Role Of PDGFs/PDGFRs Signaling Pathway In Myocardial Fibrosis Of DOCA/salt Hypertensive Rats

Background and Objective: MF is a pathological process of excessive deposition ofcollages due to their abnormal metabolism and a major cause of heart failure. Inmultiple cardiovascular diseases at end stage, MF is a major pathological feature ofmyocardium. In the heart, excessive accumulation of extracellular matrix (ECM)components, especially the typeⅠ and Ⅲcollagens, is responsible for myocardialfibrosis. Changes in tissue structure and cardiac dysfunction following myocardialfibrosis contribute to heart failure, arrhythmia, sudden cardiac death and other seriouscardiovascular events. Now that myocardial fibrosis related to renin-angiotensin-aldosterone system (RAAS), fibroblasts, oxidative stress, inflammation and growthfactors, it is the ultimate outcome of various stimuli-induced chronic inflammatoryresponse. As we all know that myocardial fibrosis is closely related to the role of renin-angiotensin-aldosterone system (RAAS) in vivo. Related studies showAngiotensin-converting enzyme inhibitors (ACEI) and Ang Ⅱreceptor antagonist(ARB) both have certain role of mitigating myocardial fibrosis, ventricular remodelingand improving prognosis of cardiovascular diseases. This shows that the RAAS playan important role in the occurrence and development process of myocardial fibrosis. Inrecent years, the view of mineralocorticoid induced myocardial fibrosismineralocorticoid(aldosterone and deoxycorticosterone) has been confirmed by moreresearch. However, the roles of some molecular signaling pathways inmineralocorticoid induced myocardial fibrosis remain unclear, and related research hasbecome a hot spot of the prevention and treatment of myocardial fibrosis. Previous work on myocardial fibrosis of desoxycorticosterone (DOCA) inducedsalt-sensitive hypertensive rats demonstrated that the increased mineralocorticoid caninduce myocardial fibrosis, and found that the PDGF/PDGFR signaling pathway isinvolved in the occurrence of MF, and the inflammatory response deteriorates in themyocardium. To inhibit infiltration of monocytes/macrophages is helpful to attenuateMF, which suggests that inflammation plays an important role in MF. However, nostudy has been conducted to investigate the relationship between inflammatoryresponse and PDGF/PDGFR signaling pathway. There is evidence showing that thefibroblasts in the heart have the potential to differentiate into myofibroblasts. In studyon myocardial fibrosis after myocardial infarction, investigators proposed thatPDGFs/PDGFRs signaling pathway is involved in the pathogenesis of myocardialfibrosis via stimulating fibroblasts to proliferate and transform into myofibroblasts andto secret massive collagens. However, this has not been confirmed in vivo.1) Thisstudy aimed to investigate infiltration of monocytes/macrophages, expressions ofPDGF and PDGFR, cells with PDGFR expression, changes in fibroblasts andmyofibroblasts in the myocardium of rats with DOCA/salt induced MF. At the sametime, therapy was done in these rats with fasudil or imatinib.2) To investigate whetherinflammation could excessively activate platelet-derived growth factor (PDGF)signaling pathway in desoxycorticosterone (DOCA) induced salt sensitivehypertensive rats with myocardial fibrosis (MF).3) To investigate the role ofPDGF/PDGFR signaling pathway in myocardial fibrosis of desoxycorticosterone(DOCA) induced salt-sensitive hypertensive rats.Methods: A total of70male SD rats underwent right nephrectomy and bred with1%sodium chloride and0.1%potassium chloride for4weeks. Then, these rats wererandomly divided into4groups:1) CON group: animals were subcutaneously treatedwith soybean oil once every4days and intragastrically treated with distilled water once daily;2) DOCA group: animals were subcutaneously treated with DOCA at60mg/kg/4d and intragastrically treated with distilled water once daily;3) DOCA+IMAgroup: animals were subcutaneously treated with DOCA at60mg/kg/4d andintragastrically treated with imatinib at60mg/kg/d once daily;4)DOCA+FAS:animals were subcutaneously treated with DOCA at60mg/kg/4d andintragastrically treated with fasudil at10mg/kg/d once daily. Systolic blood pressurewas measured once every2weeks. HE staining was done to observe myocardialinflammation; Picric acid-sirius red staining was employed to determine the severityof myocardial fibrosis and the perivascular fibrotic area (PVCA)/vascular area (VA)ratio. Real-time fluorescence quantitative PCR was done to determine the mRNAexpressions of FSP-1, S100A4(two fibroblast specific proteins), α-SMA, procollagenI, procollagen III, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFRα and PDGFRβ.Immunohistochemistry was done to detect the protein expressions ofmonocyte-macrophage antigen (ectodermal dysplasia1, ED-1), vimentin, α-SMA,PDGFRα, PDGFRβ and p-PDGFRβ. Immunofluorescence staining was performed todetermine the cell types with the expressions of PDGFRα and PDGFRβ.Results:1)14days after intervention, the SBP in DOCA group and DOCA+FASgroup increased markedly when compared with CON group (P<0.01), but there wasno marked difference between DOCA group and DOCA+FAS group (P>0.05).14and28days after intervention, the SBP in DOCA and DOCA+IMA group wassignificantly higher than that in CON group (P<0.01), but there was no significantdifference between DOCA group and DOCA+IMA group (P>0.05).2) At14days, inDOCA group, the myocardial inflammation was obvious, ED-1expression increasedmarkedly, the mRNA expressions of PDGF-A, PDGF-B, PDGF-C, PDGFRα andPDGFRβ increased to different extents, protein expressions of PDGFRα and PDGFRβalso elevated markedly (P<0.01), but the PDGF-D mRNA expression remained unchanged, when compared with CON group. After treatment with fasudil (a drug withanti-inflammatory activity), myocardial inflammation was significantly attenuated,mRNA expressions of PDGF-A, PDGF-B, PDGF-C and PDGFRα as well as PDGFRαprotein expression reduced dramatically (P<0.01), but the mRNA and proteinexpressions of PDGFRβ remained unchanged (P>0.05) when compared with DOCAgroup.3) At28days, the severity of myocardial fibrosis and PVCA/VA ratio inDOCA group were significantly increased when compared with CON group (P<0.01).The severity of myocardial fibrosis and PVCA/VA ratio in DOCA+IMA group weremarkedly lower than those in DOCA group (P<0.01) although they were higher thanthose in CON group (P<0.05).4) At days14, the mRNA expressions of PDGFRα andPDGFRβ in DOCA group were significantly higher than those in CON group (P<0.01)and DOCA+IMA (P<0.01). At days28, the mRNA expressions of PDGFRβ, FSP-1,α-SMA, procollagen I and procollagen III in DOCA group were significantly higherthan those in CON group (P<0.01), but the PDGFRα mRNA expression remainedunchaged. In addition, in a specific group, the PDGFRβ mRNA expression was higherthan the PDGFRα mRNA expression. In DOCA+IMA group, the mRNA expressionsof PDGFRβ, FSP-1, α-SMA, procollagen I and procollagen III were markedly reducedwhen compared with DOCA group (P<0.01).5) At14days, the protein expressions ofPDGFRα and PDGFRβ in DOCA group were significantly higher than those in CONgroup (P<0.01). The PDGFRα protein expression in DOCA+IMA group wasmarkedly lower than that in DOCA group (P<0.01). At days28, the proteinexpressions of PDGFRα and PDGFRβ in DOCA group were significantly increasedwhen compared with CON group (P<0.01), but the PDGFRα protein expressionremained unchanged (P>0.05). The protein expressions of PDGFRα and PDGFRβ inDOCA+IMA group were significantly lower than those in DOCA group (P<0.01). Atday28, the cardiac interstitium mainly contained vimentin positive fibroblasts, andα-SMA positive cells were less identified in CON group. In DOCA group, α-SMA positive fibroblasts (spindle-shaped) increased significantly (P<0.01), but themyofibroblasts reduced significantly in DOCA+IMA group when compared withDOCA group (P<0.05).6) PDGFRα protein expression was observed in fibroblastsand myofibroblasts, but not in VSMCs. PDGFRβ protein expression was noted in notonly fibroblasts and myofibroblasts but VSMCs.Conclusion:1) PDGFRα acts at early stage, but PDGFRβ functions in the wholeprocess.2) PDGFRα and PDGFRβ expressions increase in fibroblasts and myofibroblasts.3) In DOCA/salt induced hypertensive MF rats, the fibroblasts proliferated and themyofibroblasts also increased markedly.4) Monocyte-macrophages, fibroblasts and myofibroblasts play a key role in theDOCA-induced myocardial fibrosis in rats.5) In DOCA induced salt sensitive hypertensive rats, the massive collagen deposition,and tyrosine kinase receptor inhibitors can inhibit the activity of PDGFRs, then reducefibroblasts, myofibroblasts and collagen content.6) When there was no difference in SBP before experiment, and the CVF and PVCA inthe DOCA+IMA group were dramatically decreased when compared with those in theDOCA group, indicating that imatinib is able to relieve myocardial fibrosisindependent of lowering SBP.7) Inflammation and PDGF/PDGFR signaling pathway are involved in themineralocorticoid induced MF, and to attenuate myocardial inflammation may inhibitthe expressions of components of PDGF/PDGFR signaling pathway. These suggestthat the excessive activation of PDGF/PDGFRα signaling pathway is closely related tothe myocardial inflammation.8) In DOCA induced salt sensitive hypertensive rats, we speculate that DOCA inducesthe myocardial inflammation, activates MR on the monocytes/macrophages, and recruits the monocytes/macrophages into the myocardium leading to the productionof a large amount of PDGFs, at the same time, fibroblasts also cause the secretion ofPDGFs. The activated PDGFs binds to PDGFRs, leading to the activation ofPDGFs/PDGFRs signaling pathway. PDGF/PDGFR signaling pathway can induce themassive collagen deposition via stimulating fibroblasts to proliferate and transforminto myofibrlblasts, resulting in myocardial fibrosis. After treatment with imatinib orfasudil, the inflammatory response or PDGF/PDGFR signaling pathway are blocked,and the myofibrlblasts reduced, leading to the attenuation of myocardial fibrosis.