Experimental Study On The Effect Of Endogenous Vascular Endothelial Growth Factor In Flap Surgical Delay

Perforator flap transplantation is the most commonly used surgical method to repair skin and soft tissue defects caused by resection of various tumors(breast tumors,head and neck tumors,and melanoma),trauma,various chronic diseases(diabetic foot,acne)and congenital malformations.Although with the development of microsurgical technique and the improvement of the anatomical knowledge,the necrosis rate of perforator flap transplantation has been greatly reduced,the distal part of the perforator flap necrosis is still an important problem of the plastic surgery.Flap necrosis not only brings heavy psychological pressure to patients,but also increases hospital period and financial burden.Current studies indicate that inadequate blood supply and ischemia-reperfusion injury are major factors in the necrosis of skin flap,so it is particularly important to seek solutions to these problems.Flap surgical delay is the longest and clinically proven to be effective in improving the flap survival.Although its mechanism is still not entirely clear,many cytokines such as vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF),platelet-derived growth factor(PDGF),and transformation Growth factor-β(TGF-β)and placeta growth factor(PIGF)have been proved to be effective in improving flap survival.VEGF is a powerful angiogenic cell growth factor,which plays an important role in normal physiological and pathological conditions.Its effects are mainly promote endothelial cell proliferation or apoptosis,promote inflammation and increased vascular permeability,etc.At present,most of the studies on VEGF’s ability to improve flap survival have been studied by injecting exogenous VEGF into the flap model.The role of endogenous VEGF produced during flap surgical delay is still unclear.Axitinib is an effective and selective inhibitor of VEGFR tyrosine kinase,which can effectively inhibit the production of endogenous VEGF.It has been proved to inhibit the effect of various substantial tumors,but it has not yet been reported in the flap.We use Axitinib as an inhibitor of endogenous VEGF during the flap surgical delay,and investigate the biological effect of endogenous VEGF in flap surgical delay by inhibiting the expression of VEGF by Axitinib.This study will be elaborated from the following three parts.Part 1: the construction of the multi-vessel perforator flap modelIn this part of the study,we first observed the living habits and skin flap condition of rats after the establishment of the rat skin flap model in vitro,which was the basis of this study.The results showed that in the 6 days delay group,the skin flap was normal and soft,and the capillary reaction was good,The rat flaps in the control group and the 3 days delay group had different degrees of necrosis.Furthermore,we observed the change of choke vessels area after the surgical delay by using infrared camera imaging,an result that TD-PIC choke vessels and PIC-DCI choke vessels of white hot spots were significantly increased in the skin flap model of rats on the sixth day,suggests that surgical delay can change the choke vessels of blood vessels into direct connection.To confirm that the surgical delay can effectively improve flap survival ability,we observed the survival area of the flap after surgical delay in a preliminary experiment.It was found that the area of the flap necrosis was large in the control group and the survival area of the flap was significantly higher in the 6 days delay group than the control group.The results found that flap survival area increased significantly in the 6-day surgical delay.In summary,the results of this study show that surgical delay can improve flap survival by altering the vascular status of the choke area.In addition,the optimal delay for the flap model used in this experiment may be 6 days.Since the change of vessels in the choke vessels area may be related to the generation of endogenous VEGF,in the second part of the study the production of endogenous VEGF during surgical delay was inhibited,and the effectiveness and inhibition rate of Axitinib were studied.Part 2: Effect of Axitinib on the inhibition of VEGF in the surgical delayIn this part of the study,we first determined the optimal dose of Axitinib in inhibiting VEGF.The study found that the injection dose of Axitinib was 10mg/kg,2times/day,which could achieve the optimal inhibiting effect.We further examined the expression of VEGF and its signaling molecules in the choke vessels of flaps after surgical delay and Axitinib inhibition was detected by western blot.The results showed that the expression of VEGF was the largest in the 3-day delay,and the expression of VEGF was significantly decreased after Axitinib.We further studied the phosphorylation of vascular endothelial growth factor receptor(phosphorylatedvascular endothelial growth factor receptor,p-VEGFR)expression,and the results showed that the expression of p-VEGFR was also the largest in the 3-day delay,and the expression of p-VEGFR decreased significantly after Axitinib.The above results indicated that that Axitinib could inhibit the production of endogenous VEGF by inhibiting the expression of p-VEGFR.To further verify the effectiveness of our inhibition,we analyzed the downstream signal of VEGF in cells.The results showed that the surgical delay can activate the VEGF-mediated Akt-mTOR-HIF-1α signaling pathway,the cell proliferation,apoptosis pathway of PI3K-Akt and the eNOS/Akt signaling pathway,and these signaling pathways were significantly inhibited by Axitinib.We used ELISA to check the inhibition rate of endogenous VEGF,and the results showed that the inhibition rate was as high as 91.6%.To sum up,the results of this study showed that the optimal dose of Axitinib inhibiting VEGF was 10mg/kg.Surgical delay can effectively increase the expression of endogenous VEGF in the flap and activate its downstream signaling pathway.Axitinib can inhibit the production of endogenous VEGF by inhibiting the phosphorylation of VEGFR,and the VEGF-mediated Akt-mTOR-HIF-1α signaling pathway,PI3K-Akt cell proliferation pathway,and eNOS/Akt signaling pathway were also effectively suppressed.The 91.6% inhibition rate of VEGF indicates the effectiveness of Axitinib inhibition.In the next part of the study,we will observe and investigate the effect of endogenous VEGF produced by Axitinib in suppressing surgical delay on the molecular level of vessels in the choke vessels of the flap,and thus confirm the role of endogenous VEGF in delayed flap surgery.Part 3: The role of endogenous VEGF in the surgical delayIn the first two parts of the study,we observed and discussed the optimal delay time of the skin flap model,the optimal inhibition time of Axitinib and the effectiveness of inhibition.In this part of the study,we first discussed the role of VEGF in the surgical delay in the molecular level.The results showed that endogenous VEGF can improve the vasodilatation of choke vessels in the flap surgical delay.Although the 3 days delay produced the vasodilatation of choke vessels,the effect reached the maximum in the 6 days delay.We detected the microvascular density of choke vessels area using the anti-CD31 antibody,the results showed that endogenous VEGF can improve the microvascular generation of choke vessels area in the flap surgical delay,this effect is not obvious in the 3 days delay.The capillary effect reached the maximum in the 6 days delay.Further,we compared the flap survival area in the delay group and the inhibition group.The survival area of the flap was significantly higher in the 6-day delay group than in the 6-day inhibition group.When the surgical delay was 6 days,the surviving area of the flap was as high as 94.6%.This indicates that endogenous VEGF can effectively improve the flap survival after the surgical delay.In summary,this study shows that endogenous VEGF not only has the effect of promoting vasodilation in the choke vessels area but also has the effect of promoting angiogenesis in the choke vessels area during the surgical delay,and can effectively improve the the flap survival.