Studies On The Effects Of Thymosin β16 In Promoting Angiogenesis And Wound Healing

Theβ-thymosins are a family of highly conserved polar 5 kDa peptides. They are present in almost every cell. Theβ-thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression ofβ-thymosins appear to be related to the differentiation of cells. Among the members of the family, thymosinβ4 is demonstrated to show many physiological effects, such as the induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. Since thymosinβ16 is highly homologous with thymosinβ4, we concentrate our interest on the possible familiar effect of thymosinβ16 with thymosinβ4.PurposeTo evaluate the effects of Tβ16 in promoting angiogenesis and wound healing.MethodsThe peptide thymosinβ16 was synthesized by Chinese Peptide Company.1."scratch"wound closure assayConfluent HUVEC monolayers in 24-well plates were"scratch"wounded using the tip of a universal blue pipette tip and rinsed withD-Hank's. Tβ₁₆ (1μg/ml in DMEM with 10% fetal bovine serum) was add to six wells and the plates were incubated for 72 hours. And DMEM with 10% fetal bovine serum only and bFGF (1μg/ml in DMEM with 10% fetal bovine serum) were applied for control. The width of the wound was observed using a microscope with a 10X objective.2. In vivo migration and angiogenesis assayFertile chicken eggs were obtained, and they were placed inside an incubator, at 373C.On the seventh day of incubation, a `window' (1.5×1.5 cm) was opened on the eggshell exposing the CAM. Tβ₁₆ (50μg/ml in 50μl NS) were add to sterile plastic discs (of 5 mm diameter) and the discs were placed on the CAM area. NS only and aFGF (5μg/ml in 50μl NS) were applied for control. Three eggs were used for each group. The window was covered with sterile cellophane tape and the eggs were returned to the incubator where they were kept until day 9 for observation.3. wound healing assayOne full thickness 3mm diameter punch biopsy wounds were made on the dorsal surface of each mouse. Tβ₁₆ (0.5μg/μl in 50μl PBS) was applied topically on the second day, and again after 48 h to the wounds. At day 7, post-wounding, wound tissue was collected and fixed in formalin. The samples were sectioned, and sections from the middle of the wound were stained with H & E. PBS only and bFGF (0.5μg/μl in 50μl PBS) was applied for control. Histological sections were used to observed the re-epithelialization and the contraction of the wound using a microscope with a 4X objective.4. wound healing following alkali injury assaySix 18mm diameter alkali injury were made on the dorsal surface of each rabbit. Tβ₁₆ (5,25,50 and 100μg/g in 0.5g hydrogel) was applied topically on the second day, and again after 48 h to the wounds. At day 15, post-wounding, wound tissue was collected and fixed in formalin. The samples were sectioned, and sections from the middle of the wound were stained with H & E. Hydrogel only and bFGF (4μg/g in 0.5g hydrogel) was applied for control. Histological sections were used to observed the re-epithelialization and the contraction of the wound using a microscope with a 4X objective.Resullts1. After 72 h the"scratch"wounds in four of the total six wells(Tβ₁₆ 1μg/ml in DMEM with 10% fetal bovine serum) closed completely, none of the six wells(DMEM with 10% fetal bovine serum only) closed and three of the six wells(bFGF 1μg/ml in DMEM with 10% fetal bovine serum) closed completely.2. The difference of the eggs treated with Tβ₁₆ (50μg/ml in 50μl NS) and NS only was significant. The density of blood vessel on the CAM treated with Tβ₁₆ were significantly higher than the three eggs treated with NS only.3. Tβ₁₆ (0.5μg/μl in 50μl PBS) promotes wound closure as observed by the increase in keratinocyte migration from the wound edges. The control mice showed little wound repair with poor formation of granulation tissue.4. At day 15, post-wounding ,all the wounds treated with Tβ₁₆ ( 20,30,50,100μg/g in 0.5g hydrogel) recovered , but the wounds treated with hydrogel only and bFGF ( 4μg/g in 0.5g hydrogel ) recovered incompletely.Conclusion1. Tβ₁₆ promote HUVEC migration in vitro.2. Tβ₁₆ stimulate angiogenesis in the CAM model.3. Tβ₁₆ promote wound healing in C57 mice.4. Tβ₁₆ promote wound healing following alkali injury in rabbits.In conclusion, it is well certified that the Tβ16 play an important role in promoting angiogenesis and wound healing through our works. Although it remains unknown that what mechanism Tβ16 follows, it seems obvious that Tβ16 may undergo such a change of functions from stimulating the migration of HUVECs to promoting the angiogenesis in wound healing. We make it sure that Tβ16 is valuable in clinic therapy in wound healing. And one of the major goals is to search for the molecular mechanisms mediating the effects attributed to extracellular Tβ16.