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February 2002

Thymosin beta 4

Chemical characterization of thymosin beta 4.

As part of our ongoing investigations on the endocrine thymus, we have isolated and purified to homogeneity a hormone-like peptide which we have termed thymosin beta 4. Thymosin beta 4 has Mr = 4982 and an isoelectric point of 5.1. The complete amino acid sequence of this polypeptide has been established by automated Edman degradation as well as by manual sequence analysis. Thymosin beta 4 is composed of 43 amino acid residues with acetylserine at the NH2 terminus. This molecule induces expression of terminal deoxynucleotidyl transferase in transferase-negative murine thymocytes in vivo and in vitro. It also exhibits ability to inhibit the migration of macrophages. Comparison of the sequence of thymosin beta 4 to other thymic hormones or other published protein sequences does not reveal any statistically significant relationship. Two helical regions were identified in the structure using data for prediction of protein conformation. It is proposed that thymosin beta 4 is one of the biologically active peptides present in thymosin fractions 5 and 5A which participate in the regulation, differentiation, and function of thymus-derived lymphocytes and may also act directly or indirectly on macrophages and perhaps other cells involved in cell-mediated immunity.

Biol Chem 1982 Jan 25;257(2):1000-6

Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids.

The possibility that glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes. Here we show that thymosin beta 4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response. In vitro, thymosin beta 4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw. Thymosin beta 4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of anti-inflammatory therapy.

Nat Med 1999 Dec;5(12):1424-7

Thymosin beta 4 accelerates wound healing.

Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta 4 (T beta 4) enhanced wound healing in a rat full thickness wound model. Addition of T beta 4 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 T beta 4 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 T beta 4 was added to the assay. These results suggest that T beta 4 is a potent wound healing factor with multiple activities that may be useful in the clinic.

J Invest Dermatol 1999 Sep;113(3):364-8

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells.

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.

FASEB J 1997 May;11(6):474-81

Thymosin beta 4 (T beta 4) in activated platelets.

When resting human blood platelets are stimulated with thrombin, 50 to 60% of the G-actin polymerizes to F-actin within 60 seconds. The increase in F-actin is correlated with a corresponding decrease in the complex of G-actin with T beta 4. Within 5 seconds after stimulation, nucleation sites for pyrene actin polymerization increase 1.5 times in Triton-lysed supernatants. Cytochalasin D, known to inhibit the increase in F-actin after thrombin, also inhibits the fall in T beta 4-actin complex and the increase in nucleation sites. Phosphorylation of T beta 4 could not be detected in either control or activated cells. Increased T beta 4 corresponding to that lost from the T beta 4-actin complex is present in lysates from activated platelets and retains the ability to complex with actin. The data, taken together with previous estimates for the dissociation constant of the T beta 4-actin complex, show that actin polymerization following platelet activation could be controlled primarily by the increased availability of free barbed ends of actin filaments which have a higher affinity for G-actin than does T beta 4 and suggest that the increased free T beta 4 may serve to limit the degree of polymerization.

Eur J Cell Biol 1993 Aug;61(2):314-20

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

Proc Natl Acad Sci U S A 1992 May 15;89(10):4678-82

The modern approach to wound treatment.

INTRODUCTION: Wound healing is a complex process involving interactions among a variety of different cell types. The normal wound repair process consists of three phases—inflammation, proliferation and remodeling that occur in a predictable series of cellular and biochemical events. Wounds are classified according to various criteria: etiology, lasting, morphological characteristics, communications with solid or hollow organs, the degree of contamination. In the last few years many authors use the Color Code Concept, which classifies wounds as red, yellow and black wounds. This paper presents conventional methods of local wound treatment (mechanical cleansing, disinfection with antiseptic solutions, wound debridement—surgical, biological and autolytic; wound closure, topical antibiotic treatment, dressing), as well as general measures (sedation, antitetanous and antibiotic protection, preoperative evaluation and correction of malnutrition, vasoconstriction, hyperglycemia and steroid use, appropriate surgical technique, and postoperative prevention of vasoconstriction through pain relief, warming and adequate volume resuscitation). The role of physiological factors and antimicrobial agents in wound healing. Growth factors play a role in cell division, migration, differentiation, protein expression, enzyme production and have a potential ability to heal wounds by stimulating angiogenesis and cellular proliferation, affecting the production and the degradation of the extracellular matrix, and by being chemotactic for inflammatory cells and fibroblasts. There are seven major families of growth factors: epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), interleukins (ILs) and colony-stimulating factor (CSF). Acute wounds contain many growth factors that play a crucial role in the initial phases of wound healing. The events of early wound healing reflect a finely balanced environment leading to uncomplicated and rapid wound healing. Chronic wounds, for many reasons, have lost this fine balance. Multiple studies have evaluated the effect that exogenously applied growth factors have on the healing of chronic wounds. In the study conducted by Knighton and colleagues, topical application of mixture of various growth factors (PDGF, TGF-beta, PDAF, PF4, PDEGF) demonstrated increased wound healing over controls. Brown and associates demonstrated a decrease in skin graft donor site healing time of 1 day using topically applied EGF. Herndon and ass. used systemic growth hormone in burned children and reduction in healing time made a significant clinical difference by allowing earlier wound coverage and decreasing the duration of hospitalization. The TGF family of growth factors is believed to be primarily responsible for excessive scar formation, especially the beta 1 and beta 2 isoforms. TGF-beta 3 isoform has recently been described and may have an inhibitory function on scar formation by being a natural antagonist to the TGF-beta 1 and TGF-beta 2 isoforms. Cytokines, especially interferon-alpha (INF-alpha), INF-alpha, and INF-alpha 2b, may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts and reduce the rate of collagen and fibronectin synthesis by reducing the production of mRNA. Expression of nitric oxide synthase (NOS) and heat shock proteins (HSP) have an important role in wound healing, as well as trace elements (zinc, copper, manganese). Applications of some drugs (antioxidants—asiaticoside, vitamin E and ascorbic acid; calcium D-pantothenate, exogenous fibronectin; antileprosy drugs—oil of hydnocarpus; alcoholic extract of yeast) accelerate wound healing. Thymic peptide thymosin beta 4 (T beta 4R) topically applicated, increases collagen deposition and angiogenesis and stimulates keratinocyte migration. Thymosin alpha 1 (T alpha 1R), peptide isolated from the thymus, is a potent chemoattractant which accelerates angiogenesis and wound healing. On the contrary, steroid drugs, hemorrhage and denervation of wounds have negative effect on the healing process.

Med Pregl 2000 Jul-Aug;53(7-8):363-8

Thymosin beta 4 is a shared antigen between lymphoid cells and oligodendrocytes of normal human brain.

In the normal human brain, immunoreactive thymosin beta 4, a well-characterized thymic extract, was demonstrated specifically in the cell bodies and processes of a subset of interfascicular and satellite oligodendrocytes with their stained processes terminating around myelin sheaths. Antisera directed against two other thymic polypeptides, thymosin alpha 1 and alpha 7, did not react. In lymphoid tissues, thymosin beta 4 was present in macrophages, Langerhans’ cells of the skin, and the interdigitating cells of the thymus. Thus, a subset of oligodendrocytes shares a common antigen of thymic origin with the reticular-dendritic and phagocytic lymphoid cells—all Ia+ immunocompetent cells that participate in the presentation of antigens to T cells. The subset of thymosin beta 4-positive oligodendrocytes is antigenically distinct and may play a role in the immune surveillance of the central nervous system or the demyelinating processes induced by antigen-presenting activated macrophages.

Ann Neurol 1986 Apr;19(4):349-55

Biochemical and antibacterial analysis of human wound and blister fluid.

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11,1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

Eur J Biochem 1996 Apr 1;237(1):86-92