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(Circulation. 1998;98:2553-2559.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Expression of Functional Angiotensin-Converting Enzyme and AT₁ Receptors in Cultured Human Cardiac Fibroblasts

Sassan Hafizi, BSc; John Wharton, PhD; Kevin Morgan, PhD; Sean P. Allen, PhD; Adrian H. Chester, PhD; John D. Catravas, PhD; Julia M. Polak, FRCPath; Magdi H. Yacoub, FRCS

From the Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine at the Heart Science Centre, Harefield Hospital, Middlesex, UK (S.H., S.P.A., A.H.C., M.H.Y.); the Department of Histochemistry, Division of Investigative Science, Imperial College School of Medicine at Hammersmith Hospital, London, UK (J.W., K.M., J.M.P.); and the Vascular Biology Center, Medical College of Georgia, Augusta (J.D.C.).

Correspondence to Professor Sir Magdi Yacoub, FRCS, Harefield Hospital, Hill End Road, Harefield, Middlesex, UB9 6JH, UK.

	Abstract

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Background—Angiotensin II (Ang II) has been implicated in the development of cardiac fibrosis. The aims of the present study were to examine expression and activity of ACE and of angiotensin receptors in human cardiac fibroblasts cultured from dilated cardiomyopathic and ischemic hearts. The effects of Ang II on fibroblasts were also investigated.

Methods and Results—Human cardiac fibroblasts were cultured from ventricular and atrial myocardium and characterized immunohistochemically. Expression of ACE and the angiotensin AT₁ receptor was demonstrated in cardiac fibroblasts by reverse transcriptase–polymerase chain reaction and radioligand binding. Functional ACE activity, measured by radiolabeled substrate conversion assay, was detected in both ventricular (V_max · K_m^-1 · mg^-1, 0.031±0.010; n=13) and atrial (0.034±0.012; n=6) fibroblasts. Fibroblast ACE activity was increased after 48 hours of treatment with basic fibroblast growth factor, dexamethasone, and phorbol ester. Ang II did not affect DNA synthesis but stimulated [³H]proline incorporation in cardiac fibroblasts (20.0±4.0% increase above control by 10 µmol/L; P<0.05, n=7), which was abolished by losartan 10 µmol/L but not PD123319 1 µmol/L. Ang II also stimulated a rise in intracellular calcium (basal, 56±1 nmol/L; Ang II, 355±24 nmol/L) via the AT₁ receptor, as shown by complete inhibition with losartan.

Conclusions—We have demonstrated expression and activity of ACE and AT₁ receptor in cultured human cardiac fibroblasts. In addition, cardiac fibroblasts respond to Ang II with AT₁ receptor–mediated collagen synthesis. The presence of local ACE and AT₁ receptors in human fibroblasts suggests their involvement in the development of cardiac fibrosis.

Key Words: angiotensin • receptors • enzymes • collagen • cells

	Introduction

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Myocardial fibrosis is a pathological feature associated with cardiac hypertrophy, myocyte necrosis, and hypertension.¹ Components of the renin-angiotensin system, including ACE and angiotensin II (Ang II), have been implicated in the development of cardiac fibrosis.^{1 2} Significantly, numerous studies have provided evidence for the presence of a local tissue ACE, and expression of ACE has been demonstrated in both rat^{3 4} and human^{5 6} cardiac tissues. Furthermore, the cardiac renin-angiotensin system has been shown to be functional in humans,⁷ ACE being the predominant pathway for the local generation of Ang II in human cardiac tissues.⁸ The microvascular endothelium is considered to be the main site of ACE expression in both rat^{9 10} and human^{9 11} hearts, although other cardiac cells may express the enzyme. ACE binding and immunostaining, for example, have been localized to interstitial cells at sites of myocardial infarction (MI) in the rat,^{9 10} and ACE expression has been demonstrated in isolated rat cardiac fibroblasts.¹² In contrast, immunohistochemical studies on human tissues have provided conflicting information concerning the cellular localization of ACE immunoreactivity after MI.^{9 13} We have shown that interstitial cells in the failing human heart display ACE binding, albeit at a relatively low level compared with the vascular endothelium.¹¹ However, the expression of ACE in human cardiac fibroblasts is still uncertain. In addition, little is known about the regulation of fibroblast ACE activity, and possible regulatory factors include glucocorticoids and basic fibroblast growth factor (bFGF), both of which have been demonstrated to enhance ACE activity in other cell types.¹⁴

Several studies have suggested that Ang II is a trophic agent with the ability to influence myocardial fibrosis¹⁵ and modulate cardiac fibroblast growth and collagen synthesis.^{16 17 18 19} The effects of Ang II are mediated through specific receptors, AT₁ and AT₂ being the main subtypes.²⁰ Both receptors occur in rat²¹ and human^{11 22} myocardium, whereas the AT₁ subtype predominates in isolated adult rat cardiac fibroblasts.^{16 17} The expression of angiotensin receptors in cultured human cardiac fibroblasts and the effects of Ang II on cell growth and collagen metabolism remain to be established.

In the present study, we have determined the expression and activity of ACE in isolated human ventricular and atrial fibroblasts, studied the effects of Ang II on cardiac fibroblast collagen synthesis and proliferation, and characterized the receptor subtype mediating such responses.

	Methods

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Materials
Human Ang II, platelet-derived growth factor AB (PDGF-AB), bFGF, dexamethasone, phorbol myristate acetate (PMA), DMEM, HBSS, FCS, PBS, L-glutamine, penicillin/streptomycin, HEPES, EDTA, SDS, BSA, trichloroacetic acid, and collagenase II were purchased from Sigma Chemical Co. ¹²⁵I-labeled (1-sarcosine,8-isoleucine)angiotensin II {[¹²⁵I]-(S¹,I⁸)Ang II, 2200 Ci/mmol} was purchased from NEN Life Science Products. A ¹²⁵I-labeled tyrosyl derivative of lisinopril {N-[(s)-1-carboxy-3-phenylpropyl]-L-lysyl-tyrosyl-L-proline, [¹²⁵I]-351A} was iodinated (2000 Ci/mmol) as previously described.²³ Losartan (DuP 753) and PD123319 were gifts from DuPont Merck, Wilmington, Del, and Parke-Davis, Ann Arbor, Mich, respectively. Tissue culture plastics (Falcon) were from Marathon Laboratory Supplies. [Methyl-³H]thymidine 64 Ci/mmol and [3,4-³H]L-proline 52 Ci/mmol were from ICN Biomedicals, and [³H]benzoyl-phenylalanine-alanine-proline ([³H]BPAP, 400 µCi/mL) was synthesized and radiolabeled in our laboratory. Liquid and toluene scintillation cocktails were from Canberra Packard.

Human Cardiac Fibroblast Culture and Characterization
Samples of left ventricular and atrial myocardium were obtained from recipient hearts (age range, 2 to 59 years, with dilated cardiomyopathy or ischemic heart disease) at the time of cardiac transplantation and stored at 4°C for 12 hours in HBSS containing 20 mmol/L HEPES (pH 7.4). Myocardium from the free walls of left ventricles and atria, dissected free of epicardial and valvular regions, was minced fine and incubated in a sterile-filtered dissociation solution containing collagenase II 1000 U/mL for 2 hours under shaking in a water bath at 37°C. After 1 hour, the mixture was passed through a syringe several times to aid cell dissociation and then incubated for another hour. Dispersed cells were filtered (100-µm nylon mesh), washed in fibroblast growth medium, centrifuged (1200 rpm, 5 minutes), resuspended in growth medium, and incubated in 25-cm² plastic flasks in a humidified atmosphere of 5% CO₂ in air. Fibroblast growth medium (DMEM supplemented with L-glutamine 2 mmol/L, penicillin 100 U/mL, streptomycin 100 µg/mL, and 20% FCS) was replaced every 3 days. Fibroblasts were identified by positive immunostaining for fibroblast-specific antigen,²⁴ prolyl-4-hydroxylase, vimentin, tubulin, and filamentous actin. No immunoreactivity for endothelial (CD31) and muscle (desmin) markers was exhibited. Varying proportions of cells displayed -smooth muscle actin immunoreactivity, also a feature of cardiac fibroblasts in culture,¹⁶ which may reflect either variation in myocardial scarring between patients or possible phenotypic transformation of some cells in culture. Human endothelial cells, isolated from unused portions of donor aorta and recipient coronary artery as previously described,²⁵ exhibited the endothelial cobblestone morphology and CD31 immunostaining. Cells of passages 1 through 6 were used for all experiments.

Expression of ACE and the AT₁ Receptor Genes
Expression of ACE and AT₁ receptor was confirmed by reverse transcription–polymerase chain reaction (RT-PCR) analysis. Total RNA was prepared from cell pellets according to the method of Chomczynski and Sacchi,²⁶ and 5 µg was reverse transcribed into cDNA with random primers (In Vitrogen). Oligonucleotide primers were synthesized according to the nucleotide sequences and genomic organization of human AT₁ receptor and ACE genes. The AT₁ receptor primer sequences were 5'-GATGGGGAGCGGCT-GGAGCGG-3' (sense) and 5'-TGCCAAAGGGCCAGCGGTAT-TC-3' (antisense), the PCR amplimer spanning exons 1, 2, 3, 4, and 5 over a region of 755 bp. For human ACE, the sense primer was 5'-ACTGGTGGTATCTTCGAACC-3' and the antisense primer 5'-GACCATGTCCTTCAGCACC-3', the PCR amplimer spanning a region of 296 bp. The RT-PCR reaction and analysis were carried out as previously described.¹¹

Radioligand Binding to ACE and AT₁ Receptor
The presence of specific Ang II and ACE binding sites on human cardiac fibroblasts was examined by use of [¹²⁵I]-(S¹,I⁸)Ang II and [¹²⁵I]-351A, respectively. Subconfluent cardiac fibroblasts in 24-well plates were washed twice with DMEM and then incubated in 400 µL of DMEM containing either 0.2 nmol/L [¹²⁵I]-(S¹,I⁸)Ang II or 0.3 nmol/L [¹²⁵I]-351A, with 0.1% BSA for 90 minutes at 37°C. After a washing with ice-cold DMEM, cells were solubilized in 0.2 mol/L NaOH and 0.1% SDS for 30 minutes, and ligand present in the lysate was measured with a gamma counter. Nonspecific [¹²⁵I]-(S¹,I⁸)Ang II and [¹²⁵I]-351A binding was defined as that obtained in the presence of 1 µmol/L unlabeled (S¹,I⁸)Ang II and either 1 mmol/L EDTA or 1 µmol/L lisinopril, respectively. [¹²⁵I]-(S¹,I⁸)Ang II binding was characterized by inhibition studies with increasing concentrations (1 pmol/L to 1 µmol/L) of nonselective [Ang II, (S¹,I⁸)Ang II], AT₁-selective (losartan), or AT₂-selective (PD123319) competitors.

ACE Activity Assay
Confluent cardiac fibroblasts in 24-well plates were incubated in DMEM containing 0.4% FCS for 48 hours before assay for ACE activity or treatment with either PDGF-AB 15 ng/mL, bFGF 50 ng/mL, dexamethasone 100 nmol/L, or PMA 1 µmol/L for 24 to 48 hours. Before assay, cells were rinsed with PBS and incubated in serum-free DMEM (1 mL/well), and enzyme activity was measured with the tripeptide [³H]BPAP as substrate, as previously described.²⁷ Half of all samples were incubated with captopril 1 µmol/L for 1 hour before the start of the reaction (addition of [³H]BPAP 0.1 µCi/mL), the ACE inhibitor remaining present throughout the reaction. Enzyme activity (U/mg protein) was calculated by the formula V_max/K_m=ln([S_o]/[S])/t, where [S_o] and [S] are the initial and final substrate concentrations, respectively, and t is time of incubation. One unit of ACE activity is the V_max/K_m value equivalent to 1% substrate metabolism in 1 minute under first-order conditions.

Collagen Synthesis and DNA Synthesis Assays
Collagen synthesis was assessed by measurement of the cellular uptake of [³H]proline. Fibroblasts were seeded into 24-well plates at 3x10⁴ cells per well (1 mL/well) in growth medium and incubated overnight. Cells were then incubated in DMEM containing 0.4% FCS for 48 hours before addition of Ang II 1 nmol/L to 10 µmol/L. For experiments using selective antagonists, the cells were incubated with either losartan 10 µmol/L or PD123319 1 µmol/L for 1 hour before addition of Ang II and remained present throughout the experiment. [³H]Proline was added to each well at a final concentration of 1 µCi/mL, and cells were incubated for 48 hours. After incubation, the supernatant in each well was replaced with ice-cold 10% trichloroacetic acid for 20 minutes at 4°C. The acid-precipitable material was rinsed with deionized water, then solubilized in 0.25 mL of 0.3 mol/L NaOH–0.1% SDS at 37°C for 2 hours. The cell lysate was added to 3 mL of liquid scintillant, and the incorporated radioactivity (cpm) was measured. The same protocol was used for assessing DNA synthesis, except that [methyl-³H]thymidine incorporation between 20 and 24 hours was measured, in response to Ang II 10 nmol/L to 10 µmol/L and PDGF-AB 15 ng/mL.

Measurement of Intracellular Calcium Concentration
Cardiac fibroblasts on glass coverslips were loaded with the calcium indicator fura 2-AM 1 µmol/L for 30 minutes at room temperature in Krebs-Henseleit buffer containing (in mmol/L) NaCl 120, KCl 4.8, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, glucose 25, CaCl₂ 1.3, and HEPES 25 (pH 7.4), and 0.1% BSA. Coverslips were then placed in a temperature-controlled holder (34°C) and mounted onto the stage of an epifluorescence microscope (Zeiss Axiovert 35). Cells were challenged with Ang II 1 µmol/L and monitored visually over time as previously described.²⁸ When used, losartan 10 µmol/L and PD123319 1 µmol/L were present at all stages of the experiment. The intracellular calcium concentration ([Ca²⁺]_i) was determined from fluorescence values by the formula described by Grynkiewicz et al.²⁹

Statistics
Data are presented as mean±SEM. Multiple groups of data underwent a 1-way ANOVA followed by Bonferroni t test. Student's t test was used to compare paired observations, and a value of P<0.05 was considered significant.

	Results

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Expression of AT₁ Receptor and ACE mRNA and Radioligand Binding
Specific PCR products corresponding to human AT₁ receptor and ACE cDNA sequences were generated from both atrial and ventricular fibroblasts (Figure 1). Expression of the AT₁ receptor gene displayed alternative splicing of the 5' untranslated exons, with 2 transcripts being detected, one encoding exons 1 and 5 (466-bp PCR product) and the other exons 1, 2, and 3 (551-bp PCR product).

View larger version (39K): [in this window] [in a new window]   Figure 1. Expression of AT1 receptor and ACE in human cardiac fibroblasts (fibs). Autoradiogram of specific products corresponding to AT1 receptor (551 and 466 bp; lanes 1 through 6) and ACE cDNA sequences (296 bp; lanes 7 through 9) after RT-PCR amplification of transcripts isolated from cultured human atrial (lanes 1 through 3) and ventricular fibroblasts (lanes 4 through 8) and aortic endothelial cells (EC, lane 9). Lane 3 represents a control amplification (no cDNA).

Atrial and ventricular fibroblasts exhibited specific [¹²⁵I]-(S¹,I⁸)Ang II binding with characteristics of the AT₁ receptor subtype. Binding was selectively inhibited in the presence of losartan, whereas PD123319 had no apparent effect (Figure 2A). Binding was competitively inhibited by unlabeled (S¹,I⁸)Ang II and Ang II, as well as by losartan, and nonspecific binding represented <10% of total binding (Figure 2B). Cardiac fibroblasts also exhibited specific binding of the radiolabeled ACE inhibitor [¹²⁵I]-351A, albeit at a lower level than that displayed by endothelial cells (Figure 3), and this was abolished in the presence of either EDTA (data not shown) or 1 µmol/L lisinopril.

View larger version (19K): [in this window] [in a new window]   Figure 2. Human cardiac fibroblasts exhibited specific [125I]-(S1,I8)Ang II binding sites (0.2 nmol/L, 90 minutes at 37°C) characteristic of AT1 receptor subtype. Top, Binding was significantly reduced (**P<0.001) in presence of unlabeled (S1,I8)Ang II 1 µmol/L and losartan 1 µmol/L but was unaffected by PD123319 1 µmol/L. Values represent mean±SEM of ligand bound (cpm/µg protein, n=4 ventricular cultures). Comparisons between values were made by ANOVA followed by Bonferroni's correction. Bottom, Competitive inhibition of [125I]-(S1,I8)Ang II binding to ventricular fibroblasts in presence of increasing concentrations of nonselective [, (S1,I8)Ang II; , Ang II], AT1-selective (, losartan), and AT2-selective (, PD123319) competitors. Points represent mean of 3 determinations, expressed as percentage of maximum binding.

View larger version (15K): [in this window] [in a new window]   Figure 3. Cardiac fibroblast and coronary endothelial cells exhibited specific binding of radiolabeled ACE inhibitor [125I]-351A. Binding to both fibroblasts (***P<0.001) and endothelial cells (**P<0.0001) was significantly reduced in presence of lisinopril 1 µmol/L. Values represent mean±SEM amount of [125I]-351A bound (cpm/well), derived from 4 separate fibroblast cultures and an endothelial cell line, assayed in quadruplicate. Comparisons between values were made by unpaired Student's t test (2-tailed).

ACE Activity of Cardiac Fibroblasts
Ventricular and atrial fibroblasts exhibited functional ACE activity, as determined by cleavage of the radiolabeled substrate [³H]BPAP and its blockade in the presence of captopril 1 µmol/L (Figure 4). There was no significant difference between the ACE activities (U · mg^-1 · min^-1 ±SEM) of ventricular (0.031±0.010, n=13) and atrial (0.034±0.012, n=6) fibroblasts. However, human aortic and coronary endothelial cells exhibited 6-fold greater ACE activity per milligram total protein (0.213±0.034, P<0.001; n=4). Quiescent fibroblasts exhibited a significant increase in ACE activity after exposure for 48 hours to 100 nmol/L dexamethasone (152.6±5.3%, P<0.01; n=6), 50 ng/mL bFGF (156.8±14.0%, P<0.01; n=4), or 1 µmol/L PMA (170.0±20.0%, P<0.05; n=3). Increased ACE activity was also detected at 24 hours, albeit at a lower level (data not shown), but no change was detected after treatment with 15 ng/mL PDGF-AB.

View larger version (17K): [in this window] [in a new window]   Figure 4. ACE activity in human cardiac fibroblasts. Ventricular (n=13 separate cultures) and atrial (n=6 separate cultures) fibroblasts displayed ACE activity, as demonstrated by [3H]BPAP cleavage and its inhibition by captopril 1 µmol/L. Data are expressed as mean±SEM ACE activity (U · mg-1 · min-1), with each determination in quadruplicate. **P=0.01 and *P<0.05 vs respective control by unpaired Student's t test.

Effect of Ang II on Collagen and DNA Synthesis and Intracellular Calcium Concentration
Cardiac fibroblasts responded to Ang II 1 nmol/L to 10 µmol/L with a concentration-dependent increase in [³H]proline incorporation (Figure 5A). A maximum 20±4% increase in [³H]proline incorporation was achieved by 10 µmol/L Ang II (P<0.05, data from 7 experiments pooled together), and this was abolished by preincubation with losartan 10 µmol/L but not PD123319 1 µmol/L (Figure 5B). The response to Ang II was essentially the same in the 33 different cell cultures isolated from explanted human hearts, irrespective of the varying levels of scarring. Ang II 10 nmol/L to 10 µmol/L exhibited no effect on [methyl-³H]thymidine incorporation at 24 hours, whereas PDGF-AB caused a 7-fold increase (data not shown). Neither was any stimulation of DNA synthesis observed between 18 and 42 hours, which was measured to account for possible delayed mitogenesis.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effect of Ang II on collagen synthesis in human cardiac fibroblasts. Top, Ang II 1 nmol/L to 10 µmol/L stimulated an increase in [3H]proline incorporation in ventricular fibroblasts. Values represent mean±SEM [3H]proline incorporation (0 to 48 hours) in quadruplicate treatments from a representative of 7 experiments with similar results; *P<0.05 vs control. Similar results were obtained in 5 experiments on atrial fibroblasts. Bottom, Ang II–induced collagen synthesis was selectively inhibited by 1 hour of preincubation with losartan 10 µmol/L but not PD123319 1 µmol/L. Bars represent mean±SEM [3H]proline incorporation (0 to 48 hours) in quadruplicate treatments from a representative of 4 experiments with similar results; ***P<0.001 vs control and vs all others unmarked. Similar results were obtained in experiments on atrial fibroblasts.

Human cardiac fibroblasts responded to Ang II with a rapid increase in [Ca²⁺]_i (basal, 56±1 nmol/L to Ang II, 355±24 nmol/L; P<0.001; n=66 cells) (Figure 6). The peak response occurred 16 seconds after stimulation, returning to baseline levels thereafter (Figure 6B). The Ang II–induced calcium increases were completely inhibited by pretreatment with losartan 10 µmol/L, whereas no inhibition was observed with PD123319 1 µmol/L (Figure 6A and 6C).

View larger version (34K): [in this window] [in a new window]   Figure 6. Effect of Ang II on [Ca2+]i in human cardiac fibroblasts. Top, Fluorescent fura 2 imaging of Ang II–triggered intracellular calcium transients in ventricular fibroblasts over time. Images shown are 4 snapshots taken at 0, 16, 32, and 72 seconds after addition of Ang II 1 µmol/L alone (A through D) and in presence of losartan 10 µmol/L (E through H) or PD123319 1 µmol/L (I through L). On pseudocolor scale, dark blue (low pixel intensity) represents low [Ca2+]i and red represents high [Ca2+]i. Bottom left, Time course of Ang II stimulation of [Ca2+]i in human cardiac fibroblasts. Points represent mean±SEM [Ca2+]i (nmol/L) in ventricular (n=66) and atrial (n=55) fibroblasts at each time point (seconds). Bottom right, Losartan but not PD123319 abolishes Ang II–induced calcium transients in cardiac fibroblasts. Bars represent mean±SEM of maximal response to Ang II (n=number of cells from 4 separate cultures). Bars shown are basal [Ca2+]i (n=130) and Ang II (1 µmol/L)–stimulated [Ca2+]i in absence (n=66) and presence (n=88) of losartan 10 µmol/L and PD123319 1 µmol/L (n=58). ***P<0.001 vs control and vs Ang II+losartan.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have demonstrated expression of both ACE and the AT₁ receptor subtype in cultured human cardiac fibroblasts and have shown that the AT₁ receptor subtype mediates Ang II–induced increases in collagen synthesis and intracellular calcium in these cells. Together, these findings implicate ACE and Ang II in the development of myocardial fibrosis, independent of changes in hemodynamics.

The demonstration of ACE expression and activity in human cardiac fibroblasts supports the findings of several recent studies. In the rat heart, ACE expression has been detected at sites of fibrosis after Ang II infusion¹⁵ and after MI,¹⁰ as well as in myofibroblasts isolated from cardiac scar tissue.¹² Expression of ACE has also been detected in human cardiac tissues^{5 30} and a correlation found between the levels of ACE and fibrillar collagen type I mRNA.³⁰ More specifically, increased ACE activity has been found in aneurysmal left ventricular tissue³¹ and ACE immunoreactivity localized to myocytes as well as other cell types, including fibroblasts, adjacent to scar tissue¹³ in patients after MI. In the present study, no differences were detected between the level of ACE activity in fibroblasts cultured from failing (dilated cardiomyopathy or ischemic heart disease) and normal donor hearts. However, we cannot exclude the possibility that the culture conditions influenced the level of ACE expression. Conversely, apparent differences in ACE activity and binding between cultured human coronary endothelial cells and cardiac fibroblasts correspond with results obtained in intact tissue sections. Previous observations from this group as well as others indicate that although ACE binding¹¹ and immunostaining¹³ are localized predominantly to the microvascular endothelium in the failing and normal human heart, both are detectable on other cell types in the human myocardium, including myocardial, interstitial, and fibroblast-like cells.^{11 13} Although the potential of human cardiac fibroblasts to generate Ang II may be less than that of endothelial cells, it should not be underestimated and may be particularly significant in the diseased heart, such as after MI, in which enzyme-bearing cells are prevalent at sites of scarring and in the border zone adjacent to scar tissue.^{2 13}

Among potential modulators of ACE activity, dexamethasone, bFGF, and PMA induced an increase in cardiac fibroblast ACE activity. Dexamethasone has previously been shown to stimulate ACE activity in rat aortic smooth muscle cells,³² and the effective concentration used in this study is equivalent in glucocorticoid potency to levels of cortisol elevated in human plasma during physiological stress in vivo.³³ The stimulatory effect of bFGF suggests that the growth factor may influence ACE activity at sites of myocardial injury, where it can be released from damaged cells.³⁴ The positive effect of PMA may be attributed to either short-term activation or long-term downregulation of protein kinase C (PKC) in cardiac fibroblasts. Together, these results provide evidence for the dynamic regulation of ACE activity and potential autocrine control of cardiac fibroblasts.

Ang II induced a net stimulation of collagen synthesis, this being in agreement with the results of previous studies on isolated human¹⁹ and rat cardiac fibroblasts.^{16 18} The Ang II–induced collagen synthesis and intracellular calcium transients in human cardiac fibroblasts were both shown to occur via the AT₁ receptor, the expression of which was confirmed by RT-PCR analysis and radioligand binding. The AT₂ receptor antagonist PD123319 had no apparent effect on either ¹²⁵I-(S¹,I⁸)Ang II binding, collagen synthesis, or intracellular calcium transients, which suggests the absence of AT₂ receptors. These results contrast with those obtained by ourselves and others in tissue sections and myocardial membrane preparations, indicating the presence of both receptor subtypes in human myocardium and the predominance of the AT₂ subtype in regions of fibrosis.^{11 22 35 36} This apparent discrepancy between in vivo and in vitro findings probably reflects changes in the proportion of Ang II receptors, particularly downregulation of AT₂ receptors, after isolation of cells and in response to culture conditions.^{16 17 37 38} Thus far, AT₂ receptor expression has not been demonstrated in human primary cell cultures, and the relative instability of AT₂ receptor expression in isolated cells represents a significant limitation of in vitro investigations. Knowledge about the function of the AT₂ receptor is limited to studies using animal models and isolated rodent cells, in which the receptor mediates effects such as inhibition of collagen synthesis³⁹ and DNA synthesis,⁴⁰ thereby opposing responses mediated by the AT₁ subtype. Recently, however, blockade of the AT₂ receptor has been shown to inhibit DNA synthesis in interstitial cells after MI in the rat.⁴¹ Therefore, although the present findings implicate the AT₁ receptor in human cardiac fibrosis, the potential involvement of the AT₂ receptor in vivo cannot be excluded and requires further investigation.

In conclusion, the present results indicate that cardiac fibroblasts represent a site for the local generation and action of Ang II in the human heart and therefore may contribute to the development of cardiac fibrosis. This highlights the potential of ACE inhibition and AT₁ receptor antagonism in strategies for prevention as well as possibly regression of fibrosis.

	Acknowledgments

 
This work was supported by British Heart Foundation project grant PG95017. The authors are grateful to Dr Patricia Taylor for the immunohistochemical characterization of cells.

Received May 29, 1998; revision received August 11, 1998; accepted August 21, 1998.

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