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(Hypertension. 1997;30:209-216.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II and Myocyte Growth

Role of Fibroblasts

Parames Sil Subha Sen

From the Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation (Ohio).

Correspondence to Subha Sen, PhD, DSc, Department of Molecular Cardiology/FF40, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail sens{at}cesmtp.ccf.org

	Abstract

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Abstract Angiotensin II (Ang II) has been implicated in stimulating myocyte growth in vitro, but the mechanism for such stimulation is still an open question. To understand the role of Ang II, we studied its effect on protein synthesis in rat neonatal and adult myocytes. Ang II (10^-8 mol/L) stimulated protein synthesis in neonatal myocytes by 43±3.5% over control. To prevent the proliferation of fibroblasts, bromodeoxyuridine was added, and protein synthesis in neonatal myocytes was reduced to 21±2.2% over control. In adult myocytes (cultured without bromodeoxyuridine), Ang II stimulated [³H]leucine incorporation by 24±2.3% over control; with bromodeoxyuridine, that stimulation was reduced significantly (13±0.93% over control). These data suggest that the presence of fibroblasts in the cultures may control myocyte growth. When supernatant from pure fibroblast culture was added to myocyte preparations, a significant increase (49.8±3.5% over control) in protein synthesis occurred. Pretreatment of these fibroblasts with Ang II (10^-8 mol/L) further stimulated protein synthesis, suggesting that Ang II directly stimulates the production of a factor from fibroblasts. The stimulatory effect of Ang II on the release of the factor can be completely blocked by pretreatment with losartan, an Ang II receptor (AT₁) blocker. Our data are the first to demonstrate a paracrine effect of a fibroblast-derived factor that modulates myocyte growth. Fibroblast-derived factor loses its biological activity by (1) tryptic digestion, (2) exposure to pH below 4.0 and above 9.0, and (3) heating to 95°C. The molecular weight of the factor is approximately 65 kD. The antibodies against fibroblast growth factor (both acidic and basic) could not inhibit this factor's stimulatory effect. Furthermore, this factor is heart specific and is produced at least up to the 16th passage of neonatal rat heart fibroblasts. Skin fibroblasts, aortic endothelial cells, and aortic smooth muscle cells do not produce this protein. Our data suggest that the observed myocyte growth by Ang II comes about via fibroblast-derived factor, which is increased by Ang II. Cross talk between fibroblasts and myocytes is an important factor in stimulating myocyte growth by Ang II.

Key Words: fibroblast-derived factor • angiotensin II • fibroblasts • myocyte growth

	Introduction

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The renin-angiotensin system has been shown to be integrally involved in cardiovascular homeostasis.¹ Ang II is believed to modulate cardiac function, as evidenced by effects on myocardial contractility and metabolism and hypertrophic growth.^{2 3 4 5 6 7 8} The ventricular remodeling and compensatory hypertrophy that are seen after myocardial infarction are also believed to be related to a local increase in production of Ang II.⁹ Furthermore, several in vivo studies have implicated Ang II in cardiac hypertrophy associated with hypertension.^{10 11} In spontaneously hypertensive rats, administration of angiotensin-converting enzyme inhibitor was shown to prevent and even regress cardiac hypertrophy, while normalization of blood pressure with vasodilators had the opposite effect.^{10 11} Baker et al¹² have shown that infusion of Ang II into rats increased left ventricular mass even when pressor activity of Ang II was blocked or when a subpressor dose of Ang II was used.^{12 13} Recently, in rat models of pressure-overload hypertrophy (cardiac hypertrophy induced by abdominal aortic constriction), treatment with angiotensin-converting enzyme inhibitor completely prevented an increase in left ventricular mass, with no effect on cardiac afterload. These observations suggest that Ang II has a hypertrophic action on the heart tissue in addition to its direct effect of increasing blood pressure and vascular resistance.

Baker et al¹⁴ and others^{15 16} recently showed in vitro that Ang II has a direct hypertrophic effect on cardiomyocytes. However, the biochemical process by which Ang II stimulates cardiomyocyte hypertrophy is not known. In fact, the direct effect of Ang II on myocyte growth is still controversial, on the basis of findings by Saito et al¹⁷ that adult rat ventricles have nearly no Ang II receptors (<10 pmol/mg). Therefore, very few receptors are available to which Ang II can bind.¹⁷ Moalic et al¹⁸ demonstrated that phenylephrine and vasopressin induced the proto-oncogenes c-myc and c-fos coding for nuclear proteins, which play a role in regulatory growth and differentiation after IP injection of 6 mg/kg phenylephrine or 12 IU/kg vasopressin. However, continuous or discontinuous injections of Ang II at a dose of 7.5 mg · kg^-1 · min^-1 for 1 to 2 hours failed to turn on c-myc and c-fos but induced the expression of these two proto-oncogenes in the aorta. The lack of ventricular response to Ang II in rat ventricles has been attributed to the lack of Ang II receptors in this tissue. These data suggest that in addition to other factors that have common use of the phosphatidylinositol pathway, Ang II may activate the expression of various genes coding for regulatory proteins. These factors may play a role in the genesis of both ventricular and aortic hypertrophy.¹⁸ The present study was undertaken to reevaluate the effect of Ang II on both neonatal and adult rat myocyte growth using cultured rat myocytes as a model system. Our data showed that the effect of Ang II on myocyte growth is primarily via its effect on fibroblasts and possibly not a direct effect on myocytes.

	Methods

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Materials and Experimental Procedures
Timed pregnant rats were obtained from Hilltop Farms, Scottsdale, Pa. All normal Wistar rats used for the preparation of adult myocytes and fibroblasts were purchased from Taconic Farms, Germantown, NY. The rats were fed Purina rat chow, given water ad libitum, and housed under sanitary conditions. The experimental procedures for animals were in accordance with institutional guidelines. DVF₁₂, nonessential amino acids, and antibiotic solution used for the culture of neonatal myocytes were purchased from GIBCO-BRL. All other media and reagents used for preparation and culture of the neonatal and adult myocytes and fibroblasts, including Joklik's minimum essential medium, medium 199, FBS, BSA, insulin, transferrin, fetuin, hydrocortisone, and laminin were purchased from Sigma Chemical Company. Collagenase, type II, was purchased from Worthington Biochemicals. [³H]Leucine was obtained from Amersham Corp. Heparin and pentobarbital were purchased from LyphoMed, Inc, and from Abbott Laboratories, respectively. ¹²⁵I-Labeled protein A was purchased from ICN Biomedicals.

Preparation of Neonatal Myocytes
Neonatal myocytes were isolated and cultured on laminin-coated wells following the procedure described by Sen et al,¹⁹ with some modifications. Briefly, hearts from 2- to 3-day-old normal Wistar rat pups were aseptically taken in DVF₁₂ medium; ventricles were separated, minced in DVF₁₂ medium containing collagenase (80 U/mL), and incubated at 37°C for 10 minutes in a water bath. The first supernatant was discarded. The residual tissue was minced and incubated as before. The supernatant was collected and centrifuged at 1000 rpm for 2 minutes. The residue (myocytes) was collected in a 50-mL sterile tube and kept on ice. The procedure was repeated at least five times and the residues were combined. The cells were then suspended in DVF₁₂ medium containing 5% FBS and incubated at 37°C for 1 hour. Myocytes did not attach to the surface of the flask. The supernatant was collected and plated on laminin-coated wells (20 µg laminin per 35-mm well) having a density of 1 to 2x10⁶ cells per 35-mm well. The myocytes were allowed to grow in an incubator at 37°C for the next 24 hours either in the presence or absence of 100 µmol/L BrdU in an atmosphere of 95% O₂ and 5% CO₂. On culture day 2, old medium was aspirated and the myocytes were incubated in fresh DVF₁₂ medium containing fetuin (1 mg/mL), transferrin (25 µg/mL), and hydrocortisone (25 ng/mL) either in the presence or absence of 100 µmol/L BrdU. On culture day 3, myocytes were incubated in DVF₁₂ medium alone.

Preparation and Maintenance of Adult Myocytes in Culture
Calcium-tolerant adult myocytes were isolated, purified, and maintained in culture following the combination of perfusion techniques²⁰ and an attachment procedure²¹ as described by Sil et al.²² Briefly, after rats were killed, the hearts were aseptically excised and residual blood was removed. The heart was perfused in Joklik's medium (containing Joklik's minimal essential medium, 25 mmol/L glutamic acid, 30 mmol/L taurine, and 1 mmol/L adenosine) without recirculation on a modified Langendorff apparatus for approximately 10 minutes at 37°C. The perfusion was then continued for 30 minutes at the same temperature, with recirculation in Joklik's medium containing collagenase type II (100 U/mL). After perfusion, the ventricles were cut into small pieces and the myocytes isolated and cultured on laminin-coated (20 µg per well) 35-mm six-well plates in medium 199 containing 5% FBS.

Effect of [Sar¹]Ang II on Neonatal and Adult Myocyte Growth
To study the effect of [Sar¹]Ang II on protein synthesis in both neonatal and adult myocytes, we cultured the adult myocytes following the procedure described before (in the absence and presence of BrdU). On culture day 3, neonatal myocytes were allowed to grow in DVF₁₂ medium without FBS. [Sar¹]Ang II (10^-8 mol/L) was then added and incubated for 20 hours in the presence of 5 µCi of [³H]leucine per well. The same amount of [Sar¹]Ang II was added every 8 hours. The cells were then lysed by using 1 mL 0.1% SDS solution. A 50-µL aliquot (in duplicate) was taken from each well for the measurement of DNA. DNA was measured following the procedure described by Labarca and Paigen.²³ The lysed samples were then brought to 1N with NaOH solution. The plates were incubated at room temperature for 1 hour. One milliliter of 0.5% BSA solution was then added to each well and incubated for 30 minutes. One milliliter of 20% trichloroacetic acid solution was then added per well and kept for 30 minutes. The protein precipitate from each well was then collected on individual filter paper using a Millipore filter. The collected protein was washed thoroughly with 5% trichloroacetic acid until it was free from unbound radioactivity. Each filter paper was air dried for 1 hour and then counted in a beta counter after adding scintillation fluid. Data were expressed as disintegrations per minute per nanogram DNA. For control wells, instead of Ang II, buffer was added and the assay performed following the usual procedure. We used norepinephrine as a positive control to validate our bioassays. All the bioassays using adult myocytes were performed on culture day 2 following the same procedure as described for neonatal myocytes.

Preparation of Fibroblasts and Fibroblast-Conditioned Medium
Neonatal rat cardiac fibroblasts were isolated from 2- to 3-day-old normal Wistar rat pups. Briefly, hearts from 2- to 3-day-old rat pups were aseptically taken in DVF₁₂ medium and ventricles were separated, minced in DVF₁₂ medium containing collagenase (80 U/mL), and incubated at 37°C for 10 minutes in a water bath. The first supernatant was discarded. The residual tissue was minced and incubated as before. The supernatant was collected and centrifuged at 1000 rpm for 2 minutes. The residue (mixture of fibroblasts and myocytes) was collected in a 50-mL sterile tube and kept on ice. The procedure was repeated at least three times, and the residues were combined. The cells were then suspended in DVF₁₂ medium containing 5% FBS and incubated for 1 hour at 37°C in a sterile flask. Supernatant was collected and used for the myocyte preparation. Fibroblasts were attached on the surface of the flask and allowed to grow in 10% FBS until they were confluent. Cells were then split by trypsinization and grown again (passage 2) in 10% FBS until confluent. The cells were then kept in serum-free medium for 24 hours. That medium was aspirated and fresh serum-free medium added. After 24 hours, the medium was collected and used as conditioned medium.

Effect of Neonatal Fibroblast–Conditioned Medium on Protein Synthesis in Neonatal Myocytes and Neonatal Fibroblasts
To study the effect of the neonatal fibroblast–conditioned medium on protein synthesis in neonatal myocytes and neonatal fibroblasts, we cultured neonatal myocytes following the procedure as described before. Neonatal fibroblasts were also grown in six-well plates following the procedure as described before. On culture day 3, neonatal myocytes and fibroblasts were allowed to grow in DVF₁₂ medium without FBS. An appropriate amount of the conditioned medium was then added per well (of both myocytes and fibroblasts) and incubated for different periods of time (up to 24 hours) in the presence of 5 µCi of [³H]leucine per well. The cells were lysed and [³H]leucine incorporation into myocyte or fibroblast protein was determined following the procedures as described before.

Effect of Skin Fibroblast–, Aortic Endothelial Cell–, and Aortic Smooth Muscle Cell–Conditioned Media on Myocyte Growth
To find out the effect of the above-mentioned conditioned media on neonatal myocyte growth, we cultured those cells in DVF₁₂ medium containing 10% FBS until they were confluent. After thorough washing, the cells were kept in serum-free DVF₁₂ medium for 24 hours. The medium was aspirated and the cells were kept again in the same serum-free medium for 24 hours. The resulting conditioned media were collected and used for the bioassay as described before to study their effect on [³H]leucine incorporation into myocyte protein.

Effect of the Antibodies of Acidic and Basic FGFs on FDF–Induced Myocyte Growth
Neonatal myocytes were isolated and cultured as described before. On culture day 4, myocytes were preincubated separately with the IgG of both the acidic and basic FGFs for 2 hours in serum-free DVF₁₂ medium. FDF was then added and [³H]leucine incorporation into myocyte protein measured as described before.

Effect of Ang II on the Stimulation of FDF
To find the effect of Ang II on the stimulation of FDF, neonatal rat heart fibroblasts were cultured in FBS until they were confluent. The cells were then kept 24 hours in serum-free medium and treated with 10^-8 mol/L [Sar¹]Ang II for 24 hours. [Sar¹]Ang II was added every 8 hours. Conditioned medium was then collected, dialyzed exhaustively using a 5-kD cut-off dialysis bag, and used for the bioassays. The bioassays were performed with neonatal rat cardiac myocytes following the procedure as described before to study [³H]leucine incorporation into myocyte protein.

Effect of Losartan on Ang II–Induced Myocyte Growth
Neonatal rat heart fibroblasts were cultured as described before. In serum-free DVF₁₂ medium, the fibroblasts were treated with 10^-8 mol/L losartan for 2 hours. [Sar¹]Ang II (10^-8 mol/L) was then added and the same procedure followed for the collection of the conditioned medium and bioassays as described for the previous experiment.

Purification and Characterization of FDF
The conditioned medium from pure fibroblast culture was concentrated by using different-molecular-weight cut CENTRICON filters (3 kD, 10 kD, 30 kD, 50 kD, and 100 kD). The biological activity of all the fractions was determined. The active fraction was collected and precipitated by using 85% saturation with (NH₄)₂SO_4. The precipitate was collected by centrifugation at 10 000g and dialyzed exhaustively against water and PBS. A fraction was run on a 4% to 20% SDS-polyacrylamide gel to determine the molecular weight of FDF. The biological activity was also determined by using another fraction of this preparation.

Measurement of Protein
Protein measurements were performed following the Bradford²⁴ protein microassay method using BioRad reagents. The standard curve was drawn by using BSA at different concentrations (5 to 25 µg of protein) and absorbance at 595 nm.

Statistical Analysis
Statistical analysis was done by Student's paired t test and ANOVA where appropriate. For protein synthesis studies, four to six culture plates (six wells per plate) were used in each experiment. Experimental values for the treated groups were normalized to the control values (vehicle treated) in each experiment. Results were expressed as mean±SEM. The difference between two groups was tested by an unpaired Student's t test. Differences among more than two groups were tested by ANOVA for multiple sample comparison.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Ang II on Neonatal Myocyte Growth
The effect of Ang II on the protein synthesis of neonatal myocytes is summarized in Fig 1, bar A. When 10^-8 mol/L [Sar¹]Ang II was added to neonatal myocytes in the absence of BrdU, a significant increase of protein synthesis was observed in neonatal myocyte protein, as defined by the incorporation of [³H]leucine into neonatal myocyte protein over control (43±3.5% over control). Fig 1, bar A shows the result. This stimulatory effect of Ang II on neonatal myocyte protein synthesis was significantly reduced when myocytes were cultured in the presence of 100 µmol/L BrdU (21±2.2%; Fig 1, bar B).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effect of [Sar1]Ang II on neonatal myocyte growth. Neonatal myocytes were cultured both in the presence and absence of 100 µmol/L BrdU as described in "Methods." On culture day 3, the cells in serum-free medium were incubated with 10-8 mol/L [Sar1]Ang II for 24 hours in the presence of 5 µCi of [3H]leucine. The cells were lysed with SDS solution, and the incorporation of [3H]leucine into myocyte protein was measured as described in "Methods." In a parallel set of experiments, myocytes treated with serum-free medium were used as controls. Data shown are the average of five different sets of results and are expressed as mean±SEM. [Sar1]Ang II significantly stimulated [3H]leucine incorporation into myocyte protein (bar A). Treatment with BrdU in myocyte culture significantly reduced that stimulation (bar B).

Effect of Ang II on Adult Myocyte Growth
The effect of [Sar¹]Ang II on the protein synthesis of adult myocytes is shown in Fig 2. When 10^-8 mol/L [Sar¹]Ang II was added to adult myocytes in the absence of BrdU, a significant increase of protein synthesis into adult myocyte protein was observed, as defined by the incorporation of [³H]leucine into adult myocyte protein (24±2.3% over control; Fig 2, bar A). The stimulatory effect of Ang II on [³H]leucine incorporation into adult myocyte protein was significantly reduced when myocytes were cultured in the presence of 100 µmol/L BrdU (13±0.93%; Fig 2, bar B).

View larger version (42K): [in this window] [in a new window]   Figure 2. Effect of [Sar1]Ang II on adult myocyte growth. Adult myocytes were isolated and cultured both in the presence and absence of 100 µmol/L BrdU as described in "Methods." The cells in serum-free medium were incubated with 10-8 mol/L [Sar1]Ang II for 24 hours in the presence of 5 µCi of [3H]leucine. The cells were lysed with SDS solution, and the incorporation of [3H]leucine into myocyte protein was measured as described in "Methods." Bar A shows that [Sar1]Ang II significantly stimulated the incorporation of [3H]leucine into adult myocyte protein over control. The stimulatory effect of [Sar1]Ang II on the [3H]leucine incorporation into the adult myocyte protein was significantly reduced when the adult myocytes were cultured in the presence of 100 µmol/L BrdU. Bar B shows the result. Data shown are the average of five different sets of results and are expressed as mean±SEM.

Effect of Neonatal Heart Fibroblast–Conditioned Medium on Neonatal Myocyte and Neonatal Fibroblast Growth
The effect of the neonatal heart fibroblast–conditioned medium on the incorporation of [³H]leucine into neonatal myocyte and neonatal fibroblast protein is shown in Fig 3. Neonatal myocytes were cultured in the presence of 100 µmol/L BrdU. Neonatal fibroblasts used for this assay were obtained from the third subculture (passage 3). The conditioned medium obtained from fibroblasts significantly stimulated [³H]leucine incorporation into neonatal myocyte protein (49.8±3.5% over control). On the other hand, the same conditioned medium did not show any significant stimulatory effect on [³H]leucine incorporation into neonatal fibroblast protein (5±1.29% over control).

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of neonatal rat heart fibroblast–conditioned medium on neonatal myocyte and neonatal fibroblast growth. Neonatal myocytes were cultured in the presence of 100 µmol/L BrdU. All the assays were performed in serum-free medium. Conditioned medium (100 µL per well; 1 to 2x106 myocytes per well) was used for these experiments. Neonatal myocytes and separately neonatal fibroblasts were incubated with FDF in the presence of 5 µCi of [3H]leucine for 24 hours. The cells were lysed, and [3H]leucine incorporation into cellular protein was measured. The FDF significantly stimulated the protein synthesis in myocytes but did not show any significant stimulatory effect on [3H]leucine incorporation into neonatal fibroblast protein. Myo indicates myocyte; Cont, control; and Fib, fibroblast.

Effect of Neonatal Myocyte–Conditioned Medium on Neonatal Myocyte Growth
The effect of conditioned medium from myocytes on protein synthesis in neonatal myocytes is shown in Fig 4. To evaluate whether conditioned medium obtained from myocytes in culture would show a stimulatory effect similar to that of fibroblast supernatant, neonatal myocytes were cultured in the presence of 100 µmol/L BrdU. The serum-free conditioned medium was collected and its effect on protein synthesis in neonatal myocytes assessed. Myocyte-conditioned medium did not show any stimulatory effect on [³H]leucine incorporation into neonatal myocyte protein (3±0.97% over control).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of neonatal myocyte supernatant on neonatal myocyte growth. Neonatal myocytes were cultured in the presence of 100 µmol/L BrdU as described previously. The supernatant was collected and its effect on the protein synthesis in neonatal myocyte was determined. In a parallel set of experiments, FDF was used instead of myocyte supernatant. Myocyte-conditioned medium did not show any stimulatory effect on [3H]leucine incorporation into neonatal myocyte protein. Myo indicates myocyte; Cont, control; and Sup, supernatant.

Time-Dependent and Dose-Dependent Stimulatory Effect of FDF on Myocyte Growth
When FDF was added to myocytes and incubated for 2 to 24 hours, a linear time-dependent stimulation of [³H]leucine incorporation into myocyte protein was observed up to 24 hours (Fig 5). When FDF was added to neonatal myocytes at various concentrations from 10 µL to 1 mL, a dose-dependent stimulation of [³H]leucine incorporation was also observed by FDF into neonatal myocyte protein (Fig 6).

View larger version (14K): [in this window] [in a new window]   Figure 5. Time-dependent stimulatory effect of FDF on myocyte growth. Myocytes were isolated and cultured as described before. The study was conducted up to 24 hours. For experimental detail, see "Methods."

View larger version (35K): [in this window] [in a new window]   Figure 6. Dose-dependent effect of [3H]leucine incorporation into myocyte protein by FDF. The experimental procedure was described in the legend to Fig 3. FDF showed a dose-dependent stimulatory effect of [3H]leucine incorporation into myocyte protein.

Effect of Skin Fibroblast–, Aortic Endothelial Cell–, and Aortic Smooth Muscle Cell–Conditioned Media on Myocyte Growth
Fig 7 shows the effect of media conditioned with skin fibroblasts, endothelial cells, and aortic smooth muscle cells on [³H]leucine incorporation into myocyte protein. These conditioned media did not show any stimulatory effect on the protein synthesis of neonatal myocytes.

View larger version (25K): [in this window] [in a new window]   Figure 7. Effect of skin fibroblast–, aortic endothelial cell–, and aortic smooth muscle cell–conditioned media on neonatal myocyte growth. The experimental procedure for this study was described in the legend to Fig 3. These conditioned media did not show any stimulatory effect on [3H]leucine incorporation into myocyte protein. HT indicates heart; FIB, fibroblast; CM, conditioned medium; SK, skin; ASMC, aortic smooth muscle cell; and AEC, aortic endothelial cell.

Effect of Antibodies of Both Acidic and Basic FGFs on FDF-Induced Myocyte Growth
Fig 8 shows the effect of FGF (acidic and basic) antibodies on FDF-induced protein synthesis in neonatal myocytes. These antibodies did not prevent any FDF-induced stimulation of [³H]leucine incorporation into neonatal myocyte protein. These data suggest that FDF is not similar to FGFs (acidic or basic).

View larger version (45K): [in this window] [in a new window]   Figure 8. Effect of the antibodies of both acidic and basic FGFs on FDF-induced myocyte growth. On culture day 4, myocytes were preincubated separately with the IgG of both acidic and basic FGFs for 2 hours in serum-free DVF12 medium. FDF was then added and [3H]leucine incorporation into myocyte protein was measured as described before. These antibodies did not show any inhibitory effect on the stimulation of [3H]leucine incorporation into myocyte protein induced by FDF. aFGF indicates acidic FGF; bFGF, basic FGF.

Effect of Ang II on the Stimulation of FDF
To evaluate the role of Ang II on fibroblasts, Ang II (10^-8 mol/L) was added to pure fibroblast culture (passage 3) and its effect on myocyte protein synthesis evaluated. The effect of Ang II on the stimulation of FDF in neonatal rat cardiac myocyte protein synthesis is shown in Fig 9. FDF (made without Ang II treatment) showed considerable stimulation of [³H]leucine incorporation into neonatal cardiac myocyte protein (49±3.5% over control). Treatment of fibroblasts with Ang II significantly enhanced the stimulatory effect of FDF on the protein synthesis of neonatal cardiac myocytes (76.3±3.1% over control). Data suggest that Ang II potentiated the release of FDF from fibroblasts, which in turn increased protein synthesis in the neonatal cardiac myocytes.

View larger version (42K): [in this window] [in a new window]   Figure 9. Effect of [Sar1]Ang II on the stimulatory effect of FDF on neonatal rat cardiac myocyte protein synthesis. The bar graph shows the effect of FDF alone and the effect of FDF stimulated by Ang II. Conditioned medium (100 µL per well; 1 to 2x106 myocytes per well) was added. FDF alone without [Sar1]Ang II treatment showed approximately 50% stimulation of [3H]leucine incorporation into neonatal cardiac myocyte protein over control. Treatment of fibroblasts with [Sar1]Ang II significantly enhanced the stimulatory effect of FDF on the protein synthesis of neonatal cardiac myocytes. Data suggest that [Sar1]Ang II potentiated the release of FDF from fibroblasts, which in turn showed the increased protein synthesis in the neonatal cardiac myocytes.

Effect of Losartan on Ang II–Induced Release of FDF
Fig 10 shows the effect of losartan on the Ang II– potentiated release of FDF from rat neonatal cardiac fibroblasts. FDF alone increased [³H]leucine incorporation into neonatal cardiac myocyte protein. The stimulatory effect of FDF was significantly increased when the conditioned medium was collected after treatment of the fibroblasts with Ang II. However, when the fibroblasts were pretreated with losartan followed by Ang II treatment, the enhanced increase of the [³H]leucine incorporation into the myocyte protein was inhibited to the level of that of FDF alone (FDF alone, 43±2.8%; Ang II treatment+ FDF, 75±3.49%; and losartan pretreatment+Ang II treatment+FDF, 45±3.23% over control). These results show that Ang II has little direct effect on myocyte protein synthesis. Instead, it potentiates the release of FDF.

View larger version (39K): [in this window] [in a new window]   Figure 10. Effect of losartan on [Sar1]Ang II induced release of FDF from rat neonatal cardiac fibroblasts. Conditioned medium (100 µL) was used. FDF alone increased the [3H]leucine incorporation into neonatal cardiac myocyte protein approximately 45% over control. The stimulatory effect of FDF was significantly increased (76.3%) when the conditioned medium was collected after the treatment of the fibroblasts with [Sar1]Ang II. However, the enhanced increase of the [3H]leucine incorporation into the myocyte protein was completely inhibited when the fibroblasts were pretreated with losartan followed by [Sar1]Ang II treatment. These results show that [Sar1]Ang II has very little direct effect on the protein synthesis into myocytes. Los indicates losartan.

Purification and Characterization of FDF
Fig 11 shows the partial purification and characterization of FDF. On the left, bioassays with different-molecular-weight fractions are shown and on the right, the results of SDS-polyacrylamide gel electrophoresis. These data suggest that FDF is a protein molecule with an approximate molecular weight of 65 kD. Some of the properties of FDF are summarized in the Table.

View larger version (35K): [in this window] [in a new window]   Figure 11. The partial purification and characterization of FDF. Left, Bioassays with different-molecular-weight fractions. Right, SDS-polyacrylamide gel electrophoresis. For experimental detail, see "Methods." These data suggest that FDF is a protein molecule with an approximate molecular weight of 65 kD.

View this table: [in this window] [in a new window]   Table 1. Properties of Fibroblast-Derived Factor (Molecular Weight 65 kD)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we evaluated the effect of Ang II on myocyte growth. We have shown that Ang II acts on fibroblasts and that a factor produced by fibroblasts, FDF, stimulates myocyte growth. We have also shown²⁵ that FDF has no effect on pure fibroblast protein synthesis (5±1.29% over control) and that it exerts its effect on myocytes only. In addition, we have shown that FDF is a protein molecule with a molecular weight of approximately 65 kD and that it has no apparent similarity to either acidic or basic FGF. This is the first demonstration of a paracrine effect of fibroblasts on myocyte growth. Our data suggest that Ang II affects myocyte growth primarily via its effect on fibroblasts.

The renin-angiotensin system has been shown to be involved in cardiovascular homeostasis.¹ The physiological action of the renin-angiotensin system is through the octapeptide Ang II. Angiotensinogen, a precursor of Ang II, is hydrolyzed by an enzyme, renin, resulting in the formation of a decapeptide, Ang I. Angiotensin-converting enzyme splits the two amino acids from the carboxy-terminal of Ang I; thus the octapeptide Ang II, the most potent vasoconstrictor agent known to date, is formed. The cardiac effect of Ang II has been viewed in the context of both circulating Ang II and locally produced peptide, models that necessitated a specific receptor and signal-transduction system.^{26 27 28}

Ang II has been shown to induce a positive inotropic response in many species, including dog,^{29 30} cat,^{6 31} rabbit,³² cattle,³³ chicken,³⁴ human,³⁵ and cardiomyopathic hamster.^{35 36} In contrast, Ang II has been shown to have no effect on the hearts of guinea pigs^{34 37} or adult rats,³⁸ although Ang II has been shown to stimulate contractility of isolated adult rat ventricular myocytes³⁹ in culture. This inotropic effect of Ang II is dose dependent and can be blunted by Ang II receptor antagonists, which suggests that the effect of Ang II is a receptor-mediated mechanism.^{2 3 34 35} Therefore, how Ang II affects myocyte growth remains an open question. Although several studies have claimed that Ang II directly affects myocyte growth, other reports do not support this notion. Saito et al¹⁷ have shown that Ang II binding sites in rat hearts are very small and have demonstrated the existence of a very small number of receptors in adult myocytes. Moalic et al¹⁸ have shown also that infusion of Ang II (whether continuous or discontinuous) failed to increase the gene expression of two heat-shock proteins (HSP 68 and HSP 70) and two oncogenes, c-myc and c-fos, in the ventricle but stimulated both oncogene and heat-shock protein gene expression in the aorta.¹⁸ These authors attributed the lack of response in the heart to the absence of Ang II receptors. All these studies opened up the question of how Ang II promotes myocyte growth.

In the present study, we have shown that when neonatal myocytes are contaminated with fibroblasts, addition of Ang II (10^-8 mol/L) stimulates myocyte protein synthesis, but in the presence of BrdU, which inhibits proliferation of the fibroblasts, a significant reduction of stimulation in protein synthesis is observed. When myocytes were cultured in the absence of BrdU, an increased number of fibroblasts were found, and especially on day 4 of the myocyte culture (approximately 10% to 15% fibroblasts), a significant increase in myocyte protein synthesis (Fig 1) was noted. On the other hand, when the same experiment was performed using myocytes cultured in the presence of BrdU with a reduced number of fibroblasts, the protein synthesis was reduced to 21% from 43% (Fig 1). Therefore, the presence of fibroblasts appears to play an important role in the degree of stimulation of myocyte growth observed due to addition of exogenous Ang II. We have shown that when Ang II¹⁰ is added to pure fibroblasts and the supernatant from the fibroblasts is added to myocytes in culture, a significant increase in stimulation of protein synthesis is obtained. This finding suggests that a factor from the fibroblasts is stimulating myocyte growth. This observation will help to explain the reason for the variability in the effect of Ang II on myocyte growth between laboratories. The quality of the culture (including the number of fibroblasts present as a contaminant) is an important factor, which may vary from one laboratory to another.

Our studies have demonstrated that the stimulatory effect of FDF can be enhanced by Ang II in a dose-dependent fashion (Fig 6). This response can be blocked by losartan, a specific Ang II receptor blocker. This observation suggested that the effect of Ang II on myocytes is a receptor-mediated mechanism. Dostal et al⁴⁰ have shown that Ang II receptors are present in fibroblasts. Our studies have also shown that FDF is specific for myocytes and had no effect on fibroblasts, as FDF did not show any change in protein synthesis in pure fibroblast culture (Fig 3). The source of fibroblasts is also an important criterion. In a preliminary study we showed that FDF obtained from skin fibroblasts had no effect on myocyte growth. Although the exact nature of this factor has not been fully elucidated yet, preliminary characterization suggests that it is a protein molecule, as its activity is destroyed by trypsin digestion, heating at high temperature, and exposure to either high or low pH. The molecular weight of this factor has been estimated to be approximately 65 kD by the molecular sieve exclusion technique.

Other factors that may modulate cardiovascular structure have been demonstrated to be released by fibroblasts. Long et al^{41 42} have shown the existence of a factor produced by nonmyocytes (mainly fibroblasts) after stimulation with norepinephrine that also stimulates myocyte growth. They have shown that the growth response of myocytes to that factor was a function of nonmyocyte number and conditioning time. The nonmyocyte-derived factor that showed myocyte growth-promoting activity bound to heparin-Sepharose and could be eluted with 0.75 mol/L NaCl. That factor was named NMDGF. Long et al^{41 42} showed that myocytes in cultures containing 37% nonmyocytes appeared larger than myocytes in the control cultures containing 10% nonmyocytes. They observed that in addition to playing an active role in the process of cardiac myocyte growth under control conditions, the nonmyocyte fraction of the heart is capable of augmenting the myocyte hypertrophic response to adrenergic stimulation via a paracrine mechanism. Specifically, cardiac nonmyocytes treated with the ß-adrenergic agonist isoproterenol produced a conditioned medium whose growth-promoting effects for cardiac myocytes exceeded those of medium conditioned in the absence of this adrenergic stimulation. Furthermore, the response appeared to be ß-adrenergic specific, since the -adrenergic agonist phenylephrine did not reproduce the effect. Long et al^{41 42} have also shown that treatment of myocytes with NMDGF did not change [³H]inositol phosphate production. They suggested that this factor might work through the action of a member of the transforming growth factor-ß family of growth factors. However, they have not yet identified and characterized the factor.

It is difficult to determine whether NMDGF is similar to or different from the FDF that we identified in our laboratory. The effect of Ang II on the factor identified by Long et al^{41 42} has never been studied. Baker and Aceto² and Baker et al³ have demonstrated the effect of Ang II on neonatal myocytes in culture. The number of fibroblasts present in the studies by Baker et al¹⁴ is difficult to estimate, but it is possible that the presence of fibroblasts is responsible for the myocyte growth and the signal-transduction mechanism is due to its effect on fibroblasts.

Finally, our studies have convincingly demonstrated that a factor released from the fibroblasts is responsible for myocyte growth and that the potency of this factor can be increased by Ang II. Although the exact structure of FDF is not known yet, it appears from the molecular weight that it is a novel factor. Work is in progress to identify its structure. It can be concluded that to prevent or regress hypertrophy, the objective should be not only to block the Ang II receptors but also the FDF receptors, which appear to be an important pathway for stimulation of myocyte growth. Perhaps a combination of an Ang II receptor on the fibroblast and an FDF receptor blocker on the myocytes would be the best way to prevent the development of hypertrophy or to achieve its regression.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II BrdU = bromodeoxyuridine FBS = fetal bovine serum FDF = fibroblast-derived factor FGF = fibroblast growth factor NMDGF = nonmyocyte-derived growth factor

	Acknowledgments

 
This study was supported in part by the National Institutes of Health grants HL-47794 and HL-27838. We are grateful to Vijaya Kandaswamy for her expert assistance in culturing myocytes and fibroblasts, David Young for technical assistance, JoAnne Holl for typing the manuscript, and Christine Kassuba for editorial assistance.

Received December 2, 1996; first decision December 6, 1996; accepted January 3, 1997.

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