TGF-β1-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

TGF-β1-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages Victor V. Petrov a,⁎, Jos F. van Pelt b , Joris R. Vermeesch c , Viktor J. Van Duppen d , Katrien Vekemans b , Robert H. Fagard a , Paul J. Lijnen a a

Department of Heart Diseases, University of Leuven (KULeuven), Leuven, Belgium Department of Liver and Pancreatic Diseases, University of Leuven (KULeuven), Leuven, Belgium c Center for Human Genetics, University of Leuven (KULeuven), Leuven, Belgium d Department of Hematology, University of Leuven (KULeuven), Leuven, Belgium b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

TGF-β1 induces differentiation and total inhibition of cardiac MyoFb cell division and DNA

Received 25 July 2007

synthesis. These effects of TGF-β1 are irreversible. Inhibition of MyoFb proliferation is

Revised version received

accompanied with the expression of Smad1, Mad1, p15Ink4B and total inhibition of

3 December 2007

telomerase activity. Surprisingly, TGF-β1-activated MyoFbs are growth-arrested not only at

Accepted 9 January 2008

G1-phase but also at S-phase of the cell cycle. Staining with TUNEL indicates that these cells

Available online 26 January 2008

carry DNA damages. However, the absolute majority of MyoFbs are non-apoptotic cells as established with two apoptosis-specific methods, flow cytometry and caspase-dependent

Keywords:

cleavage of cytokeratin 18. Expression in MyoFbs of proliferative cell nuclear antigen even in

Myofibroblasts

the absence of serum confirms that these MyoFbs perform repair of DNA damages. These

TGF-β1

results suggest that TGF-β1-activated MyoFbs can be growth-arrested by two checkpoints,

Cell differentiation

the G1/S checkpoint, which prevents cells from entering S-phase and the intra-S checkpoint,

Cell proliferation

which is activated by encountering DNA damage during the S phase or by unrepaired

Telomerase

damage that escapes the G1/S checkpoint. Despite carrying of the DNA damages TGF-β1-

Cell cycle

activated MyoFbs are highly functional cells producing lysyl oxidase and contracting the

Cytoskeleton

collagen matrix.

DNA synthesis

© 2008 Elsevier Inc. All rights reserved.

DNA damage DNA repair Apoptosis Flow cytometry Lysyl oxidase Collagen matrix contraction

Introduction Myofibroblasts (MyoFbs) are absent in normal myocardium and appear in pressure overloaded hearts [1], at cardiomyo-

pathy [2] or after myocardial infarction (MI) [3–5]. In contrast to MyoFbs from other organs, the intrinsic features of the cardiac MyoFbs have not been intensively studied. Cardiac MyoFbs actively proliferate early after MI but cease proliferation in old

⁎ Corresponding author. Hypertension and Cardiovascular Rehabilitation Unit, Herestraat 49 Box 702, room 07.420, 3000 Leuven, Belgium. Fax: +32 16 34 71 06. E-mail address: [email protected] (V.V. Petrov). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.01.014

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maturated scars [5–7]. The reason for the termination of their proliferation is unknown, although the disappearance of an unidentified mitogen was suggested [5]. The amount of MyoFbs during scar maturation diminishes via apoptosis [6,8]. Nevertheless, MyoFbs do not disappear entirely and persist in the scar over extended periods of time if not during the entire life span of individuals or animals [4–6,9]. The signals regulating proliferation, inducing apoptosis and responsible for the survival of the cardiac MyoFbs are poorly understood. MyoFb occurrence in the infarcted myocardium is preceded by a temporary increase in tissue content of transforming growth factor-β1 (TGF-β1) [9]. In vitro studies have shown that TGF-β1 induces differentiation of cardiac MyoFbs, controls apoptosis and inhibits proliferation of various cell types from other organs either reversibly or irreversibly [10,11]. Various mechanisms could be involved in the induction of non-proliferating MyoFb by TGF-β1. TGF-β1-induced expression of a marker of senescence, senescent-associated β-galactosidase (SA-β-Gal), in prostate fibroblasts (Fbs), suggests a possible role of senescence in the inhibition of cardiac MyoFb [12,13]. TGF-β1induced inhibition of telomerase reverse transcriptase (TERT) expression and telomerase activity in pulmonary Fbs suggests a possible role of telomerase in the termination of the cardiac MyoFb growth [14]. Apoptotic death of a part of MyoFb during scar maturation and survival of others could also be related to TGF-β1. Indeed, TGF-β1-induced pro-apoptotic responses were previously observed in numerous cell types whereas TGF-β1induced survival was observed in many others [15]. The aim of the present study was to investigate whether TGF-β1 irreversibly induces the appearance of MyoFbs, which fail to proliferate, even in the absence of this growth factor and to elucidate the mechanism(s) for the termination of their proliferation.

Materials and methods Cell culture The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85–23, revised 1996). The research protocol is approved by the Ethical Committee for Animal Experiments of the Katholieke Universiteit Leuven, Belgium. Cardiac ventricular fibroblasts (Fbs) were isolated from hearts of male Wistar rats by collagenase digestion and were cultured as described previously [16]. Fbs spontaneously differentiated into proliferative MyoFbs (p-MyoFbs). Fbs cultured for 7 days with TGF-β1 (400 pmol/l or 10 ng/ml) performed further differentiation into MyoFbs, which were named TGF-β1activated MyoFbs (a-MyoFbs). Previously we have shown that TGF-β1 inhibited cell proliferation and induced differentiation of cardiac MyoFbs in a dose- and time-dependent manner [16,17]. Concentration of TGF-β1 (400 pmol/l) and time of incubation (7 days) are based on the results of these studies. To evaluate the reversibility of the TGF-β1-induced effects we reseeded a-MyoFbs and cultured them for 7–21 days in either medium with serum (10% fetal bovine serum) without exogenous TGF-β1 or in serum free medium, containing epidermal growth factor (EGF). In another experiments t-MyoFbs were

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grown for 4–5 days in medium with serum and then for 3– 2 days in serum free medium with 1.7 nmol/l (10 ng/ml) of EGF. These cells were named terminally differentiated MyoFbs (tMyoFbs).

Proliferation of MyoFbs After incubation for 1–21 days with or without TGF-β1 (400 pmol/l) or EGF (1.7 nmol/l) cells were harvested, stained with trypan blue and then viable cells were counted. 3H-thymidine incorporation was measured as previously described [17].

Protein electrophoresis and Western blot Western blotting was performed as previously described [16]. Rat cardiac fibroblasts from cultures were trypsinized, counted and stored at −80 °C until use. Cells were mixed with reducing sample buffer (pH = 6.8), boiled (5 min) and centrifuged (10 min, 13,000 ×g). The supernatant was collected; equal samples (50,000 cells in 30 μl/lane) were loaded onto a 15% polyacrylamide gel and separated according to manufacturer's instructions. After blotting onto a nitrocellulose membrane (Protran BA85, Schleicher & Schuell) and blocking of non-specific binding sites with non-fat milk powder (5% w/v in 10 mM phosphate buffer pH 7.4 containing 0.05% Tween 20 v/v (PBSTween)), the membrane was incubated with the primary antibody in PBS-Tween for 16 h. The blot was washed 6 times with PBS-Tween, followed by horseradish peroxidase-conjugated secondary anti-mouse antibody (Dako, 1:5000). Immunoreactivity was visualized by chemoluminescent detection using the ECL system (Amersham Biosciences, Belgium). The membrane was washed, blocked with non-fat milk powder (5% w/v PBSTween) and reproved with mouse anti-β actin followed by horseradish peroxidase-conjugated anti-mouse antibody this to confirm equal protein loading. Experiments were performed in triplicate. Antibodies for p15INK4b (Biosource International, dilution 1:200), p16INK4a (Santa Cruz Biotechnology, dilution 1:200), Smad1 (Santa Cruz Biotechnology, dilution 1:100), and β-actin (Sigma, dilution 1:15,000) were used.

Reverse transcription and semiquantitative PCR analysis Total RNA was isolated as previously described [18]. Briefly, total RNA was extracted from cells in a single step procedure with Trizol (Invitrogen). The precipitated RNA was dissolved in 20-μl DEPC-treated water and the concentration was measured using the Ribogreen RNA quantitation kit (Molecular Probes, Eugene, Oregon USA) with ribosomal RNA as standard. One μg of this RNA was used for cDNA synthesis with M-MLV reverse transcriptase (GibcoBRL, Life Technologies, Belgium) and random primers (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) in a volume of 20 μl, 1 h at 37 °C. The reaction was stopped by heating in boiling water for 1 min. 10 μl of PCR product was separated on 1% agarose gel. After digital recording of the image, a densitometric analysis of the signals was performed and the intensity was calculated relative to the house keeping gene GAPDH. Results are the mean of 3 separate cell culture experiments and the subsequent PCR analysis. Primers and PCR conditions are given in

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Table 1. mRNA expression of α-SMA, p15Ink4B, p16Ink4A, Smad1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene were studied.

Immunostaining Immunostaining has been performed with EnVision Systems (Dako). Antibodies for α-SMA (Clone 1A4, Sigma, dilution 1/400), vinculin (Clone H-300, Santa Cruz Biotechnology, dilution 1:500), Mad1 (Clones C-19, Santa Cruz Biotechnology, dilution 1:100), Ki-67 (polyclonal, Santa Cruz Biotechnology, dilution 1:10), and Proliferative cell nuclear antigen (PCNA) (Clone PC-10, Santa Cruz Biotechnology, dilution 1:10) were used. 200 cells from six randomly chosen fields were analyzed. Stress fibers were visualized with staining for α-SMA. This is possible, because short time after synthesis α-SMA becomes incorporated into actin filaments [19,20].

cytokeratin 18 by activated caspases (M30 CytoDeath; Roche Applied Science), determination of the fraction of cells in subG1phase with flow cytometry and conventional electrophoresis of the DNA extracted from MyoFbs (Apoptotic DNA ladder kit; Roche Applied Science) were used.

Telomerase activity Telomerase activity was determined using a telomeric repeat amplification protocol (TeloTAGGG Telomerase PCR ELISA kit, Roche Diagnostics).

Lysyl oxidase (LO) LO activity was evaluated with a fluometric assay utilizing cadaverine as enzyme substrate [22]. Released hydrogen peroxide was detected with an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes).

Flow cytometry Senescent associated β-galactosidase (SA-β-Gal) Cultured cells were trypsinized and washed twice with PBS and carefully resuspended in 0.5 ml PBS with Pasteur pipette to achieve a single-cell suspension. Cells were fixed by dropping them slowly into thoroughly mixed ice-cold 70% ethanol (4.5 ml) and stored at −20 °C. Fixed cells were washed twice in PBS, permeabilized and stained with propidium iodide (PI) by re-suspending them in a freshly prepared solution containing 0.025% Triton X-100, 100 μg/ml RNAse A and 25 μg/ml PI (2 × 106 cells/2 ml), and incubated for 45 min at room temperature. Cells were analyzed for DNA content by flow cytometry (FACScanto, BD). In each sample 50,000 cells were counted. Data were calculated with the Modfit software (Verity Software House, Inc.). Data are presented as histograms (cell number– cell DNA content or dot plots SSC-FCS, where SSC is sidescattered light that is proportional to cell granularity or internal complexity, and FSC is forward scattered light that is proportional to cell-surface area. Measurements of SSC-FSC allow differentiation of cell phenotypes in a heterogeneous cell population. [21]

DNA breaks and apoptosis determination For detection of DNA breaks and apoptosis a kit based on TUNEL technology (In situ cell death detection kit; Roche Applied Science), a kit for the detection of a specific cleavage of

SA-β-Gal was measured according to the method of Dimri et al. [13].

3-Dimensional collagen matrices (3-DCM) contraction The ability of cardiac Fbs and MyoFbs with different phenotypes to contract extracellular matrix was evaluated with contraction of 3-dimensional collagen matrices (3-DCM) populated with these cells. 3-DCM were prepared from rat tail collagen. A 12.55 ml collagen solution was prepared by mixing 4.25 ml collagen in 0.02N acetic acid (4.38 mg/ml), 1.65 ml 100 mmol/l NaOH, and 1.25 ml 10× MEM and 5.4 ml of bidistilled water was added to obtain a final concentration of 1.5 mg collagen per ml. The solution was kept on ice until addition of cells. 0.5 ml of this collagen solution was added to each BSAtreated well (24-well dish). MyoFbs in DMEM (100,000 cells) were added to this solution in the wells and incubated for 1 h at 37 °C for gel polymerization. Subsequently, 1 ml of DMEM was added to each well. The gels were detached spontaneously from the bottom of the wells and placed in a tissue culture incubator for 1–3 days [23]. The volume of the 3-DCM was estimated by measurement of the 3H2O distribution between 3-DCM and medium according [24]. The gel volumes of the 3-DCM populated with MyoFbs and of the 3-DCM without cells were compared.

Table 1 – Primers and reverse transcription PCR conditions primer 5′ → 3′ α-SMA-F α-SMA-R p15INK4b-F p15INK4b-R p16INK4a-F p16INK4a-R Smad-1-F Smad-1-R GAPDH-F GAPDH-R

CGC-CGC-TGA-ACC-CTA-AGG-CCA-AGG-G GCT-GGA-AGA-GGG-TCT-CCG-GGC-AGC-G ATG-ATG-ATG-GGC-AGC-GCC TCC-CGA-GCT-GCA-TCA-TGC-A TCT-GCA-GAT-AGA-CTA-GCC-A CTC-GCA-GTT-CGA-ATC-TGC-A GCA-TCA-ATC-CCT-ACC-ACT-ATA-AGC GTC-GGC-TGG-CAT-CTG-AAA-AG TCA-TCA-TCT-CCG-CCC-CTT-CCG-C AGG-CGG-CAT- GTC-AGA-TCC-ACA-ACG

Temperature °C Extension time Number of of cycles Size bp Reference 58

1 min

40

467

58

30 s

40

109

This study X06801 [14]

59

30 s

45

180

[14]

52

1 min

55

293

56

1 min

55

390

[57] NM_013130 This study NM_017008

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Fluorescent in situ hybridization (FISH) Fluorescent in situ hybridization was essentially performed as described [25]. Before FISH, cells were air dried on slides and pretreated with pepsin followed by fixation with a 1% free formaldehyde solution and subsequently dehydrated with ethanol. After hybridization overnight at 37 °C with a fluoresceine-labeled rat chromosome X painting probe (Cambio) using the concentration recommended by the manufacturer, the slides were washed for 1 min in 0.4 × SSC/0.3% NP40 solution at 72 °C, 1 min at 2× SSC/0.1% NP40 solution at RT and 1 min at 2× SSC (where 2× SSC is solution containing 0.15 mol/l NaCl + 0.03 mol/l sodium citrate). The cells were counterstained with DAPI and the slides were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The signal was visualized by digital imaging microscopy with Cytovision capturing software (Applied Imaging, Santa Clara, CA).

Statistical analysis All data are shown as mean ± SEM. The statistical methods used were repeated measures analysis of variance (Tukey's), or Student's two-tailed test for paired data when appropriate. A two-tailed p-value ≤ 0.05 was considered statistically significant.

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Results MyoFb morphology Up to 24 h after seeding, the cardiac Fbs were elongated and stained negative for α-SMA (Fig. 1A). However, after three days, in agreement with previous observations [26–29], the majority of cells became more spread, larger and lightly stained for α-SMA (Fig. 1B) indicating their moderate differentiation into proliferative p-MyoFbs. TGF-β1 greatly extended myofibroblastic differentiation and the TGF-β1-activated MyoFbs (a-MyoFbs) became considerably larger and showed much more intensive staining for α-SMA compared to the p-MyoFbs (Table 2, Fig. 1C). This is compatible with our previous results, which showed that TGF-β1 induced α-SMA expression in cultured cardiac Fbs in a dose- and time-dependent manner [16]. To evaluate the reversibility of the TGF-β1-induced effects we did not only remove TGF-β1 from the medium of the confluent cultures of a-MyoFbs, but also reseeded these cells and cultured their progenies for 7–21 days in a medium supplemented with 10% FBS in the absence of exogenous TGF-β1. The reason of that was to avoid effects induced by cell contacts in confluent cultures of a-MyoFbs, such as inhibition of cell proliferation and telomerase activity [30].

Fig. 1 – TGF-β1-induced expression of α-SMA and vinculin in cardiac MyoFb. (A–D) Immunostaining of rat ventricular Fbs and MyoFbs for α-SMA. (A) Fbs: 6 h after seeding; (B) p-MyoFbs spontaneously differentiated from Fbs after 3 days of culturing; (C) a-MyoFbs differentiated from Fbs, which were cultured for 7 days in the presence of TGF-β1; (D) t-MyoFbs that are progenies of a-MyoFbs, which were reseeded and cultured for seven days without exogenous TGF-β1; (A–C) Bar, 50 μm. (D) Bar, 80 μm. (E) α-SMA mRNA expression in p-MyoFbs and t-MyoFbs; (F–G) Immunostaining of p-MyoFbs and t-MyoFbs for vinculin. Bar, 150 μm. (n = 6).

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Table 2 – Incidence of MyoFbs with high cell size and protein content; cells in metaphase; cells with aberrant lobed nuclei; PCNA-positive and Ki-67-positive MyoFbs; Intensity of immunostaining for α-SMA, vinculin, and Mad1 Cells

p-MyoFbs

a-MyoFbs

t-MyoFbs

Aberrantly shaped nuclei (%)

PCNA-positive cells (%)

Ki-67positive cells (%)

Intensity of immunostaining

Cell size (arbitrary units)

Cell protein content (μg/106 cells)

Metaphases (%)

49.4 ± 5.0 Max. 92.1 Min. 21.0 92.2 ± 11.7⁎ Max.317.9 Min. 46.1 518 ± 105⁎# Max.1260.0 Min. 136.4

286 ± 19

2.4 ± 1.3

0

21.8 ± 3.1

61 ± 3

+

+

−

726 ± 133⁎

0

0.5 ± 0.1

3.3 ± 0.2⁎

ND

+++

ND

ND

1569 ± 467⁎

0

17.0 ± 4.2

In 10% FBS 88.3 ± 4.3⁎⁎⁎ In 0.5% FBS 95.0% ± 0.9⁎⁎⁎

0.3 ± 0.1⁎⁎⁎

+++++

+++

+

α-SMA

Vinculin

Mad1

mean ± SEM. MyoFbs; #p b 0.05 vers. a-MyoFbs; *p b 0.01 vs. p-MyoFbs; ***p b 0.001 vs. p-MyoFbs; ND \ not determined; (+) positive immunostaining; (−) negative immunostaining.

Reseeded a-MyoFbs cultured without exogenous TGF-β1 did not return to the p-MyoFb phenotype. Instead, they demonstrated advanced intensity of immunostaining for α-SMA incorporated into very dense network of stress fibers (Fig. 1D), which was accompanied by a significant increase in the α-SMA mRNA expression as compared to the p-MyoFbs (Fig. 1E). These cells were named terminally differentiated MyoFbs (t-MyoFbs), because they did not proliferate (Fig. 2A). p-MyoFbs and t-MyoFbs (Figs. 1F,G) were stained positive for vinculin and the staining of t-MyoFbs was much more intensive compared to p-MyoFbs, indicating an increase in the number and/or size of focal adhesions. As it was abovementioned, t-MyoFbs exhibit higher levels of α-SMA than a-MyoFbs despite culturing in the absence of exogenous TGF-β1, which is a strong inducer of α-SMA [11]. This can be explained by the influence of cell contacts on the α-SMA expression. After seven days incubation with TGF-β1 cultures of a-MyoFbs are confluent. To avoid cell contactinduced inhibition of the cell proliferation and of the cell growth we reseeded these cells at low density. Therefore, in contrast to a-MyoFbs the majority of non-proliferating t-MyoFbs grow as single cells without intercellular contacts even after three weeks of the culturing. However, cell density in culture has been found to control the expression of α-SMA in fibroblasts. Particularly, seeding of fibroblasts at low density induces α-SMA expression [31]. In the recent study it has been elucidated that disruption of the intercellular contacts induces Rho GTP-ase activation and a consequent nuclear accumulation on the main α-SMAinducing transcription factors, serum response factor and its coactivator myocardin-related transcription factor [32]. a-MyoFbs had significantly larger cell area than p-MyoFbs, while t-MyoFb, the growth of which was much less restricted by cell contacts, had a 10-fold large cell area than p-MyoFbs (Table 2, Figs. 1D, 2D); this was associated with a 5-fold elevation of the cell protein content (Table 2). 17% of t-MyoFbs and 0.5% of a-MyoFbs show aberrant shaped, crenated nuclei (Table 2, Fig. 5B), which are enlarged compared to the normal shaped nuclei. In contrast to t-MyoFbs and a-MyoFbs, aberrant shaped nuclei were not found in p-MyoFbs.

Cell proliferation Previously, we have shown TGF-β1-induced inhibition of cell proliferation in cultures of rat cardiac Fbs [17]. Here we studied the reversibility of this growth inhibition. Cell proliferation in cultures of cardiac Fbs was seen over the whole period (7 days) of the experiment despite spontaneous differentiation of Fbs to p-MyoFbs within 3 days (Fig. 2A, p-MyoFbs; Fig. 1B), indicating that p-MyoFbs are proliferative cells. TGF-β1 inhibited time-dependently the proliferation of MyoFbs. During the first 2 days of culturing, TGF-β1 did not affect the cell number, but considerably inhibited the proliferation by days 3 to 5, leading to a complete arrest of a-MyoFb proliferation by days 6 to 7 (Fig. 2A, a-MyoFbs). Comparable time-dependent inhibition of proliferation was found for valvular interstitial cells during their differentiation into MyoFb induced by TGF-β1 [29]. TGF-β1-induced growth arrest was irreversible, because t-MyoFbs cultured without exogenous TGF-β1 in medium supplemented with 10% of FBS performed 1.4 population doublings in the first three days and then the amount of cells in culture was not significantly changed up to 7 days (Fig. 2A, t-MyoFbs) as well as up to 21 days (data not shown) of culturing. This is compatible with the absence of metaphases in colcemid-treated cultures of t-MyoFbs. However, 2.4% of p-MyoFbs was found in metaphase (Table 2; Fig. 5B). TGF-β1 almost completely inhibited 3H-thymidine incorporation in a-MyoFbs (Fig. 2B) and their progenies, t-MyoFbs, incorporated 3H-thymidine with a markedly reduced activity (∼ 1/9-fold) compared to p-MyoFbs (Fig. 2B). Exogenously added TGF-β1 inhibits proliferation of cardiac a-MyoFb (Fig. 2A). It is known that serum contains endogenous TGF-β1 [33,34] and this TGF-β1 presented in serum might be able to inhibit proliferation of t-MyoFbs despite active proliferation of p-MyoFbs in serum containing medium. To address this question we have compared proliferation of t-MyoFb (an increase in the cell amount and 3H-thymidine incorporation) in the medium with serum (10% FBS) and in serum free medium containing a strong mitogen, EGF [35]. We observed that EGF at concentration of 1.7 nmol/l (10 ng/ml) did not stimulate quiescent t-MyoFbs. There were not significant differences in both, 3H-thymidine

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Fig. 2 – Proliferation, telomerase activity and cell cycle in TGF-β1-induced cardiac MyoFbs. (A) Time-dependent increase in cell amount in cultures;(n = 4) (B) 3H-thymidine incorporation. (n = 6) (C) Telomerase activity. (n = 4). (D–H) flow cytometry. (n = 3) (D) histogram: number of events \ FSC; FSC — forward scattered light that is proportional to cell-surface area. (E – H) histograms: number of events \ cell DNA content. (H) t-MyoFbs were cultured for 7 days in the presence of aphidicolin (10 μmol/l).

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incorporation (Fig. 2B) and cell number (data not shown) between t-MyoFbs cultured in either medium with serum or serum free medium with EGF.

Cell cycle profile Both p-MyoFbs as well as TGF-β1-activated a-MyoFbs showed G1, S and G2 phases of the cell cycle (Figs. 2E,F). TGF-β1 changed the cell cycle profile of MyoFbs. In cultures of a-MyoFbs the fraction of cells in G1-phase was increased. However, the fractions of cells in S-phase and in G2-phase were considerably decreased compared to the p-MyoFbs (Figs. 2E,F). These effects of TGF-β1 are comparable with those obtained with other cells [12]. In t-MyoFbs the G2-phase was completely inhibited whereas the S-phase was increased 2-fold compared to p-MyoFbs

(Fig. 2G). The cell cycle profile was not significantly changed when the t-MyoFbs were cultured further, for 14 or 21 days. The G2 phase was still absent and the fractions of cells in G1 and S phases were 47.7% and 52.3%, respectively. We examined the effect of aphidicolin, which arrests cell cycle at G1 phase [36]. Aphidicolin, after 7 days of culturing at a concentration of 10 μmol/l, greatly decreased the fraction of t-MyoFb in the G1 phase but increased the S phase. Treated t-MyoFbs still did not show G2 phase (Fig. 2H).

PCNA and Ki-67 PCNA is an auxiliary protein for polymerases α, δ and ε and is involved in both the DNA replication and repair [37,38]. Immunostaining showed that, in a medium with a high level of

Fig. 3 – TGF-β1-induced expression of p15INK4b (A–B) and p16INK4a (C) in cardiac MyoFbs. (A) Western blotting of p15Ink4B (n = 3). (B) p15Ink4B mRNA expression (n = 3). (C) p16Ink4A mRNA expression (n = 3).

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serum (10% FBS), 22% of proliferative p-MyoFbs were PCNApositive (Table 2). TGF-β1 diminished the population of PCNApositive cells to 3.3% (Table 2). Surprisingly, in cultures of nonproliferative t-MyoFbs the population of PCNA-positive cells was 4-fold larger compared to cultures of p-MyoFbs (Table 2). Culturing of t-MyoFbs for additional two or three days in medium containing a low level of serum (0.5% FBS) did not changed (p b 0.05) this cell fraction, ∼95% (Table 2). However, in these conditions normal proliferating cells do not stain for PCNA [37]. Ki-67 is a replication-associated antigen [39]. In a medium with serum (10% FBS), 61% of proliferative p-MyoFbs were Ki67-positive, whereas almost all t-MyoFbs were Ki-67-negative (Table 2).

Telomerase activity Contrary to human cells, somatic cells of rats demonstrate telomerase activity. Inhibition of the enzyme activity can be

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associated either with cell senescence or cell differentiation [14,40,41]. High telomerase activity was found in p-MyoFbs, which was considerably inhibited in a-MyoFbs and was completely absent in the t-MyoFbs (Fig. 2C). Cultures of t-MyoFbs were always far from confluence. Therefore, we conclude that the loss of telomerase activity in t-MyoFbs cannot be explained by the formation of intercellular contacts.

SA-β-Gal, p15INK4b, p16INK4a To evaluate possible senescence of t-MyoFbs we studied in cardiac MyoFbs SA-β-Gal activity, a marker of senescent cells [13], and the expression of inhibitors of G1 phase cyclin kinases, p16INK4a and p15INK4b. Senescent cells express p16Ink4A whereas TGF-β1 induces normally p15Ink4B [42]. We have not found SA-βGal activity in all three cell phenotypes, p-MyoFbs, a-MyoFbs or t-MyoFbs (data not shown). Figs. 3 (A, B) shows a significant stimulation of the p15Ink4B mRNA expression and protein

Fig. 4 – Mad1 expression in TGF-β1-induced cardiac MyoFb. (A–B) Immunostaining of cardiac MyoFbs for Mad1. Bar, 70 μm (n = 4). (C) Smad1 mRNA expression in cardiac MyoFbs (n = 4). (D) Western blotting of Smad1. The top panel represents Western blotting and lower panel is densitometric evaluation of the blot (n = 4).

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synthesis in a-MyoFbs and t-MyoFbs as compared to p-MyoFbs. Surprisingly, p16Ink4A mRNA expression was stimulated in both a-MyoFbs and t-MyoFbs (Fig. 3C) while synthesis of p16Ink4A protein was not changed significantly (data not shown).

Mad1 TGF-β1 induces in various cell types the expression of Mad1, which is related to the induction of the cell differentiation and to the inhibition of the cell proliferation [43,44]. Immunostaining did not reveal Mad1 in control p-MyoFbs (Fig. 4A), which is in agreement with previous studies showing that proliferative cells do not show Mad1 protein [45]. In t-MyoFbs, Mad1 was located around but not in nuclei (Fig. 4B), in agreement with studies performed with terminally differentiated chondrocytes [46]. Mad1 becomes active after binding with Max [44]. Therefore, we studied mRNA expression of Max. However, there was

no significant difference in the levels of Max mRNA between p-MyoFbs and t-MyoFbs.

Smad1 Smad1 mRNA expression levels were significantly higher (3-fold increase) in t-MyoFbs than in p-MyoFbs (Fig. 4C). Stimulation of the Smad1 mRNA expression is accompanied by induction (5-fold increase) of Smad1 protein (Fig. 4D indicating irreversible induction of Smad1 expression during TGF-β1-induced MyoFb differentiation).

DNA fragmentation — TUNEL labeling, DNA electrophoresis, sub-G1 phase Cell differentiation is associated with breaks of the nuclear DNA. Despite this pro-apoptotic process, the differentiated

Fig. 5 – (A) TUNEL labeling of TGF-β1-induced cardiac MyoFbs (n = 3). (B) Fluorescent in situ hybridization with a fluoresceine-labeled rat chromosome X painting probe (green). Cells were counterstained with DAPI (blue) (n = 3). Bar, 20 μm. (C) Conventional DNA electrophoresis in 1% agarose gel. Positive control: L-937 cells treated with camptothecin (n = 3).

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cells remain to be living cells and do not show apoptotic morphology [47–51]. TUNEL technique labels single strand breaks (SSB) and double strand breaks (DSB) of DNA [52–54]. TUNEL labeling of a-MyoFbs and especially of p-MyoFbs was very low. In contrast, 96 ± 3% of the t-MyoFb nuclei were TUNEL positive with variable intensities of fluorescence (Fig. 5,A), although these intensities were considerably lower than observed in control apoptotic t-MyoFbs treated with DNase 1.

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Experiments with DNA electrophoresis in 1% agarose gel have shown DNA fragmentation in all three phenotypes of cardiac MyoFbs. There is no considerable difference in DNA fragmentation between proliferating p-MyoFbs and a-MyoFbs or t-MyoFbs. All three phenotypes of cardiac MyoFbs, p-MyoFbs, aMyoFbs and t-MyoFbs show a single band with the same high molecular weight: HMW-DNA fragment (Fig. 5C). t-MyoFbs show faint second HMW-DNA band. These HMW-DNA fragments bands were not seen in DNA isolated from apoptotic L-937 cells used as the positive control (Fig. 5C, line P). All MyoFb phenotypes show two strong bands with low molecular weight, LMW-DNA fragments, one between 1500 and 2072 bp and another one 800 bp. a-MyoFbs and t-MyoFbs show faint bands with molecular weight lower than 800 bp, whereas p-MyoFbs do not. It is important to underscore that molecular weights of DNA fragments in all MyoFb phenotypes are different from molecular weights of DNA fragments after internucleosomal fragmentation in the apoptotic L-937 cells. L-937 cells show 185 bp integer DNA fragments whereas DNA fragments in MyoFbs have intermediate values (Fig. 5C). It indicates that a mechanism of the DNA fragmentation in MyoFbs can be different from that in apoptotic cells. Faint bands of the LMW-DNA fragments belong most likely to the minority of apoptotic cells, which are present in cultures of all MyoFb phenotypes (Figs. 2E–H). Appearance of the sub-G1 phase of the cell cycle indicates DNA fragmentation and loss of DNA fragments by cells. Flow cytometry of MyoFbs specified the existence of only very small cell populations at the sub-G1 phase, indicating that only 0.7%, 0.1% and 0.3% of each cell population had abnormally low cell DNA content (apoptotic cells) in cultures of p-MyoFbs, a-MyoFbs and t-MyoFbs, respectively (Figs. 2E,F,G). In agreement with very low level of the apoptotic MyoFbs determined with the flow cytometry, labeling of cleavage site within cytokeratin 18 specific for activated caspases [55,56] showed a very low level of apoptosis (b0.1%) in p-MyoFbs, aMyoFbs and t-MyoFbs.

Lysyl oxidase (LO) and 3-DCM contraction by MyoFbs To elucidate if t-MyoFbs are still functional cells, we studied the activity of LO and the t-MyoFb-induced 3-DCM contraction. The low LO activity in control p-MyoFbs was 2-fold increased in a-MyoFbs. Non-proliferating t-MyoFbs showed a 10-fold higher level of LO activity than p-MyoFbs (Fig. 6A). p-MyoFbs slightly contracted the 3-DCM while both a-MyoFbs and t-MyoFbs contracted 3-DCM almost 3-fold more intensive than p-MyoFbs (Fig. 6B), indicating that TGF-β1 irreversibly induces highly contractible MyoFbs.

Discussion

Fig. 6 – (A) Lysyl oxidase activity in cultures of TGF-β1-induced cardiac MyoFbs (n = 6). (B) Contraction of 3-DCM by p-MyoFbs, a-MyoFbs and t-MyoFbs (n = 6). (−cells) \ 3-DCMs are not populated with cells.

The aim of this study was to elucidate the relationship between TGF-β1-induced irreversible differentiation of MyoFb and their proliferative capacity and to find a possible explanation for the loss of their proliferation. Present data and data previously obtained by us, which showed that TGF-β1-induced expression of α-SMA in the rat cardiac MyoFbs is dose- and time-dependent [16], indicate that TGF-β1 induces gradual and finally irreversible differentiation

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of rat cardiac MyoFbs. Gradual myofibroblastic differentiation is associated with gradual, time-dependent inhibition of a-MyoFb proliferation and with complete loss of the proliferative capacity of their progenies, t-MyoFbs that is proved by: 1) constant cell number in cultures of t-MyoFbs for at least three weeks; 2) negligible 3H-thymidine incorporation into t-MyoFbs compared to proliferative p-MyoFbs (9-fold less); 3) absence of the Ki-67 expression, a replication-associated antigen; 4) t-MyoFbs either treated or non-treated with colcemide do not show metaphase as it was shown by FISH; 5) t-MyoFbs do not show G2 phase of the cell cycle for at least three weeks as it was shown by flow cytometry; 6) Addition of the inhibitor of DNA replication, aphidicolin, which inhibits polymerases (α, δ, ε) and arrests cell growth at G1 phase [36], to the cultures immediately after t-MyoFb seeding decreased G1 phase. 7) t-MyoFbs demonstrate very low activity if at all of the telomerase; 8) stimulated expression of p15INK4b, which arrests cells in the G1 phase of the cell cycle via inhibition of the G1-phase cyclin kinases, Cdk4/ Cdk6; 9) induction of Mad1, a transcriptional factor related to the terminal cells differentiation [44]; 10) absence of the proliferation in serum free medium containing strong mitogen EGF. Furthermore, the inability of a strong mitogen, EGF to induce the cell division and 3H-thymidine incorporation in the serum free medium indicates that the termination of the t-MyoFb proliferation is their intrinsic fundamental feature but not related to the inhibition by TGF-β1, which presents in the serum. Inhibition of telomerase activity during cell differentiation, particularly during TGF-β1-induced differentiation, was earlier shown for pulmonary Fbs [14,41]. The inhibition of telomerase in t-MyoFbs can be mediated by Mad1, which suppress the activity of the TERT promoter and inhibits TERT expression and telomerase activity in other cells [57,58]. Recently it has been found another way by which TGF-β1 repress TERT via Smad3 [59]. Both, Mad1 and Smad3 are related to the cell differentiation [44,60]. Transfection of cells with Smad3-expressing plasmid markedly increased differentiation and α-SMA expression in fibroblasts [60]. It is of interest that TGF-β1 considerably induces (5-fold increase) in cardiac MyoFbs Smad1 (Figs. 4C–D), which is also related to the cell differentiation and is able to induce α-SMA expression [61]. It is generally known that Smad1 mediate bone morphogenetic protein signaling [62]. However, TGF-β1 may also signal through Smad1 pathway in some cells [63]. Particularly, Smad1 seems to be able to regulate α-SMA expression through TGF-β1 responsible CArG elements on promoter [64]. The loss of the telomerase activity could be a basis for cell senescence [40]. However, TGF-β1 does not induce senescence in cardiac t-MyoFbs. Indeed, t-MyoFbs do not stain for SA-βGal, a marker of the cell senescence, express p15Ink4B, α-SMA and show G1 and S phases of the cell cycle. Our previous data showed that these cells produce elevated levels of collagen [16]. However, senescent cells stain for SA-β-Gal [13], express p16Ink4A but not p15Ink4B [12,42], do not express α-SMA [42], are arrested at exclusively G1 phase [12] and show very little activity of collagen production [65,66]. 51% of t-MyoFbs are found to be in S phase of the cell cycle suggesting that these t-MyoFbs still proliferate but the cell cycle is retarded and the culturing for 7 days is not enough long for the cells to proceed up to G2 phase. If this were true, a culturing of t-MyoFbs for the extended period of time would be

resulted in the appearance of G2 phase and cell division. However, cell number and the cell cycle profile of t-MyoFbs are not changed and G2 phase is still absent after 21 days of culturing. On the other hand, loss of the telomerase activity in t-MyoFbs despite augmentation of the S phase suggest an inability of S-phase t-MyoFbs to proliferate, because in proliferating cells the telomerase activity is present throughout the entire cell cycle and its level is the highest in S phase [67]. The incapability of G1 t-MyoFbs to proliferate is supported by an inability of aphidicolin, which arrests cells at G1 phase [36], to increase number of G1 MyoFbs. The incapability of tMyoFbs to replicate DNA at both, G1- and S-phase is further supported by the deficiency to express Ki-67. This indicates that t-MyoFbs are permanently arrested either at G1 or at S phases and the S phase is not related to DNA replication. To explain the appearance of S phase in non-proliferating t-MyoFbs we suggest that the arrest of t-MyoFbs at S phase can be related to the appearance of multiple DNA breaks. To recognize DNA breaks we used TUNEL technique. TUNEL is not specific for apoptosis, because besides DNA breaks in apoptotic cells it also recognizes functional cells carrying DNA breaks and performing DNA repair [52,53,68]. Therefore, it has earlier been concluded that TUNEL technique may not be used alone for the determination of apoptosis [52–54,68,69]. TUNEL labels 96 ± 3% of t-MyoFbs (although intensity of fluorescence was considerably lower than in control apoptotic cells) indicating that absolute majority of t-MyoFbs carry DNA breaks, either SSB or DSB. Despite considerable TUNEL labeling mainly of t-MyoFbs, conventional DNA electrophoresis in agarose gel has shown one clear band of the HMW-DNA fragments (exact MW is not determined) and two bands of the LMW-DNA fragments (800 bp and between 1500–2000 bp) in all three MyoFb phenotypes, including actively proliferating p-MyoFbs. The reason of discrepancy between the data of TUNEL labeling and DNA electrophoresis is unknown. It is possible to suggest that DNA is not fragmented in the intact cells but the fragmentation of SSB-DNA takes place during cell lysis and DNA extraction. This suggestion is supported by the low level of the DNA fragmentation determined as sub-G1 phase (0.7, 0.1, and 0.3% for p-MyoFbs, a-MyoFbs and t-MyoFbs, respectively) by flow cytometry. However, presence of faint bands of DNA fragments with MW lower than 800 bp is in agreement with very small sub-G1 phase. An appearance of DNA fragment bands during DNA electrophoresis was previously shown to be dependent on the lysis buffer [70,71]. The data obtained with TUNEL labeling and DNA electrophoresis suggest that t-MyoFbs, a-MyoFbs as well as proliferative p-MyoFbs can be apoptotic. However, another data obtained in this study argue against this suggestion. Cells need a short time to complete apoptosis, from several hours to 24 h [50,72]. Despite this short time, the cell amount in t-MyoFbs cultures is constant for at least three weeks. Of course, we have to take into account that there is some equilibrium between cell proliferation and cell death [72]. Constant number of the non-proliferating functional t-MyoFbs indicates that these cells do not die. Very low level of the apoptotic death was found with flow cytometry (b0.7%) and with immunostaining of the cleavage site of cytokeratin 18 specific for caspases [56] (b0.1%) that manifest caspase activation. Absence of apoptosis in t-MyoFb was also

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proved by cell morphology examination, which is probably the most reliable method of the cell apoptosis verification. Indeed, cell can proceed to apoptosis without changes that are believed to be markers of the apoptosis, particularly: 1) without internucleosomal fragmentation (DNA laddering) [73–79]; 2) without activation of caspases [80,81]; 3) without induction of proapoptotic genes, such as Bax [81] etc. Despite this, apoptotic cells always show the typical morphological features of apoptosis. Absent of apoptosis in t-MyoFbs was confirmed: 1) by the absence of morphological signs of apoptosis, such as membrane blebbing and the appearance of small retractile apoptotic bodies [82]; 2) by a remarkable increase in size of t-MyoFbs (Table 2, Figs. 1D and 2D), whereas the size of apoptotic cells decreases markedly [72]. Moreover, apoptotic cells are functionally and metabolically inactive [72] whereas t-MyoFbs are highly active in LO secretion, in the 3-DCM contraction and, as we previously showed, in collagen production [16]. p-MyoFbs, a-MyoFbs and tMyoFbs are differentiated cells which are at the different levels of the differentiation. t-MyoFbs are terminally differentiated cells whereas p-MyoFbs are between fibroblasts and t-MyoFbs. Therefore, we can conclude that the differentiation of cardiac MyoFbs is associated with DNA breaks, which are not lethal, because non-proliferating t-MyoFbs were alive and functionally very active for all periods of time used in the study and pMyoFbs actively proliferate. Despite widespread opinion that DNA fragmentation is obligatory related to apoptosis, DNA breaks (both SSB and DSB) appear in the intact cells early at the beginning of the cell differentiation, such as erythropoietic, granulocytic, myelocytic or myoblasts to myotubes [48–51,83]. DNA breaks during differentiation are not due to an incapability of cells to repair DNA. These cells are still competent in the DNA repair [83–85]. Moreover differentiated cells are resistant to apoptosis after DNA breaks induced by various factors, whereas non-differentiated cells die in these circumstances [49,86,87]. It explains surviving of the differentiated cells after DNA breaking induced during the differentiation. For that reason, DNA breaking was considered as an intrinsic part of the cell differentiation [84]. To check whether t-MyoFbs perform DNA repair, we immunostained these cells for PCNA, which is involved in DNA repair as well as in DNA replication and for Ki-67, which is involved in DNA replication [37–39]. It was previously found that normally proliferating cells cultured in a medium with a low level of serum (0.5% FBS) are quiescent (in G0 phase) and do not show PCNA staining, whereas cells performing DNA repair are in these conditions PCNA positive [37]. Positive staining for PCNA of the majority of t-MyoFbs (88%–95% of cells) in the medium containing a high level (10% FBS) or even a low level (0.5% FBS) of serum as well as without of serum and negative staining for Ki-67 indicates that t-MyoFbs do not replicate DNA but perform PCNA-managed DNA repair. During DNA repair non-proliferating cells carrying damaged DNA still incorporate 3H-thymidine, but with much less activity than during DNA replication [88]. In agreement with this, the nonproliferating t-MyoFbs incorporate 3H-thymidine with a rate that is 9-fold less than in p-MyoFbs. It is known, that cells carrying DNA damages do not proliferate and are withdrawn from the cell cycle via several checkpoints. G1/S checkpoint prevents cells from entering the S phase by inhibiting the initiation of replication. The intra-S

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checkpoint is activated by encountering DNA damage during the S phase or by unrepaired damage that escapes the G1/S checkpoint and leads to a block in replication, the G2/M checkpoint and the replication checkpoint, S/M [89]. Arrest of t-MyoFb at G1- and S-phase suggests that in t-MyoFbs can be activated G1/S and intra-S checkpoints. Coincidence of DNA breaking with high functionality of the TGF-β1-induced t-MyoFbs is remarkable. However, t-MyoFbs are not the solely cells in these regard. Cardiac myocytes in the heart with cardiomyopathy were found to be TUNEL labelled but functional living cells that persist in the myocardium continuously, carry DNA damages and perform DNA repair. They have large aberrantly shaped nuclei with crenated edges [68,90] that remind nuclei in a fraction of t-MyoFbs. So far, only proliferative MyoFbs taken from the myocardium at early stages of healing are sufficiently characterized [6,7], whereas characteristics of non-proliferative MyoFbs from old maturated scars remain to be elucidated. We can only suggest that they might be similar to t-MyoFbs. In conclusion, our data show that TGF-β1 induces irreversible growth arrest of rat cardiac MyoFbs either at G1- and S-phase of the cell cycle, which is associated with: 1) the appearance and long-term existence of DNA breaks; 2) total inhibition of telomerase activity and of Ki-67 expression; 3) expression of the transcriptional factor Smad1; 4) expression of the transcriptional factor Mad1 and of inhibitor cyclin kinases cdk4/cdk6, p15Ink4B, both of which arrest various cells at G1/S transition of the cell cycle [91,92]. Therefore, we suggest that the myofibroblastic differentiation is tightly associated with DNA breaks, which induce growth arrest of t-MyoFbs via activation of the G1/ S- and intra-S-checkpoint. Mechanisms involved in the appearance of DNA damages and in the DNA damage-induced cell differentiation and total inhibition of the t-MyoFb proliferation have to be further elucidated.

Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Mrs. Tamara Coenen, Mrs. Yvette Piccart, Mrs. Petra Windmolens and Mrs. Reinhilde Thoelen. This work was supported by an educational grant from AstraZeneca (Belgium). K. Vekemans is a postdoctoral reseacher of the “Fund for Scientific Research-Flanders (FWO)".

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