Fibrocalcific Mechanism Studies Of Idiopathic And Post–cardiac-injury Constrictive Pericarditis

Objective: Idiopathic and post-cardiac-injury constrictive pericarditis (IAPCP) has become increasingly prevalent in recent years. Some studies showed that pericardial fibrosis and/or calcification played an important role in the initiation and progression of IAPCP. The objective of the present study is to examine the mechanism underlying the initiation and progression of IAPCP, and explore possible methods to prevent or delay the occurrence of IAPCP.Methods: The present study was divided into six sections. 1. Molecular pathology of IAPCP pericardia: To evaluate histological features, data were collected from 45 consecutive IAPCP patients (idiopathic constrictive pericarditis [ICP], 43 cases; post–cardiac-injury constrictive pericarditis [PCP], two cases) who had archived paraffin-embedded pericardial specimens available for study, including fresh pericardial specimens from 18 patients (all ICP). Twelve fresh pericardial specimens free of pericarditis were obtained at autopsy or during cardiac surgery as controls. The histological features and the degree of fibrosis and calcification were observed by Hematoxylin and Eosin (HE), Victoria blue-van Gieson (VG) and Von Kossa staining respectively. Cellular phonotype and apoptosis were determined by immunohistochemistry and TUNEL staining respectively. Total RNA was extracted from the fresh samples, and change of extracellular matrix (ECM)-related genes was examined by quantitative RT-PCR analysis. 2. TGF-β1 expression in IAPCP pericardia and effect on PICs: Expression and distribution of transforming growth factor-β1 (TGF-β1) in IAPCP samples were examined by western blot and immunohistochemistry. Pericardial interstitial cells (PICs) were isolated from normal pericardia and treated with TGF-β1. Changes in ECM-related genes before and after TGF-β1 treatment were compared by quantitative RT-PCR. The activity of gelatinases was determined by Gelatin zymography. The course of TGF-β1-induced nodules in confluent PICs was monitored. Cellular phonotype, apoptosis and calcium deposition were examined by immunocytochemistry, TUNEL and Von Kossa staining respectively. The degree of calcium deposition and the index of apoptosis were quantified colorimetrically and by flow cytometry respectively. 3. FGF-2, PDGF and BMP-2 expression in IAPCP pericardia and effect on PICs: Expression of fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and bone morphogenetic protein-2 (BMP-2) in IAPCP pericardia was examined by immunohistochemistry. PICs were isolated from normal pericardia and treated with FGF-2, PDGF-AA and BMP-2. Cellular phonotype was determined by immunocytochemistry. 4. Aortic valve replacement with autologous pericardium: pathological examination of surgically removed pericardial valves: Fifteen patients who underwent aortic valve replacement with autologous pericardium were followed up for the prevalence of reoperation. The pathological features of pericardial valves and the phenotype of PICs were analyzed by HE, VG and immunohistochemical staining. 5. Biological characteristics of PICs: PICs and mesenchymal stem cells (MSCs) were isolated and cultured. Senescent cells in each passpage were determined byβ-galactosidase staining, and the immunophenotype of PICs was analyzed by flow cytometry. PICs were induced to differentiate into osteoblasts, chondroblasts and adipocytes with appropriate inducing media. The differentiation capacity of PICs was determined by immunocytochemistry, immunohistochemistry and oil O staining. Cell proliferation, cell-cycle distribution and self-renewal were examined by MTT, flow cytometry and fibroblastic colony-forming unit (CFU-f) assay. Expression differences in self-renewal related genes between MSCs and PICs were compared by RT-PCR. 6. Transdifferentiation of pericardial myofibroblasts to PICs by Klf4: Pericardial myofibroblasts derived from IAPCP pericardia were isolated and cultured. Biological characteristics of myofibroblasts were determined by usingβ-galactosidase staining, flow cytometry and in-situ apoptosis. Infection efficiency and changes in morphology, cellular phonotype and collagen synthesis were examined by flow cytometry and western blot after infection of myofibroblasts with adenovirus-mediated Krüppel-like factor 4 (AdKlf4) and adenovirus-mediated enhanced green fluorescent protein (AdEGFP). In addition, the effect of Klf4 on cellular senescence and the expression of senescent genes were examined byβ-galactosidase staining and RT-PCR.Results: 1. Molecular pathology of IAPCP pericardia: HE and VG staining showed that the architecture of IAPCP pericardia was distorted markedly due to moderate-to-severe fibrosis and calcification (bone formation in 5 cases). Immunohistochemical staining ofα-smooth muscle actin (α-SMA) and alkaline phosphatase (ALP) showed that some PICs in IAPCP pericardia were myofibroblasts and osteoblasts. Even PICs in three cases without calcification also expressed ALP protein. The index of osteoblast transdifferentiation of PICs significantly correlated with the degree of calcification (rs=0.7579, P<0.05). Strong positive signals of apoptosis were present in PICs as shown by TUNEL staining. The apoptosis rate increased gradually with the degree of calcification increasing (rs=0.4826, P<0.05). Compared with the normal pericardia, the expression of collagenⅠand collagenⅢwas significantly increased in ICP pericardia, whereas the expression of elastin decreased. Furthermore, mRNA expression of matrix metalloproteinase-2 (MMP-2), MMP-8, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 increased significantly in ICP as compared with normal pericardia, whereas the expression of MMP-1 and MMP-13 decreased. Importantly, the MMP-1/TIMP-1 and MMP-13/TIMP-1 ratios decreased significantly in ICP compared with normal pericardia, while the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios were significantly higher in ICP pericardia. In contrast, the MMP-8/TIMP-1 radio did not differ significantly between ICP and normal pericardia (all P<0.05). 2. TGF-β1 expression in IAPCP pericardia and effect on PICs: IAPCP pericardia expressed TGF-β1 protein, and TGF-β1 protein abundance significantly correlated with an increase in the collagen/elastin ratio (r=0.6585, P<0.05) and the degree of calcification (rs=0.5328, P<0.05). Cellular specific immunohistochemistry confirmed the expression of TGF-β1 mainly in PICs. The in vitro model showed thatα-SMA mRNA expression increased in a concentration- and time-dependent manner after treatment of PICs with TGF-β1. Meanwhile, there was an increase in the expression of collagen I and collagen III mRNA. TGF-β1 increased the expression of MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2, although MMP-1 and MMP-13 expressions were reduced. Gelatinolytic activity in the conditioned medium of the co-culture model was measured by gelatin zymography. By day 3, TGF-β1 elevated the proteolytic band around 130 kDa, which represents the heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL), and this gelatinolytic activity increased in a time-dependent manner. Furthermore, TGF-β1 promoted the formation of apoptotic-enriched calcified nodules in confluent PICs. These nodules shared certain properties with the bone, including increased ALP activity, the development of osteoblast phenotype, high calcium deposition and increased apoptosis (all P<0.05). 3. FGF-2, PDGF and BMP-2 expression in IAPCP pericardia and effect on PICs: Immunohistochemistry confirmed that IAPCP pericardia expressed FGF-2, PDGF and BMP-2. In vitro experiments showed that FGF-2, PDGF-AA and BMP-2 induced transdifferentiation of PICs to myofibroblasts with the expression ofα-SMA, and BMP-2 also induced transdifferentiation of PICs to osteoblasts with the expression of ALP. 4. Aortic valve replacement with autologous pericardium: pathological examination of surgically removed pericardial valves: The freedom from commissural tear, endocarditis, valve degeneration (fibrosis and calcification) and the prevalence of re-operation at the end of follow-up was 100%, 93%, 80% and 67%, respectively. Valve-related mortality was 0%. Pathological examination confirmed partial endothelialization of pericardial valves as early as five months, and some cells under the endothelium were positive forα-SMA. PICs in degenerated pericardial valves due to fibrosis and calcification (n=3) were positive forα-SMA, and some cells were positive for ALP. VG staining revealed a band of elastic tissue of the excised autologous pericardium after long term adaptation (> nine years). 5. Biological characteristics of PICs: PICs showed a fibroid spindle-shaped appearance in vitro. The cellular morphology changed dramatically during propagation, from spindle-shaped cells to large and flat cells with increased senescent cells and higherα-SMA expression. Cell cycle analysis revealed that the number of cells in G2/M to S phase of cell cycle was increased significantly. Furthermore, PICs possessed similar surface markers as did MSCs. In vitro experiments showed that PICs had the ability to differentiate into osteoblasts, chondroblasts and adipocytes. Cell cycle analysis showed that there were more than 90% of the MSCs in the G0/G1 phase of cell cycle but there were only≈64% of the PICs in G0/G1. MSCs were able to produce CFU-f continously, but the CFU-f formation efficiency of PICs was lower than that of MSCs (P<0.05). Although PICs expressed self-renewal related genes, the transcript level of Oct3/4 and Bmi-1 was lower than that of MSCs. 6. Transdifferentiation of pericardial myofibroblasts to PICs by Klf4: As compared with PICs, confluent myofibroblasts spontaneously retracted from neighboring areas and grouped into aggregates that progressed to form nodules with increased senescent cells and higher apoptotic cells. Flow cytometry showed that Klf4 resulted in the cell cycle arrest on day five. RT-PCR confirmed that Klf4 induced the expression of p53 and inhibited the expression of collagen I and collagen III. However, Klf4 had no effect on the expression ofα-SMA. Myofibroblasts infected with AdKlf4 for five weeks, both RT-PCR and western blot showed that Klf4 significantly decreased the expressed ofα-SMA and p53.Conclusion: 1. Fibrosis and calcification are the most important histological features of IAPCP pericardia, of which fibrosis is characterized by myofibroblast transdifferentiation, abnormal collagen deposition and elastin degradation accompanied by MMPs and TIMPs imbalance; pericardial calcification correlated with apoptosis and osteoblast transdifferentiation, suggesting that pericardial calcification is a PICs-mediated active process, regulated by multiple mechanisms. 2. Although TGF-β1, FGF-2, PDGF and BMP-2 are all involved in the pathogenesis of fibrocalcification, the transdifferentiation of PICs to myofibroblasts and osteoblasts is the main mechanism for pericardial fibrocalcification, suggesting that pericardial fibrosis and calcification are active processes induced by multiple signal transduction pathways. 3. Pathological examination of the autologous pericardial valves confirmed that the abnormal transdifferentiation of PICs to myofibroblasts and osteoblasts is the main mechanism for pericardial fibrocalcification. 4. Compared with MSCs, PICs have the similar immunophenotype and differentiation potential (plasticity), whereas the proliferation activity of PICs is significantly higher than that of MSCs, and the self-renewal capacity of PICs is lower than that of MSCs. Moreover, the plasticity of adult cells may provide a substrate for growth factors or cytokines that promote inappropriate differentiation of these cells. 5. Klf4 induced-transdifferentiation from myofibroblasts to PICs is a progressive, multiple-step and highly-regulated process. Klf4 can reduce collagen secretion even before cell transdifferentiation, suggesting that phenotypic reversion of pericardial myofibroblasts is expected to prevent pericardial fibrosis.