pretreatment of the segments with the protein kinase C (PKC) inhibitor17 Ro 31-8220 (0.1 μmol/L) or the c-Src family–specific tyrosine kinase inhibitor18 herbimycin A (0.1 μmol/L) for 1 hour had no significant effect on ECE-1 or ETA-R expression both under basal conditions and in the presence of an elevated perfusion pressure (not shown). In contrast, Ro 31-8220 significantly attenuated the ETB-R mRNA level after 6-hour perfusion both at 2 mm Hg (to 43±15% of control, n=5, P<0.05) and 160 mm Hg (Figure 5b⇑). In contrast, Ro 31-8220 did not affect basal and only marginally inhibited the pressure-dependent expression of ppET-1 (Figure 5b⇑). Herbimycin A, on the other hand, abolished the pressure-induced increase in ppET-1 mRNA but had no effect on ETB-R mRNA abundance both under basal conditions and after 6-hour exposure to 160 mm Hg (Figure 5c⇑).
Among several transcription factors that are potentially involved in the control of ppET-1 and ETB-R expression (analysis of the 5′-flanking region of the rat ppET-1[GenBank accession No. S76970] and human ETB-R gene [GenBank accession No. D13162] with the use of the Matinspector V2.2 software package19 ), only activator protein-1 (AP-1) and 2 members of the C/EBP family of transcription factors, β and δ (according to supershift analysis, not shown), revealed a prominent translocation to the nucleus when perfusion pressure was raised from 2 to 160 mm Hg for up to 2 hours (Figure 6⇓). In contrast, there was no appreciable difference in CREB, egr-1, ets-1, GATA, NF-κB, or Sp1 activity between pressurized and nonpressurized segments over this period of time (not shown). Activation of AP-1 and C/EBP was transient with a nadir at 30 to 60 minutes, and there was no difference in the pressure sensitivity of both transcription factors between rostral and caudal segments. Both basal and pressure-induced AP-1 activity was strongly inhibited by Ro 31-8220 (Figure 7a⇓), whereas the PKC inhibitor had no significant effect on the activity of C/EBP (not shown). Herbimycin A, on the other hand, had no significant effect on AP-1 activity (not shown) but completely abrogated the pressure-induced activation of C/EBP (Figure 7b⇓).
Although the chosen experimental conditions do not precisely match the situation in the rabbit carotid artery in vivo, the newly developed model allowed us to study the effect of a pathophysiological increase in intraluminal pressure on gene expression in the vessel wall in situ. With the aid of this model, we were able to substantiate earlier findings in cultured EC of an increase in ppET-1 expression6 and ET-1 release5 in response to a presumably undirected deformation of these cells. This deformation-induced gene expression did not follow the pattern of an all-or-nothing response but was clearly dependent both on the duration and intensity of the increase in perfusion pressure. Of note was that it occurred only at supra-physiological pressure levels (>90 mm Hg), whereas circumferential (tensile) strain at 90 mm Hg was nearly maximal. This suggests that in addition to circumferential strain, another hemodynamic force (ie, radial strain or the stiffening of the vessel wall when collagen takes over from elastin to balance the distending pressure)13 14 must contribute to the deformation of the cells in the vessel wall. This hypothesis is substantiated by our finding of an additional pressure-induced increase in smooth muscle ETB-R expression only in the rostral part of the rabbit carotid artery, even though the dilatability of the caudal segments was very similar. Moreover, the finding of a virtually indistinguishable pressure-dependent increase in ppET-1 expression in both parts of the carotid artery suggests that certain thresholds exist for deformation-induced gene expression in the vessel wall and that this threshold is lower for ppET-1 than for ETB-R expression. Alternatively, it may be argued that EC are either more easily deformed than SMC or deformed differently with other components of the cytoskeleton being affected. In contrast to ppET-1 and the ETB-R, neither expression of the ETA-R nor that of ECE-1 appeared to be pressure-sensitive per se.
Because the deformation-induced increase both in ppET-1 and ETB-R mRNA and ET-1 peptide content was sensitive to actinomycin D, it would appear that it is controlled at the level of mRNA synthesis. In a first attempt to elucidate the signal transduction pathway involved therein, Ro 31-8220, a potent and highly selective inhibitor of various PKC isoforms,17 was used. PKC blockade, however, did not affect the pressure-induced expression of ppET-1 in the endothelium, whereas smooth muscle ETB-R expression was completely inhibited. Pressure-induced ppET-1 but not ETB-R expression was abolished, on the other hand, by the c-Src family tyrosine kinase inhibitor18 herbimycin A. It would appear, therefore, that the pressure-dependent deformation of the carotid artery wall is sensed differently by EC and SMC, involving activation of the c-Src and protein kinase C pathways, respectively.20
Moreover, the differential inhibition by Ro 31-8220 and herbimycin A of the activation of AP-1 and C/EBP suggests that C/EBP (β and/or δ) is an essential transcription factor for the pressure-induced expression of ppET-1 in the venous EC, whereas AP-1 is involved in the pressure-dependent expression of the ETB-R in the SMC. Hitherto, a cooperative interaction of GATA-2 and AP-1 is thought to regulate transcription of the ppET-1 gene,21 whereas a functional role for C/EBP in ppET-1 gene expression has not yet been recognized. The transcriptional control of ETB-R expression has not been investigated thus far. Our data, therefore, provide the first indirect evidence for a role of AP-1 in ETB-R expression.
In addition to providing a basis for a more detailed investigation of the nuclear signaling mechanisms involved in hypertension-induced gene expression in the vessel wall in situ, the aforementioned findings raise the question as to what the functional consequences of the pressure increase in ET-1 synthesis and ETB-R expression are. With the superfusion bioassay technique, no ETB-R–mediated vasoconstriction could be detected (compare with References 15 and 1615 16 ). On the other hand, we noted a pressure-dependent ETB-R–mediated increase in apoptosis of the medial SMC, a finding that clearly points to a functional role of the ETB-R in the rabbit carotid artery other than influencing SMC tone, as does the ETA-R. In this context, it was interesting to note that arterial SMC have been reported to undergo apoptosis in experimental hypertension and that this response is aggravated by ETA-R blockade.22 Provided that the observed enhanced rate of apoptosis represents a general phenotypic change of the SMC and is transferable to the situation in human blood vessels, it may ultimately lead to the media hypertrophy that is characteristic for hypertension-induced remodeling in conduit arteries.
In summary, the aforementioned findings reinforce the notion of a hypertension-induced rise in ET-1 synthesis in the endothelium along with an increased expression of the ETB-R in the smooth muscle of large conduit arteries. These blood pressure–induced changes in gene expression may contribute to arterial remodeling in hypertension, hence promoting the manifestation of this disease.
This work was supported by the Deutsche Forschungsgemeinschaft (He 1587/5-2 and 7-1). The authors are indebted to Drs Andreas Wagner and Ekkehard Schütz (Department of Clinical Chemistry, University of Goettingen) for their help with the electrophoretic mobility shift analysis and the sequencing of the PCR amplification products, respectively.
- Received September 2, 1999.
- Revision received September 24, 1999.
- Accepted October 6, 1999.
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