Anti-hypertrophic Scarring Effect Of Recombinant Human Endostatin In A Rabbit Ear Model And Its Mechanisms

Objective:To explore the possible cellular and molecular mechanisms of action involved, and to provide experiment data for the development of new therapeutic agents and targets of hypertrophic scar(HS). we investigated the antiscaring effects of recombinant human endostatin(rhEndostatin) on HS in rabbit ears induced by excision of full-thickness skin.Methods:①Grouping and Hypertrophic Scar Model:A total of21New Zealand white rabbits were randomly and equally divided into normal group, model group, normal saline group, rhEndostatin1.25mg/ml group, rhEndostatin2.5mg/ml group, rhEndostatin5.0mg/ml group and triamcinolone40mg/ml group. A rabbit ear model with hypertrophic scars was established. Full-thickness wounds down to cartilage were created in New Zealand White rabbits, except the normal group.②Treatments:Normal group and model group left untreated, treatment wounds were administrated with0.9%normal saline, triamcinolone, or rhEndostatin (1.25,2.5,5mg/ml) on postwounding day28. Each scar was injected with a100u1solution once every other day for a total of six treatments.③Gross observation of HS and histological assay:The healing of each wound was observed and the hypertrophic scars were examined in terms of color, texture and thickness. The SEI of each scar was measured by histological analyses of HE staining.④The expression of type Ⅰ, Ⅲ collagens, VEGF, TGF-β1, bFGF and microvascular quantification:The expression level of type Ⅰ,Ⅲ collagens VEGF, TGF-β1and bFGF was measured by the immunohistochemistry S-P method. MVD were evaluated histologically with CD34immunohistochemical staining to assess vessel ingrowth.⑤The effects of rhEndostatin on proliferation and cell cycle of hypertrophic scar fibroblasts (HSFs):Primary cultures of HSFs were isolated, characterized and established from fresh HS tissues in rabbit ears. The cultured cells were identified by cell morphology and immunocytochemical staining. MTT assay and flow cytometric detection were performed to investigate HSFs proliferation and cell cycle distribution induction, respectively.Result:①Effects of rhEndostatin on Hypertrophic Scarring in the Rabbit Ear Model: All wounds healed well and were re-epithelialized on postoperation days28, the rabbit ear hypertrophic scars were successfully established. Compared with normal saline group and model group, group treated with rhEndostatin2.5mg/ml and5.0mg/ml showed significant reduction in height, volume and hardness. Histomorphometric quantification after treatment showed that the SEI values of model group and normal saline group were high.The difference between the two groups failed to be significant (P>0.05). Significant decreases in SEI values were observed in rhEndostatin (2.5,5mg/ml) compared to model group and normal saline group. The mean SEI of scars treated with rhEndostatin5mg/ml was the lowest, which was lower than that of triamcinolone group, and statistically different (P<0.05).②Effects of rhEndostatin on the expression of type I, III collagens, VEGF, TGF-β1and bFGF in the scar tissue:The results of immunohistochemical detection demonstrated the levels of typeⅠ, Ⅲ collagens,VEGF,TGF-β1,and MVD were notably decreased (p<0.05),and the expression of bFGF was markedly increased in rhEndostatin-treated(2.5mg/ml and5.0mg/ml) groups conpared with those of normal saline group and model group. Recombinant human endostatin1.25mg/ml did not significantly exert effect either on the levels of type Ⅰ,Ⅲ collagens,VEGF,TGF-β1,bFGF or on MVD.③Effect of rhEndostatin on proliferation and cell cycle of HSFs:Elongated spindle-shape cells could be seen migrating from the tissue pieces7～9d after explanting. After subcultured, the cells were in a fibroblast-like form with long fusiform shape, homogeneous and had good refractive index. The cells stained positive for vimentin. MTT assay showed that rhEndostatin inhibited HSFs proliferation in a concentration-and time-dependent manner, its IC50value toward HSFs was100mg/ml. Compared with the control group, the proliferation of HSFs treated with rhEndostatin at the low-dose (6.25ug/ml) showed no statistical significance at all time points. As co-culture continued and concentration increased, the proliferation-suppressing effect of rhEndostatin increased. After72h of treatment, however, the effect of rhEndostatin on HSFs proliferation decreased. Analysis of the cell cycle by flow cytometry showed significant changes in the distribution of the cell cycle. Compared with normal group and HS group, rhEndostatin induced G0/G1arrest of HSFs, thus increasing the G0/G1population and decreasing the S phase population of HSFs, respectively(P<0.05, p<0.01).Conclusion:Recombinant human endostatin has a markedly inhibitive effect on the formation of hypertrophic scar. And the possible mechanism of action is that rhEndostatin suppresses angiogenesis, reduces the excessive deposition of collagen and the expression of TGF-β1,induces HSFs cell cycle arrested in G1phase, and subsequently inhibits the in vitro proliferation of HSFs.