The Roles Of Fibrocytes In The Myocardial Fibrosis Of Coronary Heart Disease And Cardiac Shock Wave Therapy

[Background]Cardiac remodeling caused by coronary heart disease (CHD), in particular myocardial fibrosis, seen in both the infracted and non-infarcted myocardium is recognized to be a major determinant of the development of postinfarction heart failure. At present, there is still no effective treatment for myocardial fibrosis. Therefore, the formative mechanism and treatment of myocardial fibrosis in CHD remain an area of active research.Fibrosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix (ECM). Fibroblasts, and related myofibroblasts, are the principle producers of ECM and contribute significantly to fibrosis. Recent researches indicate that a potential source of fibroblasts is the fibrocytes, a distinct circulating bone marrow-derived population. It has been revealed that fibrocytes may contribute to the development of fibrosis in many fibrotic diseases. However, the contribution of fibrocytes to the myocardial fibrosis of patients with CHD has not been demonstrated.Cardiac shock wave therapy (CSWT), using a low-energy shock wave (about10%of the energy density used for urolithiasis), is an effective, safe, and noninvasive therapy in cardiovascular medicine. Recent researches show that CSWT, inducing angiogenesis in the border zone of infarcted myocardium, can improve the symptom of patients with CHD and ameliorate cardiac remodeling after myocardial infarction. This suggests that CSWT may also be an effective therapy for improving myocardial fibrosis after acute myocardial infarction (AMI). To our knowledge, it is not reported that CSWT has been used to treat myocardial fibrosis after AMI.[Objective]We hypothesized that fibrocytes might be associated with the myocardial fibrosis in CHD, and CSWT might ameliorate myocardial fibrosis through reducing the amount of fibrocytes. In order to elucidate the roles of fibrocytes in myocardial fibrosis of CHD, the presence of fibrocytes and the localization of SDF-1/CXCL12were examined in the heart of patient with CHD and the normal heart. The effect of CSWT on myocardial fibrosis and the relationship between fibrocytes and CSWT were evaluated at the border zone of myocardial infarction in Diannan miniature pig. The aim of this study was to test our hypothesis and to provide a therapeutic cellular target and novel strategy for the myocardial fibrosis after AMI.[Methods]1. The methods of fibrocytes identification The presence of CD45+/aSMA+fibrocytes in5paraffin-embedded heart tissues with CHD were identified by three double immunostaining methods, including double immunohistochemistry staining, combination labelling of immunohistochemistry and immunofluorescence and double immunofluorescence labelling. The results were observed by light microscopy, immunofluorescence microscopy and confocal microscopy. The sensitiveness of the three double immunostaining methods was compared.2. Human cardiac specimens Human tissue specimens from27CHD hearts and11normal hearts as control following forensic autopsy were included in this study. Diagnosis of CHD includes coronary atherosclerosis, fatal definite myocardial infarction and/or myocardial scarring and without evidence of non-coronary cardiac diseases.(1) Formalin-fixed and paraffin-embedded specimens were stained with hematoxylin and eosin.(2) The presence of fibrocytes was identified by double immunofluorescence labelling and confocal microscopy.(3) The collagen volume fraction was examined by Masson’s Trichrome staining, and the correlation between fibrocytes and myocardial fibrosis was evaluated.(4) The location of CXCL12was examined by immunohistochemistry.3. Animal experiments25domestic Diannan miniature pigs were divided into three groups:AMI group (n-5), AMI+CSWT group (n=15), and CSWT group (n=5). A low-energy shock wave (0.09mJ/mm2, approximately10%of the energy used for the lithotripsy treatment,200shots/spot for9spots) was applied to the border zone around the infarcted myocardium. CSWT was performed three times in the first week (day3,5, and7). The animals were euthanized by an overdose of pentobarbital sodium, and the paraformldehyde-fixed and paraffin-embedded heart tissues were taken for further research. The presence of fibrocytes, collagen volume fraction and the location of CXCL12were examined as the methods described above.[Results]1. Comparison of three double immunostaining methodsThe comparison of three methods to identify the presence of fibrocytes in cardiac tissue:(1) CD45+/αSMA+fibrocytes could be clearly identified by double immmunofluorescence labelling.(2) Combination staining of immunohistochemistry and immunofluorescence could identify the CD45+/αSMA+fibrocytes, but, was prone to produce many false positives.(3) Double immunohistochemistry staining was very difficult to identify the CD45+/aSMA+fibrocytes.2. Studies in human cardiac specimens(1) Examination of the fibrocytes Fibrocytes identified by5labelling-combinations including CD34+/aSMA+, CD34+/procollagen-I+, CD45+/aSMA+, CXCR4+/procollagen-I+and CXCR4-7aSMA+, were present in26out of27CHD hearts and10out of11normal hearts. All of the labelling-combinations stained significantly more fibrocytes in CHD hearts compared with those in normal hearts (CD34+/aSMA+:5.65±5.52vs.1.6±1.58; CD34+/procollagen-I+:4.33±2.68vs.1.7±1.57; CD45+/aSMA+:4.0±3.43vs.1.5±1.20; CXCR4+/procollagen-I+:4.59±3.84vs.0.67±1.00; CXCR4+/aSMA+:4.21±3.53vs.0.91±1.30, P<0.05).(2) Correlation analysis There was a positive correlation between the collagen volume fraction and the amount of fibrocytes (r=0.558; P<0.05) in CHD hearts. There was also a positive correlation between the number of CXCR4+fibrocytes and the CXCL12+cells (r=0.741; P<0.05) in CHD hearts.3. Studies in animal experiments(1) Collagen volume fraction The fibrosis degree, in terms of collagen volume fraction, was significantly higher in AMI group than that in CSWT group (27.21±8.13vs.1.78±0.57, P<0.05), and was significantly lower in AMI+CSWT group than that in AMI group (10.13±4.96vs.27.21±8.13, P<0.05).(2) The amount of fibrocytes Both of the labelling-combinations (CD34+/aSMA+and CXCR4+/aSMA+) stained significantly more fibrocytes in AMI group compared with those in CSWT group (CD34+/aSMA+:35.40±11.72vs.1.40±1.14; CXCR4+/aSMA+:40.80±8.96vs.1.80±1.30, P<0.05), and less fibrocytes in AMI+CSWT group compared with those in AMI group (CD34+/aSMA+:12.27±7.71vs.35.40±11.72; CXCR4+/aSMA+:16.54±6.38vs.40.80±8.96, P<0.05).(3) Correlation analysis There was a positive correlation between the collagen volume fraction and the number of fibrocytes(r=0.936; P<0.05). There was also a positive correlation between the number of CXCR4+fibrocytes and the SDF-1/CXCL12+cells (r=0.802; P<0.05).[Conclusions] 1. Double immunofluorescence labelling is the optimal technical approach to identify the presence of fibrocytes in routinely paraffin-embedded heart tissues.2. Fibrocytes, likely in response to SDF-1/CXCL12, migrate into the CHD heart and may be the principal contributors to the development of myocardial fibrosis in patient with CHD. Fibrocytes may be a potential cell targets for the therapy of myocardial fibrosis.3. CSWT may ameliorate myocardial fibrosis after AMI in vivo, which is associated with the inhibitory effects of CSWT on SDF-1and fibrocytes. CSWT may also be a novel therapeutic strategy for myocardial fibrosis after AMI.