GM-CSF Regulates Microvascular Permeability During Cutaneous Wound Healing

Background and objectiveWound healing is an intricate progress,including inflammatory,proliferative and remodeling phases.In wound beds,oxygen,nutrition and some bioactive molecules are all transported via mature and functional blood vessels reconstructed by interaction between pericytes and endothelial cells.The blood vessel is lined by endothelial cells,which comprise the primary component of the microvascular permeability barrier.Such barrier is mainly maintained by the junctions between cells,functioned as gatekeepers controlling the passage of macromolecules through vessel wall.Plasma leakage and edema are key features of impaired wound healing,and regulation of vascular permeability has clinical importance during wound healing.It is suggested that granulocyte macrophage colony stimulating factor?GM-CSF?is a growth factor with pleiotropic functions,favoring wound healing.Our previous results also demonstrated that wound healing is significantly compromised in GM-CSF KO mice,accompanied with reduced infiltration of neutrophils and macrophages.Furthermore,insufficient vascularization is observed in the wound beds of GM-CSF KO mice.On the other hand,wound healing is enhanced by exogenous GM-CSF,particularly in diabetes.Therefore,GM-CSF plays a key role during wound healing via promoting angiogenesis in wounds bed.In the preliminary experiment,we found GM-CSF gel reduced the microvascular permeability of wound beds during wound healing.In the support of previous reference,Ang-1 has unequivocal effect on regulating vascular permeability.We proposed our hypothesis that there would be an underlying mechanism,that is“GM-CSF-Ang1derived from pericytesé-regulate vascular permeability in wound healing”.To investigate our hypothesis,the anti-leakage effect of GM-CSF was detected using Evans blue assay and FITC-dextran permeability assay in vivo and in vitro,respectively.The mediation effect of Ang-1 in GM-CSF protecting vascular permeability during wound healing was confirmed by RNAi technology.In addition,the possible signal pathway of GM-CSF elicits Ang-1 was explored.Our research might provide a new insight into the underlying mechanisms of GM-CSF regulating wound healing.Methods1.A full-thickness skin wound with size of 1.0x1.0 cm² was generated on the back of the mouse.Mice were randomly grouped and applied with GM-CSF gel and vehicle gel,respectively.Wound closure was monitored daily by photograph and calculated using imageJ.Evans blue was injected via tail vein to observe the permeability of microvascular barrier in the wound beds and for quantitation analysis.2.Monoculture and co-culture models were using to detect the endothelium permeability in vitro.Firstly,GM-CSF was added into medium of insert for 24h.Followed by VEGF treatment for 10min,as a stimulator of permeability,FITC-dextran of 70 kDa was applied to detect the trans-endothelial permeability in two models,respectively.Additionally,the gaps among endothelial cells were detected using immunofluorescent stain with anti-PECAM antibody followed by GM-CSF protection and VEGF stimulation.The images were captured under fluorescent microscope.3.The wound tissues samples from either GM-CSF gel or mock treated mice on the days 7 post-wounding,were stained with anti-NG2?pericyte marker?conjugated with Alexa Fluor?594 and anti-Ang-1 antibody conjugated with Alexa Fluor?488.Immunofluorescence images were captured.The Ang-1 production,as well as the relationship between pericytes and Ang-1 in the wounds,was detected.In vitro,HBVP were treated with GM-CSF for 3,6,12 or 24 hours,the protein levels of Ang-1 and VEGF in the supernatant of HBVP culture were determined using ELISA kits.4.A specific siRNA sequence targeting Ang-1 was selected using WB and Real time PCR,among four siRNAs corresponding to different coding regions of the Ang-1gene.In FITC-dextran permeability assay,pericytes seeded on the lower chamber of transwell were transfected with siRNA^Ang-1 in co-culture models.In vivo,a mixture of siRNA/PEI/pluronic gel was prepared according to a previous described method.The same full-thickness skin defect wound models were generated as above.The siRNA^Ang-1 gel and the siRNA^NC gel were applied to the wounds every other day up to7d post-injury.To confirm the effect of siRNA in inhibiting Ang-1 expression in vivo,the transfected wounds were harvested for Ang-1 detection using WB on the days 7after Evans blue assay.Furthermore,GM-CSF gel plus siRNA^NC gel or siRNA^Ang-1was applied on the skin defect wounds every other day up to 5d post-wounding,in turn.After Evans blue injection,the blue dyed wounds were photographed and harvested for further quantification analysis as described above.5.HBVP were lysed after GM-CSF or JAK inhibitor?Ruxolitinib?treatment.Aliquots of cell lysates were subjected to 10%SDS-PAGE gel and Western blot with anti-Ang-1,anti-pJAK2 and total JAK antibodies overnight at 4 ^oC.Protein bands were visualized using the enhanced chemiluminescence detection kit.Images were taken by fusion-capture software?Fusion FX7,France?Results1.Treatment of GM-CSF promotes wound repair and decreases skin microvascular permeability in vivo.The wound closure rate of mock treated mice is lower than GM-CSF treated mice.The mock gel group has a darker blue staining than GM-CSF gel ones.2.GM-CSF abolishes VEGF induced endothelium permeability in both monoculture and co-culture models.In monoculture model,GM-CSF reduced the endothelial permeability by 69%,while the reduction in co-culture model was 78%,compared to positive control group in each models,respectively.GM-CSF closed the gaps among endothelial cells under the stimulation of VEGF using immunofluorescence.3.GM-CSF increases Ang-1 levels derived from pericytes in vivo and in vitro.By ELISA,the Ang-1 level was up regulated with the treatment of GM-CSF.The result of RT-PCR is similar to the result of ELISA.4.GM-CSF maintained vascular integrity via Ang-1 derived from pericytes in wound healing.Inhibition of Ang-1 increased the endothelial permeability in endothelial cell/pericyte co-culture model,as well as in the wound beds of full-thickness skin defect models in mice.5.GM-CSF elicited Ang-1 production is JAK2 dependent in pericytes.In WB,pJAK2 was visible after GM-CSF treatment,while the control group had no band. Additionally,JAK2 inhibitor down-regulated Ang-1 level in pericytes.ConclusionGM-CSF reduces microvascular permeability via Ang-1 derived from pericytes.Our investigation of the mechanism that GM-CSF protects wound vascular integrity and stability is a supplement study of wound healing mechanism and has some clinical importance.