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(Hypertension. 1997;30:1035-1040.)
© 1997 American Heart Association, Inc.

Articles

Effect of Nitric Oxide on DNA Replication Induced by Angiotensin II in Rat Cardiac Fibroblasts

Toshikazu Takizawa; Miaofen Gu; Aram V. Chobanian; Peter Brecher

From the Department of Biochemistry and The Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.

Correspondence to Peter Brecher, PhD, Boston University School of Medicine, 80 East Concord St W507, Boston, MA 02118. E-mail pbrecher{at}acs.bu.edu

	Abstract

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Abstract Our previous in vivo studies (Hou et al. J Clin Invest. 1995;96:2469-2477.) demonstrated that chronic inhibition of nitric oxide synthase led to an exaggerated response to relatively low doses of angiotensin II, resulting in a rapid and marked cardiac fibrosis. To examine further the importance of angiotensin II in inducing cardiac fibrosis and the possibility that nitric oxide serves as a modulator of the proliferative effects of angiotensin II, we used cultured rat cardiac fibroblasts to study the interrelationships between these substances. Angiotensin II induced a delayed DNA synthetic response in quiescent cells that occurred 30 hours after exposure to the hormone. The most pronounced effect of angiotensin II on thymidine uptake occurred 36 to 42 hours after the addition to cells. This response was inhibited in a dose-dependent manner by the addition of either S-nitroso-N-acetylpenicillamine or sodium nitroprusside, each a source of nitric oxide. The nitric oxide donor was most effective in reducing thymidine incorporation when added 12 hours after angiotensin II, whereas the metabolite N-acetylpenicillamine had no effect at any time. The inhibitory effect of S-nitroso-N-acetylpenicillamine was mimicked by 8-bromoguanosine 3':5'-cyclic monophosphate but not by 8-bromoadenosine 3':5'-cyclic monophosphate. Nitric oxide donors did not appear to inhibit the induction of c-fos, Egr-1, or other immediate-early genes in response to angiotensin II. The results suggest that nitric oxide affects the cell cycle following the transition into G₁ and modulates the proliferation of fibroblasts during cardiac fibrosis induced by angiotensin II.

Key Words: cardiac fibrosis • fibroblasts • angiotensin II • nitric oxide • cell proliferation

	Introduction

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Cardiac fibrosis is a consequence of myocardial remodeling and occurs following varied pathophysiological conditions, including hypertension. The renin-angiotensin system has been implicated in the pathogenesis of cardiac fibrosis, and this relationship has been the subject of several recent reviews.^{1 2 3} In a previous study,⁴ we had shown that angiotensin (Ang) II, when infused into rats via an osmotic minipump, caused a relatively rapid induction of cardiac fibrosis but to a moderate degree. In a subsequent study,⁵ we found that a far more rapid and massive cardiac fibrosis ensued if the rats were pretreated with an inhibitor of nitric oxide synthase prior to administration of a relatively low infusion dose of Ang II. This effect was not dependent solely on hypertension and suggested that nitric oxide might antagonize effects of Ang II on cardiac fibroblasts. Studies with cultured cells had suggested that nitric oxide (NO) might have antiproliferative effects on fibroblasts and smooth muscle cells,^{6 7 8} but the mechanism for such effects of NO did not depend solely on guanylate cyclase activation⁹ and remain unclear.

Ang II has been shown to cause proliferation of cardiac fibroblasts in culture^{10 11 12} and to stimulate fibrosis as evidenced by enhanced extracellular matrix production both in vivo⁴ and in vitro.^{13 14} To examine the importance of Ang II in inducing cardiac fibrosis and the possibility that NO might normally serve as a modulator of the proliferative effects of Ang II, we used cardiac fibroblasts obtained from neonatal rats to study the effects of Ang II and NO on cell DNA synthesis. The data showed a delayed response to Ang II with respect to labeled thymidine incorporation and indicated that NO donors could modulate this response through a mechanism that appeared to be mediated by cyclic GMP (cGMP).

	Methods

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Materials
DMEM/F12, penicillin/streptomycin/amphotericin B, and fetal calf serum (FCS), were purchased from Life Technologies, Inc. Ang II, insulin/transferrin/selenium, bovine serum albumin (BSA), 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, N^G-nitroso arginine methyl ester (L-NAME), sodium nitroprusside (SNP), isosorbide dinitrate (ISDN), and 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ) were obtained from Sigma Chemical Co. Platelet-derived growth factor-BB (PDGF-BB) was purchased from Genzyme Corporation, and S-nitroso-N-acetylpenicillamine (SNAP) was obtained from Calbiochem. [³H]Methylthymidine was purchased from DuPont NEN Research Products. Losartan was generously provided by DuPont Merck. Mouse tumor necrosing factor- (TNF-) and mouse interferon- (IFN-) were purchased from Genzyme Corporation. Human recombinant transforming growth factor-ß1 (TGF-ß) was purchased from Austral Biologicals.

Cell Culture
Primary cultures of neonatal rat cardiac fibroblasts were prepared and characterized as described recently.¹⁵ For the present studies, cells were used in the third or fourth passage, and shortly before confluency was reached, the cells were made quiescent by maintenance in DMEM containing 0.4% BSA. For analysis of [³H]thymidine incorporation, cells were plated in 24-well multiwell plates (Corning) at approximately 5000 cells per well. After 3 days, the cells were almost confluent and were rendered quiescent by a 48-hour serum deprivation period. The cells were incubated for 6 to 24 hours with tritiated thymidine (0.5 to 1 µCi/ml) and the other designated additions.

The procedure for quantitating the incorporation of labeled thymidine was essentially as described.¹⁶ At the end of the incubation period, the medium was aspirated, and the cells were washed twice with cold phosphate-buffered saline, pH 7.0, washed once with ice-cold 10% trichloroacetic acid, and then incubated at 4°C for 30 minutes in 10% trichloroacetic acid. Following aspiration, the cell residue was then rinsed in 95% ethanol and dissolved in 0.25N NaOH at room temperature for 4 hours. After neutralizing with HCl, the radioactivity was measured by liquid scintillation spectrometry. All experiments were performed in triplicate and are representative of two to three separate experiments with different preparations of cells. Cell number was quantified by direct counting of trypsinized cells using a hemocytometer.

Isolation and Analysis of RNA
Total cellular RNA was isolated from fibroblasts by the acid guanidinium thiocyanate-phenol-chloroform method.¹⁷ Typically, two confluent Petri dishes of 100-cm² surface area yielded 50 to 80 µg of total RNA. RNA was quantified by absorbance at 260 nm. Northern blot analysis was carried out essentially as described previously.¹⁸

Statistical Analysis
All values are expressed as mean±SEM. Measurements were compared using a one-way ANOVA. Subsequent comparisons were performed using a two-tailed, unpaired Student t test.

	Results

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Experiments were performed to determine if Ang II had a proliferative effect on the cardiac fibroblasts used in our laboratory. Table 1 shows the effects of Ang II on the number of cells following treatment of subconfluent cells for 48 to 72 h. When Ang II was added in conjunction with 0.5% FCS, cell number increased progressively following 48 and 72 hours of Ang II treatment, whereas no change in cell number was observed when only 0.5% serum was present. This increase was about one-half of the proliferative response observed when 10% FCS was added. Thus, Ang II was a proliferative growth factor under defined culture conditions.

View this table: [in this window] [in a new window]   Table 1. Effect of Ang II and Fetal Calf Serum on Cell Proliferation of Subconfluent Cardiac Fibroblasts

Further experiments were performed to define the effects of Ang II on DNA synthesis in the presence of low serum levels. Quiescent and confluent cardiac fibroblasts were incubated with labeled thymidine for 24 hours in the absence or presence of Ang II (100 nmol/L) and the designated amount of FCS. Fig 1 summarizes the effects of serum and Ang II on thymidine incorporation over a 48-hour time period, with thymidine being present either for the initial 24 hours or between 24 and 48 hours after Ang II addition. During the initial 24-hour exposure to Ang II, thymidine incorporation was not increased by the presence of hormone, whether or not FCS was present. In fact, there was a small but significant (P<.01, n=8) decrease during the initial 24-hour period. However, during the 24 to 48-hour time interval, Ang II had a clear effect on thymidine incorporation when low levels of FCS were present (between 0.5% and 1% FCS). In seven sets of separate experiments using different cell preparations over a 14-month period, the increase in thymidine incorporation due to Ang II addition ranged from 1.3 to 2.2-fold greater than control levels between 24 and 48 hours after Ang II addition in the presence of 0.5% FCS (P<.005). In the absence of FCS, Ang II was consistently ineffective, and if the concentration of serum was high, any effect of Ang II was masked by the response to serum growth factors. Based on these data, conditions were standardized so that 0.5% FCS was included unless otherwise designated. In separate experiments, changes in cell number were examined during the 48-hour time course of the experiments. There was no significant change in cell number in the absence or presence of Ang II by the confluent cell preparations. When PDGF-BB (50 ng/mL) was compared with Ang II, PDGF induced a two-fold increase in thymidine incorporation both in the initial and subsequent 24-hour period (P<.01). Comparable effects also were seen with basic fibroblast growth factor (50 ng/mL) addition, whereas when phenylephrine (1 mmol/L) was added, no effect on thymidine incorporation was observed at any time up to 48 hours. Thus, Ang II was relatively specific in its ability to produce a delayed transition into S phase.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effects of Ang II and FCS on thymidine incorporation into cultured cardiac fibroblasts. Confluent and quiescent cells were incubated with Ang II (100 nmol/L) for 24 or 48 hours in the absence or presence of the designated amount of FCS. Labeled thymidine was included for the last 24 hours of incubation. Control levels (100%) were arbitrarily set as the amount of incorporation by cells incubated without serum and without Ang II for the initial 24-hour period.

Fig 2A shows that the effect of Ang II on thymidine incorporation during the 24 to 48 hour time period was dose dependent and most effective at concentrations of 10 to 100 nmol/L. Losartan completely blocked the effects of Ang II under these conditions, indicating an effect mediated through the AT₁ receptor. Losartan alone (10 µmol/L) had no influence on thymidine incorporation. When thymidine was added at 6-hour intervals during the 24 to 48-hour period following addition of 100 nmol/L Ang II, incorporation was greatest between the 36 and 42-hour interval (Fig 2B).

View larger version (13K): [in this window] [in a new window]   Figure 2. Characterization of the delayed proliferative response to Ang II by cultured cardiac fibroblasts. A, Confluent and quiescent cells were incubated with labeled thymidine between 24 and 48 hours after addition of the designated concentration of Ang II. All culture dishes contained 0.5% FCS. The data represent triplicate determinations, and the data are expressed relative to the incorporation of thymidine by cells not treated with Ang II. B, Cells were treated with Ang II (100 nmol/L) in the presence of 0.5% FCS. Labeled thymidine was added for the designated 6-hour period following the addition of Ang II and FCS.

The delayed response to Ang II was influenced by the addition of various agents and was selectively reduced by certain NO donors (Table 2). The experiments in Table 2 summarize data where thymidine was included between 24 and 48 hours after the addition of Ang II (100 nmol/L), and each indicated agent was added initially with Ang II. The NO donors SNAP and SNP effectively and consistently suppressed the Ang II-induced increase in a dose-dependent manner, and both drugs were effective at concentrations as low as 10 µmol/L. ISDN was considerably less effective, even at relatively high concentrations. SNP and SNAP each release NO by chemical decomposition, whereas ISDN is thought to require enzymatic steps for NO release, which may not be prevalent in the cell culture system. 8-Bromo-cyclic GMP also reduced thymidine incorporation in the 24 to 48-hour time period, whereas the cAMP analogue was without effect. At higher concentrations, the cAMP analogue was inhibitory but less effective than equimolar amounts of cGMP. Addition of L-NAME, an inhibitor of nitric oxide synthases, also did not affect thymidine incorporation. IFN- and TGF-ß1 opposed the Ang II-induced increase in thymidine incorporation, and the cells were particularly sensitive to IFN- addition, whereas TNF- had little effect, even at relatively high doses. In separate experiments, each of the NO donors were added at different concentrations to cells in the absence of Ang II. Each of the drugs had an inhibitory effect (5-20% inhibition) on the baseline levels of thymidine incorporation that were present due to the presence of 0.5% serum. A similar reduction was found for cGMP addition, whereas L-NAME had no effect. Further evidence for a role of cGMP in mediating the effect of NO donors was provided by experiments where the soluble guanylate cyclase inhibitor ODG was added in either the presence or absence of 100 µmol/L SNAP. ODQ (3 µmol/L) completely reversed the inhibitory effects of SNAP on thymidine incorporation, whereas the drug was ineffective when added to cells containing Ang II alone.

View this table: [in this window] [in a new window]   Table 2. Effect of Nitric Oxide and Cytokines on Ang II–Induced Thymidine Incorporation

The experiments summarized in Fig 3 provide additional information on the effects of NO on the delayed response to Ang II. NO addition was accomplished by adding SNAP (100 µmol/L) at 12-hour intervals following Ang II addition. All of these experiments were performed with the addition of labeled thymidine between 36 and 48 hours after Ang II addition. The left panel in Fig 3A shows that maximum suppression of Ang II-induced thymidine incorporation by SNAP occurred when the NO donor was added 12 hours after Ang II, although significant attenuation of the response (P<.01) was also seen when SNAP was added together with Ang II or up to 24 hours after the hormone. N-Acetyl penicillamine (NAP), an analogue of SNAP with no ability to donate NO, was completely ineffective in modulating Ang II-induced chAnges in thymidine incorporation. The data shown in Fig 3B demonstrate that the temporal pattern of SNAP inhibition was mimicked by the addition of 8-bromo-cyclic GMP, but not by cyclic AMP, implicating increased cGMP in the inhibition of cell growth mediated by SNAP. The inhibition of thymidine incorporation was also significant at 10 µmol/L SNAP (20% inhibition, data not shown), and comparable inhibition also was seen when SNP was added.

View larger version (42K): [in this window] [in a new window]   Figure 3. Effect of SNAP and 8-bromo-cyclic GMP on the delayed proliferative response to Ang II. A, SNAP or the analogue NAP was added at the designated time interval following addition of Ang II and 0.5% FCS to quiescent cells. B, 8-bromo-cyclic GMP or 8-bromo-cyclic AMP were added at the designated time interval following the addition of Ang II and 0.5% FCS to quiescent cells. Labeled thymidine was added 36 to 48 h following Ang II addition in all experiments shown.

Despite the delayed DNA synthetic response to Ang II, a rapid change in immediate early gene expression was noted (Fig 4). Fig 4A shows a representative Northern blot of c-fos and Egr-1 mRNA at different times following the addition of Ang II. For both substances, steady-state mRNA levels increased markedly by 1 hour, but the change was transient and gone by 3 hours. Although the temporal pattern differed slightly, transient increases also were found when c-jun and c-myc mRNA were estimated (data not shown). Densitometric analysis for the change in mRNA levels relative to controls and expressed as a ratio to after 1 hour of Ang II treatment were 18-fold, 5-fold, 12-fold and 4-fold for c-fos, Egr-1, c-jun, and c-myc, respectively. These changes were all detectable within 1 hour of Ang II addition and are thought to characterize a transition from G₀ to G₁ of the cell cycle. In Fig 4B, the increases in c-fos and Egr-1 mRNA that occurred after 30 min of Ang II treatment were completely prevented by the inclusion of losartan but were essentially unchanged in the presence of either SNAP, SNP, or 8-bromo-cGMP at concentrations that effectively blocked the delayed proliferative response characterized by labeled thymidine incorporation. In separate experiments (data not shown), induction of c-myc and c-jun mRNA by Ang II also was unaffected by the NO donors, and when each of the NO donors were added to cells not treated with Ang II, there was no change in any of the mRNA moieties for the immediate-early genes.

View larger version (34K): [in this window] [in a new window]   Figure 4. Northern blot analysis showing the effects of Ang II and NO donors on immediate-early gene expression. A, Time course for changes in steady-state mRNA of Egr-1 and c-jun following Ang II addition (100 nmmol/L) to quiescent cells. B, Effects of losartan (10 µmol/L) SNAP (100 µmol/L) SNP (100 µmol/L), and 8-bromo-cGMP (100 µmol/L) on immediate-early gene expression 30 min after Ang II addition. All drugs were added to quiescent cells 15 minutes prior to the addition of Ang II (100 nmol/L).

	Discussion

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These studies define a delayed response to Ang II in quiescent cardiac fibroblasts and show that NO may influence this cellular response even when added 12 hours following Ang II addition, most likely through a mechanism involving cGMP. Thymidine incorporation, which occurs during the S phase of the cell cycle, was maximal between 36 and 42 h after Ang II addition and occurred only when a small amount of serum was present in combination with Ang II. In another study¹⁹ with cultured smooth muscle cells from spontaneously hypertensive rats, Ang II was shown to stimulate cell proliferation only in combination with a submaximal concentration of FCS, analogous to the effect reported in the present study.

The temporal relationship between the addition of Ang II and entry into S phase was similar to the response to Ang II reported by Weber et al²⁰ in rat aortic smooth muscle cells. In those studies, the mechanism involved in the delayed proliferative response to Ang II implicated synthesis or activation of undefined growth factor(s) sensitive to suramin addition, and those latter events were presumably responsible for the subsequent proliferation. Subsequent studies indicated that endothelin and Ang II both mediated their proliferative effects on smooth muscle cells by similar intracellular mechanisms involving inositol phosphate generation, protein kinase C activation, and tyrosine phosphorylation of several protein kinases.²¹ Delayed mitogenesis in response to thrombin activation was studied recently in smooth muscle cells,²² and although a mechanism was not defined, it was ascertained that tyrosine phosphorylation patterns distinct from those found with more classical mitogens occurred rapidly in response to thrombin, and de novo expression of autocrine mitogens was involved.

We found that NO donors did not influence the relatively rapid induction of several immediate-early genes by Ang II. Immediate-early gene induction is a characteristic response of cardiac fibroblasts to Ang II¹¹ and represents early events in the transition from quiescence into the G₁ phase of the cell cycle. The transient time course of induction for c-fos, c-jun, c-myc, and Egr-1, which we observed are typical of changes in immediate-early gene expression following a stimulus to growth, and such increases in steady-state mRNA represent the net effect of many intracellular signaling pathways. Recent studies have indicated that there are diverse intracellular signaling pathways that are activated by Ang II in cardiac fibroblasts, and those events occur within minutes following Ang II addition.³ Tyrosine kinase activity is clearly associated with angiotensin binding to the AT₁ receptor and has been suggested to mediate activation of phospholipase C and increase intracellular inositol triphosphate and calcium ion.²³ The mitogenic response follows activation of the mitogen-activated protein kinase cascade.²⁴ Other studies have shown that the Janus-activated kinase/signal transducer and activator of transcription signaling system can be activated following Ang II addition to cardiac fibroblasts and vascular smooth muscle cells, and this pathway could influence c-fos induction.³ Clearly, there are multiple pathways induced rapidly by Ang II that could ultimately lead to proliferation; multiple targets exist for the observed modulatory role of NO, but these were not obvious in the present study. NO did reduce the expression of the Egr-1 gene when induced by cytokines in rat lung macrophages, although relatively high concentrations of an NO donor were used.²⁵

Because of the effectiveness of NO when added after 12 hours, it appears plausible that the effects occur after the cells have entered into the cell cycle (possibly during G₁) rather than solely influencing the transition from quiescence into the cell cycle (G₀-G₁). An antiproliferative effect for NO has been described for certain fibroblast cell types⁹ and for vascular smooth muscle cells,^{6 8} but the issue is complex, because proliferative effects ascribed to NO have been described as well.^{26 27} Our studies in cardiac fibroblasts have indicated an antiproliferative effect occurring after the G₀-G₁ transition (eg, immediate-early gene transcription) but before S phase. A study describing a specific effect of NO on the cell cycle suggested that NO blocked macrophage-like cells in the G₂ or M phase²⁸ following stimulation with interleukin, but those effects were distinct from that presented in the current study.

Counterregulatory roles for Ang II and NO have been suggested at several levels of physiological control. Both substances can be generated by the vascular endothelial cell and can either induce vasoconstriction or vasodilation. Studies have suggested that treatment with Ang II can lead to increased NO production by endothelial cells as a form of feedback control.²⁹ In the kidney, opposing effects on glomerular and tubular function have been described.³⁰ NO recently was shown to inhibit Ang II-induced migration of rat aortic smooth muscle cells, and this effect was mimicked by 8-bromo-cyclic GMP.³¹ In a model of experimental fibrosis in the rat, aortic rings from cirrhotic rats showed reduced vascular reactivity to Ang II by a mechanism mediated by NO.³² The possibility that Ang II might influence vascular cell function by inducing oxidative stress has recently been suggested,³³ and the relative availability of NO in regions of the aorta where free radicals are being generated could influence the potentially harmful role of oxidative stress in the vasculature.

We have shown⁵ that the in vivo effects of Ang II on the heart were dramatically enhanced by concurrent treatment with an inhibitor of nitric oxide synthase activity. In that study, cardiac fibrosis, characterized in part by fibroblast proliferation, occurred when a subpressor dose of Ang II and NO inhibitor were given together but not when either drug was given alone. Recently, we showed how cardiac fibroblasts, when acted upon by cytokines, would produce NO through the induction of inducible NO synthase.¹⁵ Thus, it is possible that fibroblasts in a region of progressing fibrosis could produce NO and thus influence cellular events in an autocrine or paracrine manner. The present study reports a counterregulatory relationship between Ang II and NO on the cell cycle of cardiac fibroblasts and suggests that local changes in the levels of these important hormones could influence cell growth in pathological situations where fibrosis occurs. Although the molecular basis for this counterregulatory effect remains unresolved, one possible site of action could be on the system of cyclin-dependent kinases or phosphatases that control the cell cycle transition from G₁ into S phase. NO has been reported to influence cell function both indirectly through its ability to enhance cGMP production or more directly by nitrosylation of regulatory proteins containing susceptible sulfhydryl groups.³⁴ It is plausible that the effects reported in the present study are mediated by indirect or direct effects of NO on cyclin-dependent kinases.

Received April 14, 1997; first decision May 12, 1997; accepted May 12, 1997.

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