The Mechanism Of A Mitochondria-targeted Hypochlorous Acid Probe Inducing Human Dermal Fibroblasts To Differentiate Into Vascular Endothelial Cells

BACKGROUND AND TARGETThe skin is the largest organ in the human body,accounting for about 16%of body weight.When the skin tissue is traumatized,the fibroblasts proliferate,divide and migrate to the damaged site,which produce extracellular matrix,form scar tissue,and fill in the wound cut.At this time,the granulation tissue gradually becomes fibrotic,forming dense fibrous connective tissue.Then the capillaries are closed,degenerated,and disappear.The formation of scar tissue not only affects people’s normal and mental life,but also affects the normal physiological functions of the skin.Dermal fibroblasts are the loose connective tissues’ main cellular components,which are differentiated from embryonic mesenchymal cells.Dermal fibroblasts have strong division and proliferation ability,which can promote the proliferation and maturation of epidermal cells,and regulate epidermal cells.It plays an important role in morphogenesis and dermal reorganization.Endogenous hypochlorous acid regulates signal transduction and cell fate by modifying proteins,and the imbalance of endogenous hypochlorous acid to protein modification level will cause various diseases.At the study’s beginning,we screened the hypochlorous acid probes with different organelle targeting in dermal fibroblasts.We found a mitochondria-targeted hypochlorous acid probe CPP which has the ability to induce dermal fibroblasts to differentiate into vascular endothelial cells.While,hypoxia-inducible factor 1-alpha(HIF-1α)is a transcriptionally active nuclear protein that regulates the expression of a series of target genes and is involved in the regulation of vascular endothelial cell survival,proliferation and migration.Therefore,in this study,we intend to use the small molecule compound CPP as the main molecular tool to explore the molecular mechanism of endogenous mitochondrial hypochlorous acid-induced differentiation of dermal fibroblasts into vascular endothelial cells.This study provides new clues for promoting skin angiogenesis and remodeling,and lays the foundation for the development of new drug lead compounds that promote skin angiogenesis.RESARCH CONTENT1.The morphological identification of the compound CPP inducing differentiation of dermal fibroblasts into angiogenesis.2.To detect changes in vascular endothelial cells and dermal fibroblast-related biomarkers levels to identify that compound CPP is capable of inducing differentiation of dermal fibroblasts into vascular endothelial cells.3.To detect changes in levels of all pro-angiogenic factors after dermal fibroblasts are treated with compound CPP.4.To explore the molecular mechanism of up-regulation of pro-angiogenic factors in dermal fibroblasts induced by compound CPP.5.To explore the molecular mechanism of compound CPP-induced up-regulation levels of HIF-1α in dermal fibroblasts.6.To explore the molecular mechanism of translocation of HIF-1α protein from nucleus to mitochondria mediated by compound CPP.7.To investigate whether compound CPP regulates cell differentiation by promoting the expression of mitochondrial function related genes.METHODS1.Observe cell morphology with inverted phase contrast microscopy.2.Using SRB assay for cell viability.3.Matrigel test is applied to detect angiogenesis in vitro.4.Detect the co-localization of related proteins with point scanning confocal microscopy.5.Using point scan confocal microscopy to detect the levels of vascular endothelial cells and dermal fibroblast-associated markers.6.Western blot is used to analyzethe levels of vascular endothelial cells and dermal fibroblast-associated markers.7.Western blot is used toanalyze the level of downstream related pro-angiogenic factors in dermal fibroblasts.8.Western blot analysis of the HIF-la protein level in dermal fibroblasts is adopted.9.RT-PCR is used to detect mRNA levels of HIF-1α and downstream pro-angiogenic factors.EXPERIMENTAL RESULTS1.Compound CPP co-localizes with dermal fibroblast mitochondria.2.CPP induces human dermal fibroblasts into blood vessels.3.CPP induces the differentiation of dermal fibroblasts to vascular endothelial cells.4.CPP induces the expression of pro-angiogenic factors in dermal fibroblasts.5.CPP induces the expression of HIF-1α protein in dermal fibroblasts in a concentration-and time-dependent manner.