Mechanism And Effects Of Recombinant Human Endostatin On Apoptosis Of Hypertrophic Scar Fibroblasts In A Rabbit Ear Model

Hypertrophic scar?HS?typically occurs as a result of a full-thickness injury to the dermis with an abnormal healing response.Previous studies reported that incidence rates of HS vary from 40% to 70% following surgery and up to 91% following burns.It is characterized by abnormal hyperplasia in the normal skin,accompanied by pain,itching and other symptoms,which not only affects the appearance,but also leads to scar contracture.However,the exact mechanism of HS formation is poorly elucidated.Also,there is no single treatment modality that produces consistently favorable results.The surgical ablation can not inhibit the growth of HS,whereas causes new scars.In clinical,it is widely believed that hypertrophic scar fibroblasts?HSFs?are the major effector cells during wound healing,and that there may be an imbalance between the growth and death of HSFs,which leads to scar hyperplasia.Therefore,inhibit proliferation and induction of apoptosis in HSFs has been proposed as one of the critical strategies for the treatment of HS by way of reducing scar hypertrophy in situ.Endostatin,a 20 k Da C-terminal proteolytic fragment,is a member of a group of endogenous antiangiogenic proteins that was isolated from conditioned murine hemangioendothelioma?EOMA?cell line.Previous studies have suggested that endostatin can inhibit the proliferation of vascular endothelial cells,further research confirmed that endostatin can direct inhibit the proliferation,migration and induce apoptosis of some tumor cells so as to exert the anti-tumor effect.Our previous studies have shown that recombinant human endostatin?rh Endostatin?can inhibit the proliferation and promote apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats;Ren et al.confirmed that endostatin can inhibit the hyperplasia of scar tissue by decreasing the expression of bcl-2 in scar tissues and inhibiting the formation of new blood vessels.Our previous study found that rh Endostatin?6.25,12.5,25,50,100 ?g/ml?can directly inhibit the proliferation of HSFs,this study aims to understand the potential mechanisms,and to provide an experimental data for the development of new therapeutic interventions and targets for HS.Objective: To clarify the effects of rh Endostatin on HSFs apoptosis,and to elucidate part of its cell and molecular mechanisms.Methods:?1?Induction and Identification of HS model in a rabbit ear model: Eight New Zealand white rabbits were divided into normal?two?and HS control group?six?.The rabbits in the control group were used for HS modeling.HS began to appear on day28 post-surgery.Two normal and control rabbit ears were choosed at random and stained with hematoxylin and eosin?HE?for histopathological analyses.?2?Isolation and Identifaction of HSFs in vitro: Cells were isolated and extracted from fresh normal skin and HS tissues of rabbit ears.The cells?passage 3?were identified using immunohistochemical staining of vimentin and used in the subsequent experiments.?3?Analysis of apoptosis in HSFs: HSFs were divided into HSFs-untreated,rh Endostatin-treated?100 ?g/ml?and 5-fluorouracil-treated?500?g/ml?.Flow cytometry?FCM?were used to detect the apoptosis of HSFs.?4?Detection of apoptosis regulatory m RNAs and proteins: The c-fos,c-jun,NF-?B,fas,caspase-3,bcl-2 and p53 m RNA in various groups were quantified by RT-FQ-PCR.The above mentioned proteins were detected by western blot analysis.?5?Measurements of the cytosolic Ca²⁺ in HSFs: The cultured HSFs were loaded with the calcium ion-sensitive fluorescent indicator fluo-4/AM,and perfused with rh Endostatin?100 m g/ml?.Confocal laser scanning microscope?CLSM?was used to measure the change of cytosolic Ca²⁺ fluorescence intensity?FI?in HSFs when cells were immersed in the buffer with or without Ca²⁺,as well as to investigate the effect of rh Endostatin on cytosolic free calcium concentration([Ca²⁺]i)in HSFs.Results:?1?HS formed 28 days later after operation.HE staining showed that the scar tissue was thicken than the surrounding normal skin,with fibroblasts and small vessel proliferation.The thick,spiral or nodular collagen fibers formed collagen nodules.?2?The vimentin immunohistochemical showed that a large number of brown granules were found in the cytoplasm of the cells,with characteristics of fibroblasts.?3?FCM analysis showed that rh Endostatin?100 ?g/ml?promotes early and late apoptosis of HSFs compared to the untreated HSF controls?P<0.01?.The cell populations in early and late apoptosis were 10.78 ± 0.06% and 2.12 ± 0.04%,respectively.?4?Real-time PCR and Western blot showed that the m RNA and protein expression levels of c-fos,c-jun,NF-?B,fas,caspase-3,p53 and bcl-2 in rh Endostatin treatment were decreased compared with the untreated HSFs controls?P < 0.01?.?5?CLSM analysis showed that rh Endostatin?100 m g/ml?did not change the cytosolic Ca²⁺ FI in HSFs when cells were immersed in non-calcium extracelluar solution.The cells were immersed in the buffer with Ca²⁺ and perfused with rh Endostatin at the concentration of 100 m g/ml.The cytosolic Ca²⁺ FI in HSF was rapidly increased to maximum and then gradually declined.Conclusion:?1?rh Endostatin is sufficient to induce apoptosis in HSFs.?2?rh Endostatin induces apoptosis in HSFs which associates with the downregulation of bcl-2,NF-?B,c-jun and c-fos expression,and independent of fas,caspase-3 and p53 activation.?3?rhEndostatin can elicit the extracellular calcium influx in intact HSFs.Imbalance of Ca²⁺ steady state induced by rh Endostatin is the very early events occur prior to HSFs damage.Cytoplasmic Ca²⁺ overload may occur prior to any other molecular events of apoptosis in HSFs.