Hexarelin suppresses cardiac fibroblast proliferation and ... - CiteSeerX

Page 1 Articles of 26 in PresS. Am J Physiol Heart Circ Physiol (August 31, 2007). doi:10.1152/ajpheart.00004.2007

Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat

Xiangbin Xu1, PhD; Jinjiang Pang1, MD, PhD; Hongchao Yin2, MD, Meixiu Li3, MD; Wei Hao1, BS; Chen Chen4, MD, PhD; Ji-Min Cao1, MD, PhD 1

Department of Physiology and Pathophysiology and 2Department of Pathology, Institute of

Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medical Sciences Peking Union Medical College, Beijing, China; 3

Department of Regional Anatomy, College of Basic Medicine, Jiamusi University, Jiamusi,

China; 4

Endocrine Cell Biology, Prince Henry’s Institute of Medical Research, Melbourne, Australia.

Short title: Xu, et al. Hexarelin and fibroblast

For correspondence: Chen Chen, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia (Tel: 61 3 95944371; Fax: 61 3 95946125; Email: [email protected]); or Ji-Min Cao, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medical Sciences Peking Union Medical College, Beijing 100005, People’s Republic of China. Tel & Fax: 8610 65296959. Email: [email protected].

This work supported by NSFC and NKBRP of China and NHMRC of Australia.

Copyright Information Copyright © 2007 by the American Physiological Society.

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Abstract Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are however not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with Ang II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing MTT assay, 3H-thymidine incorporation and 3H-proline incorporation. Level of mRNA of TGF- was evaluated by RT-PCR, and active TGF- 1 release from cardiac fibroblasts was evaluated by ELISA. Level of cellular cAMP was measured by radioimmunassay. In addition, the effects of DMPX (a specific adenosine receptor A2R antagonist) and DPCPX (a specific A1R antagonist) were tested. It was found that incubation with 10-7 mol/L hexarelin for 24 hours 1) inhibited the Ang II-induced proliferation and collagen synthesis, and the 5% FCS and TGF- induced increase of DNA synthesis in cardiac fibroblast; and 2) reduced Ang II-induced upregulation of TGF- mRNA expression and active TGF- 1 release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10-8 mol/L), but not DPCPX, abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A2R and diminishment of Ang II-induced increase in TGFexpression and release.

Key words: GHS, GHRP, fibroblast, heart, collagen

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Introduction Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac remodeling induced by hypertension and myocardial ischemia/reperfusion injury (7). Cardiac fibroblasts , which constitute 60% of the total heart cells, contribute to the pathologic structural changes in the heart by undergoing proliferation, deposition of extracellular matrix (ECM) proteins such as collagen, and replacing myocytes with fibrotic scar tissue (17). Thus, fibroblastinduced cardiac remodeling may participate in diastolic and systolic dysfunction, leading to congestive heart failure (7). Hexarelin is one of the synthetic growth hormone secretagogues (GHS). GHS possess strong growth hormone–releasing effects and other neuroendocrine activities, such as stimulatory effects on prolactin and adrenocorticotropic hormone secretion, and influence on sleep and food intake (21, 43). These activities are mediated by specific receptors, GHSR, which have also been identified in several peripheral tissues other than the hypothalamus-pituitary system, particularly in the myocardium, where they probably mediate GH-independent activities (36). Recent evidence indicates that GHS feature a variety of cardiovascular activities, including increase of myocardial contractility (4, 5, 44), improvement of left ventricular dysfunction and left ventricular pathological remodeling (6, 25, 34), protection of cardiomyocytes from Angiotensin II (Ang II)-induced apoptosis (35) and myocardial infarction- or pressure overload-induced heart failure in vivo (33, 37, 45). However, the cellular and molecular mechanisms underlying the effect of GHS on cardiac fibroblasts have not been investigated. Using cultured 8-day-old rat cardiac fibroblasts, we tested whether hexarelin 1) inhibits cardiac fibroblast proliferation (3H-thymidine incorporation and MTT assay) when stimulated with fetal calf serum (FCS) or Ang II; 2) blocks collagen synthesis by cardiac fibroblasts stimulated with

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Ang II in vitro (3H-proline incorporation into collagenase-sensitive proteins); or 3) affects the expression and release of transforming growth factor- (TGF- ) by cardiac fibroblasts. We also tested whether DPCPX, a selective A1 adenosine receptor (A1R) inhibitor, or DMPX, a selective A2 adenosine receptor (A2R) inhibitor, blocks FCS-induced DNA synthesis. Methods Reagents Hexarelin was provided by Dr. R Deghenghi (Europeptide, France). DPCPX, DMPX, and Ang-II were purchased from Sigma (USA). Cardiac fibroblast culture Primary cultures of rat cardiac fibroblasts were prepared and characterized as described recently with a minor modification (19). Briefly, hearts were removed under aseptic conditions from 8day-old Sprague-Dawley rats that were overdosed by sodium pentobarbital. All animal experiments were conducted in compliance with the regulations of the Ethics Committee of Peking Union Medical College. The cardiac ventricles were minced into 2- to 3-mm3 fragments. Digestion was performed by four to six 15-minute periods of incubation at 37°C with HEPESbuffered saline solution containing (mmol/L) HEPES-NaOH 20, pH 7.6, NaCl 130, KCl 3, NaH2PO4 1, and glucose 4, along with 3.3 µmol/L phenol red containing 0.1% collagenase II (Sigma), 0.1% trypsin (GIBCO), 15 µg/mL DNase I (Sigma), and 1.0% chicken serum (GIBCO) at 37°C. At the end of each cycle, the supernatant was stored on ice after the addition of newborn calf serum (10% [vol/vol]) to neutralize trypsin. The dissociated cells were collected by centrifugation at 1000g for 10 minutes at 4°C and resuspended in DMEM/F-12 Ham supplemented with 5% horse serum, 3 mmol/L pyruvic acid, 100 µmol/L ascorbic acid, 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL sodium selenite, 100 U/mL penicillin, 100 µg/mL

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streptomycin, and 0.25 mg/L amphotericin B. Cell suspensions were passed through a 200µ mesh screen (Sigma) into a 100-mm culture dish and incubated for 45 minutes at 37°C. Unattached cells were discarded; attached cells were washed twice with 10% fetal calf serum (FCS)/DMEM and allowed to grow to confluence before passage in 1:3 dilutions of trypsin-based solution. All cells used in these experiments were taken from passage 2-3. MTT assay Fibroblast proliferation was evaluated by MTT assay, which is based on the transformation of tetrazolium salt MTT by active mitochondria to an insoluble formazan salt. Cardiac fibroblasts (plating density, 120 cells/mm2) were treated in 96-well plates and grown until 70% to 80% confluent, synchronized in medium containing 0.1% FCS, and then stimulated to grow synchronously by adding 10-7 mol/L Ang II to the culture medium. After 24 hours of treatment with and without hexarelin, MTT was added to each well under sterile conditions (with a final concentration of 0.5 mg/mL), and the plates were incubated for 4 h at 37°C. Untransformed MTT was removed by aspiration, and formazan crystals were dissolved in dimethyl sulfoxide (150 µL/well). Formazan was quantified spectroscopically at 540nm using a Bio-Rad automated EIA Analyzer (Bio-Rad Laboratories, USA). The experiments were performed in triplicate with different preparations of fibroblasts. 3

H-thymidine incorporation

Fibroblasts were seeded onto 24-well plates containing DMEM supplemented with 10% FCS at a density of 2×104 cells per well and allowed to grow until subconfluent, occupying 60% to 70% of the total surface of the plate. Cells were cultured in serum-free DMEM for 24 hours and then treated with 5% FCS either alone or combined with hexarelin (10-7 mol/L). After 20 hours, the

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treatments were repeated with freshly prepared solutions but supplemented with 3H-thymidine (1 mCi/mL, NEN) for an additional 4 hours. The procedure for quantitating the incorporation of labeled thymidine was essentially as described by Itoh et al. (23). 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 icecold 10% trichloroacetic acid, then incubated at 4°C for 30 minutes in 10% trichloroacetic acid. Following aspiration, the cell residue was 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. Triplicate experiments were performed with different preparations of cells. 3

H-proline incorporation

Collagen synthesis by confluent cardiac fibroblasts was measured according to the method described by Brilla et al. (10). Briefly, fibroblasts from passage 2 (1×105 cells/well) were seeded onto 24-well plates containing 10% FCS/DMEM and allowed to grow until confluent. Cells were cultured in serum-free DMEM for 24 hours before the medium was replaced with 0.5% FCS/DMEM. Cells were treated with hexarelin (10-7 mol/L) and/or Ang II (10-7 mol/L) for 18 hours, followed by 6-hour exposure to 3H-proline (14 µCi/well) in fresh serum-free DMEM containing 10-7 mol/L Ang II either alone or combined with hexarelin. After incubation, cells were sonicated on ice, and TCA (final concentration 10% wt/vol) was used to precipitate proteins in the presence of 0.04% proline and 0.1% BSA. The samples were allowed to stand overnight at 4°C before centrifugation. Protein pellets were washed 3 times with 1 mL of 5% TCA/1 mmol/L proline, and the final pellet was dissolved in 1 mL of 0.2 mol/L NaOH. Fibroblast proteins were incubated with 1 mmol/L CaCl2 and 2.5 mmol/L N-ethylmaleimide in the presence of either

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collagenase type III (50 U/mL; Calbiochem) or 2 mmol/L Tris (pH 7.6) and 0.2 mmol/L CaCl2 for 90 minutes at 37°C. The vials were placed on ice, and 0.5 mL of 20% TCA/0.5% tannic acid was added to precipitate protein for 1 hour. Supernatants were transferred to scintillation vials together with 0.5 mL 5% TCA after centrifugation at 10 000 rpm for 5 minutes. Scintillation fluid (10 mL) was added to each sample, and radioactivity was determined with a liquid scintillation counter. Triplicate experiments were performed with different cell preparations. RT-PCR Total RNA was isolated from cardiac fibroblasts by Trizol reagent (Gibco BRL, Life Technologies). Two µg of total RNA from each sample were reverse-transcribed into cDNA with SUPPERSCRIPTTM First-Strand Synthesis System (Gibco BRL). The PCR was performed as previously described (35) in a 50µL reaction volume containing 0.5µmol/L-1 of each primer. TGF- , sense: 5'- GCC CTG GAC ACC AAC TAT TGC T -3', antisense: 5'- AGG CTC CAA ATG TAG GGG CAG G -3’; GAPDH, sense: TGA AGG TCG GTG TGA ACG GAT TTG G, antisense: ACG ACA TAC TCA GCA CCA GCA TCA C. The reaction system included 10×PCR buffer 5µL, MgCl2 (25mmol·L-1) 5µL, dNTP (10 mmol·L-1) 1µL, Taq polymerase 2U, 1µL of each primer and 2µL of each cDNA sample. In our preliminary experiment, 30 cycles of amplification of GAPDH generated PCR products in a linear increasing phase. Thirty cycles of amplification were therefore performed for GAPDH in a TC-96AE programmable thermal controller (MJ Research, Inc., watertown, Mass). The experiments were performed in triplicate with different preparations of cells. Measurement of active TGF- 1 release from cardiac fibroblasts Cardiac fibroblasts were placed in 24-well plates at density of 2×104 cells per well with DMEM supplemented with 10% FCS and allowed to grow until subconfluent. Cells were synchronized in

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medium containing 0.1% FCS, and were then stimulated to proliferate by adding 10-7 mol/L Ang II either alone or combined with different concentrations of hexarelin to the culture medium. After a 24-hour incubation, the protein levels of bioactive TGF- 1 in the culture medium were measured using an ELISA Kit (TGF-beta1 Emax ImmunoAssay System, Promega). Briefly, a 96-well ELISA plate (Corning Costar, Cat # 3590) was coated with 100 µl/well monoclonal mouse anti-human TGF- 1 antibody overnight at 4°C without shaking. After washing once with TBST wash buffer containing 20mM Tris-HCl (pH7.6), 150mM NaCl and 0.05% (v/v) Tween 20, the plate was incubated with 100 µL of sample or standard at room temperature for 90 minutes with shaking (500 rpm). After a 5-minute wash, 100 µl of polyclonal anti-human TGF1 antibody was added into each well and incubated for 2 hours at room temperature with shaking. The plate was then washed and incubated with 100 µl diluted TGF- 1 HRP conjugate at room temperature for 2 hours with shaking. After a further wash, the TMB One Solution was added for 15 minutes at room temperature in the dark to generate color, and then the reaction was terminated by adding an equal volume of 1N hydrochloric acid to the well. Absorbance at 450 nm was immediately read with the use of an ELISA microtiter plate reader (Vector II, PerkinElmer), and active TGF- 1 concentrations were determined from a standard curve, with human recombinant TGF- 1 used as the standard. The active TGF- 1 concentrations were calculated by reference to the total cellular protein contents of the corresponding samples and reported as pg/µg cellular protein. Duplicate quantitative experiments were performed for each sample. Measurement of cAMP level in cardiac fibroblasts The fibroblasts were seeded onto 24-well plates containing DMEM supplemented with 10% FCS at a density of 2×104 cells per well and allowed to grow until sub-confluent. Cells were synchronized in medium containing 0.1% FCS and then stimulated by hexarelin at different

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concentrations. After incubation with hexarelin for 10 minutes, the cAMP level in cardiac fibroblast was determined by radioimmunassay. Brifly, the cells were lysed in 6% trichloroacetic acid, and the acid was removed by water-saturated diethyl ether extraction. After lyophilization of the aqueous phase, intracellular cAMP was measured with an RIA kit (Amersham, England). Triplicate experiments were performed with different cell preparations. Statistical Analysis Data were expressed as mean ± SD, and differences in mean values were analyzed by student’s t test and 1-way ANOVA with pair wise multiple comparisons and further analyzed by the Newman-Keuls method. P90%) of non-myocyte cells in the heart (17). Cardiac fibroblasts increase the production of fibronectin and collagen when the heart is exposed to a variety of injuries, such as myocardial infarction, pressure overload, and myocarditis. It is believed that an increase in both the number of cardiac fibroblasts and the content of extracellular matrix proteins during cardiac remodeling is a major cause of cardiac dysfunction (9, 31, 32). GHS, of which hexarelin is one, have myocardial protective effects, including improving ventricular function in cardiac diseases and cardiac remodeling (25, 34). Based on these observations, we raised the hypothesis that GHS may directly inhibit cardiac fibroblast proliferation and collagen turnover. Our results have shown that Ang II significantly promoted the cardiac fibroblast proliferation and collagen synthesis, whereas incubation with Ang II and hexarelin abolished Ang II-induced proliferation. Meanwhile, FCS significantly increased rat cardiac fibroblast DNA synthesis, but following incubation with both FCS and hexarelin for 24h, FCS-increased DNA synthesis was inhibited. These findings may throw light on the mechanisms of action of GHS on the heart. Hexarelin maximally inhibited Ang II–induced collagen synthesis at the same dose that blocked DNA synthesis in fibroblasts. The mechanisms of the inhibitory effects of hexarelin on fibroblasts are still unknown. Possible mechanisms are through GHSR and/or adenosine receptor directly, or indirectly by down-regulation of TGF- expression and release, and inhibition of the cardiac fibroblast proliferation. In addition, hexarelin does not seem to have a cytotoxic effect on fibroblasts, because cell numbers were not reduced when quiescent fibroblasts was incubated with hexarelin alone.

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The inhibitory effects of hexarelin on DNA and collagen synthesis in rat cardiac fibroblasts are mediated by activating both GHSR and A2R Hexarelin has been found to act via GHSR. The present study shows that a specific GHSR antagonist, D-Lys(3)-GHRP-6, dose-dependently abolished the inhibitory effect of hexarelin on DNA synthesis. This result strongly supports the hypothesis that one of the mechanisms of hexarelin-induced inhibition of DNA and collagen, and synthesis and proliferation of cardiac fibroblasts, is through activation of GHSR. Cardiac fibroblast growth is regulated by several autocrine/paracrine factors (7), including adenosine (26), which has long been known as a "retaliatory" metabolite, particularly in the heart, where it induces cardioprotective effects (26). The biological effects of adenosine are mediated via adenosine receptors, which exist in multiple subtypes (A1, A2A, A2B, and A3 receptors) (26). The currently accepted view is that within the heart, mainly A1 and A2A adenosine receptors are cardioprotective. For example, activation of A1 receptors attenuates sympathetic nerve activity, inhibits renin release from juxtaglomerular cells and opens cardiac K+ channels (26). By means of activating A2B receptor, adenosine causes vasodilation, inhibits platelet aggregation, diminishes neutrophil adhesion to vascular endothelial cells, attenuates neutrophil-induced endothelial cell damage, and stimulates nitric oxide release from vascular endothelial cells and vascular smooth muscle cells (15, 20). Although the standard view is that A1 and A2A receptors are the most important with regard to adenosine-mediated cardioprotection, some indirect evidence suggests that adenosine inhibits cardiac fibroblast growth by means of activation of A2B receptors (14, 16). Tullin et al (41) reported that adenosine is an agonist of the growth hormone secretagogue receptor. Our study demonstrated that hexarelin dose-dependently increased the cellular level of cAMP in cardiac fibroblasts. Based on these findings, we speculate that the

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inhibitory effect of hexarelin on rat cardiac fibroblast proliferation is possibly partially mediated by the adenosine receptor. The present study showed that the specific A2R antagonist DMPX abolished the inhibitory effect of hexarelin on FCS-induced increase of DNA synthesis, while specific A1R antagonist DPCPX had no effect on these outcomes. These results suggest that the inhibitory effect of hexarelin on DNA and collagen production in rat cardiac fibroblasts is partially mediated by means of activating the A2R, possibly by the A2BR subtype. The inhibitory effect of hexarelin on DNA and collagen synthesis in rat cardiac fibroblasts is associated with reduced TGF- expression and release in response to Ang II Ang II has a mitogenic effect in neonatal rat cardiac fibroblasts (38, 39). Ang II also exerts mitogenic effects on adult cardiac fibroblasts (13). As in neonatal heart, Ang II increases mRNA levels for c-fos, c-jun, jun B, Egr-1, and c-myc in cardiac fibroblasts via AT1 receptor (38). Furthermore, Ang II increases collagen type I mRNA and the synthesis and secretion of collagen (13), as well as mRNA expression and protein secretion of fibronectin (24). Ang II also increases cardiac fibroblast osteopontin expression and DNA synthesis that are completely blocked by antibodies against osteopontin and 3 integrin, suggesting that Ang II-induced cardiac fibroblast proliferation may require osteopontin engagement of 3 integrin (2). In this experiment, we observed stimulation of cardiac fibroblast proliferation and collagen production by Ang II in vitro. In addition to these effects, Ang II increased cardiac fibroblast mRNA expression and protein secretion of transforming growth factor-1 (TGF-1) (11), which acted in synergy to interfere with the normal structure and function of the surrounding myocardium (11, 12, 28). TGF- has been implicated in several fibrotic disorders, including glomerulonephritis, liver cirrhosis, lung fibrosis, and vascular restenosis (8). In vitro observations indicate that TGF- 1

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stimulates the expression of fibronectin and collagen and their incorporation into the ECM from cardiac fibroblasts (1, 18, 22, 40), indicating that TGF- 1 plays a crucial role in the myocardial remodeling process, particularly in cardiac fibrosis. The present studies show that administration of Ang II for 24h significantly increases mRNA expression of TGF- and active TGF- 1 release from cardiac fibroblasts. However, hexarelin abolished Ang II-induced up-regulation of TGFexpression and active TGF- 1 release. It is therefore suggested that the effect of hexarelin on collagen production in rat cardiac fibroblasts is associated with a decrease in Ang II-induced TGF- expression and active TGF- 1 release. In summary, hexarelin inhibits cardiac fibroblast proliferation and collagen synthesis. These inhibitory effects are mediated by A2BR and probably also GHS-R, and associated with downregulation of TGF- expression. Our study suggests that the inhibitory effects of hexarelin on DNA and collagen synthesis in rat cardiac fibroblasts may be considered to be a new mechanism for hexarelin’s protective effect on cardiac diseases. Acknowledgements This work was supported by the Distinguished Young Investigator Awards from the Natural Science Foundation of China (NSFC) (30125016 to J.-M.C. and 30028007 to C.C.), NSFC grants (30670863, 30370565 and 30313902 to J.-M.C.), the National Key Basic Research Program (NKBRP; 973 Program) funded by MOST (2006CB503806 and 2006CB933202 to J.M.C.) and Australian NHMRC (to C.C.). Hexarelin was supplied by Dr. R Deghenghi (Europeptide, France). X Xu and J Pang contributed equally to this work. JM Cao and C Chen are both corresponding authors in this work.

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Figure Legends Figure 1. The inhibitory effect of hexarelin on Ang II-induced proliferation of rat cardiac fibroblasts. The survival of 8-day-old rat cardiac fibroblasts was measured by (3,4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24h treatment with Ang II or Ang II +Hex. Values are means ± SD of 10 observations. **P