No abstract

Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound healing in a rat full thickness wound model. Addition of Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61% at 7 d post-wounding. Treated wounds also contracted at least 11% more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds. We also found that Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay. These results suggest that Tbeta4 is a potent wound healing factor with multiple activities that may be useful in the clinic.

Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.

The amino acid sequence of thymosinj,84, a polypeptide isolated from calf thymus, was determined. Thymosin 184 is composed of 43 amino acid residues and has a molecular weight of 4982 and an isoelectric point of 5.

In recent years, investigations involving thymic ablation and replacement by thymic grafts or extracts in several species have suggested a central role for the thymus gland in the maturation, proliferation, and immunological competence of the lymphocyte.1 Two major types of observations imply that the influence of the thymus is in part endocrine. The first is the in vivo effect of thymic extracts on lymphopoiesis, lymphocytosis, and immunological competence;2-ll the second is the effect of thymus grafts, enclosed in cell-impermeable Millipore diffusion chambers, on thymectomized animals.12-'4 Previous attempts to isolate and purify "thymic hormones" have been hampered by the lack of a satisfactory, rapid assay method. In two earlier publications, Klein, Goldstein, and White10' 11 have described the preparation of thymic extracts and an in vivo method for testing their biological activity. In this communication, we wish to report the preparation and partial purification from calf thymic tissue of a product which stimulates incorporation of H3-thymidine into mesenteric lymph node cells. The lymphocytopoietic factor, which we term thymosin, is active when administered in vivo, as well as when added directly to a lymphocyte suspension incubated in vitro. This in vitro activity has permitted the development of a new assay procedure for thymosin and has facilitated purification studies.Materials.-Animals: Used for the in vitro and in vivo experiments were 29-day-old male Swiss Webster CD1 mice, 8-10-week-old male Sprague-Dawley rats, and 8-10-week-old male New Zealand white rabbits. All animals received food and water ad libitum until sacrificed.Radioactive precursors: H3-thymidine (3.0 c/mmole) and H3-deoxycytidine (2.4 c/mmole) were purchased from Schwarz BioResearch, Inc. H3-uridine (1.8 c/mmole), C'4-lysine (222 mc/mmole), C'4-leucine (223 mc/mmole), and CL4-phenylalanine (333 mc/mmole) were purchased from New England Nuclear. Chemicals and reagents: Trypsin (2 X crystallized, salt-free), soybean trypsin inhibitor (5 X crystallized), RNase (5 X crystallized), bovine serum albumin, and the nucleosides used were all purchased from Nutritional Biochemicals Corp. Phytohemagglutinin in a powdered form was obtained from the Burroughs Wellcome Co. Pooled calf serum (sterile and filtered) was purchased from the Pentex Corp. Eagle's medium for spinner cultures (MEM, without phosphate and calcium) was purchased in powdered form from General Biochemical Corp. Phosphate was added to the powder during preparation of the medium. Bio-Gel P-10 (50-150 mesh) was purchased from Bio-Rad Laboratories.All other chemicals used in this study were of analytical or reagent grade and were used without further purification.Fractionation procedure: Figure 1 presents a diagram of the fractionation procedure. Fresh or frozen calf thymus, obtained from a local abattoir, was cleaned, defatted, and homogenized in 0.15 M NaCl (tissue: saline = 1:3) at 0-50C in a Waring Blendor. All isolation procedures were carried out in the cold. The homogenate w...