Ewald Hannappel

beta-Thymosins, small acidic peptides with multiple functions.

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-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 of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohns disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohns disease led to detection of intestinal mTNF+ immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF+ cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF+ cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohns disease and autoimmune or inflammatory disorders.

Thymosin β4 Is an Essential Paracrine Factor of Embryonic Endothelial Progenitor Cell–Mediated Cardioprotection

Background— Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin β4 (Tβ4) as a mediator of eEPC-mediated cardioprotection. Methods and Results— In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tβ4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5×106 cells) or cells transfected with Tβ4 shRNA when indicated or 15 mg Tβ4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32±4% to 90±2% after eEPC application, an effect sensitive to shRNA transfection compared with Tβ4 (45±7%). In vivo, infarct size decreased with eEPC application (38±4% versus 54±4% of area at risk; P<0.01), an effect abolished by Tβ4 shRNA (62±3%). Segmental subendocardial shortening improved after eEPC treatment (22±3% versus −3±4% of control area) unless Tβ4 shRNA was transfected (−6±4%). Retroinfusion of Tβ4 mimicked eEPC application (infarct size, 37±3%; segmental endocardial shortening, 34±7%). Myeloperoxidase activity (3323±388 U/mg in controls) was decreased by eEPCs (1996±546 U/mg) or Tβ4 alone (1455±197 U/mg) but not Tβ4 shRNA–treated eEPCs (5449±829 U/mg). Conclusion— Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tβ4.

The actin binding site on thymosin β4 promotes angiogenesis

Thymosin β4 is a ubiquitous 43 amino acid, 5 kDa polypeptide that is an important mediator of cell proliferation, migration, and differentiation. It is the most abundant member of the β‐thymosin family in mammalian tissue and is regarded as the main G‐actin sequestering peptide. Thymosin β4 is angiogenic and can promote endothelial cell migration and adhesion, tubule formation, aortic ring sprouting, and angiogenesis. It also accelerates wound healing and reduces inflammation when applied in dermal wound‐healing assays. Using naturally occurring thymosin β4, proteolytic fragments, and synthetic peptides, we find that a seven amino acid actin binding motif of thymosin β4 is essential for its angiogenic activity. Migration assays with human umbilical vein endothelial cells and vessel sprouting assays using chick aortic arches show that thymosin β4 and the actin‐binding motif of the peptide display near‐identical activity at ~50 nM, whereas peptides lacking any portion of the actin motif were inactive. Furthermore, adhesion to thymosin β4 was blocked by this seven amino acid peptide demonstrating it as the major thymosin β4 cell binding site on the molecule. The adhesion and sprouting activity of thymosin β4 was inhibited with the addition of 5–50 nM soluble actin. These results demonstrate that the actin binding motif of thymosin β4 is an essential site for its angiogenic activity.

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

Nuclear localisation of the G-actin sequestering peptide thymosin β4

Thymosin β4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin β4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin β4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin β4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

Thymosin β4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen

The β‐thymosins constitute a family of highly conserved and extremely water‐soluble 5 kDa polypeptides. Thymosin β4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G‐actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin β4. For example, thymosin β4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin β4 can be cross‐linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin β4 is not cross‐linked to many other proteins and its cross‐linking to fibrin is competed by another family member, thymosin β10. After activation of human platelets with thrombin, thymosin β4 is released and crosslinked to fibrin in a time‐ and calcium‐dependent manner. We suggest that thymosin β4 cross‐linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin β4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.

The Thymosins: Prothymosin α, Parathymosin, and β-Thymosins: Structure and Function

The studies on thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immuno-deficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide beta 1, thymosin alpha 1, prothymosin alpha, parathymosin, and thymosin beta 4) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the thymosins is discussed in this review.

Thymosin β4: a multi-functional regenerative peptide. Basic properties and clinical applications.

Introduction: Thymosin β4, a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin β4, is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin β4 binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue. Thymosin β4 also decreases the number of myofibroblasts in wounds, resulting in decreased scar formation and fibrosis Areas covered: This article will cover the many thymosin β4 activities that directly affect the repair and regeneration cascade with emphasis on its therapeutic uses and potential. Our approach has been to evaluate the basic biology of the molecule as well as its potential for clinical applications in the skin, eye, heart and brain. Expert opinion: The considerable advances in our understanding of the functional biology and mechanisms of action of thymosin β4 have provided the scientific foundation for ongoing and projected clinical trials in the treatment of dermal wounds, corneal injuries and in the regeneration and repair of heart and CNS tissue following ischemic insults and trauma.

Biosynthesis rates and content of thymosin β4 in cell lines

Abstract The content and relative biosynthetic rates of thymosin β4 have been determined in 28 different cell lines. The highest content of thymosin β4 as well as the highest rate of biosynthesis was observed in Epstein-Barr virus-transformed human B-cell lines. The levels observed in these cells are 1 pg thymosin β4 per cell, which is three times higher than that in rat peritoneal macrophages. Thus, these B-cell lines have the highest content of thymosin β4 of any cell type yet described. Since all of the Epstein-Barr virus-transformed B-cells described here grow in suspension, it is unlikely that the presence of thymosin β4 is related to anchorage in these cells. Thymosin β4 is not secreted by viable Epstein-Barr virus-transformed B cells in culture, suggesting some intracellular function of the peptide. These results indicate that these B-cell lines may be suitable for the study of thymosin β4 function.