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(Circulation. 1997;95:473-478.)
© 1997 American Heart Association, Inc.

Articles

Upregulation of Vascular Angiotensin II Receptor Gene Expression by Low-Density Lipoprotein in Vascular Smooth Muscle Cells

G. Nickenig, MD; A. Sachinidis, PhD; F. Michaelsen, PhD; M. Bohm, MD; S. Seewald, MD; H. Vetter, MD

the Medizinische Universitats-Poliklinik Bonn (M.B.) and the Klinik III fur Innere Medizin, Universitat Koln (M.B., G.N.).

Correspondence to Prof Dr H. Vetter, Medizinische Universitats-Poliklinik, Wilhelmstr 35-37, 53111 Bonn, Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Because LDL and the angiotensin II type 1 (AT1) receptor are thought to be involved in the pathogenesis of chronic vascular disease, we studied possible interactions between these two biological systems in cultured rat vascular smooth muscle cells.

Methods and Results Incubation of vascular smooth muscle cells with 100 µg/mL LDL profoundly increased AT1 receptor mRNA to 250% of control levels as assessed by Northern hybridization analysis. This effect is maximal 12 hours after addition of LDL to the culture medium and is sustained for up to 24 hours. The LDL-induced upregulation is dose dependent, with a maximal effect obtained with 100 µg/mL LDL. There is a correlative increase of cell surface–associated AT1 receptors as assessed by saturation radioligand binding assays. The half-life of AT1 receptor mRNA is increased substantially by LDL compared with that of cells treated only with 5,6-dichlorobenzimidazole to block transcription. Angiotensin II–induced elevation of cytosolic calcium concentration is significantly increased in vascular smooth muscle cells pretreated with LDL to 368±41 nmol/L compared with control cells pretreated with vehicle (248±33 nmol/L). Moreover, angiotensin II–induced DNA synthesis is markedly enhanced when cells are coincubated with 100 µg/mL LDL.

Conclusions These data reveal a significant upregulation of AT1 receptor gene expression by LDL in vascular smooth muscle cells through mechanisms that involve posttranscriptional mRNA stabilization. Ultimately, this AT1 receptor upregulation leads to an elevated functional response of vascular smooth muscle cells on angiotensin II stimulation.

Key Words: genes • lipoproteins • angiotensin • arteriosclerosis • hypertension

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The pathogenesis of chronic vascular diseases such as hypertension and arteriosclerosis is associated with abnormalities in VSMCs.^{1 2} These can be manifested as changes in the state of VSMC differentiation, gene expression patterns, and VSMC morphology.³ A pathophysiological role for numerous factors such as polypeptide growth factors, Ang II, and lipoproteins is becoming increasingly clear.^{1 4}

Because LDL is one of the most important risk factors for cardiovascular diseases,^{5 6} the effects of LDL on VSMCs with respect to signaling pathways, state of contraction, and mitogenesis have been subject of intense investigations. Recent studies have shown that LDL induces elevation of cytosolic calcium concentration ([Ca²⁺]_i) and the expression of the early growth response gene-1 in VSMCs.^{7 8 9} Furthermore, stimulation with LDL leads to proliferation of VSMCs and vasoconstriction of rat aortic strips.^{10 11} Despite numerous studies describing intracellular effects of LDL on VSMCs, the exact molecular events that mediate LDL-caused development and progression of cardiovascular diseases remain poorly understood.

The AT1 receptor is a G protein–coupled receptor expressed in various tissues that mediates most of the known biological effects of Ang II.^{12 13} However, VSMCs are the principal physiological effector targets for circulating Ang II. Indeed, the pressor response of Ang II results from activation of AT1 receptors expressed on VSMCs.^{12 14} In addition to its role in the control of blood pressure and fluid and electrolyte regulation,^{12 13} Ang II, along with the AT1 receptor, has been implicated in chronic vascular disease that may be due to reported growth-promoting effects of Ang II on VSMCs in vivo and in vitro. In vascular injury and cell culture models, Ang II has been reported to enhance VSMC proliferation,^{15 16} whereas ACE inhibitors and the selective AT1 receptor antagonists Dup 753 and TCV-116 have been shown to attenuate blood vessel smooth muscle cell proliferation.^{17 18 19}

Because it is currently believed that the pathogenesis of chronic vascular diseases is not dependent on one particular pathogenic factor, the cross talk of various pathophysiologically important systems has been the subject of recent studies.^{20 21 22 23} We investigated the effect of LDL on AT1 receptor gene expression in cultured VSMCs to elucidate putative interactions between lipoproteins and the renin-angiotensin system.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Angiotensin peptides, salts, DRB, and other chemicals were purchased from Sigma Chemical Co. [Methyl-³H]-thymidine, [³²P]-dCTP, Hybond N-nylon membranes, and [¹²⁵I]–Ang II were obtained from Amersham. Antibiotics, serum, and cell culture medium were purchased from GIBCO BRL. TRI reagent was obtained from the Molecular Research Center, and TCV-116 (candesartan cilexetil) was a gift from Takeda. Oligonucleotides were synthesized by use of Pharmacia chemicals with an automated DNA synthesizer (Gene Assembler Plus, Pharmacia LKB). Fura-2/AM was obtained from Calbiochem.

Cell Culture
VSMCs were isolated from rat thoracic aorta (female Wistar-Kyoto rats, 6 to 10 weeks old, Charles River Wega GmbH, Sulzfeld, Germany) by enzymatic dispersion as described previously²⁴ and cultured over several passages according to the method of Ross.²⁵ Cells were grown in a 5% CO₂ atmosphere at 37°C in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 1% nonessential amino acids (100x), and 20% FBS. Experiments were performed with cells from passages 5 through 15.

Isolation of LDL and Measurement of Lipid Peroxidation
LDL (d=1.019 to 1.063 g/mL) was isolated from the plasma of normocholesterolemic subjects (serum cholesterol <6.2 mmol/L) by ultracentrifugation according to the method of Redgrave et al.²⁶ Oxidation of LDL was prevented by addition of 10 mmol/L BHT to all LDL preparations. Quantification of LDL was performed as previously described.^{27 28} To exclude lipid peroxidation, malondialdehyde contamination was ruled out by high-performance liquid chromatography as recently described.^{29 30}

Measurement of DNA Synthesis
VSMCs were seeded onto 24-well culture plates and grown to confluence. Cells were deprived of serum for 24 hours, and subsequently Ang II, LDL, or TCV-116 was added. After 20 hours, 3 µCi/mL [³H]-thymidine was added. Experiments were terminated 4 hours after the addition of [³H]-thymidine by aspirating the medium and subjecting the cells to sequential washes with PBS containing 1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 10% trichloracetic acid, and ethanol:ether (2:1 by volume). Acid-insoluble [³H]-thymidine was extracted by adding 0.5 mL per dish of 0.5 mol/L NaOH. Then 0.05 mL of this solution was mixed with 5 mL scintillator and quantified (Beckman LS 3801). Aliquots were saved for protein determinations.²⁷

mRNA Isolation and Northern Analysis
After the indicated treatments, culture medium was aspirated, and the cells were lysed with 1 mL TRI reagent, scraped, and processed according to the manufacturer's protocol to obtain total cellular RNA. Aliquots (10 µg) were electrophoresed through 1.2% agarose/0.67% formaldehyde gels and stained with ethidium bromide to verify the quantity and quality of the RNA. After capillary transfer on Hybond N-nylon membranes in 20x SSC (3 mol/L sodium chloride, 300 mmol/L sodium citrate). The RNA was cross-linked to the membranes with a Stratalinker 1800 (Stratagene). Northern blots were prehybridized for 2 hours at 42°C in a buffer containing 50% deionized formamide, 0.5% SDS, 6x SSC, 10 mg/mL denatured salmon sperm DNA (Sigma Chemical Co), and 5x Denhardt's solution and were then hybridized for 15 hours at 42°C with a random-primed, [³²P]-dCTP–labeled, rat AT1 receptor cDNA probe in the same buffer but without Denhardt's solution. The rat AT1 receptor cDNA probe was a 824-bp fragment generated from an AT1 receptor cDNA template³¹ by polymerase chain reaction with the primer pair 5'GTCATGATCCCTACCCTCTACAGC-3' and 5'-CCGTAGAACAGAGGGTTCAGGCAG-3' and Taq polymerase.

Radioligand Binding Assays
Cells (seeded on 24-well culture plates and pretreated as indicated) were washed three times with PBS. Binding assays were performed in 25 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl₂, and 100 mmol/L NaCl in a final volume of 250 µL. Saturation binding assays were conducted with increasing amounts of [¹²⁵I]–Ang II (Amersham). Total and nonspecific binding points were measured in duplicate. Nonspecific binding was determined in the presence of 10 µmol/L TCV-116. The samples were incubated for 90 minutes at 22°C, followed by three washes with ice-cold PBS. Cells were lysed with 250 µL of 0.5 N NaOH for 30 minutes at 22°C and collected. Samples were counted in a Beckmann -counter, and protein concentrations were determined.²⁷

Measurement of Free [Ca²⁺]_i
VSMCs were cultured on round glass microscope slides (diameter, 12 mm) and at confluence were incubated with 2 µmol/L Fura-2/AM at 37°C for 30 minutes in 20 mmol/L Hepes, 16 mmol/L glucose, 130 mmol/L NaCl, 1 mmol/L MgSO₄, and 0.5 mmol/L CaCl₂. Before the measurements, cells were rinsed gently with the same buffer containing 1 mmol/L CaCl₂ instead of 0.5 mmol/L CaCl₂. The glass slides were positioned diagonally in the cuvette, and [Ca²⁺]_i was measured in an LS50 luminescence spectrofluorometer (Perkin Elmer) at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. Maximum (R_max) and minimum (R_min) fluorescence was determined by adding digitonin at a final concentration of 30 mmol/L, followed by the addition of Tris-base/EGTA (final concentration, 0.1 mmol/L per 25 mmol/L). Fluorescence was corrected for cellular autofluorescence. Fluorescent signals were calibrated according to the method of Grynkiewisz et al.³²

Statistical Analysis
Data are presented as means±SE. Statistical analysis was performed by use of the Mann-Whitney U test. The data generated in the DNA synthesis experiments were analyzed by the one-factor ANOVA test with Scheffe's procedure.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cells were grown to confluence, and serum was removed from the culture medium 24 hours before initiation of experimental treatments to obviate its effects. Fig 1 (top) illustrates autoradiographic results from Northern hybridization of a rat vascular AT1 receptor cDNA probe to 10 µg of electrophoretically separated, total cellular RNA extracted from VSMCs at the indicated time points after addition of LDL to the culture medium. The probe hybridizes to an abundant 2.2-kb transcript and a minor 3.2-kb transcript as observed previously.^{31 33} This autoradiogram reveals a time-dependent elevation of the transcript level. The AT1 receptor mRNA signal appears significantly increased 12 hours after exposure to LDL, and this increase is sustained for up to 24 hours. Fig 1 also shows hybridization of a GAPDH cDNA probe to the same Northern blot. Compared with the AT1 receptor mRNA, GAPDH mRNA appears relatively stable over the time course of the experiment. Autoradiographic data, generated from five separate experiments with different VSMC cell lines and various LDL preparations, were analyzed by laser densitometry. Fig 2A shows the LDL-induced upregulation of AT1 receptor mRNA hybridization signal relative to vehicle-treated control levels at 0 hour. A 12-hour incubation with 100 µg/mL LDL causes an upregulation of AT1 receptor mRNA levels to 253±23.4%. After 24 hours of LDL stimulation, the AT1 receptor mRNA signals are measured at 245±21.6% relative to the control level at 0 hour (100%). GAPDH mRNA expression is not significantly regulated by LDL. The LDL-induced enhancement of AT1 receptor mRNA expression is dose dependent. VSMCs were incubated for 12 hours with 0, 5, 25, 50, 100, and 200 µg/mL LDL. Fig 2B shows autoradiographic data from three separate experiments. A significant upregulation of AT1 receptor mRNA expression is detectable with 50 µg/mL LDL. The maximal effect is reached with 100 µg/mL LDL. In a set of control experiments, cells were deprived of serum for 24 hours; subsequently, RNA was isolated after 0, 4, 12, and 24 hours without further treatment. As demonstrated in Fig 2C, neither the AT1 receptor mRNA nor the GAPDH mRNA is significantly altered during the time course of the assay, which suggests that the AT1 receptor mRNA expression level remains stable for up to 24 hours after withdrawal of serum. These data demonstrate that LDL induces specifically the upregulation of AT1 receptor mRNA in VSMCs. Radioligand binding assays were performed to assess whether the increased level of AT1 receptor mRNA is coincident with an elevation of AT1 receptor protein expression. Therefore, AT1 receptor binding sites were measured after a 24-hours treatment of VSMCs with 100 µg/mL LDL in an intact cell binding assay. Linear Scatchard blots demonstrated that ¹²⁵I–Ang II bound to a single population of sites, as expected for VSMCs (data not shown). Fig 3 shows graphically the [¹²⁵I]–Ang II saturation binding to VSMCs treated with either LDL or vehicle. Binding to vehicle-treated cells reveals a K_d value of 0.61±0.1 nmol/L and a B_max value of 1.47±0.13 pmol/mg protein. Binding to LDL-treated cells shows an increase in the B_max value to 2.76±0.21 pmol/mg protein without changes in the affinity for the radioligand (K_d, 0.6±0.03 nmol/L). These binding data indicate that LDL markedly elevates AT1 receptor protein expression. We further reasoned that upregulation of AT1 receptor gene expression should consequently lead to an enhanced functional response of VSMCs on Ang II stimulation. To test this, we examined Ang II–induced elevation of intracellular calcium concentration. Fig 4 illustrates a representative time course of [Ca²⁺]_i of VSMCs pretreated for 24 hours with either 100 µg/mL LDL or vehicle. Basal [Ca²⁺]_i was measured at 50 nmol/L. After 1 minute, the cells were challenged with 100 nmol/L Ang II. Calculation of four separate experiments reveals that 100 nmol/L Ang II induces in vehicle-treated VSMCs a maximal [Ca²⁺]_i increase of 248±33 nmol/L, whereas the same dose of Ang II causes in LDL-pretreated VSMCs a maximal [Ca²⁺]_i increase of 368±41 nmol/L. These data suggest that the LDL-induced upregulation of AT1 receptor mRNA and protein leads to the expected elevated functional response of VSMCs with respect to AT1 receptor–mediated Ang II stimulation. Because abnormal growth of VSMCs is thought to be a central event in the pathogenesis of chronic vascular disease, we performed another set of experiments to investigate whether LDL stimulation of VSMCs also causes an increase of Ang II–induced DNA synthesis in VSMCs. Fig 5 shows that a 24-hour stimulation of VSMCs with either 100 µg/mL LDL (166±4%) or 100 nmol/L Ang II (166±20%) induces a significant increase in DNA synthesis as assessed by [³H]-thymidine incorporation assays. Interestingly, coincubation of 100 µg/mL LDL and 100 nmol/L Ang II causes a marked increase of DNA synthesis to 272±52%. This strong effect is obviously not simply explainable by the addition of Ang II– and LDL-induced mitogenesis but suggests a mutual potentiation of LDL and Ang II. Experiments that used 1 µmol/L of the selective AT1 receptor antagonist TCV-116 revealed that TCV-116 inhibits the Ang II–induced mitogenesis. When TCV-116 is coincubated with LDL and Ang II, the potentiated effect on the DNA synthesis is blocked, which suggests that the activation of AT1 receptors by Ang II potentially mediates this significant increase in DNA synthesis after coincubation of Ang II and LDL. Experiments were performed to gain insight into general mechanisms participating in the LDL-caused elevation of AT1 receptor expression. After a 12-hour treatment with either vehicle or 100 µg/mL LDL, gene transcription of VSMCs was inhibited by incubation with 50 mg/mL DRB. Northern hybridizations were then performed on RNA extracted from VSMCs at the indicated time points (Fig 6). In vehicle-treated cells, AT1 receptor mRNA levels are reduced to 50% of control levels 5 hours after the addition of DRB. Therefore, this rate of AT1 receptor mRNA decay is taken to represent a measure of mRNA stability under otherwise normal conditions. This decay rate contrasts markedly with the degradation for AT1 receptor mRNA in cells pretreated with LDL; preincubation of VSMCs with LDL leads to a marked increase in AT1 receptor mRNA stability. Indeed, there is no significant decay in AT1 receptor mRNA over the time course of the assay. These data suggest that LDL-induced enhancement of AT1 receptor mRNA stability is involved in the LDL-caused upregulation of AT1 receptor gene expression.

View larger version (57K): [in this window] [in a new window]   Figure 1. Representative Northern hybridization autoradiography. VSMCs were grown to confluence, deprived of serum for 24 hours, and exposed to 100 µg/mL LDL. Hybridization of an AT1 receptor cDNA probe to Northern blots of 10 µg total RNA extracted from VSMCs is shown for the indicated time points. Hybridization of a GAPDH cDNA probe to the same blot stripped of the AT1 receptor cDNA probe also is shown. Findings are representative for five separate experiments.

View larger version (14K): [in this window] [in a new window]   Figure 2. Quantification of Northern hybridization signal intensity. A, Time course of the AT1 receptor () and GAPDH mRNA () in the presence of 100 µg/mL LDL. Northern hybridizations were performed as described in "Methods." Each point represents the relative hybridization signal (mean±SE) normalized to the 0-hour treatment with vehicle (100%) from five separate experiments. *P<.05. B, Dose-response curve of LDL-induced upregulation of AT1 receptor mRNA () and GAPDH mRNA () expression. Each point represents the relative hybridization signal (mean±SE) from three separate experiments. *P<.05. C, AT1 receptor mRNA () and GAPDH mRNA () levels in VSMCs that were deprived of serum for 24 hours. RNA was isolated at the indicated time points and analyzed as described in "Methods." Each point represents the relative hybridization signal (mean±SE) of three separate experiments.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of LDL on membrane receptors by saturation binding with [125I]–Ang II. Confluent cells on 24-well culture plates were exposed to either vehicle () or 100 µg/mL LDL (). Saturation binding assays with [125I]–Ang II were performed on intact cells. AT1 receptor antagonist TCV-116 (10 µmol/L) was used to define nonspecific binding. Each curve represents specific binding of the radioligand (cpm radioligand bound minus cpm bound in the presence of 10 µmol/L TCV-116). Kd and Bmax values reported in the text were derived from nonlinear regression of the specific bound vs free data. Each point represents binding data of three independent experiments±SE.

View larger version (12K): [in this window] [in a new window]   Figure 4. Time course of Ang II–caused elevation in [Ca2+]i. Cells were seeded on round glass slides, grown to confluence, and preincubated with either vehicle (----) or 100 µg/mL LDL (—) for 24 hours. After pretreatment of the cells according to the description in "Methods," VSMCs were challenged with 100 nmol/L Ang II, and [Ca2+]i was measured. Data represent four separate experiments.

View larger version (48K): [in this window] [in a new window]   Figure 5. DNA synthesis of VSMCs in response to LDL and Ang II. VSMCs were seeded on 24-well culture plates and deprived of serum for 24 hours. Incorporation of [3H]-thymidine into VSMCs and the amount of protein per culture dish were measured after incubation with either 100 nmol/L Ang II or 100 µg/mL LDL or coincubation of 100 nmol/L Ang I and 100 µg/mL LDL for 24 hours. TCV-116 (TCV; 1 µmol/L) was added simultaneously with vehicle, LDL, or Ang II. Each point represents mean±SE of three separate experiments. *P<.05, control vs either LDL or Ang II treatment. **P<.05, either Ang II or LDL treatment vs coincubation of LDL and Ang II.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of transcriptional blockade on VSMC mRNA levels in the presence or absence of LDL. Confluent cells and cells deprived of serum for 24 hours on 6-cm culture dishes were pretreated with either vehicle () or 100 µg/mL LDL () for 12 hours. Then VSMCs were exposed to 50 mg/mL DRB, and total RNA was isolated at the indicated time points. Northern hybridizations were performed with an AT1 receptor (top) and a GAPDH cDNA (bottom) probe. Each point represents the relative hybridization signal (mean±SE of three separate experiments) normalized to the mRNA level obtained from cells pretreated for 12 hours before the addition of DRB to the culture medium.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our study demonstrates that stimulation with native LDL increases AT1 receptor mRNA stability in cultured VSMCs. This stabilization of AT1 receptor mRNA is a mechanism of AT1 receptor upregulation during exposure to LDL. Enhanced expression of AT1 receptors leads ultimately to an increased functional response of VSMCs on Ang II stimulation. It was previously reported that lipoproteins and Ang II cause synergistic effects in VSMCs with respect to phosphoinositide and [Ca²⁺]_i responses.³⁴ In this particular study, cells were challenged simultaneously with lipoproteins and Ang II, which caused synergistically increased elevations in [Ca²⁺]_i and phosphoinositide turnover. This may be due to LDL-induced modulations on signal transduction components such as phospholipase C and intracellular calcium mobilization. The effect of LDL on AT1 receptor gene expression was not examined; however, it seems unlikely that a short-term incubation with LDL leads to changes in AT1 receptor gene expression. Because most of the known biological effects of Ang II are mediated by the AT1 receptor subtype, regulation of the responsivity of this receptor has been a prominent subject of recent research. Indeed, it is well established that the AT1 receptor is regulated in vivo and in vitro. Conditions of increased renin-angiotensin system activity cause downregulation of AT1 receptors,^{35 36} whereas a decrease in the activity of the renin-angiotensin system upregulates the AT1 receptor.^{37 38} Recently, it has been shown that various growth factors and Ang II induce a profound downregulation of AT1 receptor gene expression in cultured VSMCs.^{39 40} A principal mechanism underlying this downregulation is thought to be the inducible destabilization of AT1 receptor mRNA. In this work, we provide evidence once again that AT1 receptor regulation is closely dependent on modification of mRNA turnover because LDL causes AT1 receptor upregulation by means of AT1 receptor mRNA stabilization. Although native LDL and Ang II are known to cause elevation of [Ca²⁺]_i in VSMCs,^{7 12 13 14} it is not conceivable that this second messenger pathway causes a differential regulation of the AT1 receptor. To date, however, it is unclear which intracellular signals mediate AT1 receptor regulation. Nevertheless, the observation that native LDL is capable of upregulating the AT1 receptor in vitro is very intriguing because numerous epidemiological studies have shown that elevated levels of LDL are associated with the onset of hypertension and arteriosclerosis.^{41 42 43 44 45 46 47} Several molecular mechanisms have been suggested to account for this correlation of elevated LDL serum levels with the development of chronic vascular diseases. First, native LDL is known to cause elevation of intracellular calcium concentration in cultured VSMCs, and putatively related to that, it was described that native LDL induces vasoconstriction of rat aortic rings and enhanced growth of VSMCs.^{7 10} Second, it has been reported that arterial tissue is increasingly sensitive to vasoconstrictor substances after exposure to cholesterol, and it has been speculated that this hypersensitivity may be mediated by an enhanced calcium influx into VSMCs through receptor-dependent calcium channels that are influenced by excessive cholesterol.^{48 49 50} LDL also is capable of increasing production of platelet-derived growth factor-AA and expression of platelet-derived growth factor receptors in human VSMCs.⁵¹ Here, we provide evidence that native LDL upregulates AT1 receptor gene expression in cultured VSMCs. It is attractive to speculate that this occurrence resembles another pathway by which elevated LDL serum levels may interfere with the course of hypertension and arteriosclerosis. This is especially presumable because activation of AT1 receptors by Ang II leads to vasoconstriction and has been implicated in abnormal growth of VSMCs.^{12 13 14 15 16} The following observations strengthen this notion. Elevation of [Ca²⁺]_i in VSMCs on Ang II stimulation, which closely correlates to vasoconstriction, is significantly increased by treatment with LDL. Furthermore, LDL-induced upregulation of AT1 receptors leads to an enhancement of Ang II–induced DNA synthesis in VSMCs. Finally, our hypothesis is supported by recent animal studies that showed a regression of atherosclerosis in hyperlipidemic hamsters and minipigs by ACE inhibitors.^{52 53} In any event, it is important to note that the aforementioned data were generated in an in vitro system on cultured VSMCs of a rat aorta. Further research is necessary to find out whether this LDL-induced upregulation also occurs in humans with elevated LDL serum levels. If so, ACE inhibitors and AT1 receptor antagonists could be part of a new supportive therapeutic concept in the treatment of hypercholesterolemia-associated cardiovascular diseases.

	Selected Abbreviations and Acronyms

 

Ang = angiotensin AT1 = Ang II type 1 DRB = 5,6-dichlorobenzimidazole Fura-2/AM = fura-2-pentaacetoxymethyl ester VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by the grants Sa 568/2-1 and Ni 398/2-1 from the Deutsche Forschungs-Gemeinschaft, BonFOR grant 110/05, and a Takeda basic research grant. We are very grateful to H. Feltkamp (Takeda Euro R&D Centre, Frankfurt, Germany) for providing TCV-116. The excellent technical assistance of Claudia Seul, Petra Epping, and Maria-Katharina Meyer zu Brickwedde is greatly appreciated.

Received April 25, 1996; revision received August 28, 1996; accepted September 4, 1996.

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