(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
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.
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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 (Vmax · Km-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
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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 rat3 4 and human5 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 rat9 10 and human9 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 human11 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.
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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
Vmax/Km=ln([So]/[S])/t,
where [So] and [S] are the initial and final
substrate concentrations, respectively, and t is time of incubation.
One unit of ACE activity is the
Vmax/Km 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.
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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).
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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.
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,
(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.
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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.
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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.
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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).
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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 tissues5 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.
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Acknowledgments |
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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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