Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2014, Vol. 8 Issue (4) : 445-455
Chronic inhibition of cyclic guanosine monophosphate-specific phosphodiesterase 5 prevented cardiac fibrosis through inhibition of transforming growth factor β-induced Smad signaling
Wei Gong,Mengwen Yan,Junxiong Chen,Sandip Chaugai,Chen Chen,Daowen Wang()
Department of Internal Medicine and Gene Therapy Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
 Download: PDF(949 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Recent evidences suggested that cyclic guanosine monophosphate-specific phosphodiesterase 5 (PDE5) inhibitor represents an important therapeutic target for cardiovascular diseases. Whether and how it ameliorates cardiac fibrosis, a major cause of diastolic dysfunction and heart failure, is unknown. The purpose of this study was to investigate the effects of PDE5 inhibitor on cardiac fibrosis. We assessed cardiac fibrosis and pathology in mice subjected to transverse aortic constriction (TAC). Oral sildenafil, a PDE5 inhibitor, was administered in the therapy group. In control mice, 4 weeks of TAC induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation to myofibroblasts). Sildenafil treatment markedly prevented TAC-induced cardiac dysfunction, cardiac fibrosis and cardiac fibroblast activation but did not block TAC-induced transforming growth factor-β1 (TGF-β1) production and phosphorylation of Smad2/3. In isolated cardiac fibroblasts, sildenafil blocked TGF-β1-induced cardiac fibroblast transformation, proliferation and collagen synthesis. Furthermore, we found that sildenafil induced phosphorylated cAMP response element binding protein (CREB) and reduced CREB-binding protein 1 (CBP1) recruitment to Smad transcriptional complexes. PDE5 inhibition prevents cardiac fibrosis by reducing CBP1 recruitment to Smad transcriptional complexes through CREB activation in cardiac fibroblasts.

Keywords PDE5      cardiac fibrosis      TGF-β      CREB     
Corresponding Author(s): Daowen Wang   
Just Accepted Date: 24 October 2014   Online First Date: 24 November 2014    Issue Date: 18 December 2014
 Cite this article:   
Daowen Wang,Wei Gong,Mengwen Yan, et al. Chronic inhibition of cyclic guanosine monophosphate-specific phosphodiesterase 5 prevented cardiac fibrosis through inhibition of transforming growth factor β-induced Smad signaling[J]. Front. Med., 2014, 8(4): 445-455.
Fig.1  PDE5 inhibition attenuated TAC-induced cardiac hypertrophy and improved the function of failing hearts. (A) Representative images of hearts. Scale bar, 2 mm. (B) Ratio of heart weight to body weight (HW/BW). (C,D) H&E staining of mouse hearts and cardiomyocyte cross-sectional area (mm). Scale bar, 50μm. (E) Expression of BNP and β-MHC analyzed by real-time polymerase chain reaction. n = 6. *P<0.05 vs. Sham. #P<0.05 vs. TAC. Data are presented as mean±SEM. Sil: sildenafil.
Sham Sil TAC TAC+ Sil
n 5 6 6 7
BW (g) 22.3±0.41 21.8±0.43 22.44±0.46 22.16±0.64
HW (mg) 116.0±4.3 118.4±4.56 148.3±3.09* 120.82±4.76#
Tab.1  Anatomic data
Sham Sil TAC TAC+ Sil
n 6 6 8 8
LVEF (%) 89.74±6.52 89.51±7.03 60.44±8.56* 78.16±8.34#
LVFS (%) 59.28±4.76 58.66±6.56 37.98±5.09* 47.16±5.76#
LVPW;d (mm) 0.75±0.14 0.78±0.11 1.08±0.08* 0.86±0.10#
LVPW;s (mm) 1.31±0.06 1.33±0.05 1.46±0.09* 1.40±0.06
Tab.2  Echocardiographic characteristics of Sham, Sil, TAC, and TAC+Sil mice
Fig.2  PDE5 inhibition prevented TAC-induced cardiac fibrosis in mice. (A, B) Representative staining for collagen deposition in LV is presented. Collagen deposition was stained with a saturated solution of picric acid containing 1% Sirius Red as red color and was quantified as percent of cardiac area. Scale bar, 100μm. (C) Expression of collagen I and III analyzed by real-time polymerase chain reaction. n = 6. (D) Immunoblot analysis showed α-SMA was increased in hearts of TAC mice and decreased in PDE5 treatment mice. α-SMA was normalized to GAPDH. n = 4. *P<0.05 vs. Sham. #P<0.05 vs. TAC. Results are presented as mean±SEM.
Fig.3  PDE5 inhibition did not block TAC-induced TGF-β1 production or phosphorylation of Smad2/3. (A) Immunoblot analysis showed TGF-β1 was increased in TAC mice, and sildenafil treatment did not reduce TGF-β1 expression. TGF-β1 was normalized to GAPDH. n = 5. (B) Gene expression of TGF-β1 in LV was analyzed by real-time polymerase chain reaction. n = 6. (C) Expression of TGF-β1 in serum measured by ELISA. n = 6. *P<0.05 vs. Sham. #P<0.05 vs. TAC. (D) Level of p-Smad2, p-Smad3 and total Smad2/3 measured by Western blot after TAC. n = 4. (E) Densitometric quantification of Smad2 and Smad3 phosphorylation after TAC. *P<0.05 vs. Sham. #P<0.05 vs. TAC. Data are presented as mean±SEM.
Fig.4  PDE5 inhibition prevented the TGF-β1-stimulated fibrotic response in cardiac fibroblasts. Cells were pretreated with sildenafil (1μM) for 1 h, and then exposed to TGF-β1 (10ng/ml) for 24 h. (A) Sildenafil alleviated TGF-β1-induced α-SMA expression in cardiac fibroblasts. n = 4. (B) Sildenafil alleviated TGF-β1-induced cardiac fibroblast proliferation. n = 6. (C) Sildenafil pretreatment markedly decreased collagen synthesis (collagen I and III) by TGF-β1 incubation. Results are shown as mean±SEM. n = 5. *P<0.05 vs. control. #P<0.05 vs. TGF-β1.
Fig.5  PDE5 inhibition increasd phosphorylation of CREB. Cells were pretreated with sildenafil (0 μM, 0.5 μM, 1 μM) for 24 h. (A) Sildenafil pretreatment markedly increased p-CREB. (B) The level of cGMP and cAMP in cardiac fibroblasts. Results are shown as mean±SEM. n = 6, *P<0.05 vs. control.
Fig.6  PDE5 inhibition disrupted the interaction between Smads and the coactivator CBP1. (A) CBP1 expression was detected by Western blot. (B) Immunoprecipitates of the indicated proteins were separated by SDS-PAGE and probed with antibodies specific for CBP1, p-Smad2, p-Smad3, or p-CREB were used to examine levels of the indicated proteins. Results are shown as mean±SEM. n = 4.
1 Mudd JO, Kass DA. Tackling heart failure in the twenty-first century. Nature<?Pub Caret?>2008; 451(7181): 919–928 pmid: 18288181
2 Berk BC, Fujiwara K, Lehoux S. ECM remodeling in hypertensive heart disease. J Clin Invest2007; 117(3): 568–575 pmid: 17332884
3 Chen J, Shearer GC, Chen Q, Healy CL, Beyer AJ, Nareddy VB, Gerdes AM, Harris WS, O’Connell TD, Wang D. Omega-3 fatty acids prevent pressure overload-induced cardiac fibrosis through activation of cyclic GMP/protein kinase G signaling in cardiac fibroblasts. Circulation2011; 123(6): 584–593 pmid: 21282499
4 Leask A. TGFbeta, cardiac fibroblasts, and the fibrotic response. Cardiovasc Res2007; 74(2): 207–212 pmid: 16919613
5 Kai H, Kuwahara F, Tokuda K, Imaizumi T. Diastolic dysfunction in hypertensive hearts: roles of perivascular inflammation and reactive myocardial fibrosis. Hypertens Res 2005; 28(6): 483–490 pmid: 16231753
6 Kuwahara F, Kai H, Tokuda K, Kai M, Takeshita A, Egashira K, Imaizumi T. Transforming growth factor-beta function blocking prevents myocardial fibrosis and diastolic dysfunction in pressure-overloaded rats. Circulation2002; 106(1): 130–135 pmid: 12093782
7 Li P, Wang D, Lucas J, Oparil S, Xing D, Cao X, Novak L, Renfrow MB, Chen YF. Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts. Circ Res2008; 102(2): 185–192 pmid: 17991884
8 Shi X, Yang X, Chen D, Chang Z, Cao X. Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling. J Biol Chem1999; 274(19): 13711–13717 pmid: 10224145
9 Kass DA, Champion HC, Beavo JA. Phosphodiesterase type 5: expanding roles in cardiovascular regulation. Circ Res2007; 101(11): 1084–1095 pmid: 18040025
10 Omori K, Kotera J. Overview of PDEs and their regulation. Circ Res2007; 100(3): 309–327 pmid: 17307970
11 Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, Bedja D, Gabrielson KL, Wang Y, Kass DA. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med2005; 11(2): 214–222 pmid: 15665834
12 Hassan MA, Ketat AF. Sildenafil citrate increases myocardial cGMP content in rat heart, decreases its hypertrophic response to isoproterenol and decreases myocardial leak of creatine kinase and troponin T. BMC Pharmacol2005; 5(1): 10 pmid: 15813973
13 Pérez NG, Piaggio MR, Ennis IL, Garciarena CD, Morales C, Escudero EM, Cingolani OH, Chiappe de Cingolani G, Yang XP, Cingolani HE. Phosphodiesterase 5A inhibition induces Na+/H+ exchanger blockade and protection against myocardial infarction. Hypertension2007; 49(5): 1095–1103 pmid: 17339532
14 Nagendran J, Archer SL, Soliman D, Gurtu V, Moudgil R, Haromy A, St Aubin C, Webster L, Rebeyka IM, Ross DB, Light PE, Dyck JR, Michelakis ED. Phosphodiesterase type 5 is highly expressed in the hypertrophied human right ventricle, and acute inhibition of phosphodiesterase type 5 improves contractility. Circulation2007; 116(3): 238–248 pmid: 17606845
15 Pokreisz P, Vandenwijngaert S, Bito V, Van den Bergh A, Lenaerts I, Busch C, Marsboom G, Gheysens O, Vermeersch P, Biesmans L, Liu X, Gillijns H, Pellens M, Van Lommel A, Buys E, Schoonjans L, Vanhaecke J, Verbeken E, Sipido K, Herijgers P, Bloch KD, Janssens SP. Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice. Circulation2009; 119(3): 408–416 pmid: 19139381
16 deAlmeida AC, van Oort RJ, Wehrens XH. Transverse aortic constriction in mice. J Vis Exp2010; (38): e1729
pmid: 20410870
17 Ni L, Zhou C, Duan Q, Lv J, Fu X, Xia Y, Wang DW. β-AR blockers suppresses ER stress in cardiac hypertrophy and heart failure. PLoS ONE2011; 6(11): e27294 pmid: 22073308
18 Villarreal FJ, Kim NN, Ungab GD, Printz MP, Dillmann WH. Identification of functional angiotensin II receptors on rat cardiac fibroblasts. Circulation1993; 88(6): 2849–2861 pmid: 8252698
19 Wang H, Lin L, Jiang J, Wang Y, Lu ZY, Bradbury JA, Lih FB, Wang DW, Zeldin DC. Up-regulation of endothelial nitric-oxide synthase by endothelium-derived hyperpolarizing factor involves mitogen-activated protein kinase and protein kinase C signaling pathways. J Pharmacol Exp Ther2003; 307(2): 753–764 pmid: 12975498
20 Soeki T, Kishimoto I, Okumura H, Tokudome T, Horio T, Mori K, Kangawa K. C-type natriuretic peptide, a novel antifibrotic and antihypertrophic agent, prevents cardiac remodeling after myocardial infarction. J Am Coll Cardiol2005; 45(4): 608–616 pmid: 15708711
21 Hammermeister KE, DeRouen TA, Dodge HT. Variables predictive of survival in patients with coronary disease. Selection by univariate and multivariate analyses from the clinical, electrocardiographic, exercise, arteriographic, and quantitative angiographic evaluations. Circulation1979; 59(3): 421–430 pmid: 761323
22 Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFbeta activation. J Cell Sci2003; 116(2): 217–224 pmid: 12482908
23 Ruiz-Ortega M, Rodríguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J. TGF-beta signaling in vascular fibrosis. Cardiovasc Res2007; 74(2): 196–206 pmid: 17376414
24 Isono M, Chen S, Hong SW, Iglesias-de la Cruz MC, Ziyadeh FN. Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells. Biochem Biophys Res Commun2002; 296(5): 1356–1365 pmid: 12207925
25 Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J1998; 17(11): 3091–3100 pmid: 9606191
26 Chen SJ, Yuan W, Lo S, Trojanowska M, Varga J. Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta. J Cell Physiol2000; 183(3): 381–392<381::AID-JCP11>3.0.CO;2-O pmid: 10797313
27 Yang YC, Piek E, Zavadil J, Liang D, Xie D, Heyer J, Pavlidis P, Kucherlapati R, Roberts AB, B?ttinger EP. Hierarchical model of gene regulation by transforming growth factor beta. Proc Natl Acad Sci USA2003; 100(18): 10269–10274 pmid: 12930890
28 Massagué J, Seoane J, Wotton D. Smad transcription factors. Genes Dev2005; 19(23): 2783–2810 pmid: 16322555
29 Buxton IL, Duan D. Cyclic GMP/protein kinase G phosphorylation of Smad3 blocks transforming growth factor-beta-induced nuclear Smad translocation: a key antifibrogenic mechanism of atrial natriuretic peptide. Circ Res2008; 102(2): 151–153 pmid: 18239144
30 Lu Z, Xu X, Hu X, Lee S, Traverse JH, Zhu G, Fassett J, Tao Y, Zhang P, dos Remedios C, Pritzker M, Hall JL, Garry DJ, Chen Y. Oxidative stress regulates left ventricular PDE5 expression in the failing heart. Circulation2010; 121(13): 1474–1483 pmid: 20308615
31 Nagayama T, Hsu S, Zhang M, Koitabashi N, Bedja D, Gabrielson KL, Takimoto E, Kass DA. Sildenafil stops progressive chamber, cellular, and molecular remodeling and improves calcium handling and function in hearts with pre-existing advanced hypertrophy caused by pressure overload. J Am Coll Cardiol2009; 53(2): 207–215 pmid: 19130990
32 Hsu S, Nagayama T, Koitabashi N, Zhang M, Zhou L, Bedja D, Gabrielson KL, Molkentin JD, Kass DA, Takimoto E. Phosphodiesterase 5 inhibition blocks pressure overload-induced cardiac hypertrophy independent of the calcineurin pathway. Cardiovasc Res2009; 81(2): 301–309 pmid: 19029137
33 Schiller M, Verrecchia F, Mauviel A. Cyclic adenosine 3′,5′-monophosphate-elevating agents inhibit transforming growth factor-beta-induced SMAD3/4-dependent transcription via a protein kinase A-dependent mechanism. Oncogene2003; 22(55): 8881–8890 pmid: 14654784
34 Gonzalez GA, Montminy MR. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell1989; 59(4): 675–680 pmid: 2573431
35 Gonzalez GA, Yamamoto KK, Fischer WH, Karr D, Menzel P, Biggs W 3rd, Vale WW, Montminy MR. A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence. Nature1989; 337(6209): 749–752 pmid: 2521922
36 Liu X, Sun SQ, Hassid A, Ostrom RS. cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts. Mol Pharmacol2006; 70(6): 1992–2003 pmid: 16959941
37 Zhang X, Yan G, Ji J, Wu J, Sun X, Shen J, Jiang H, Wang H. PDE5 inhibitor promotes melanin synthesis through the PKG pathway in B16 melanoma cells. J Cell Biochem2012; 113(8): 2738–2743 pmid: 22441938
[1] Supplementary Material 1 Download
Full text