Inhibition of cardiac fibroblast proliferation and
myofibroblast differentiation by resveratrol
Erik R. Olson,1Jennifer E. Naugle,1Xiaojin Zhang,1Joshua A. Bomser,2and J. Gary Meszaros1
1Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown;
and2Department of Human Nutrition, The Ohio State University, Columbus, Ohio
Submitted 28 July 2004; accepted in final form 15 October 2004
Olson, Erik R., Jennifer E. Naugle, Xiaojin Zhang, Joshua A.
Bomser, and J. Gary Meszaros. Inhibition of cardiac fibroblast
proliferation and myofibroblast differentiation by resveratrol. Am J
Physiol Heart Circ Physiol 288: H1131–H1138, 2005. First published
October 21, 2004; doi:10.1152/ajpheart.00763.2004.—Cardiac fibro-
blasts (CFs) regulate myocardial remodeling by proliferating, differ-
entiating, and secreting extracellular matrix proteins. Prolonged acti-
vation of CFs leads to cardiac fibrosis and reduced myocardial
contractile function. Resveratrol (RES) exhibits a number of cardio-
protective properties; however, the possibility that this compound
affects CF function has not been considered. The current study tests
whether RES directly influences the growth and proliferation of CFs
and differentiation to the hypersecretory myofibroblast phenotype.
Pretreatment of CFs with RES (5–25 ?M) inhibited basal and ANG
II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK
kinase activation. This inhibition by RES reduced basal proliferation
and blocked ANG II-induced growth and proliferation of CFs in a
concentration-dependent manner, as measured by [3H]leucine and
[3H]thymidine incorporation, respectively. RES pretreatment attenu-
ated ERK phosphorylation when CFs were stimulated with 0.2 nM
epidermal growth factor (EGF), a concentration at which EGF-
induced ERK activation over basal was similar to the phosphorylation
induced by 100 nM ANG II. Akt phosphorylation in CFs was
unaffected by treatment with either 100 nM ANG II or 25 ?M RES.
Pretreatment of CFs with RES also reduced both ANG II- and
transforming growth factor-?-induced CF differentiation to the myo-
fibroblast phenotype, indicated by a reduction in ?-smooth muscle
actin expression and stress fiber organization in CFs. This study
identifies RES as an anti-fibrotic agent in the myocardium by limiting
CF proliferation and differentiation, two critical steps in the patho-
genesis of cardiac fibrosis.
angiotensin II; fibrosis; remodeling; mitogen-activated protein kinase
CARDIAC FIBROBLASTS (CFs) are the predominant secretory cells
of extracellular matrix (ECM) proteins in the heart and the key
mediators of normal and pathological cardiac remodeling.
Prolonged activation of CFs, defined by increased proliferation
and subsequent ECM secretion, is a direct consequence of
hypertension and heart failure and leads to cardiac fibrosis, a
condition characterized by excess ECM deposition and a stiff
myocardium. The impaired compliance of the fibrotic heart
ultimately compromises contractile performance (3). In addi-
tion to proliferation, CFs differentiate into myofibroblasts, a
cell type with an increased capacity to secrete ECM proteins
(30). The proliferation and differentiation of new ECM-pro-
ducing cells enhance the deposition of ECM proteins, and thus,
limiting these parameters represents a potential therapeutic
avenue to reduce pathological myocardial fibrosis.
ANG II levels are elevated during hypertension and heart
failure. This peptide hormone has direct and potent effects on
CF function, including activation of the mitogen-activated
protein kinase (MAPK)/extracellular signal-regulated kinase
(ERK) cascade and cellular proliferation via direct stimulation
of the ANG II type 1 (AT1) receptor (1, 2, 6, 23, 24, 28). We
have demonstrated that proliferation of CFs is dependent on
ERK kinase (MEK) and ERK1/2 activation via pharmacolog-
ical inhibition of MEK (25). In addition to activating ERK,
ANG II regulates collagen type I expression, at least in part, by
inducing CFs to produce and secrete transforming growth
factor-? (TGF-?; see Refs. 11 and 16). TGF-? is a potent
paracrine mediator of differentiation and contributes to the
development of cardiac fibrosis by increasing the number of
myofibroblasts in the heart (22, 26).
Resveratrol (RES; trans-3,4?,5-trihydroxystilbene), a phy-
toalexin found in the skins of grapes, has been identified as a
key biologically active ingredient in red wine. RES has been
credited with mediating a number of beneficial effects in the
cardiovascular system that accompany moderate red wine con-
sumption. In rat aortic smooth muscle cells, RES suppresses
ANG II-induced Akt (protein kinase B) and, to a lesser extent,
ERK1/2 activation, both of which are required for ANG
II-induced hypertrophy (13). RES reduces ERK and JNK
phosphorylation in coronary artery smooth muscle cells (10)
and induces vasorelaxant responses by activating membrane-
bound guanylyl cyclase (9) and nitric oxide release (21).
Together, these findings depict RES as a potential therapeutic
agent in a multitude of cardiovascular diseases.
The studies focusing on the cardiovascular effects of RES
have primarily examined the coronary artery and vascular
smooth muscle cells (VSMCs), whereas the effects of RES on
CFs have not yet been examined. Our initial hypothesis was
that RES would inhibit both ANG II-induced proliferation and
differentiation of CFs. The proliferation of CFs via ANG II and
the ERK1/2 cascade (24) plays a key role in the development
of cardiac fibrosis, and inhibiting prolonged CF activity repre-
sents a viable method for preventing the long-term loss of
cardiac function that accompanies this condition. We report
that RES does inhibit ANG II-induced proliferation and growth
of CFs, an effect mechanistically controlled through direct
blockade of the ERK1/2 cascade. The study also demonstrated
that RES pretreatment inhibited ANG II-induced CF differen-
tiation to the hypersecretory myofibroblast phenotype. In ad-
Address for reprint requests and other correspondence: J. Gary Meszaros,
Northeastern Ohio Universities College of Medicine, Dept. of Physiology and
Pharmacology, 4209 State Route 44, Rootstown, OH 44272-0095 (E-mail:
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Heart Circ Physiol 288: H1131–H1138, 2005.
First published October 21, 2004; doi:10.1152/ajpheart.00763.2004.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society http://www.ajpheart.orgH1131
16. Lee AA, Dillmann WH, McCulloch AD, and Villarreal FJ. Angiotensin
II stimulates the autocrine production of transforming growth factor-beta
1 in adult rat cardiac fibroblasts. J Mol Cell Cardiol 27: 2347–2357, 1995.
17. Lijnen P, Petrov V, Rumilla K, and Fagard R. Transforming growth
factor-beta 1 promotes contraction of collagen gel by cardiac fibroblasts
through their differentiation into myofibroblasts. Methods Find Exp Clin
Pharmacol 25: 79–86, 2003.
18. Lijnen PJ, Petrov VV, and Fagard RH. Induction of cardiac fibrosis by
transforming growth factor-beta(1). Mol Genet Metab 71: 418–435, 2000.
19. Meszaros JG, Gonzalez AM, Endo-Mochizuki Y, Villegas S, Villarreal
F, and Brunton LL. Identification of G protein-coupled signaling path-
ways in cardiac fibroblasts: cross talk between Gq and Gs. Am J Physiol
Cell Physiol 278: C154–C162, 2000.
20. Meszaros JG, Raphael R, Lio FM, and Brunton LL. Protein kinase C
contributes to desensitization of ANG II signaling in adult rat cardiac
fibroblasts. Am J Physiol Cell Physiol 279: C1978–C1985, 2000.
21. Orallo F, Alvarez E, Camina M, Leiro JM, Gomez E, and Fernandez
P. The possible implication of trans-Resveratrol in the cardioprotective
effects of long-term moderate wine consumption. Mol Pharmacol 61:
22. Petrov VV, Fagard RH, and Lijnen PJ. Stimulation of collagen pro-
duction by transforming growth factor-beta1 during differentiation of
cardiac fibroblasts to myofibroblasts. Hypertension 39: 258–263, 2002.
23. Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, and Baker
KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ
Res 72: 1245–1254, 1993.
24. Schorb W, Conrad KM, Singer HA, Dostal DE, and Baker KM.
Angiotensin II is a potent stimulator of MAP-kinase activity in neonatal rat
cardiac fibroblasts. J Mol Cell Cardiol 27: 1151–1160, 1995.
25. Stockand JD and Meszaros JG. Aldosterone stimulates proliferation of
cardiac fibroblasts by activating Ki-RasA and MAPK1/2 signaling. Am J
Physiol Heart Circ Physiol 284: H176–H184, 2003.
26. Tharaux PL, Chatziantoniou C, Fakhouri F, and Dussaule JC. An-
giotensin II activates collagen I gene through a mechanism involving the
MAP/ER kinase pathway. Hypertension 36: 330–336, 2000.
27. Tian B, Liu J, Bitterman P, and Bache RJ. Angiotensin II modulates
nitric oxide-induced cardiac fibroblast apoptosis by activation of AKT/
PKB. Am J Physiol Heart Circ Physiol 285: H1105–H1112, 2003.
28. Villarreal FJ, Kim NN, Ungab GD, Printz MP, and Dillmann WH.
Identification of functional angiotensin II receptors on rat cardiac fibro-
blasts. Circulation 88: 2849–2861, 1993.
29. Voisin L, Foisy S, Giasson E, Lambert C, Moreau P, and Meloche S.
EGF receptor transactivation is obligatory for protein synthesis stimulation
by G protein-coupled receptors. Am J Physiol Cell Physiol 283: C446–
30. Weber KT, Sun Y, and Katwa LC. Myofibroblasts and local angiotensin
II in rat cardiac tissue repair. Int J Biochem Cell Biol 29: 31–42, 1997.
31. Yu R, Hebbar V, Kim DW, Mandlekar S, Pezzuto JM, and Kong AN.
Resveratrol inhibits phorbol ester and UV-induced activator protein 1
activation by interfering with mitogen-activated protein kinase pathways.
Mol Pharmacol 60: 217–224, 2001.
RESVERATROL INHIBITS CARDIAC FIBROBLAST FUNCTION
AJP-Heart Circ Physiol • VOL 288 • MARCH 2005 • www.ajpheart.org