The Mechanism Of Hypochlorous Acid Probe CPP In Regulating The Differentiation Of Human Dermal Fibroblasts Into Vascular Endothelial Cells

BACKGROUND AND OBJECTIVEVascular endothelial cells(VECs)play a crucial role in regulating vascular permeability and tension,as well as maintaining the transport logistics of the body.Damage to VECs can result in various diseases.As a result,current research is focused on promoting VECs regeneration and function,and repairing damage in regenerative medicine.However,the limited sources of adult stem cells and endothelial progenitor cells have hindered the clinical application of these methods.Human dermal fibroblasts(HDFs)are abundant in the body.Studies have demonstrated that HDFs possess multi-directional differentiation potential.Therefore,current research is exploring the induction of HDFs to differentiate into VECs,thereby restoring tissue blood supply and treating related diseases.Reprogramming technology and direct reprogramming have been widely used to induce other adult cells to differentiate into VECs.However,research has shown that reprogramming has serious cell lethality and tumorigenicity,limiting its clinical application.As a result,small chemical molecules have become an alternative research method for inducing HDFs to differentiate into VECs due to their cost-effectiveness and fast effects.Therefore,finding small chemical molecules that can efficiently induce HDFs to differentiate into VECs is urgently needed.Currently,the mechanism of differentiation of HDFs to VEC is unclear.Endogenous HOCl can regulate signal pathways by oxidatively modifying biomacromolecules,thereby playing a role in cell growth,proliferation,apoptosis,and other life processes.However,whether HOCl plays a role in modulating HDFs differentiation into VECs remains unclear.Previous studies have demonstrated that the HOCl probe ZBM-H regulates autophagy by binding HOCl.Therefore,the HOCl probe CPP((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3carbonyl)piperazin-1-yl)styryl)-1 methylpyridin-1-ium iodide)was used as a tool to investigate the mechanism of HDFs differentiation into VECs.This study demonstrates that the HOCl probe CPP can induce the differentiation of HDFs into VECs with certain functions.The mechanism of HDFs differentiation into VECs was investigated using CPP as a tool,which showed that CPP bound HOCl to inhibit the activity of PHD2 through the regulation of Cys302.This resulted in the increased level of HIF-1α that efficiently induced the expression of HEY1 and the differentiation of HDFs into VECs.In summary,this study not only discovered a HOCl probe,CPP,that can induce HDFs to differentiate into VECs but also utilized CPP as a tool to investigate the mechanism of HDFs differentiation into VECs.From the perspective of chemical biology,the study uncovered a new signaling pathway that regulates the differentiation of HDFs into VECs using a small chemical molecule CPP.This provides experimental evidence for the development of new technologies and lead compounds for repairing vascular function,laying the foundation for the development of new strategies for treating various ischemic diseases in the future.STUDY CONTENTS1.HOCI probe CPP induced HDFs to differentiate into VEC.1.1 Identification of CPP-induced differentiation of HDFs into VEC.1.2 To study the function of VEC derived from HDFs.2.Mechanism of CPP-induced differentiation of HDFs into VEC.2.1 To study the role of HIF-1α in the differentiation of HDFs into VEC.2.2 To study the role of PHD2 in the differentiation of HDFs into VEC.2.3 To study the role of HEY1 in the differentiation of HDFs into VEC.3.Application of VEC derived from CPP-treated HDFs in the treatment of hindlimb ischemia in BALB/C nude mice.3.1 Establishment of hindlimb ischemia model in BALB/C nude mice.3.2 To study the therapeutic effect of CPP-induced HDFs on hindlimb ischemia model in BALB/C nude mice.METHODS1.RT-qPCR was used to detect the expression of related genes.2.Flow cytometry was used to determine the differentiation efficiency of CPP-induced HDFs into VEC.3.Dil-ac-LDL uptake assay was used to detect the function of VEC derived from CPP-treated HDFs.4.the chicken embryo allantoic membrane model(CAM)proved that the VEC derived from CPP-treated HDFs could participate in the contribution to vessel formation.5.Western blot was used to detect the level of related proteins.6.human PHD2 ELISA detection kit was used to test the activity of PHD2.7.Constructing related plasmid to clarify the site of the CPP on PHD2.8.RNA-sequence assay was used to investigate the entry point of CPP to induce HDFs to differentiate into VEC after increasing the protein level of HIF-1α.9.Inhibitor and RNA interference were used to detect the function of specific proteins.10.The number of Vimentin-positive cells expressing HEY1,CD31,and CDH5 were detected after injection of CPP in mice.11.Laser Doppler imaging,H&E staining,and FITC-BSL1 were used to detect the therapeutic effect of CPP-induced HDFs on hindlimb ischemia model in BALB/C nude mice.RESULTS1.Identification of CPP-induced differentiation of HDFs into VEC.1.1 CPP promoted the expression of CD133 and CD31,reduced the level of Vimentin.1.2 CPP promoted the expression of VEC genes,ERG,vWF,KDR,TEK,and CDH5.1.3 CPP upregulated the protein levels of KDR and CDH5 in HDFs.2.Function of VEC derived from CPP-induced HDFs.2.1 CPP promoted the expression of pro-angiogenic factors VEGF,FGF-2,and PDGF-BB.2.2 Approximately 51.9%of CPP treated-HDFs took up Dil-ac-LDL.2.3 CAM indicated the contribution of VEC derived from HDFs to vessel formation in vivo.3.To study the role of HIF-la in the differentiation of HDFs into VEC.3.1 CPP induced the differentiation of HDFs into VEC by elevation of HIF-1α.3.2 CPP failed to induce HDFs to differentiate into VEC in the presence of the HIF-1α inhibitor LW6.3.3 CPP co-localized with PHD2,not HIF-1α.4.To study the role of PHD2 in the differentiation of HDFs into VEC.4.1 CPP inhibited the activity of PHD2.4.2 Exogenous HOCl could significantly inhibit the function of CPP,so that CPP couldn’t inhibit the enzyme activity of PHD2,and up-regulate the protein level of HIF-1α.4.3 CPP inhibited the activity of PHD2 to increase the level of HIF-la through the modulation of PHD2 at cysteine 302 by HOCl in HDFs,which resulted in the differentiation of HDFs into VEC.5.To study the role of HEY1 in the differentiation of HDFs into VEC.5.1 We analyzed the results of RNA-seq and found that CPP promoted the expression of HEY1,the target gene of HIF-1α.5.2 RT-qPCR and western blot to verify the expression of HEY 1.53 We knockdown the mRNA of HEY1 and found that CPP failed to induce HDFs to differentiate into VEC.6.The model of hindlimb ischemia in BALB/C nude mice was established successfully.7.To study the therapeutic effect of CPP-induced HDFs on hindlimb ischemia model in BALB/C nude mice.7.1 CPP-treated HDFs significantly enhanced blood perfusion.7.2 CPP-treated HDFs significantly promoted morphological recovery of the hindlimb ischemia model in BALB/C nude mice.7.3 CPP-treated HDFs reduced fibrosis of gastrocnemius at the ischemic site.7.4 CPP-treated HDFs significantly promoted the capillary density at the ischemic site.CONCLUSIONS1.CPP-induced HDFs could differentiate into functional VEC.2.CPP induced the differentiation of HDFs into VEC by the increase of HIF-1α.3.CPP inhibited the enzyme activity of PHD2 by inhibiting the oxidation of PHD2 at Cys302.4.CPP induced the differentiation of HDFs into VEC by promoting the expression of HEY1,a key transcription factor of VEC.5.The transplantation of HDFs-derived VEC enhanced recovery from hindlimb ischemia and increased the number of capillaries.