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Originally published online as doi:10.2353/ajpath.2008.080365 on October 2, 2008

Published online before print October 2, 2008
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(American Journal of Pathology. 2008;173:1275-1285.)
© 2008 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2008.080365

Transforming Growth Factor-β Regulates in Vitro Heart Valve Repair by Activated Valve Interstitial Cells

Amber C. Liu and Avrum I. Gotlieb

From the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto; and the Department of Pathology, Toronto General Research Institute, University Health Network, Toronto, Canada


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The regulation of valve interstitial cell (VIC) function in response to tissue injury and valve disease is not well understood. Because transforming growth factor-β (TGF-β) has been implicated in tissue repair, we tested the hypothesis that TGF-β is a regulator of VIC activation and associated cell responses that occur during early repair processes. We used a well-characterized wound model that was created by mechanical denudation of a confluent VIC monolayer to study activation and repair 24 hours after wounding. VIC activation was demonstrated by immunofluorescent localization of {alpha}-smooth muscle actin ({alpha}-SMA), and {alpha}-SMA mRNA levels were quantified by real-time polymerase chain reaction. Proliferation and apoptosis were quantified by bromodeoxyuridine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Repair was quantified by measuring VIC extension into the wound, and TGF-β expression was shown by immunofluorescent localization of intracellular TGF-β. Compared with nonwounded monolayers, VICs at the wound edge showed {alpha}-SMA staining, increased {alpha}-SMA mRNA content, elongation into the wound with stress fibers, proliferation, and apoptosis. VICs at the wound edge also showed increased TGF-β and pSmad2/3 staining with co-expression of {alpha}-SMA. Addition of TGF-β neutralizing antibody to the wound decreased VIC activation, {alpha}-SMA mRNA content, proliferation, apoptosis, wound closure rate, and stress fibers. Conversely, exogenous addition of TGF-β to the wound increased VIC activation, proliferation, wound closure rate, and stress fibers. Thus, wounding activates VICs, and TGF-β signaling modulates VIC response to injury.

Valve interstitial cells (VICs) are the predominant cell type in the heart valve.1 Under normal circumstances, VICs are quiescent and maintain the structural integrity and function of the valve.2,3 In response to valve injury, VICs undergo phenotypic changes and become activated.4,5 {alpha}-Smooth muscle actin ({alpha}-SMA), a cytoskeletal isoform of actin not normally found in the quiescent VICs of normal heart valves, is a marker for activated VICs. Diseased heart valves show up-regulation of {alpha}-SMA staining in VICs.6-10 Activated VICs have features of myofibroblasts showing increased contraction, actin stress fibers, and other contractile proteins.3-5,11-13 Myofibroblast-type cells regulate wound repair in many organs14 and it is likely that activated VICs also regulate wound repair in the heart valve.15 Thus, understanding the regulation of VIC activation and the associated cellular responses that occur in early wound repair is critical to understanding the pathobiology of heart valve diseases.

Transforming growth factor (TGF)-β,16 a 25-kDa protein that is a member of the TGF-β superfamily, is a well-studied regulator of extracellular matrix deposition in wound repair. It is secreted by numerous cell types17 including VICs with potent autocrine effects.18,19 It is known to promote differentiation of mesenchymal cells into myofibroblasts20,21 and to regulate multiple aspects of the myofibroblast phenotype through transcriptional activation of {alpha}-SMA, collagen,22 matrix metalloproteinases,23 and other cytokines such as connective tissue growth factor24 and basic fibroblast growth factor.25-27 TGF-β is present in mitral valve prolapse6,28 and calcific aortic stenosis.7,29,30 Heart valves of carcinoid syndrome patients show VIC activation and increased expression of TGF-β, which is associated with increased collagen deposition, changes in the organization of extracellular matrix components, and calcification.31,32

The regulation of the early stages of VIC wound repair are less well understood than the later stages of fibrosis and wound contracture. Because TGF-β has been implicated in several tissue repair conditions, we tested the hypothesis that TGF-β regulates VIC activation and associated cell functions that are implicated in early wound repair including VIC activation, extension of elongated stress fiber-rich VICs into the wound, proliferation, and apoptosis. We choose to use an in vitro model that has been extensively used to study endothelial, smooth muscle cell, and epithelial wound repair. Wounding is achieved by mechanical denudation of a confluent monolayer.1,33-36 We demonstrate that injury to a confluent VIC monolayer leads to TGF-β and VIC activation. VIC cultures treated with TGF-β neutralizing antibodies and exogenous TGF-β alter VIC activation and the associated cellular activities that occur in the early stages of wound repair. We examine changes in VIC proliferation and apoptosis, which are processes intrinsic to repair and remodeling that contribute directly to wound closure and show that TGF-β is required to maintain VIC activation and is a key regulator of wound repair by VICs.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture

Porcine hearts were obtained from a local abattoir, and explants were prepared from the distal third of the anterior leaflet of porcine mitral valves as previously described.37 Briefly, the atrial and ventricular surfaces of the explants were scraped with a scalpel blade and rinsed with phosphate-buffered saline (PBS), pH 7.4, to remove valve endothelial cells. The explants were cut into 4 x 5-mm pieces, placed in 35-mm tissue culture dishes (Falcon; BD Biosciences, San Jose, CA), and grown in medium 199 (M-199) supplemented with 10% fetal bovine serum (FBS), and 2% penicillin, streptomycin, and Fungizone (Life Technologies Inc., Rockville, MD) in a humidified 95% air and 5% carbon dioxide atmosphere in an incubator at 37°C. VICs that grow out of the explants were detached with TrypLE Express (Invitrogen Corp., Carlsbad, CA) and subcultured. In all experiments, VICs were cultured in 10% or in 0.5% FBS M-199 with 2% penicillin, streptomycin, and Fungizone. VICs of passages two to four were used.

Concentration of TGF-β in Culture Media

Conditioned media was collected from wounded and control nonwounded monolayers (NWMs) cultured in parallel in 0.5% or 10% FBS containing media. VIC-conditioned media was collected at time 0 after wounding and at 24 hours after wounding and enzyme-linked immunosorbent assay (ELISA) for TGF-β1 was performed. Porcine TGF-β1 immunoassay kit (R&D Systems, Minneapolis, MN) was used to measure the concentration of active porcine TGF-β1 in culture supernatants. Following the manufacturer’s instructions, before ELISA, conditioned media were treated with 1 N HCl for 10 minutes followed by 1.2 N NaOH/0.5 mol/L HEPES to activate TGF-β before performing the ELISA measurements. Measurements were performed in duplicate. Three independent experiments were performed. To determine the concentration of active TGF-β in conditioned media collected at 24 hours after wounding from cultures treated with TGF-β neutralizing antibody, Dynabeads Protein G and Dynal MPC-S Magnet (Invitrogen) were used to first precipitate complexes of TGF-β/TGF-β-neutralizing antibodies from media. Then the supernatants were assayed for TGF-β by ELISA.

Experimental Wounds

VICs were platted at a density of 1700 cells per well onto 18-mm diameter glass coverslips (Fisher Scientific, Pittsburgh, PA) in 12-well tissue culture dishes. VICs were then cultured in 1 ml of standard media per well for 6 to 8 days until a confluent monolayer was formed on top of the glass coverslip. This allowed the VICs to deposit their own matrix.36 A 1-mm-wide flat blade was dragged across the monolayer in a linear manner completely removing a strip of VICs and associated extracellular matrix in the center of the monolayer to form a linear wound containing two straight edges. Approximately 10% of cells were removed from the monolayer to form the wound. The wounded monolayer was washed three times with serum-free M-199 media to remove cell debris. NWMs were cultured in parallel to wounded monolayers as controls. Throughout all of the experiments, the morphology of the monolayer in the wounded cultures away from the wound edge (WE) was similar to the monolayer in the nonwounded control cultures.

Treatment with TGF-β Neutralizing Antibody

A pan-TGF-β neutralizing antibody (R&D Systems) was reconstituted in sterile PBS to a concentration of 10 mg/ml. After wounding and washing of VIC monolayers, the cultures were incubated in 10% FBS containing media with 15 µg/ml TGF-β neutralizing antibody. At 24 hours after wounding, the coverslips were washed in serum-free M-199 media and immunofluorescently stained for {alpha}-SMA. Apoptosis, proliferation, and extent of wound closure were also quantified. Three independent experiments were performed in triplicate coverslips.

Treatment with Exogenous TGF-β

In preliminary experiments, addition of exogenous TGF-β to cultures in 10% FBS-containing media led to focal areas of contraction with disruption of the confluent monolayer and retraction of the WE. By lowering the serum concentration, these effects disappeared and the monolayer was not perturbed. Therefore, for the experiments involving treatment with exogenous TGF-β, VICs were incubated in 0.5% FBS media that contains 0.1 ± 0.07 ng/ml of TGF-β as measured by ELISA. Porcine TGF-β1 (R&D Systems) was reconstituted in a vehicle solution of 4 mmol/L HCl and 0.1% bovine serum albumin. Media containing vehicle or 0.5 ng/ml of active porcine TGF-β1 was added to cultures after wounding and washing. This allowed us to add five times the concentration of TGF-β that was available for activation in the 0.5% FBS media. At 24 hours after wounding, the coverslips were washed in serum-free M-199 media and immunofluorescently stained for {alpha}-SMA. Apoptosis, proliferation, and extent of wound closure were also quantified. Three independent experiments were performed in triplicate coverslips.

Immunofluorescent Staining

Nonwounded and wounded monolayers were immunofluorescently stained at 24 hours after wounding. VICs on glass coverslips were fixed with 4% paraformaldehyde, rinsed three times in PBS, incubated with 0.1% Triton X-100 in PBS for 5 minutes, and rinsed again three times in PBS at 5-minute intervals. The coverslips were incubated with mouse anti-{alpha}-SMA (1:500; Sigma, Oakville, Canada), rabbit anti-TGF-β (against TGF-β1, -2, -3), anti-phosphorylated Smad2/3 (Ser423/425) (1:100 and 1:50, respectively; Santa Cruz Biotechnology, Santa Cruz, CA), anti-TGF-β3 (1:100; Abcam, Cambridge, MA), anti-cleaved caspase-3 (1:50; Cell Signaling, Danvers, MA), and mouse anti-BrdU (1:100, Cell Signaling) primary antibodies at room temperature for 1 hour. The coverslips were then washed three times with PBS at 5-minute intervals. Secondary antibodies were goat anti-mouse Alexa 488 or 568 and goat anti-rabbit Alexa 488 or 568 (1:200; Molecular Probes, Invitrogen, Eugene, OR). Hoechst 33342 (1:500; Lonza, Basel, Switzerland) or propidium iodide (1:5000, Sigma) were used to counterstain nuclei. After secondary antibody, coverslips were dipped in deionized water and mounted with Prolong Gold antifade reagent (Molecular Probes, Invitrogen) allowing the samples to cure overnight. Equivalent amounts of mouse IgG and rabbit IgG protein (Jackson ImmunoResearch, West Grove, PA) were used for negative control. Coverslips were examined using the x20, x40, and x60 objective of a Nikon C1 scanning confocal laser-imaging system (Nikon Canada, Mississauga, Canada) fitted with an argon-krypton mixed-gas laser with excitation wavelengths of 488 and 568 nm. Serial optical sections were taken at 0.5-µm intervals for a total of 5 to 8 µm depending on cell or monolayer thickness. Volume rendering of optical sections were compiled using the EZ-C1 software (Nikon Canada). Images were captured at randomly selected regions within the NWM or at the WE using the FV10-ASW 1.6 software (Olympus, Markham, Canada) to avoid saturation.

Quantification of {alpha}-SMA Stress Fibers

VICs at the WE showing {alpha}-SMA stress fiber staining were quantified at 24 hours after wounding. Random counting of VICs was performed using the x40 objective of the Nikon C1 confocal microscope starting five fields away from the edge of the coverslip and was continued linearly counting cells in every other field of view for 30 fields along the WE. A total of 40 to 50 cells per field were counted. Both WEs were counted for a total of 60 fields per wound. Thirty random fields in the center of the coverslip were counted as controls in the nonwounded confluent monolayer.

Quantification of Proliferation

Bromodeoxyuridine labeling reagent (Sigma) was added in 1:500 dilution to VIC cultures at 22 hours after wounding. At 24 hours after wounding, the coverslips were washed in serum-free M-199 media, fixed in ethanol:acetic acid:H2O (90:5:5) for 20 minutes, incubated with denaturing solution (Calbiochem, La Jolla, CA) for 1 hour and immunofluorescently stained. VIC nuclei were counterstained with propidium iodide (1:5000, Sigma). Both the total number and the number of proliferating VICs at the WE and in the NWM were counted randomly as described above for the counting of VICs with {alpha}-SMA stress fibers. Proliferation was calculated as a percentage of the labeled cells out of the total number of VICs.

Quantification of Apoptosis

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using the TACS TdT-fluorescein in situ apoptosis detection kit (R&D Systems) to identify apoptotic VICs. VICs were also immunofluorescently stained for cleaved caspase-3. VICs were counterstained with propidium iodide. The total number of VICs and the number of VICs undergoing apoptosis in the NWM and at the WE were counted as described above for the counting of VICs with {alpha}-SMA stress fibers. The apoptotic index was calculated as a percentage of the labeled cells out of the total number of VICs.

Quantification of Wound Closure

Confocal images of wounded coverslips stained for {alpha}-SMA were captured using the x20 objective. Fifteen to twenty fields were captured from every other field along the WE. Confocal z-stacks were volume rendered using the FV10-ASW 1.6 software. Measurements were made using the NIS-Elements software (Nikon Instruments Inc., Melville, NY) of the distance between the original nonstained multilayered WE and the stained front row of cells at the WE at 24 hours after wounding.

RNA Extraction and Isolation

All reagents used for RNA isolation were purchased in the PicoPure RNA Isolation Kit (Arcturus Bioscience Inc., Mountain View, CA). Wounded and nonwounded VICs cultured in 10% FBS standard media with or without TGF-β neutralizing antibody treatment were washed three times with serum-free M-199 media. VICs were then observed under a phase contrast microscope at x100 magnification and a 27G1/2 PrecisionGlide needle (Becton-Dickinson, Mountain View, CA) was bent and used to capture VICs under the phase contrast microscope in the NWM or at the WE. Needle points containing VICs of interest were then immersed immediately in extraction buffer to obtain cell extract. Approximately 300 to 500 cells were captured from the NWM or WE.

RNA isolation protocols specified in the PicoPure RNA isolation kit (Arcturus Bioscience Inc.) were followed. Briefly, RNA purification columns were preconditioned with 250 µl of conditioning buffer for 5 minutes at room temperature followed by centrifugation at 16,000 x g for 1 minute. Cell extracts are mixed with 50 µl of 70% ethanol by pipetting up and down. The cell extract and ethanol mixtures were applied to the preconditioned purification columns and centrifuged for 2 minutes at 100 x g, followed immediately by a centrifugation at 16,000 x g for 30 seconds. One hundred µl of wash buffer1 was applied to each purification column followed by 8000 x g centrifugation for 1 minute. One hundred µl of wash buffer 2 was applied to each purification column followed by 8000 x g centrifugation for 1 minute. RNA was eluted with 10 µl of elution buffer by first incubating the elution buffer on the purification column for 1 minute at room temperature then centrifuging at 1000 x g for 1 minute followed by at 16,000 x g for 1 minute.

First-Strand cDNA Synthesis

First strand cDNA synthesis from RNA isolated from VICs was completed using the SuperScriptIII reverse transcriptase kit (Invitrogen). Briefly, 2 µl of random decamers (Ambion, Austin, TX), 2 µl of 10 mmol/L dNTP mix (Invitrogen), and 9 µl of RNA were mixed and incubated for 5 minutes at 65°C followed by incubation on ice for 1 minute. Four µl of 5x first-strand buffer, 1 µl of 0.1 mol/L dithiothreitol, 1 µl of RNaseOUT (40 U/µl), and 1 µl of Superscript III reverse transcriptase (2000 U) were then added to each reaction mixture, making a total reaction volume of 20 µl per sample. Each reaction mixture was incubated at 25°C for 5 minutes, 50°C for 50 minutes, and 70°C for 15 minutes.

Real-Time Polymerase Chain Reaction (PCR)

Real-time PCR primers (ACGT Inc., Wheeling, IL) were designed using Primer Express (Applied Biosystems, Foster City, CA) and span across an intron whenever possible. Primer sequences were: HPRT-F 5'-AACGGCTTGCTCGAGATGTG-3'; HPRT-R 5'-TCCAGCAGGTCAGCAAAGAA-3'; β-actin-F 5'-TGTCCACCTTCCAGCAGATGT-3'; β-actin-R 5'-TGCAACTAACAGTCCGCCTAGA-3'; {alpha}-SMA-F 5'-TCTGTAAGGCCGGCTTTGC-3'; {alpha}-SMA-R 5'-TGTCCCATTCCCACCATCA-3'. For each real-time PCR reaction, 12.5 µl of Power SYBR Green PCR Master Mix, 1.5 µl of 10 µmol/L forward and reverse primers mixture, 10 µl of first-strand cDNA, and 1 µl of H2O were mixed for a total reaction volume of 25 µl. Real-time PCR reactions were performed using a Prism 7900 HT sequence detection system (Applied Biosystems). Thermal cycling conditions were designed as follows: 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. A final dissociation stage was added at 95°C for 15 seconds followed by 60°C for 15 seconds. Fluorescent measurements of SYBR green incorporation were recorded during each annealing step. Dissociation curve analyses were performed to confirm specificity of the SYBR green signals in each experiment. Relative quantifications were performed using the comparative standard curve method (Sequence Detection Systems Software 2.0, Applied Biosystems). {alpha}-SMA values were divided by HPRT and β-actin values independently for standardization and the resulting values were averaged. The relative {alpha}-SMA mRNA content for VICs in a nontreated NWM was assigned a value of 1, and the {alpha}-SMA mRNA content in VICs at the nontreated WEs, treated WEs, and treated NWM were expressed as folds increase from that of the nontreated NWM.

Statistical Analysis

A value of P < 0.05 was considered significant. Student’s t-test was used to compare control and treated groups. One-way analysis of variance followed by Bonferroni posttest was used to reflect multiple comparisons. These statistical analyses were performed using GraphPad Prism version 5 software (GraphPad Software Inc., San Diego, CA).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Wounding Activates VICs and Leads to Increased Intracellular TGF-β Expression and TGF-β Signaling

After wounding, VICs participate in wound repair by closing the wound from both sides of the wound forming two advancing linear WEs. By 4 days after wounding, ~90% of the wound is filled in with VICs, and wound closure occurs in 6 days. The wounded area is not identifiable by phase contrast microscopy by 10 days. At 24 hours after wounding, all VICs at the WE show significantly increased {alpha}-SMA staining (Figure 1B) compared to the control NWM where {alpha}-SMA is absent (Figure 1A) . In the monolayer away from the WE, {alpha}-SMA staining is also absent. Wounded cultures were stained at 24, 48, 72, 96 hours, 6 days, and 10 days, and {alpha}-SMA staining at the WE begins to disappear at 6 days and was absent at 10 days (data not shown).


Figure 1
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  		Figure 1. Immunofluorescent confocal photomicrographs of VIC NWMs (A, C, E, and G) and wounded VIC monolayers at the WE (B, D, F, and H) at 24 hours after wounding. VICs are stained for {alpha}-SMA (A and B, green; nuclei, blue), TGF-β (C and D), phosphorylated Smad2/3 (E and F), and are double stained (G and H) for {alpha}-SMA (red) and TGF-β (green). {alpha}-SMA, TGF-β, and phosphorylated Smad2/3 are expressed at low levels in the NWM and are increased at the WE. {alpha}-SMA and TGF-β are co-expressed at the WE and not in the NWM. Note that phosphorylated Smad2/3 complexes are localized to the nucleus of VICs. Arrow indicates direction of wound closure. Original magnifications, x600.

 
TGF-β staining using the anti-TGF-β (against TGF-β1, -2, -3) is increased in VICs at the WE (Figure 1D) at 24 hours after wounding compared to VICs in the NWM (Figure 1C) . TGF-β staining is frequently concentrated in the perinuclear area (Figure 1D) . Using anti-TGF-β3 staining did not show the presence of TGF-β3 in the VIC cultures (data not shown), and TGF-β2 is not present in mesenchymal cells.38 TGF-β1 concentration in the nonconditioned 10% FBS-containing media is 1.2 ± 0.07 ng/ml measured by ELISA. VIC-conditioned media collected at 24 hours after wounding contains 1.5 ± 0.06 ng/ml of TGF-β1.

Staining for the phosphorylated Smad2/3 complex, key transcription factors forming a signaling complex in the TGF-β signaling pathway, is increased at the WE at 24 hours after wounding (Figure 1F) whereas the NWM was faintly stained (Figure 1E) . At the WE, phosphorylated Smad2/3 complexes are concentrated in the nuclear region with some perinuclear staining. VICs at the WE co-stain for {alpha}-SMA and TGF-β at 24 hours after wounding (Figure 1H) . This {alpha}-SMA and TGF-β co-expression is not observed in VICs in the NWM (Figure 1G) .

TGF-β Neutralizing Antibody Reduces VIC Wound Repair through Decreasing Activation, {alpha}-SMA mRNA Content, Proliferation, Apoptosis, Wound Closure, and Formation of {alpha}-SMA Stress Fibers in VICs at the WE

At 24 hours after wounding, {alpha}-SMA staining is significantly increased at the WE compared to the NWM (Figure 1, A and B) . When 15 µg/ml of TGF-β neutralizing antibody is applied to nonwounded and wounded VIC cultures reducing the available TGF-β from 1.2 ± 0.07 ng/ml to 0.85 ± 0.05 ng/ml through ELISA measurements, {alpha}-SMA staining intensity is significantly reduced at the WE (Figure 2, A and B) .


Figure 2
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  		Figure 2. Immunofluorescent confocal photomicrographs of wounded VIC monolayers at the WE stained for {alpha}-SMA (green) in TGF-β neutralizing antibody-treated or nontreated cultures at 24 hours after wounding. A: VICs show prominent {alpha}-SMA staining at the nontreated WE. B: Treatment with TGF-β neutralizing antibody show decreased {alpha}-SMA staining intensity. Arrow indicates direction of wound closure. C: Real-time PCR quantification of {alpha}-SMA mRNA in VICs at the WE and in the NWM in TGF-β neutralizing antibody-treated and nontreated cultures at 24 hours after wounding. An increase in {alpha}-SMA mRNA content is observed at the WE compared to NWM. In TGF-β neutralizing antibody-treated cultures, decreased {alpha}-SMA mRNA content is observed at the WE compared to nontreated cultures. No difference in {alpha}-SMA mRNA content is observed in the NWM between treated and nontreatment cultures. ** and *** denotes significance between the indicated groups. P < 0.05; n = 6. Original magnifications, x600.

 
Quantification of relative {alpha}-SMA mRNA abundance in VICs in the NWM and at the WE showed that {alpha}-SMA mRNA is 2.06 ± 0.43 times as abundant in VICs at the WE than in VICs in the NWM (Figure 2C) . When VIC cultures are treated with 15 µg/ml of TGF-β neutralizing antibody, no significant change in {alpha}-SMA mRNA is observed in the NWM. However, {alpha}-SMA mRNA content in VICs at the WE is reduced 6.90 ± 0.25-fold in 15 µg/ml of TGF-β neutralizing antibody-treated cultures compared to nontreated cultures (Figure 2C) .

At 24 hours after wounding, VIC proliferation as quantified by bromodeoxyuridine staining is 35.07 ± 2.04% higher at the WE than VIC proliferation within the NWM (Figure 3A) . After 15 µg/ml of TGF-β neutralizing antibody treatment, VIC proliferation at the WE is reduced by 22.75 ± 3.73% in treated cultures compared to nontreated cultures at 24 hours after wounding (Figure 3A) . TGF-β neutralizing antibody treatment did not show an effect on VIC proliferation in the NWM compared to the nontreated NWM.


Figure 3
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  		Figure 3. A: Percentage of VIC proliferation at the WE in TGF-β neutralizing antibody-treated and nontreated cultures as determined by BrdU staining at 24 hours after wounding. VIC proliferation is higher at the WE compared to the NWM. TGF-β neutralizing antibody treatment leads to a decrease in proliferation of VICs at the WE compared to nontreated cultures. No effect on proliferation is observed in the NWM in treated compared to nontreated cultures. ** and *** denote significant differences between groups as indicated. P < 0.05; n = 6. B: Apoptosis index of VICs at the WE in TGF-β neutralizing antibody-treated and nontreated cultures determined by TUNEL at 24 hours after wounding. TGF-β neutralizing antibody-treated cultures show lower apoptosis index of VICs at the WE compared to nontreated cultures. ***Significant differences of groups as indicated. P < 0.05; n = 7.

 
At 24 hours after wounding, 6.15 ± 0.34% of the VIC population at the WE undergo apoptosis as determined by TUNEL (Figure 3B) . When VIC cultures are treated with 15 µg/ml of TGF-β neutralizing antibody, VIC apoptosis index at the WE is decreased to 1.93 ± 0.12%, a 69% decrease relative to nontreated cultures at 24 hours after wounding (Figure 3B) . VICs in the NWM show no apoptosis both in nontreated and treated cultures (data not shown). Staining for cleaved caspase-3 show an index of 3.34 ± 0.20% at 24 hours after wounding, and treatment with neutralizing antibody show a significantly decreased index of 0.45 ± 0.05%.

When wounded VIC cultures are treated with 15 µg/ml of TGF-β neutralizing antibody, wound closure is decreased by 106.40 ± 5.36 µm at 24 hours after wounding (Figure 4A) . The percentage of VICs showing {alpha}-SMA stress fibers at the WE is reduced by 19.91 ± 1.10% at 24 hours after wounding when wounded VIC cultures are treated with 15 µg/ml of TGF-β neutralizing antibody (Figure 4B) .


Figure 4
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  		Figure 4. A: Measure of extent of VIC wound closure in TGF-β neutralizing antibody-treated and nontreated cultures at 24 hours after wounding. Treatment of wounded VIC cultures with TGF-β neutralizing antibody reduces extent of wound closure. ***Significant differences between groups as indicated. P < 0.05; n = 6. B: Percentage of VICs showing {alpha}-SMA stress fibers at the WE in TGF-β neutralizing antibody-treated and nontreated cultures at 24 hours after wounding. Treatment with TGF-β neutralizing antibody decreases the number of VICs at the WE showing {alpha}-SMA stress fibers. ***Significant differences between groups as indicated. P < 0.05; n = 6.

 
Exogenous TGF-β Enhances VIC Wound Repair through Increasing Activation, Proliferation, Wound Repair, and Formation of Stress Fibers in VICs at the WE

Treatment of VIC cultures with 0.5 ng/ml of exogenous TGF-β in 0.5% FBS-containing media at the time of wounding leads to increased staining intensity of {alpha}-SMA in VICs at the WE of treated cultures (Figure 5B) compared to vehicle (Figure 5A) at 24 hours after wounding. No {alpha}-SMA staining in the NWM is observed in treated cultures and vehicle (data not shown). At 24 hours after wounding, VIC proliferation at the WE is 7.55 ± 0.90% higher than in the NWM in 0.5% FBS-containing media (Figure 6) . In cultures treated with 0.5 ng/ml of exogenous TGF-β in 0.5% FBS-containing media, proliferation at the WE is increased by 6.67 ± 1.56% compared with that at the WE of vehicle. No difference in proliferation is observed between treated cultures and vehicle in the NWM. No difference in VIC apoptosis is observed at the WE of 0.5 ng/ml exogenous TGF-β-treated cultures compared to vehicle at 24 hours after wounding. Treatment of wounded VIC cultures with 0.5 ng/ml of exogenous TGF-β in 0.5% FBS-containing media leads to increased wound closure by 83.50 ± 5.31 µm or 35% at 24 hours after wounding (Figure 7A) . Furthermore, the number of VICs showing {alpha}-SMA stress fibers is increased by 31.16 ± 2.38% in exogenous TGF-β-treated cultures compared to vehicle at 24 hours after wounding (Figure 7B) .


Figure 5
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  		Figure 5. Immunofluorescent confocal photomicrographs of wounded VIC monolayers at the WE stained for {alpha}-SMA (green; nuclei, blue) at 24 hours after wounding. VICs in vehicle cultures (A) show {alpha}-SMA staining at the WE and VICs in exogenous TGF-β-treated cultures (B) show increased {alpha}-SMA staining intensity compared to those at the WE of vehicle. Arrow indicates direction of wound closure. Original magnifications, x600.

 

Figure 6
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  		Figure 6. Percentage of VIC proliferation at the WE in exogenous TGF-β-treated or vehicle cultures determined by BrdU staining at 24 hours after wounding. VIC proliferation is increased at the WE compared to the NWM in vehicle. Exogenous TGF-β treatment leads to a further increase in proliferation of VICs at the WE compared to vehicle. No effect on proliferation is observed in the NWM of treated cultures compared to vehicle. * and ** denote significant differences between groups as indicated. P < 0.05; n = 6.

 

Figure 7
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  		Figure 7. A: Measure of extent of VIC wound closure in exogenous TGF-β-treated or vehicle cultures at 24 hours after wounding. Treatment of wounded VIC cultures with exogenous TGF-β leads to an increase in the extent of wound closure compared to vehicle. **Significant differences between groups as indicated. P < 0.05; n = 6. B: Percentage of VICs showing {alpha}-SMA stress fibers at the WE at 24 hours after wounding. Exogenous TGF-β treatment increases the number of VICs at the WE with {alpha}-SMA stress fibers compared to vehicle. ***Significant differences between groups as indicated. P < 0.05; n = 6.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first heart valve study to investigate the regulatory role of TGF-β on several VIC functions that occur during the early stages of wound repair. We show that at 24 hours after wounding, VICs at the WE become activated, form {alpha}-SMA stress fibers, proliferate, undergo apoptosis, and demonstrate TGF-β up-regulation and signaling (Figure 8) . We also show that these cell functions are regulated by changes in TGF-β concentration in the media. Our mechanical wound model does have similarities to in vivo tissue repair because VIC activation, cell proliferation and migration, and apoptosis do occur in our model. However in this current study, VICs are not exposed to inflammation, hemodynamic and mechanical forces, and surface microthrombosis that is observed to occur at the site of in vivo valve injury and disease. These factors can be added to this type of model in future experiments as has been done successfully in experimental vascular studies.39,40


Figure 8
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  		Figure 8. Summary of the response of VICs to TGF-β in early stages of wound repair. VICs are quiescent when residing in a confluent monolayer. After wounding, VICs at the WE express {alpha}-SMA, a marker of VIC activation. Activated VICs require TGF-β to maintain activation through promoting {alpha}-SMA mRNA production and protein expression. Activated VICs at the WE undergo proliferation and apoptosis, as well as form {alpha}-SMA stress fibers, leading to wound closure and repair. Neutralization of TGF-β leads to reduced VIC activation, {alpha}-SMA mRNA production, proliferation, apoptosis, {alpha}-SMA stress fiber formation, and wound closure, decreasing VIC wound repair. Addition of exogenous TGF-β increases proliferation, stress fiber formation, and wound closures, enhancing VIC wound repair.

 
Our observations of {alpha}-SMA expression and stress fiber formation in VICs at the WE indicate that wounding in our model activates the myofibroblast phenotype of quiescent VICs. Myofibroblasts are central in wound healing in many types of tissue and are defined biochemically by {alpha}-SMA expression and morphologically by contractile structures represented by stress fibers.14,41 Stress fibers are responsible for maintaining an isometric tension applied to the extracellular matrix through focal adhesion sites and occur in mesenchymal,42,43 epithelial,44,45 and endothelial cells, especially during the early stages of repair.46,47 Myofibroblast {alpha}-SMA expression and its assembly into stress fibers are important in cell migration, force generation, and wound contracture both in vitro and in vivo.48,49 In our model it is likely the increased stress fibers at the WE is associated with elongation and migration of the VICs into the wound.

We observe that {alpha}-SMA mRNA and protein are up-regulated concurrently with increased TGF-β in VICs at the WE. We also find that TGF-β neutralization reduces {alpha}-SMA protein, mRNA, and stress fibers whereas exogenous addition of TGF-β increases {alpha}-SMA protein, mRNA, and stress fibers. These findings are consistent with data showing that in tissue fibroblasts, TGF-β directly regulates {alpha}-SMA expression and plays a pivotal role in wound repair.20,50 Our observation of {alpha}-SMA mRNA reduction when TGF-β is neutralized may be explained by the findings in fibroblasts that TGF-β stimulates {alpha}-SMA gene expression through the TGF-β control element at –42 to –61 from the transcriptional start site of the {alpha}-SMA promoter.51,52 In fibroblasts, BTEB2, GKLF, SP1, and other Kruppel-like factors are reported to bind and regulate {alpha}-SMA promoter activity.53,54 Sp1/3 and Smad3 proteins have also been shown to stimulate {alpha}-SMA enhancer activity in fibroblasts.51 Furthermore, our findings are consistent with a recent report showing that TGF-β induces {alpha}-SMA protein and stress fiber expression in porcine aortic VICs cultured at low-cell density on collagen substrates.13

TGF-β is produced by VICs in our model. We show intracellular TGF-β in VICs at the WE to be located in the perinuclear region, consistent with the known synthesis and secretion pathways of TGF-β through the endoplasmic reticulum and Golgi networks.17,55 Our ELISA measurements further support VICs as a source of TGF-β because we find that the concentration of TGF-β is increased in VIC-conditioned media collected at 24 hours after wounding compared to conditioned media collected at 0 hours after wounding.

TGF-β isoforms (TGF-β1, -2, and -3), activin, nodals, bone morphogenetic proteins, growth and differentiation factor, and Mullerian inhibitory substance are all members of the TGF-β superfamily that all contain common sequence and structural features including a unique positioning of seven cysteine residues resulting in the formation of a core cysteine knot.56 TGF-β1 is the predominant isoform of TGF-β exerting biological effects in the adult human and pig. TGF-β2 is not expressed in mesenchymal cells,38 and we do not detect TGF-β3 in our VIC cultures. Tethering of the secreted latent TGF-β complex through LTBP to matrix proteins such as fibronectin and heparin is an important cellular mechanism to tightly regulate TGF-β bioavailability, spatial gradients, and activation in the cell microenvironment.19,57-60 Direct fibronectin-TGF-β interactions or indirect fibronectin-TGF-β interactions through heparin activate porcine aortic VICs.19 TGF-β1 activation has also been shown to be integrin-mediated in vivo.61 We previously report the up-regulation of fibronectin, changes in {alpha}vβ6 integrin, and prominent fibrillar adhesion assembly by VICs at the WE.36 The tethering of latent TGF-β complexes to fibronectin is necessary to allow {alpha}vβ6 integrin-mediated TGF-β activation.58,59,62

One possibility in our model is that latent TGF-β becomes concentrated at the WE because of increased secretion by activated VICs and increased fibronectin. It is likely that our mechanical disruption of VIC-VIC and VIC-matrix interactions by mechanical wounding leads to activation of TGF-β by unknown mechanisms. Two possible mechanisms are that integrin activation and rearrangement at the WE and/or proteolytic enzymes, such as plasmin,63 thrombin,64 and matrix metalloproteinasess,65 which may be secreted by VICs at the WE activates TGF-β. This would result in active TGF-β concentrated specifically at the WE, which then signals to regulate VIC wound repair.

The most well-characterized TGF-β signaling mechanism is through the Smad2 and Smad3 proteins that are phosphorylated and activated by the type I receptor, and shuttle to the nucleus through Smad4.66,67 Our observations that phosphorylated Smad2 and Smad3 are up-regulated and accumulate in the nucleus of VICs at the WE suggest that this important TGF-β signaling pathway regulates VIC wound repair at least in part. The cellular responses to TGF-β signaling via Smad2 and Smad3 can be variable depending on cell type. TGF-β causes epithelial cells to undergo growth arrest and apoptosis, whereas in fibroblasts, it induces activation, proliferation, and differentiation.68-70 Other cellular processes regulated by TGF-β in fibroblasts include cell growth, apoptosis, migration, extracellular matrix production, and degradation.66-72 These findings are consistent with our current observations that VICs undergo activation, proliferation, apoptosis, and our previous observations of VIC migration and matrix deposition at the WE.36

We show that there is increased VIC proliferation at the WE. Our findings are consistent with previous observations in a different model in which TGF-β promotes proliferation in sheep aortic VICs at low cell densities cultured on collagen substrates.18 The signaling mechanism of TGF-β in promoting proliferation has been under study for a number of cell types.25,73,74 In addition to the well-characterized Smad pathways in promoting proliferation, non-Smad TGF-β signaling pathways are recently found to be also important in regulating fibroblasts proliferation in which TGF-β signals through the phosphoinositide-3 kinase (PI3K) pathway leading to phosphorylation of Akt and PAK2 activation.75-79 These signaling mechanisms are still under elucidation and they may occur in VICs, which are myofibroblast-like in nature. TGF-β also promotes fibroblast proliferation via transcriptionally stimulating platelet-derived growth factor and basic fibroblast growth factor-2.80 This finding in fibroblasts is consistent with our previous report of the stimulatory effect of basic fibroblast growth factor-2 on VIC wound repair.34

The presence of VIC apoptosis at the WE indicates that cell turnover is another important aspect of valve wound repair. In the healing of a dermal wound, hypertrophic scarring occurs if apoptosis is dysregulated.41 In the same manner, if activated VICs did not undergo apoptosis, excessive extracellular matrix deposition and fibrosis would likely occur leading to valve scarring and dysfunction. Apoptosis is mediated by the caspase family of proteases that are controlled through the environment-mediated extrinsic pathway and/or the mitochondria-mediated intrinsic pathway. Caspases-8, -9, and -10 are initiator caspases that cleave and activate effector caspases-3, -6, and -7.81 The regulation of initiator caspases is dependent on the balance of pro-apoptosis and anti-apoptosis cues, and apoptosis is sometimes reversible through inhibition of initiator caspases. However, once apoptosis progresses to the cleavage of effector caspases, irreversible cell death results as effector caspases fragment DNA, disrupt cytoskeleton, and lead to metabolic breakdown of the cell.82 Thus, we identify VICs in the irreversible and terminal stages of apoptosis at the WE by using both TUNEL to detect DNA fragmentation and cleaved caspase-3 staining to detect caspase-3 activation. TGF-β is known to promote apoptosis through a number of signaling pathways. In a number of carcinomas, TGF-β pro-apoptotic responses by Smad-3 is followed by caspase-8 activation via the Fas receptor83 or by activation of PI3K-Akt signaling.84 Apoptosis may also be regulated by TGF-β through its effect on the balance of pro-apoptosis BH3-only proteins and anti-apoptosis Bcl-2 family members.85,86

In conclusion, we demonstrate that wounding activates VICs that repair the wound. Wound repair is regulated by TGF-β by modulating {alpha}-SMA protein expression, mRNA expression, and stress fiber formation. TGF-β further regulates proliferation, apoptosis, and wound closure, which are critical processes in repair.


   Footnotes
 
Address reprint requests to Avrum I. Gotlieb, M.D.C.M., F.R.C.P.C., Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Banting Institute, 100 College St., Toronto, M5G 1L5, Ontario, Canada. E-mail: avrum.gotlieb{at}utoronto.ca

Supported by the Heart and Stroke Foundation of Ontario (grant NA6204 and masters’ award to A.C.L.).

Accepted for publication July 31, 2008.


   References
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 Abstract
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 References
 

  1. Mulholland DL, Gotlieb AI: Cell biology of valvular interstitial cells. Can J Cardiol 1996, 12:231-236[Medline]
  2. Mulholland DL, Gotlieb AI: Cardiac valve interstitial cells: regulator of valve structure and function. Cardiovasc Pathol 1997, 6:167-174[CrossRef]
  3. Taylor PM, Allen SP, Yacoub MH: Phenotypic and functional characterization of interstitial cells from human heart valves, pericardium and skin. J Heart Valve Dis 2000, 9:150-158[Medline]
  4. Rabkin-Aikawa E, Farber M, Aikawa M, Schoen FJ: Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves. J Heart Valve Dis 2004, 13:841-847[Medline]
  5. Liu AC, Gotlieb AI: Characterization of cell motility in single heart valve interstitial cells in vitro. Histol Histopathol 2007, 22:873-882[Medline]
  6. Rabkin E, Aikawa M, Stone JR, Fukumoto Y, Libby P, Schoen FJ: Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves. Circulation 2001, 104:2525-2532[Abstract/Free Full Text]
  7. Jian B, Jones PL, Li Q, Mohler ER, III, Schoen FJ, Levy RJ: Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis. Am J Pathol 2001, 159:321-327[Abstract/Free Full Text]
  8. Jian B, Xu J, Connolly J, Savani RC, Narula N, Liang B, Levy RJ: Serotonin mechanisms in heart valve disease I: serotonin induced up-regulation of transforming growth factor-beta1 via G-protein signal transduction in aortic valve interstitial cells. Am J Pathol 2002, 161:2111-2121[Abstract/Free Full Text]
  9. Xu J, Jian B, Chu R, Lu Z, Dunlop J, Rosenzweig-Lipson S, McGonigle P, Levy RJ, Liang B: Serotonin mechanisms in heart valve disease II: the 5-HT2 receptor and its signaling pathway in aortic valve interstitial cells. Am J Pathol 2002, 161:2209-2218[Abstract/Free Full Text]
  10. O'Brien KD: Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more). Arterioscler Thromb Vasc Biol 2006, 26:1721-1728[Abstract/Free Full Text]
  11. Filip DA, Radu A, Simionescu M: Interstitial cells of the heart valves possess characteristics similar to smooth muscle cells. Circ Res 1986, 59:310-320[Abstract/Free Full Text]
  12. Merryman WD, Lukoff HD, Long RA, Engelmayr GC, Jr, Hopkins RA, Sacks MS: Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast. Cardiovasc Pathol 2007, 16:268-276[CrossRef][Medline]
  13. Pho M, Lee W, Watt DR, Laschinger C, Simmons CA, McCulloch CA: Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves. Am J Physiol 2008, 294:H1767-H1778
  14. Darby I, Skalli O, Gabbiani G: Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990, 63:21-29[Medline]
  15. Liu AC, Gotlieb AI: The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol 2007, 171:1407-1418[Abstract/Free Full Text]
  16. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB: Type beta transforming growth factor: a bifunctional regulator of cell growth. Proc Natl Acad Sci USA 1985, 82:119-123[Abstract/Free Full Text]
  17. Sporn MB, Roberts AB, Wakefield LM, Assoian RK: Transforming growth factor-beta: biological function and chemical structure. Science 1986, 233:532-534[Abstract/Free Full Text]
  18. Walker GA, Masters KS, Shah DN, Anseth KS, Leinwand LA: Valvular myofibroblast activation by transforming growth factor-beta. Implications for pathological extracellular matrix remodeling in heart valve disease. Circ Res 2004, 95:253-260[Abstract/Free Full Text]
  19. Cushing MC, Liao JT, Anseth KS: Activation of valvular interstitial cells is mediated by transforming growth facor-beta1 interactions with matrix molecules. Matrix Biol 2005, 24:428-437[CrossRef][Medline]
  20. Desmoulière A, Geinoz A, Gabbiani F, Gabbiani G: Transforming growth factor-beta1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993, 122:103-111[Abstract/Free Full Text]
  21. Vaughan MB, Howard EW, Tomasek JJ: Transforming growth factor beta-1 promotes the morphological and functional differentiation of the myofibroblast. Exp Cell Res 2000, 257:180-189[CrossRef][Medline]
  22. Zhang W, Ou J, Inagaki Y, Greenwel P, Ramirez F: Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription. J Biol Chem 2000, 275:39237-39245[Abstract/Free Full Text]
  23. Benbow U, Brinckerhoff CE: The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 1997, 15:519-526[CrossRef][Medline]
  24. Moussad EE, Brigstock DR: Connective tissue growth factor: what’s in a name? Mol Genet Metab 2000, 71:276-292[CrossRef][Medline]
  25. Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, van Kooten C, Muller G: TGF-beta1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2). Kidney Int 2001, 59:579-592[CrossRef][Medline]
  26. Mehra A, Wrana JL: TGF-beta and the Smad signal transduction pathway. Biochem Cell Biol 2002, 80:605-622[CrossRef][Medline]
  27. Bobik A: Transforming growth factor-betas and vascular disorders. Arterioscler Thromb Vasc Biol 2006, 26:1712-1720[Abstract/Free Full Text]
  28. Ng CM, Cheng A, Myers LA, Martinez-Murillo F, Jie C, Bedja D: TGF-beta dependent pathogenesis of mitral valve prolapsed in a mouse model of Marfan syndrome. J Clin Invest 2004, 114:1586-1592[CrossRef][Medline]
  29. Fondard O, Detaint D, Iung B, Choqueux C, Adle-Biassette H, Jarraya M, Hvass U, Couetil JP, Henin D, Michel JB, Vahanian A, Jacob MP: Extracellular matrix remodeling in human aortic valve disease: the role of matrix metalloproteinases and their tissue inhibitors. Eur Heart J 2005, 26:1333-1341[Abstract/Free Full Text]
  30. Hinton RB, Jr, Lincoln J, Deutsch GH, Osinska H, Manning PB, Benson DW, Yutzey KE: Extracellular matrix remodeling and organization in developing and diseased aortic valves. Circ Res 2006, 98:1431-1438[Abstract/Free Full Text]
  31. Waltenberger J, Lundin L, Oberg K, Wilander E, Miyazono K, Heldin CH, Funa K: Involvement of transforming growth factor-beta in the formation of fibrotic lesions in carcinoid heart disease. Am J Pathol 1993, 142:71-78[Abstract]
  32. Jian B, Narula N, Li QY, Mohler ER, III, Levy RJ: Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis. Ann Thorac Surg 2003, 75:457-465[Abstract/Free Full Text]
  33. Lester WM, Gotlieb AI: In vitro repair of the wounded porcine mitral valve. Circ Res 1988, 62:833-845[Abstract/Free Full Text]
  34. Gotlieb AI, Rosenthal A, Kazemian P: FGF-2 regulates in vitro mitral valve interstitial cell repair. J Thorac Cardiovasc Surg 2002, 124:591-597[Abstract/Free Full Text]
  35. Durbin A, Nadir NA, Rosenthal A, Gotlieb AI: Nitric oxide promotes in vitro interstitial cell heart valve repair. Cardiovasc Pathol 2005, 14:12-18[CrossRef][Medline]
  36. Fayet C, Bendeck MP, Gotlieb AI: Cardiac valve interstitial cells secrete fibronectin and form fibrillar adhesions in response to injury. Cardiovasc Pathol 2007, 16:203-211[CrossRef][Medline]
  37. Lester W, Rosenthal A, Granton B, Gotlieb AI: Porcine mitral valve interstitial cells in culture. Lab Invest 1988, 59:710-719[Medline]
  38. Ghosh J, Murphy MO, Turner N, Khwaja N, Halka A, Kielty CM, Walker MG: The role of transforming growth factor-β1 in the vascular system. Cardiovasc Pathol 2005, 14:28-36[CrossRef][Medline]
  39. Luscinskas FW, Kansas GS, Ding H, Pizcueta P, Schleiffenbaum BE, Tedder TF, Gimbrone MA, Jr: Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, β1-integrins, and β2-integrins. J Cell Biol 1994, 125:1417-1427[Abstract/Free Full Text]
  40. Noria S, Xu F, McCue S, Jones A, Gotlieb AI, Langille BL: Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress. Am J Pathol 2004, 164:1211-1223[Abstract/Free Full Text]
  41. Desmouliere A, Chaponnier C, Gabbiani G: Tissue repair, contraction, and the myofibroblast. Wound Rep Regen 2005, 13:7-12[CrossRef]
  42. Jester JV, Petroll WM, Barry PA, Cavanagh HD: Expression of alpha-smooth muscle (alpha-SM) actin during corneal stromal wound healing. Invest Ophthalmol Vis Sci 1995, 36:809-819[Abstract/Free Full Text]
  43. Schoenwaelder S, Burridge MK: Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol 1999, 11:274-286[CrossRef][Medline]
  44. Soong HK, Cintron C: Different corneal epithelial healing mechanisms in rat and rabbit: role of actin and calmodulin. Invest Ophthalmol Vis Sci 1985, 26:838-848[Abstract/Free Full Text]
  45. Zhu J, Sun N, Aoudjit L, Li H, Kawachi H, Lemay S, Takano T: Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes. Kidney Int 2008, 73:556-566[CrossRef][Medline]
  46. Vyalov S, Langille BL, Gotlieb AI: Decreased blood flow rate disrupts endothelial repair in vivo. Am J Pathol 1996, 149:2107-2118[Abstract]
  47. Lee TY, Gotlieb AI: Early stages of endothelial wound repair conversion of quiescent to migrating endothelial cell involves tyrosine phosphorylation and actin microfilament reorganization. Cell Tissue Res 1999, 297:435-450[CrossRef][Medline]
  48. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C: Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 2001, 12:2730-2741[Abstract/Free Full Text]
  49. Zimerman B, Volberg T, Geiger B: Early molecular events in the assembly of the focal adhesions-stress fiber complex during fibroblast spreading. Cell Motil Cytoskel 2004, 58:143-159[CrossRef][Medline]
  50. Arora PD, McCulloch AG: The deletion of transforming growth factor-beta-induced myofibroblasts depends on growth conditions and actin organization. Am J Pathol 1999, 155:2087-2099[Abstract/Free Full Text]
  51. Roy SG, Nozaki Y, Phan SH: Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts. Int J Biochem Cell Biol 2001, 33:723-734[CrossRef][Medline]
  52. Cogan JG, Subramanian SV, Polikandriotis JA, Kelm RJ, Strauch AR: Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer. J Biol Chem 2002, 277:36433-36442[Abstract/Free Full Text]
  53. Hautmann MB, Madsen CS, Owens GK: Transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements. J Biol Chem 1997, 272:10948-10956[Abstract/Free Full Text]
  54. Adam PJ, Regan CP, Hautmann MB, Owens GK: Positive and negative-acting Kruppel-like transcription factors bind a transforming growth factor beta control element required for expression of the smooth muscle cell differentiation marker SM22alpha in vivo. J Biol Chem 2000, 275:37798-37806[Abstract/Free Full Text]
  55. Chin D, Boyle GM, Parsons PG, Coman WB: What is transforming growth factor-beta (TGF-β)? Br Assoc Plast Surg 2004, 57:215-221
  56. Sun PD, Davies DR: The cysteine-knot growth factor superfamily. Annu Rev Biophys Biomol Struct 1995, 24:269-291[CrossRef][Medline]
  57. Kaartinen V, Warburton D: Fibrillin controls TGF-beta activation. Nat Genet 2003, 33:331-332[CrossRef][Medline]
  58. Annes JP, Chen Y, Munger JS, Rifkin DB: Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1. J Cell Biol 2004, 165:723-734[Abstract/Free Full Text]
  59. Fontana L, Chen Y, Prijatelj P, Sakai T, Fassler R, Sakai LY: Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1. FASEB J 2005, 19:1798-1808[Abstract/Free Full Text]
  60. Jenkins G: The role of proteases in transforming growth factor-beta activation. Int J Biochem Cell Biol 2008, 40:1068-1078[CrossRef][Medline]
  61. Yang Z, Mu Z, Dabovic B, Jurukovski V, Yu D, Sung J: Absence of integrin-mediated TGF-beta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice. J Cell Biol 2007, 176:787-793[Abstract/Free Full Text]
  62. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J: The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999, 96:319-328[CrossRef][Medline]
  63. Yehualaeshet T, O'Connor R, Green-Johnson J, Mai S, Silverstein R, Murphy-Ullrich JE: Activation of rat alveolar macrophage-derived latent transforming growth factor beta-1 by plasmin requires interaction with thrombospondin-1 and its cell surface receptor, CD36. Am J Pathol 1999, 155:841-851[Abstract/Free Full Text]
  64. Ribeiro SM, Poczatek M, Schultz-Cherry S, Villain M, Murphey-Ulrich JE: The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta. J Biol Chem 1999, 274:13586-13593[Abstract/Free Full Text]
  65. Kaden JJ, Dempfle CE, Grobholz R, Fischer CS, Vocke DC, Kilic R, Sarikoc A, Pinol R, Hagl S, Lang S, Brueckmann M, Borggrefe M: Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis. Cardiovasc Pathol 2005, 14:80-87[CrossRef][Medline]
  66. Massagué J, Seoane J, Wotton D: Smad transcription factors. Genes Dev 2005, 19:2783-2810[Abstract/Free Full Text]
  67. Goto D, Nakajima H, Mori Y, Kurasawa K, Kitamura N, Iwamoto I: Interaction between Smad anchor for receptor activation and Smad3 is not essential for TGF-beta/Smad3-mediated signaling. Biochem Biophys Res Commun 2001, 281:1100-1105[CrossRef][Medline]
  68. Siegel PM, Massague J: Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 2003, 3:807-821[CrossRef][Medline]
  69. Ghosh AK, Yuan W, Mori Y, Varga J: Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-beta involves functional cooperation with p300/CBP transcriptional coactivators. Oncogene 2000, 19:3546-3555[CrossRef][Medline]
  70. Jinnin M, Ihn H, Asano Y, Yamane K, Trojanowska M, Tamaki K: Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1. Oncogene 2004, 23:1656-1667[CrossRef][Medline]
  71. Chen SJ, Ning H, Ishida W, Sodin-Semrl S, Takagawa S, Mori Y, Varga J: The early-immediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression. J Biol Chem 2006, 281:21183-21197[Abstract/Free Full Text]
  72. Ten Dijke P, Goumans MJ, Itoh F, Itoh S: Regulation of cell proliferation by Smad proteins. J Cell Physiol 2002, 191:1-16[CrossRef][Medline]
  73. Kay EP, Lee MS, Seong GJ, Lee YG: TGF-betas stimulate cell proliferation via an autocrine production of FGF-2 in corneal stromal fibroblasts. Curr Eye Res 1998, 17:286-293[CrossRef][Medline]
  74. Dkhissi F, Raynal S, Jullien P, Lawrence DA: Growth stimulation of murine fibroblasts by TGF-beta1 depends on the expression of a functional p53 protein. Oncogene 1999, 18:703-711[CrossRef][Medline]
  75. Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL, Arteaga CL: Phosphatidylinositol 3-kinase function is required for transforming growth factor b-mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 2000, 275:36803-36810[Abstract/Free Full Text]
  76. Cho HJ, Baek KE, Saika S, Jeong MJ, Yoo J: Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway. Biochem Biophys Res Commun 2007, 353:337-343[CrossRef][Medline]
  77. Wang H, Quah SY, Dong JM, Manser E, Tang JP, Zeng Q: PRL-3 down-regulates PTEN expression and signals through PI3K to promote epithelial-mesenchymal transition. Cancer Res 2007, 67:2922-2926[Abstract/Free Full Text]
  78. Wilkes MC, Murphy SJ, Garamszegi N, Leof EB: Cell-type-specific activation of PAK2 by transforming growth factor-beta independent of Smad2 and Smad3. Mol Cell Biol 2003, 23:8878-8889[Abstract/Free Full Text]
  79. Wilkes MC, Mitchell H, Gulati-Penheiter S, Dore JJ, Suzuki K, Edens M, Sharma DK, Pagano RE, Leof EB: Transforming growth factor-b activation of phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates fibroblast responses via p21-activated kinase-2. Cancer Res 2005, 65:10431-10440[Abstract/Free Full Text]
  80. Khalil N, Xu YD, O'Connor R, Duronio V: Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor- beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem 2005, 280:43000-43009[Abstract/Free Full Text]
  81. Taylor RC, Cullen SP, Martin SJ: Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 2008, 9:231-241[CrossRef][Medline]
  82. Fischer U, Schultze-Osthoff K: New approaches and therapeutics targeting apoptosis in disease. Pharmacol Rev 2005, 57:187-215[Abstract/Free Full Text]
  83. Kim SG, Jong HS, Kim TY, Lee JW, Kim NK, Hong SH, Bang YJ: Transforming growth factor-beta1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells. Mol Biol Cell 2004, 15:420-434[Abstract/Free Full Text]
  84. Conery AR, Cao Y, Thompson EA, Townsend CMJ, Ko TC, Luo K: Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis. Nat Cell Biol 2004, 6:366-372[CrossRef][Medline]
  85. Motyl T, Grzelkowska K, Szimowska W, Skierski J, Wareski P, Ploszaj T, Trzeciak L: Expression of bcl-2 and bax in TGF-beta 1-induced apoptosis of L1210 leukemic cells. Eur J Cell Biol 1998, 75:367-374[Medline]
  86. Ohgushi M, Uroki S, Fukamachi H, O'Reilly LA, Kuida K, Strasser A, Yonehara S: Transforming growth factor beta-dependent sequential activation of Smad, Bim and caspase-9 mediates physiological apoptosis in gastric epithelial cells. Mol Cell Biol 2005, 25:10017-10028[Abstract/Free Full Text]



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