Major Improvement In Wound Healing Through Pharmacologic Mobilization Of Stem Cells In Severely Diabetic Rats

ObjectiveCurrent therapeutic strategies for diabetic foot ulcer(DFU)have focused on developing topical healing agents,but few agents have controlled prospective data to support their effectiveness in promoting wound healing.We tested a stem cell mobilizing therapy for DFU using a combination of AMD3100 and low-dose FK506(Tacrolimus)(AF Therapy or AF)in streptozocin-induced type 1 diabetic(T1DM)rats and type 2 diabetic Goto-Kakizaki(GK)rats that had developed peripheral artery disease and neuropathy.MethodsIn this study,we used streptozotocin(STZ)to induce type 1 diabetic rats,and also used type 2 non-obese spontaneous diabetic Goto-Kakizaki(GK)rats.With severe diabetic complications,we treated rats with AF,and test the effect of drugs on healing and repair of diabetic wounds in these two models,respectively.Started from the day of the wounding,it was administered subcutaneously every other day until complete healing.The outlined methods are listed below:1.Inducing type 1 diabetic rat models with intraperitoneal injection of streptozotocin(STZ)into SD rats,creating back wound and foot wound models,observing healing time,calculating scar size,and evaluating healing quality.2.Using the diabetic characteristics of spontaneous type 2 diabetes in Goto-Kakizaki(GK)rats,we created a back wound model,observed the healing time,calculated the size of the scar,and evaluated the quality of healing.3.Collecting wound samples at early time point and analyzing changes in the number and structure of stem cells and neovascularization in granulation tissue.4.By using flow cytometry to separate the number of bone marrow-derived cells in peripheral blood before and after AF administration to determine the mobilization of stem cells or progenitor cells.5.Using immunohistochemical staining and immunofluorescence staining to observe the distribution of Ym1~+/Ym2~+type 2 macrophages in the wound granulation tissue.6.Western blot was used to detect the expression of cytokines expression of VEGFA and HGFαthat affect healing in granulation tissue.7.Using bone marrow transplantation technology,transplant bone marrow from GFP~+rat to non-GFP~+rats and then inducing type 1 diabetes.Observed the relationship between the distribution of CD133~+cells and bone marrow-derived cells in early phase of granulation tissue formation.8.By observing the effects of long-term administration on the temperature of hind limbs of GK rats and the extent of lesions in peripheral blood vessels,determining the effect of AF treatment on improving the microcirculation of diabetic rats.9.Investigating the mechanism of BMP pathway’s effect on the wound healing in AF treatment plan in rats by using new synthetic compound FKVP.Results1.We found that the healing time of the back skin wounds of type 1 diabetic rats was shortened from 27 days to 19 days,and that of the diabetic foot and back skin wounds was shortened from 25 days to 20 days.2.In the GK rat model,we also saw very similar accelerated healing phenomena.The back-wound healing time of GK rats in the treatment group was shortened from26 days to 21 days.This treatment not only accelerated the wound healing time,but also reduced the area of scar formation and promoted the regeneration of hair follicles in the scar.3.In the early wound granulation tissue,we saw that AF drugs recruited more CD133~+cells,CD34~+cells and SDF-1α~+cells,and also saw more RECA-1~+neovascularization andαSMA~+neovascularization.4.The AF treatment plan mobilized bone marrow-derived stem cells into the blood,especially CD133~+CD31~+and CD34~+stem cells or progenitor cells released into the blood.5.Ym1~+/Ym2~+2 type macrophages in early granulation tissue increased significantly.Some of these cells also co-expressed SDF-1α.6.The expression of VEGFA and HGFαin early granulation tissue was significantly increased.7.In the bone marrow transplantation diabetic models,in the early granulation tissue,a large number of bone marrow-derived GFP~+cells(without staining)could be seen colocalized with CD133~+cells.8.The temperature of hind limbs of GK rats after long-term AF treatment was significantly higher than that of the control group.PAS staining results showed that the severity of microvascular disease was significantly better than that of the control group.9.FK506 or its non-immunosuppressive analog FKVP activated BMPR1 kinase through FKBP12,activated the BMP pathway,and promoted wound healing.Conclusion1.AF combination therapy restored healing times back towards normal after full-thickness skin excision in type 1 diabetic rats.2.AF combination therapy prevented a delay of wound healing in type 2 diabetic Goto-Kakizaki(GK)rats with peripheral neuropathy and vascular disease.3.AF combination therapy mobilized monocytes and cells bearing stem cell markers for CD34 and CD133 in peripheral blood and increased SDF-1αexpressing Ym1~+/Ym2~+M2 macrophages and CD133~+and CD34~+stem cells in granulation tissues.4.Recruitment of stem cells into the wound sites increased expression of VEGF-A and HGFαand promoted angiogenesis.5.The bone marrow transplantation model demonstrated the critical role of bone marrow-derived stem cells in improving diabetic wound healing by AF combination therapy.6.AF combination therapy ameliorated diabetic angiopathy of the hind limbs.7.BMP pathway is an indispensable important pathway in AF treatment scheme to promote healing,not with immunosuppressive effect.This study provides a new systemic treatment plan for clinical treatment of DFU.