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(Hypertension. 2002;39:258.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Stimulation of Collagen Production by Transforming Growth Factor-ß₁ During Differentiation of Cardiac Fibroblasts to Myofibroblasts

Victor V. Petrov; Robert H. Fagard; Paul J. Lijnen

From the Hypertension and Cardiovascular Rehabilitation Unit, Department of Molecular and Cardiovascular Research, Faculty of Medicine, University of Leuven, Leuven, Belgium.

Correspondence to Prof Dr P. Lijnen, Hypertension Unit, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail paul.lijnen{at}med.kuleuven.ac.be

	Abstract

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The aim of the present study was to elucidate how transforming growth factor-ß₁ (TGF-ß₁) can stimulate collagen deposition in cardiac tissue by interstitial cells via stimulation of fibroblasts, via myofibroblasts, or via differentiation of fibroblasts to myofibroblasts. The dose- and time-dependent stimulation of collagen production and of expression of -smooth muscle actin (-SMA), a marker of myofibroblasts, was studied in cultures of second-passage adult rat cardiac fibroblasts. The TGF-ß₁-stimulated collagen production is positively correlated (r=0.68, P<0.001) with the appearance of -SMA. Only at high concentrations (40 to 600 pmol/L) and after a long time (24 to 48 hours) of incubation, TGF-ß₁ increases the collagen production and stimulates the differentiation of fibroblasts to myofibroblasts. The maximal stimulation of the collagen production (2-fold, P<0.001) observed after incubation of cultures of fibroblasts with 600 pmol/L TGF-ß₁ for 48 hours is accompanied by a maximal stimulation of -SMA expression (3.5-fold, P<0.001), when cultures consist mainly of myofibroblasts. The stimulation of collagen production cannot be reversed either after additional incubation of TGF-ß₁-stimulated second-passage cultures for 2 days or in their offspring in the next third passage after incubation for 7 days without TGF-ß₁. The increased collagen production in these third-passage cultures cannot be further stimulated by TGF-ß₁. Our data suggest that TGF-ß₁-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of the collagen production either in fibroblasts or in myofibroblasts. Instead, TGF-ß₁ induces the differentiation of fibroblasts to myofibroblasts, which have a higher activity for collagen production than fibroblasts.

Key Words: fibroblasts • myofibroblasts • transforming growth factors • collagen

	Introduction

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Myofibroblasts and transforming growth factor-ß₁ (TGF-ß₁) are probably key elements of cardiac tissue fibrosis development.¹ Myofibroblasts appear in pericardial,² perivascular,³ endomyocardial,⁴ and interstitial insertion³; in round foreign body insertion⁴; in places of myocardial infarction^2,3,5; and in cardiac fibrosis induced in animals by infusion of angiotensin II,³ aldosterone,³ isoproterenol,⁴ unilateral renal ischemia,³ left coronary ligation,² insertion in tissue of foreign body,⁴ or pericardiotomy.² These in vivo data suggest that myofibroblasts can be implicated in collagen turnover irrespective of the site or nature of injury within the heart. Increases in the concentration of TGF-ß₁ in cardiac tissue at places of fibrosis,^5–11 as well as the possibility to inhibit fibrosis development by decreasing the tissue TGF-ß₁ in TGF-ß₁-deficient mice¹¹ or during treatment of rats with anti-TGF-ß₁ antibodies,⁷ suggest that TGF-ß₁ and myofibroblasts can be implicated in tissue collagen turnover. TGF-ß₁ stimulates the production of collagen in cultures of cardiac fibroblasts.^12–14 Therefore, it has been concluded that TGF-ß₁ activates collagen production in fibroblasts.^12,13 The effects of TGF-ß₁ have usually been studied at high concentrations (40 to 600 pmol/L),^12,13,15 at which the cytokine also induces the differentiation of cardiac fibroblasts to myofibroblasts.^16,15 If TGF-ß₁ induces the appearance of myofibroblasts in cultures of fibroblasts then it becomes unclear in which type of cells the production of collagen is stimulated by TGF-ß₁. Therefore, the aim of the present study was to determine the types of cells responsible for the stimulation of the collagen production in cultures of adult rat cardiac ventricular fibroblasts in the presence of TGF-ß₁.

	Methods

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Cell Cultures
All animal procedures are in accordance with the laws, regulations, and administrative provisions of the Member States of the European Community (Council Directive 86/609/EEC of November 24, 1986) regarding the protection of animals for experimental and other scientific purposes. This research protocol is also approved by the Ethical Committee for Animal Experiments of the Katholieke Universiteit Leuven, Belgium.

Cardiac ventricular fibroblasts obtained from male Wistar rats were grown in Dulbecco‘s modified Eagle’s medium (DMEM) in the presence of 10% fetal bovine serum (FBS) and used in passages 2 and 3 as previously described.¹⁵ When cultures reached confluence, the medium was replaced with fresh DMEM without serum. After 24-hour incubation of cells without serum, TGF-ß₁ (0.12 to 600 pmol/L) was added and cells were additionally incubated for 2 days.

Immunocytochemistry
The cells grown in slides (Becton-Dickinson) are fixed in methanol at -20°C, blocked with bovine serum albumin, incubated with primary antibodies (Ab)—antivimentin Ab, antidesmin Ab, anti-factor VIII Ab, or anti- -smooth muscle actin Ab (-SMA)—and then incubated with an anti-IgG and conjugated with fluorescein isothiocyanate. Visualization is performed by the use of an inverted Leica-Leitz microscope (Leica AG).¹⁵

Western Blotting
Western blotting for the determination of -SMA has been performed as previously described.¹⁵

Assay of Collagen Production
Collagen production is measured according to a slight modification of method of Peterkofsky.¹⁷ Soluble collagen has been measured in the conditioned medium and nonsoluble collagen in the cell layer.¹⁸ Total cell protein content has been measured according to the method of Bradford.¹⁹

Statistical Analysis
Values are expressed as mean±SEM. The statistical methods used are repeated ANOVA (Tukey’s), Student’s 2-tailed test for paired data when appropriate, and Pearson correlation analysis. A value of P<0.05 is considered statistically significant.

	Results

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Characterization of Fibroblasts in Culture
Our cultures of rat cardiac second-passage fibroblasts have morphological characteristics typical for fibroblasts in culture and are positive for vimentin, a marker of fibroblast-like cells. These cell cultures do not contain desmin or factor VIII, markers of vascular smooth muscle and endothelial cells, and do not show structures typical for these cell cultures.

Concentration-Dependent Effect of TGF-ß₁ on Collagen Production
TGF-ß₁, at concentrations from 1 to 600 pmol/L, induces a dose-dependent increase in the production of soluble and nonsoluble collagen (Figure 1A). In the concentration range from 0.12 to 40 pmol/L, TGF-ß₁ does not, however, affect the soluble and nonsoluble collagen production. The maximal increase in collagen production was approximately 2-fold at 600 pmol/L TGF-ß₁. When neutralizing antibody to TGF-ß₁ (10 µg/mL) is added to the culture, the soluble and nonsoluble collagen production is completely blocked. TGF-ß₁ at concentrations from 40 to 600 pmol/L does not, however, change significantly the number of cells in cultures of cardiac fibroblasts. The de novo production of soluble and nonsoluble noncollagen protein is also dose-dependently increased by TGF-ß₁ (40 to 600 pmol/L), whereas no effect is observed at lower concentrations of TGF-ß₁ (Figure 1B). As shown in Figure 1A and 1B, the collagen production by fibroblasts in the presence of TGF-ß₁ is increased in parallel with the noncollagen protein, but to a greater extent. This indicates that TGF-ß₁ can induce the collagen production more specifically compared with that of total protein. Indeed, Figure 1C shows that the production of collagen relative to total protein is increased from 15% to 20% for soluble collagen and from 3.9% to 5.7% for nonsoluble collagen.

View larger version (17K): [in this window] [in a new window]   Figure 1. Dose-dependent stimulation of collagen and noncollagen protein production by TGF-ß1 in second-passage adult rat cardiac ventricular fibroblasts. A, Absolute stimulation of collagen production by TGF-ß1. B, Stimulation of noncollagen protein production. C, Stimulation of collagen production relative to total (collagenous plus noncollagenous) protein production. indicates soluble and • indicates nonsoluble collagen or noncollagen proteins.

TGF-ß₁ Induces -SMA
Western blotting (Figure 2A) shows that adult rat cardiac ventricular fibroblasts in second passage seeded at a density of 2600 cells/cm² at confluence contain a small amount of -SMA. TGF-ß₁ dose-dependently induces an increase in -SMA at concentrations of 40 to 600 pmol/L (Figure 2A and 2B). A maximal stimulation of -SMA of 256% versus control is obtained at 600 pmol/L TGF-ß₁.

View larger version (31K): [in this window] [in a new window]   Figure 2. Western blot analysis of -SMA expression in the presence of TGF-ß1. A, Dose-dependent effect of TGF-ß1 on -SMA content in second-passage rat cardiac fibroblasts. B, Dose-dependent stimulation of the intensity of -SMA bands by TGF-ß1.

Relationship Between Cell -SMA Content and Collagen Production
The TGF-ß₁-induced appearance of -SMA is positively correlated (r=0.68, P<0.001) to the TGF-ß₁-evoked stimulation of soluble collagen (y=96.7+0.0063x).

Time-Dependent Effect of TGF-ß₁ on Collagen Production, -SMA, and Total Cell Protein
After 24 and 48 hours of incubation, TGF-ß₁ (600 pmol/L) significantly stimulates soluble collagen compared with control(50%, P<0.01, and 139%, P<0.001, respectively) and nonsoluble collagen (50%, P<0.05, and 140%, P<0.001, respectively) production (Figure 3A and 3B). No significant effect of TGF-ß₁ on soluble and nonsoluble collagen production has been found after 2 and 4 hours of incubation (Figure 3A and 3B). TGF-ß₁ also increases the total cell protein content after 24 (16±5%, P<0.05) and 48 hours (50±14%, P<0.01), but not after 2 and 4 hours.

View larger version (32K): [in this window] [in a new window]   Figure 3. Time-dependent stimulation of collagen production and -SMA expression in second-passage fibroblasts cultured for 0, 2, 4, 24, and 48 hours with 600 pmol/L TGF-ß1. A, Soluble collagen. B, Nonsoluble collagen. C, Western blot analysis of -SMA. D, Intensity of bands of -SMA expressed in arbitrary units: control fibroblasts (open bars) and TGF-ß1-stimulated fibroblasts (solid bars).

The effect of TGF-ß₁ on the collagen production is accompanied by a time-dependent induction of -SMA (Figure 3C and 3D). The induction of -SMA by TGF-ß₁ after 24 and 48 hours is 3-fold compared with control cultures incubated for the same time. No induction in -SMA is found after 2 and 4 hours.

Control fibroblasts cultured in the absence of serum and TGF-ß₁ for 24 and 48 hours produce more soluble collagen than after 2 and 4 hours (Figure 3A). The nonsoluble collagen production in control cultures is not, however, changed after 24 or 48 hours incubation (Figure 3B). -SMA in control cultures of fibroblasts is increased after 48 hours of culturing (Figure 3C and 3D).

Reversibility of the Effects of TGF-ß₁
Second-Passage Fibroblasts
Soluble collagen production is similar in cultures of fibroblasts incubated in serum-free medium with 600 pmol/L TGF-ß1 for 48 hours and in cultures of fibroblasts subsequently incubated for an additional 48 hours without TGF-ß₁ (102663±1319 dpm/10⁶ cells versus 101129±1636 dpm/10⁶ cells, P>0.1). Thus, the increased collagen production in second-passage TGF-ß₁-stimulated fibroblasts is not returned to control levels after their additional incubation without TGF-ß₁.

Third-Passage Fibroblasts
Second-passage fibroblasts incubated for 7 days with TGF-ß₁ at a concentration of 400 pmol/L in the presence of FBS are replated (third-passage offspring fibroblasts). Third-passage cells are cultured for 7 days in DMEM with 10% FBS and subsequently for 2 days without serum. Figure 4 shows that third-passage offspring fibroblasts, which are cultured in the absence of TGF-ß₁, nevertheless produce more soluble collagen (P<0.001) than control second-passage fibroblasts (columns 3 and 1). However, the production of collagen in third-passage offspring fibroblasts is not significant different from the second-passage TGF-ß₁-stimulated fibroblasts (Figure 4, columns 3 and 2). TGF-ß₁ cannot further stimulate collagen production in third-passage offspring fibroblasts. Indeed, soluble collagen production in third-passage offspring fibroblasts after additional incubation for 24 hours with 600 pmol/L TGF-ß₁ is not significantly different from both second-passage fibroblasts and their third-passage offspring (Figure 4, columns 2, 3, and 4).

View larger version (37K): [in this window] [in a new window]   Figure 4. Production of soluble collagen in second and third passage of TGF-ß1-treated cultures of fibroblasts. Bar 1, Second-passage fibroblasts are cultured to confluence with 10% FBS and then for 2 days without serum. Bar 2, Second-passage TGF-ß1-treated cultures of fibroblasts. Second-passage fibroblasts are cultured to confluence with 10% FBS and then for 2 days with 600 pmol/L TGF-ß1 in the absence of serum. Bars 3 and 4, Third-passage cultures of fibroblasts. Second-passage fibroblasts are incubated with 400 pmol/L TGF-ß1 for 7 days in the presence of 10% FBS. The cells are replated for the third passage. Bar 3, Third-passage offspring of second-passage TGF-ß1-stimulated fibroblasts are cultured for 7 days in DMEM with 10% FBS and subsequently for 2 days without serum. Bar 4, Third-passage offspring of second-passage TGF-ß1-stimulated fibroblasts are cultured for 7 days in DMEM with 10% FBS, subsequently without serum for 2 days and additionally for 2 days with 600 pmol/L of TGF-ß1 in the absence of serum.

Third-passage offspring fibroblasts—like their progeny, second-passage TGF-ß₁-treated fibroblasts—contain significantly more -SMA than second-passage control fibroblasts (Figure 5). However, the content of -SMA in second-passage TGF-ß₁-stimulated fibroblasts and in third-passage offspring fibroblasts is not significant different (Figure 5, lines 2 and 3).

View larger version (16K): [in this window] [in a new window]   Figure 5. -SMA in second-passage TGF-ß1-stimulated fibroblasts and their offspring in third passage. A, Western blot analysis of -SMA expression in second passage of TGF-ß1-stimulated fibroblasts and their offspring in third passage. Line 1, Second-passage fibroblasts incubated for 7 days in the presence of 10% FBS. Line 2, Second-passage fibroblasts incubated with 400 pmol/L TGF-ß1 for 7 days in the presence of 10% FBS. Line 3, Third-passage fibroblasts, offspring of second-passage TGF-ß1-stimulated fibroblasts. Third-passage fibroblasts are cultured for 7 days in DMEM with 10% FBS and subsequently for 2 days without serum. B, The intensity of -SMA bands (folds of increase): second-passage fibroblasts incubated with 400 pmol/L TGF-ß1 for 7 days in the presence of 10% FBS (solid bars); third-passage fibroblasts, offspring of second-passage TGF-ß1-stimulated fibroblasts (striped bars). Third-passage fibroblasts are cultured for 7 days in DMEM with 10% FBS and subsequently for 2 days without serum.

	Discussion

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The presence in cultures of rat cardiac fibroblasts of -SMA (Figure 2), a marker of myofibroblasts,^20,21 suggests that these cultures contain a small number of these cells. This is in agreement with the reported myofibroblast content of 6% to 30% in cultures of fibroblasts.^15,22–26 The dose-dependent stimulation of the collagen production in cultures of fibroblasts by TGF-ß₁ is accompanied by the induction of -SMA (Figures 1 and 2). Correlation analysis has revealed a significant positive relationship between the TGF-ß₁-induced collagen production and -SMA expression, suggesting a link between cellular activity of the collagen production and the quantity of myofibroblast in cultures.

Direct activation of cellular enzymes (enzymes implicated in collagen metabolism in the present study) by hormones is usually a fast process compared with that of a global rearrangement of cells, like cell differentiation. Therefore, a direct stimulation of fibroblasts by TGF-ß₁ to produce more collagen could be performed earlier than the differentiation of fibroblasts to myofibroblasts (measured as -SMA appearance) and reversed shortly after exclusion of TGF-ß₁ from the medium. However, in contrast to this hypothesis, the increase in the collagen production and in the expression of -SMA occurs at the same time after addition of TGF-ß₁ (Figure 3). The absence of the reversion of the TGF-ß₁-stimulated collagen production after additional incubation of TGF-ß₁-stimulated fibroblasts without TGF-ß₁ also argues against a direct stimulation of fibroblasts by TGF-ß₁ to produce collagen. The effect of TGF-ß₁ on the stimulation of second-passage fibroblasts, in terms of collagen production and -SMA expression, cannot be reversed even in the next third-passage cultures incubated for 7 days without TGF-ß₁ (Figures 4 and 5). These cells, myofibroblasts, in the absence of TGF-ß₁ express -SMA and produce collagen with a higher activity compared with that of the original fibroblasts. Additional incubation of myofibroblasts with TGF-ß₁ does not further stimulate the cellular activity of the collagen production (Figure 4, bar 4). Incapacity of myofibroblasts to restore the low level of collagen production in the absence of TGF-ß₁ or to activate the collagen production in the presence of TGF-ß₁ indicates that the collagen production in these cells is insensitive to TGF-ß₁.

The present data suggest that TGF-ß₁ does not stimulate the collagen production of usual cardiac interstitial fibroblasts in all ranges of TGF-ß₁ concentrations used in the present study. Instead, TGF-ß₁ stimulates the differentiation of fibroblasts to myofibroblasts. This differentiation is apparently irreversible, and rat cardiac myofibroblasts have probably a stable phenotype. Myofibroblasts produce collagen with a higher activity than their progeny, fibroblasts. The higher activity of collagen production in rat cardiac myofibroblasts compared with fibroblasts is apparently an intrinsic property of the myofibroblasts and cannot be regulated by TGF-ß₁.

In cultures of fibroblasts, TGF-ß₁ modifies the expression of metalloproteases and tissue inhibitors of metalloproteases,^27–30 which are involved in collagen metabolism. All these modifications have been found at high concentrations of TGF-ß₁ (40 to 600 pmol/L), at which the cytokine induces the differentiation of fibroblasts. Therefore, it can be suggested that TGF-ß₁-induced modifications of various components of the collagen metabolism machinery, and the collagen production activity in TGF-ß₁-stimulated fibroblasts, could be related with cell differentiation and appearance of myofibroblasts.

Coincidence of the localization of myofibroblasts and of an excess of tissue collagen suggests myofibroblasts as a tissue extracollagen source.² The higher activity of the collagen production in cultured myofibroblasts found in the present study confirms this suggestion.

Stability of the TGF-ß₁-induced myofibroblastic phenotype found in the present study can be explained by the high activity of myofibroblast to secrete TGF-ß_1.^31,32 The concentration of TGF-ß₁ secreted in the medium by myofibroblasts can be high enough to prevent in an autocrine manner their differentiation. This mechanism can also maintain myofibroblast phenotype in fibrotic tissue.

The data obtained in the present study are in agreement with the paradigm of cellular events of fibrosis development, particularly the central role of TGF-ß₁ and myofibroblasts in the tissue collagen deposition, as suggested by Weber.¹ We have shown that tissue TGF-ß₁, independent of source (exogenous [monocytes, macrophages] or endogenous [myofibroblast]), can keep a high tissue collagen deposition via generation and maintenance of a stable myofibroblastic phenotype, but not via activation of collagen secretion in fibroblasts or in myofibroblasts.

	Acknowledgments

 
The authors gratefully acknowledge the technical assistance of Tamara Coenen, Lieve Lommelen, and Yvette Piccart. This work was supported by a grant from the Fund for Scientific Research, Flanders (Belgium), and by an educational grant of AstraZeneca (Belgium). Robert Fagard is holder of the Prof Antoon Amery Chair in Hypertension Research founded by Merck, Sharp & Dohme (Belgium). Paul Lijnen is holder of the Boehringer Ingelheim Chair in Hypertension.

Received July 9, 2001; first decision July 30, 2001; accepted November 12, 2001.

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				K. Anand, A. N Mooss, and S. M Mohiuddin Review: Aldosterone Inhibition Reduces the Risk of Sudden Cardiac Death in Patients with Heart Failure Journal of Renin-Angiotensin-Aldosterone System, March 1, 2006; 7(1): 15 - 19. [Abstract] [PDF]

				P. Rocic and P. A. Lucchesi NAD(P)H Oxidases and TGF-{beta}-Induced Cardiac Fibroblast Differentiation: Nox-4 Gets Smad Circ. Res., October 28, 2005; 97(9): 850 - 852. [Full Text] [PDF]

				H. Peng, O. A. Carretero, N. Vuljaj, T.-D. Liao, A. Motivala, E. L. Peterson, and N.-E. Rhaleb Angiotensin-Converting Enzyme Inhibitors: A New Mechanism of Action Circulation, October 18, 2005; 112(16): 2436 - 2445. [Abstract] [Full Text] [PDF]

				E. D. Lekgabe, H. Kiriazis, C. Zhao, Q. Xu, X. L. Moore, Y. Su, R. A.D. Bathgate, X.-J. Du, and C. S. Samuel Relaxin Reverses Cardiac and Renal Fibrosis in Spontaneously Hypertensive Rats Hypertension, August 1, 2005; 46(2): 412 - 418. [Abstract] [Full Text] [PDF]

				P. J Lijnen, V. V Petrov, M. Turner, and R. H Fagard Collagen Production in Cardiac Fibroblasts During Inhibition of Aminopeptidase B Journal of Renin-Angiotensin-Aldosterone System, June 1, 2005; 6(2): 69 - 77. [Abstract] [PDF]

				K. Saito, N. Ishizaka, T. Aizawa, M. Sata, N. Iso-o, E. Noiri, I. Mori, M. Ohno, and R. Nagai Iron chelation and a free radical scavenger suppress angiotensin II-induced upregulation of TGF-{beta}1 in the heart Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1836 - H1843. [Abstract] [Full Text] [PDF]

				E. R. Olson, J. E. Naugle, X. Zhang, J. A. Bomser, and J. G. Meszaros Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1131 - H1138. [Abstract] [Full Text] [PDF]

				J. S. Swaney, D. M. Roth, E. R. Olson, J. E. Naugle, J. G. Meszaros, and P. A. Insel Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase PNAS, January 11, 2005; 102(2): 437 - 442. [Abstract] [Full Text] [PDF]

				J E Toblli, G Cao, G DeRosa, and P Forcada Reduced cardiac expression of plasminogen activator inhibitor 1 and transforming growth factor {beta}1 in obese Zucker rats by perindopril Heart, January 1, 2005; 91(1): 80 - 86. [Abstract] [Full Text] [PDF]

				K. Chen, J. L. Mehta, D. Li, L. Joseph, and J. Joseph Transforming Growth Factor {beta} Receptor Endoglin Is Expressed in Cardiac Fibroblasts and Modulates Profibrogenic Actions of Angiotensin II Circ. Res., December 10, 2004; 95(12): 1167 - 1173. [Abstract] [Full Text] [PDF]

				M. Ikeuchi, H. Tsutsui, T. Shiomi, H. Matsusaka, S. Matsushima, J. Wen, T. Kubota, and A. Takeshita Inhibition of TGF-{beta} signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction Cardiovasc Res, December 1, 2004; 64(3): 526 - 535. [Abstract] [Full Text] [PDF]

				A. Orlandi, A. Francesconi, M. Marcellini, A. Ferlosio, and L. G. Spagnoli Role of ageing and coronary atherosclerosis in the development of cardiac fibrosis in the rabbit Cardiovasc Res, December 1, 2004; 64(3): 544 - 552. [Abstract] [Full Text] [PDF]

				F. See, W. Thomas, K. Way, A. Tzanidis, A. Kompa, D. Lewis, S. Itescu, and H. Krum p38 mitogen-activated protein kinase inhibition improves cardiac function and attenuates left ventricular remodeling following myocardial infarction in the rat J. Am. Coll. Cardiol., October 19, 2004; 44(8): 1679 - 1689. [Abstract] [Full Text] [PDF]

				P. Philip-Couderc, F. Smih, J. E. Hall, A. Pathak, J. Roncalli, R. Harmancey, P. Massabuau, M. Galinier, P. Verwaerde, J.-M. Senard, et al. Kinetic analysis of cardiac transcriptome regulation during chronic high-fat diet in dogs Physiol Genomics, September 16, 2004; 19(1): 32 - 40. [Abstract] [Full Text] [PDF]

				C. S. Samuel, E. N. Unemori, I. Mookerjee, R. A. D. Bathgate, S. L. Layfield, J. Mak, G. W. Tregear, and X.-J. Du Relaxin Modulates Cardiac Fibroblast Proliferation, Differentiation, and Collagen Production and Reverses Cardiac Fibrosis in Vivo Endocrinology, September 1, 2004; 145(9): 4125 - 4133. [Abstract] [Full Text] [PDF]

				P. Stawowy, C. Margeta, H. Kallisch, N. G Seidah, M. Chretien, E. Fleck, and K. Graf Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-{beta}1 involves furin-convertase Cardiovasc Res, July 1, 2004; 63(1): 87 - 97. [Abstract] [Full Text] [PDF]

				K. T. Weber From Inflammation to Fibrosis: A Stiff Stretch of Highway Hypertension, April 1, 2004; 43(4): 716 - 719. [Full Text] [PDF]

V.V Petrov, R.H Fagard, and P.J Lijnen Arginine-aminopeptidase in rat cardiac fibroblastic cells participates in angiotensin peptide turnover Cardiovasc Res, March 1, 2004; 61(4): 724 - 735. [Abstract]