Author + information
- Xiaohong Liu, MD, PhD,
- Chenguang Bai, MD, PhD,
- Dejun Gong, BSc,
- Yang Yuan, BSc,
- Lin Han, MD, PhD,
- Fanglin Lu, MD, PhD,
- Qinqi Han, MD, PhD,
- Hao Tang, MD, PhD,
- Shengdong Huang, MD, PhD and
- Zhiyun Xu, MD, PhD⁎ ()
- ↵⁎Institute of Cardiothoracic Surgery, Changhai Hospital, 168 Changhai Road, Shanghai 200433, People's Republic of China
To the Editor:
Chronic and permanent constrictive pericarditis represents a serious hemodynamic syndrome that can lead to heart failure unless surgically treated (1). Although a number of factors may cause constriction, idiopathic constrictive pericarditis (ICP) is becoming increasingly prevalent in developed countries (2). Unfortunately, the mechanisms underlying pericardial fibrosis and calcification in ICP remain poorly understood.
Transforming growth factor-β1 (TGF-β1) plays a key role in many fibrocalcific lesions, including cardiovascular diseases (3). Here, using immunoblotting, we found increased expression of TGF-β1 in ICP pericardia (n = 18), and this increased expression was associated with increased collagen/elastin ratio (r = 0.6585, p = 0.0030) and calcification (rs= 0.5328, p = 0.0228), suggesting a possible contribution of TGF-β1 to pericardial fibrosis and calcification. Real-time reverse transcription–polymerase chain reaction (RT-PCR) demonstrated that the messenger ribonucleic acid (mRNA) levels of collagen I, collagen III, matrix metalloproteinase (MMP)-2, MMP-8, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were significantly increased in ICP (n = 18) pericardia compared with control pericardia (n = 12), whereas those of elastin, MMP-1, and MMP-13 were decreased (all p < 0.05). Subsequently, ICP pericardia (n = 43, including additional 25 archived specimens) were processed for histological analysis; fibrosis was detected in 43 of 43 (100%) cases and calcification in 34 of 43 (79%). Ossification was seen in 5 of 34 (15%) calcified pericardia. The study was approved by the local ethics committee, and all of the patients gave informed consent.
We isolated pericardial interstitial cells (PICs) and found that they possessed a similar immunophenotype as mesenchymal stem cells. The PICs differentiated into myofibroblasts, chondroblasts, and osteoblasts after stimulation with TGF-β1, indicating that TGF-β1 may promote abnormal differentiation of adult PICs and lead to biological changes.
The effects of TGF-β1 on PICs were consistent with the changes in fibrosis-related genes (compared to control baseline values) in ICP pericardia as mentioned in the preceding text. Incubation of PICs with TGF-β1 (10 to 60 ng/ml) for 48 h or with 10 ng/ml TGF-β1 for 6 to 48 h increased the mRNA expression of collagen I and collagen III in a concentration- and time-dependent manner. After PICs were treated with TGF-β1 (10 ng/ml) for 48 h, MMP-2 and -9 mRNA, critical for elastin degradation, were increased by 5.19-fold and 2.68-fold, respectively. The TIMP-2 mRNA, a natural inhibitor for MMP-2, was also increased by 25.48-fold. However, TGF-β1 decreased the mRNA levels of MMP-1 (≈57%) and MMP-13 (≈53%) in PICs and increased that of MMP-8 by 6.22-fold. Three days after TGF-β1 (10 ng/ml) stimulation, zymography of conditioned media revealed a band of gelatin degradation at 130 kDa, which represents the heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin. Heterodimer can prevent MMP-9 degradation, thereby augmenting MMP-9 activity. However, gelatinolytic band of MMP-2 was not detectable until day 28, indicating that TGF-β1–induced robust transcription of TIMP-2 inhibits the activity of MMP-2.
We found TGF-β1 may also contribute to the progression of calcification by inducing apoptosis and osteogenic differentiation of PICs. The TGF-β1 treatment (10 ng/ml, 48 h) resulted in a 1.8-fold increase in apoptosis of confluent PICs compared with untreated cells (Fig. 1A). After treatment with TGF-β1 (10 ng/ml), confluent PICs (Fig. 1B1) spontaneously retracted from neighboring areas (Fig. 1B2) and grouped into aggregates (Fig. 1B3). With the formation of nodules (Fig. 1B4), cells became denser in the central areas of the nodules (Fig. 1B5) and expressed alkaline phosphatase (osteoblast marker) (Fig. 1B6). Propidium iodide counterstaining (Fig. 1B7) showed that a proportion of cells exhibited nuclear shrinkage and chromatin condensation. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining (Fig. 1B8) confirmed the presence of apoptosis. Hoechst 33342 and propidium iodide double staining (Fig. 1B9) revealed a number of cells had already died by apoptosis. Von Kossa staining (Fig. 1B10) detected calcium deposition in the nodules. These nodules displayed increased alkaline phosphatase activity (sevenfold) (Fig. 1C) and contained more calcium (6-fold) (Fig. 1D) than TGF-β1-untreated cells.
In conclusion, our results demonstrate that TGF-β1 exerts pleiotropic effects on PICs by promoting abnormal differentiation, inducing apoptosis, and regulating the expression of fibrosis-related genes in PICs, which indicates that TGF-β1 may act as a regulator of both fibrosis and calcification during the progression of ICP.
Please note: Drs. Liu and Bai are both first authors. Supported by a grant (30700809) from the National Natural Science Foundation of China. The authors have reported that they have no relationships to disclose.
- American College of Cardiology Foundation