Cardiac fibroblasts (CFs) play a key role in cardiac fibrosis by regulating the balance between extracellular matrix synthesis and breakdown. Although phosphatase and tensin homologue on chromosome 10 (PTEN) has been found to play an important role in cardiovascular disease, it is not clear whether PTEN is involved in functional regulation of CFs. In the present study, PTEN was overexpressed in neonatal rat CFs via recombinant adenovirus-mediated gene transfer. The effects of PTEN overexpression on cell-cycle progression and angiotensin II- (Ang II-) mediated regulation of collagen metabolism, synthesis of matrix metalloproteinases, and Akt/P27 signaling were investigated. Compared with uninfected cells and cells infected with green fluorescent protein-expressing adenovirus (Ad-GFP), cells infected with PTEN-expressing adenovirus (Ad-PTEN) significantly increased PTEN protein and mRNA levels in CFs (
Cardiac remodeling is a complex process that involves ultrastructural rearrangement of the heart [
Phosphatase and tensin homolog on chromosome ten (PTEN) is a 3′-lipid phosphatase that is widely expressed in various cell types including cardiomyocytes, vascular smooth muscle cells (VSMCs), and endothelial cells [
Angiotensin II (Ang II) regulates collagen synthesis and production and promotes cardiac fibrosis [
In the present study, recombinant adenovirus-mediated gene transfer was used to enhance PTEN expression over basal levels in neonatal rat CFs so as to study the effects of PTEN on Ang II-induced CF proliferation, apoptosis, cell cycle, and collagen metabolism.
Animal experiments were conducted in accordance with guidelines established by the Animal Care and Use Committee of The Third Military Medical University.
CFs were isolated from the left ventricles of 3-day-old Sprague-Dawley rats, as previously described [
Adenovirus vector carrying the wild-type PTEN gene (from human species) was constructed by cloning and homologous recombination. An adenovirus vector carrying the green fluorescent protein (GFP) reporter gene was used as the negative control. Recombinant adenoviruses were produced by transfection of homologous recombinant plasmid into the AD-293 cell line.
Fibroblasts were cultured in a 250 mL culture bottle containing complete medium and were infected with adenoviruses at 90% confluency. The multiplicity of infection (MOI) was calculated using the following equation: MOI (pfu/mL) = [number of GFP positive cells × virus dilution (GFP positive maximum dilution)]/0.1 mL. To ensure comparable multiplicity of infection (MOI) between passages with different growth rates, cells plated on 96-well culture dishes at a concentration of 105 cells/mL were trypsinized and counted. Adenovirus (MOI = 100) was resuspended in 0.5 mL of DMEM and added to each well immediately after aspirating the medium. Complete medium (1.5 mL) was added to the wells after 2 h. After 48 h of incubation, fibroblasts were treated with Ang II (10−7 mM) or DMEM for 24 h. Fibroblasts were divided into the following treatment groups: Control (DMEM), Ad-GFP, PTEN adenovirus (Ad-PTEN), Control+Ang II, Ad-GFP + Ang II, and Ad-PTEN + Ang II. Each experiment was repeated three times with 3-4 replicates each.
Total RNA was isolated from CFs using the Tripure reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions. RNA concentration and purity were measured using a UV spectrophotometer. Subsequently, first-strand cDNA was obtained from total RNA (1000 ng) using the M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random hexamer primers. PCR amplification of specific genes was conducted using a prealiquoted PCR master mix (TOYOBO, Japan) in a thermocycler (T100
Primers designed and used for the RT-PCR studies.
Gene | Primers | Product size (bp) | Annealing temperature |
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PTEN | Forward 5′-AGAACTTATCAAACCCTT-3′ | 186 | 55°C |
Reverse 5′-GTCCTTACTTCCCCAT-3′ | |||
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Col I- | Forward 5′-CTCAGGGGCGAAGGCAACAGT-3′ | 125 | 50°C |
Reverse 5′-ATGGGCAGGCGGGAGGTCT-3′ | |||
| |||
Col III- | Forward 5′-ATGGTGGCTTTCAGTTCAGC-3′ | 425 | 45°C |
Reverse 5′-TGGGGTTTCAGAGAGTTTGG-3′ | |||
| |||
MMP-2 | Forward 5′-TGG TCGCAGTGATGGCTTCCTCT-3′ | 414 | 57°C |
Reverse 5′-CCCCACTTCCGGTCATCATCGTAG-3′ | |||
| |||
TIMP-1 | Forward 5′-TCGACGCTGTGGGGAATG-3′ | 466 | 54°C |
Reverse 5′-AAAGAACGGAGGAAACAG-3′ | |||
| |||
GAPDH | Forward 5′-TGCTGAGTATGTCGTGGAGT-3′ | 289 | 55°C |
Reverse 5′-AGTCTTCTGAGTGGCAGTGAT-3′ |
CFs from each group at various time points (approximately 107 cells) were washed in cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, and complete protease inhibitor mixture for 20 min on ice, according to the manufacturer’s instructions. Protein concentration was measured using the bicinchoninic acid protein assay kit (Bio-Rad, Hercules, CA, USA), with bovine serum albumin as the standard. Equal amounts of protein extracts (2 mg/mL) were mixed with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiled at 95–100°C for 5 min. The samples were then separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding sites were blocked by incubating the membranes with 5% nonfat dry milk for 2 h at room temperature in Tris-buffered saline with Tween (TBS-T) [20 mM Tris-HCl (pH 8.0), 8 g/L NaCl, and 0.1% Tween 20]. The blots were washed in TBS-T three times for 10 min and incubated at 4°C overnight with the appropriate primary antibody: mouse anti-GAPDH (1 : 1000 dilution; KangChen Bio-tech Inc., Shanghai, China); mouse anti-PTEN (1 : 1000 dilution; Cell Signaling Technology, Boston, MA, USA); rabbit anti-P27 (1 : 1000 dilution; Cell Signaling Technology, Boston, MA, USA); or rabbit anti-Akt (Thr-308 phosphorylation site, 1 : 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
The blots were then washed with TBS-T and incubated with horseradish peroxidase-conjugated anti-mouse (1 : 1000 dilution; KangChen Bio-tech Inc., Shanghai, China) or anti-rabbit (1 : 1000 dilution; Cell Signaling Technology, Boston, MA, USA) secondary antibodies diluted in 5% nonfat dry milk for 2 h at room temperature. After washing the membranes thrice in TBS-T, the proteins were detected by the 3,3′-diaminobenzidine colorimetric method (Boster Biological Engineering Co., Ltd. Wuhan, China), according to the manufacturer’s instructions. Each experiment was performed in triplicate and repeated three times. Immunoreactive bands were quantified by using the Quantity One densitometer analysis system (model 4.6.2, Bio-Rad, USA). The protein levels in each sample were normalized to the level of GAPDH protein.
Fibroblasts were cultured in 250 mL culture bottles for 24 h, after which they were washed once in PBS and detached by adding 0.25% trypsin. The cells were resuspended in complete DMEM and centrifuged at 200 ×g for 5 min. Next, the cells were fixed in 80%–95% ethanol and stored at 4°C. The cell-cycle status was then measured by staining CFs with propidium iodide (50 mg/mL in PBS containing 0.1% Triton X-100) at 4°C. After staining for at least 24 h, flow cytometry was performed on a FACScan instrument (BD Biosciences), and data were analyzed using the CellQuest.
The 3H-proline incorporation assay was used to determine collagen synthesis, as previously described [
Gelatinase activity was detected by gelatin zymography, according to a previous study [
Data were presented as mean ± standard deviation. The SPSS 16.0 software for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. The normal distribution of data was checked by the SPSS program. For comparison of different groups, the unpaired Student’s
We used cultured fibroblasts with >95% purity for all experiments. We first established the optimal MOI and time for infection of adenovirus on the basis of GFP expression in CFs transfected with the Ad-GFP plasmid by fluorescence microscopy. At an MOI of 100, the positive rate of GFP expression exceeded 90%, with no further increase at MOI > 100. With regard to time course, maximum GFP expression was observed at 48 h. Hence, all subsequent analyses were performed at an MOI of 100 and a transfection time of 48 h.
PTEN protein expression was assessed by western blotting (Figures
PTEN overexpression in cardiac fibroblasts (CFs) by adenoviral gene transfer. Infection with Ad-GFP or Ad-PTEN for 48 h. (a, b) PTEN protein expression was assessed by western blot analysis. (c) The PTEN mRNA level was assessed by semiquantitative RT-PCR. Each experiment was repeated three times with 3-4 replicates each.
Compared with the uninfected cells, cells infected with Ad-GFP virus for 48 h showed no changes in the distribution of cell-cycle phases (Figures
Effect of PTEN overexpression on the cell cycle in CFs.
G1 | S | G1/S | |
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Ad-PTEN | 76.4% | 8.07% | 9.47 |
Ad-GFP | 59.4% | 24.5% | 2.42 |
Untreated group | 61.8% | 21.6% | 2.86 |
Effect of PTEN overexpression on the cell cycle in CFs. Infection with Ad-GFP or Ad-PTEN for 48 h. The cell cycle phases were analyzed by flow cytometry: (a) uninfected cells; (b) control Ad-GFP-treated cells; and (c) Ad-PTEN-infected cells. Each experiment was repeated three times with 3-4 replicates each.
Compared with the uninfected cells, Ang II (10−7 mM) stimulation decreased PTEN mRNA levels at 1, 2, 4, 8, 16, and 32 h (Figures
Effect of angiotensin II (Ang II) on PTEN expression in CFs. Fibroblasts were treated with Ang II (10−7 mM) for 32 h. (a, c) The PTEN mRNA level was assessed by RT-PCR. (b, d, e, f) PTEN protein expression was assessed by western blotting. Each experiment was repeated three times with 3-4 samples.
Overexpression of PTEN inhibits Ang II-induced collagen synthesis in CFs. After infection for 48 h, fibroblasts were treated with Ang II (10−7 mM) for 32 h. (a, b) The collagen type I-
In the presence of Ang II, Ad-PTEN-infected cells showed decreased MMP-2 mRNA at 32 h (Figures
Overexpression of PTEN inhibits Ang II-induced MMP-2 and TIMP-1 production in CFs. After infection for 48 h, fibroblasts were treated with Ang II (10−7 mM) for 32 h. (a, b) The MMP-2 mRNA level was assessed by RT-PCR. (c, d) The TIMP-1 mRNA level was assessed by RT-PCR. (e, f) The activity of gelatinase was assessed by gelatin zymography. Each experiment was repeated three times with 3-4 replicates.
To determine the effects of PTEN on Akt/P27 pathway, Akt and P27 expression were analyzed. Compared with Ad-GFP-infected cells, P27 expression was increased in Ad-PTEN-infected cells independent of Ang II stimulation (Figures
Effect of PTEN on Ang II-induced (10−7 mM) Akt and P27 expression in CFs. After infection for 48 h, fibroblasts were treated with Ang II (10−7 mM) for 32 h. (a, b) P27 protein expression was assessed by western blotting. (c, d) Total Akt and phospho-Akt (pAkt-308) expression levels were assessed by western blotting. Each experiment was repeated three times with 3-4 replicates.
PTEN is widely expressed in the cardiovascular system and plays an important role in cardiovascular disease [
Mammalian PTEN (molecular weight = 40–50 kDa) is a phosphoinositide 3-phosphatase that directly counteracts growth factor-stimulated PI 3-kinases by metabolizing phosphatidylinositol 3,4,5-trisphosphate (Ptd Ins(3,4,5)P(3)) [
The elevation of Ang II concentration is an important cause of cardiac hypertrophy induced by pressure overload. We use Ang II in our experiments to mimic cardiac hypertrophy induced by pressure overload. Ang II stimulation directly decreased the synthesis of PTEN mRNA and protein in cells infected with PTEN-overexpressing adenovirus. The effect of Ang II stimulation on endogenous PTEN mRNA and protein needs to be investigated to confirm the direct effect of Ang II on PTEN expression. Dong et al. demonstrated that downregulation of PTEN expression and activity by Ang II increased proliferation and migration of VSMCs [
Recent data have identified additional roles of PTEN. Parajuli et al. showed that PTEN regulates cardiac remodeling after myocardial infarction via modulating the Akt/interleukin-10 signaling pathway [
Ang II is known to downregulate P27 expression in the heart. Previous research showed that P27 protein levels were highest in the stationary phase of the cell cycle and began to decline after mitogen-stimulation in cultured myocytes. On completion of the cell cycle, P27 protein accumulated, and cells entered the stationary state [
PTEN status is inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in diffuse large B-cell lymphoma cell lines and patient samples, whereas overexpression of PTEN induced cytotoxicity in PTEN-deficient cell lines by inhibiting PI3K/AKT signaling [
A potential limitation of the present study was that Akt and p27 were not inhibited, precluding analysis of the direct role of PTEN on the Akt/P27 pathway. Because of the in vitro nature of the study, we could not clarify the role of PTEN in cardiac fibrosis. These mechanisms should be investigated in future studies.
In conclusion, PTEN could regulate collagen metabolism of neonatal rat CFs via activation of the Akt/P27 pathway. Elucidation of the mechanism of action will help thus providing a new possible target for the treatment of cardiac fibrosis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the National Natural Science Foundation of China (Grant no. 30370584).