THYMOSIN

Thymosin is a small, actin-sequestering protein belonging to the thymosin-β family that is found at high concentrations within the spleen, thymus, and peritoneal macrophages, where it is most notably responsible for the organization of cytoskeletal structure.

 

Published studies have described a number of physiological properties and cellular functions of thymosin beta4 (Tbeta4), the major G-actin-sequestering molecule in mammalian cells. Those activities include the promotion of cell migration, blood vessel formation, cell survival, stem cell differentiation, the modulation of cytokines, chemokines, and specific proteases, the upregulation of matrix molecules and gene expression, and the downregulation of a major nuclear transcription factor.

 

Such properties have provided the scientific rationale for a number of ongoing and planned dermal, corneal, cardiac clinical trials evaluating the tissue protective, regenerative and repair potential of Tbeta4, and direction for future clinical applications in the treatment of diseases of the central nervous system, lung inflammatory disease, and sepsis. A special emphasis is placed on the development of Tbeta4 in the treatment of patients with ST elevation myocardial infarction in combination with percutaneous coronary intervention.

TB4 appears to accelerate neurogenes is restoration - potential to be combined with Cerebrolysin

Expert Opin Biol Ther. 2015 Jan 22:1-4. [Epub ahead of print]

Thymosin β4 as a restorative/regenerative therapy for neurological injury and neurodegenerative diseases

Chopp M1, Zhang ZG.

Author information

  • 1Henry Ford Hospital, Department of Neurology , 2799 W. Grand Boulevard, Detroit, MI 48202 , USA chopp@neuro.hfh.edu.

Abstract

Thymosin β4 (Tβ4) promotes CNS and peripheral nervous system (PNS) plasticity and neurovascular remodeling leading to neurological recovery in a range of neurological diseases. Treatment of neural injury and neurodegenerative disease 24 h or more post-injury and disease onset with Tβ4 enhances angiogenesis, neurogenesis, neurite and axonal outgrowth, and oligodendrogenesis, and thereby, significantly improves functional and behavioral outcomes. We propose that oligodendrogenesis is a common link by which Tβ4 promotes recovery after neural injury and neurodegenerative disease. The ability to target many diverse restorative processes via multiple molecular pathways that drive oligodendrogenesis and neurovascular remodeling may be mediated by the ability of Tβ4 to alter cellular expression of microRNAs (miRNAs). However, further investigations on the essential role of miRNAs in regulating protein expression and the remarkable exosomal intercellular communication network via exosomes will likely provide insight into mechanisms of action and means to amplify the therapeutic effects of Tβ4.

PLoS One. 2015 Jun 17;10(6):e0130040. doi: 10.1371/journal.pone.0130040. eCollection 2015.

Thymosin Beta-4 Induces Mouse Hair Growth

Gao X1, Liang H1, Hou F1, Zhang Z1, Nuo M1, Guo X1, Liu D1.

Author information

  • 1National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.

Abstract

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting.

The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

Mol Oncol. 2015 Aug;9(7):1484-500. doi: 10.1016/j.molonc.2015.04.005. Epub 2015 Apr 29.

Thymosin-β4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma

Cheung BB1, Tan O2, Koach J2, Liu B2, Shum MS3, Carter DR2, Sutton S2, Po'uha ST2, Chesler L4, Haber M2, Norris MD2, Kavallaris M2, Liu T2, O'Neill GM5, Marshall GM6.

Author information

  • 1Children's Cancer Institute Australia, University of New South Wales, Sydney, Australia. Electronic address: bcheung@ccia.unsw.edu.au.

  • 2Children's Cancer Institute Australia, University of New South Wales, Sydney, Australia.

  • 3Kids Research Institute, Children's Hospital at Westmead, Sydney, Australia.

  • 4Division of Clinical Studies, Institute of Cancer Research, Sutton, Surrey, UK.

  • 5Kids Research Institute, Children's Hospital at Westmead, Sydney, Australia; Discipline of Paediatrics and Child Health, University of Sydney, Australia.

  • 6Children's Cancer Institute Australia, University of New South Wales, Sydney, Australia; Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia. Electronic address: g.marshall@unsw.edu.au.

Abstract

Retinoids are an important component of neuroblastoma therapy at the stage of minimal residual disease, yet 40-50% of patients treated with 13-cis-retinoic acid (13-cis-RA) still relapse, indicating the need for more effective retinoid therapy. Vorinostat, or Suberoylanilide hydroxamic acid (SAHA), is a potent inhibitor of histone deacetylase (HDAC) classes I & II and has antitumor activity in vitro and in vivo. Fenretinide (4-HPR) is a synthetic retinoid which acts on cancer cells through both nuclear retinoid receptor and non-receptor mechanisms. In this study, we found that the combination of 4-HPR + SAHA exhibited potent cytotoxic effects on neuroblastoma cells, much more effective than 13-cis-RA + SAHA. The 4-HPR + SAHA combination induced caspase-dependent apoptosis through activation of caspase 3, reduced colony formation and cell migration in vitro, and tumorigenicity in vivo. The 4-HPR and SAHA combination significantly increased mRNA expression of thymosin-beta-4 (Tβ4) and decreased mRNA expression of retinoic acid receptor α (RARα).

Importantly, the up-regulation of Tβ4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tβ4 knockdown in neuroblastoma cells increased cell migration and blocked the effect of 4-HPR + SAHA on cell migration and focal adhesion formation. In primary human neuroblastoma tumor tissues, low expression of Tβ4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tβ4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

 

 

Oncotarget. 2015 Apr 30;6(12):9820-33.

Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4

Lee JW1, Ryu YK1, Ji YH2, Kang JH3, Moon EY1.

Author information

  • 1Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea.

  • 2Research Center for Radiotherapy, Korea Institute of Radiological and Medical Science, Seoul 139-709, Korea.

  • 3Molecular Imaging Research Center, Korea Institute of Radiological and Medical Science, Seoul 139-709, Korea.

Abstract

Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

Expert Opin Biol Ther. 2015 Jan 22:1-4. [Epub ahead of print]

Thymosin β4 as a restorative/regenerative therapy for neurological injury and neurodegenerative diseases

Chopp M1, Zhang ZG.

Author information

  • 1Henry Ford Hospital, Department of Neurology , 2799 W. Grand Boulevard, Detroit, MI 48202 , USA chopp@neuro.hfh.edu.

Abstract

Thymosin β4 (Tβ4) promotes CNS and peripheral nervous system (PNS) plasticity and neurovascular remodeling leading to neurological recovery in a range of neurological diseases. Treatment of neural injury and neurodegenerative disease 24 h or more post-injury and disease onset with Tβ4 enhances angiogenesis, neurogenesis, neurite and axonal outgrowth, and oligodendrogenesis, and thereby, significantly improves functional and behavioral outcomes. We propose that oligodendrogenesis is a common link by which Tβ4 promotes recovery after neural injury and neurodegenerative disease. The ability to target many diverse restorative processes via multiple molecular pathways that drive oligodendrogenesis and neurovascular remodeling may be mediated by the ability of Tβ4 to alter cellular expression of microRNAs (miRNAs). However, further investigations on the essential role of miRNAs in regulating protein expression and the remarkable exosomal intercellular communication network via exosomes will likely provide insight into mechanisms of action and means to amplify the therapeutic effects of Tβ4.

 

Mol Cell Endocrinol. 2015 Feb 5;401:238-47. doi: 10.1016/j.mce.2014.12.008. Epub 2014 Dec 23.

Effect of thymosin beta 4 in the presence of up-regulation of the insulin-like growth factor-1 signaling pathway on high-glucose-exposed vascular endothelial cells

Kim S1, Kwon J2.

Author information

  • 1Department of Laboratory Animal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-156, Republic of Korea.

  • 2Department of Laboratory Animal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-156, Republic of Korea. Electronic address: jkwon@chonbuk.ac.kr.

Abstract

Thymosin beta 4 (Tβ4), which regulates vascular cell growth, can ameliorate some of the problems associated with diabetes. However, the precise signaling mechanisms by which Tβ4 protects against hyperglycemia-induced damage to endothelial cells have not been investigated in detail. Thus, the aim of this study was to elucidate the role of Tβ4 in diabetes and the possible involvement of insulin-like growth factor-1 (IGF-1), which affects cellular survival, metabolism, and glucose homeostasis in high-glucose (HG)-injured human umbilical vein endothelial cells (HUVECs). Immunoblotting assays revealed that under HG blockade conditions, Tβ4 did not alter the insulin-signaling pathway, but induced overexpression of IGF-1 protein, leading to activation of factors in alternative signaling pathway. Small interfering RNA of Tβ4 and IGF-1 were studied to clarify relationship between Tβ4 and IGF-1. These findings suggest that IGF-1 induction by Tβ4 ameliorates the damage in HG-injured HUVECs which manifest as diabetic vascular disorder.

 

Brain. 2014 Feb;137(Pt 2):433-48. doi: 10.1093/brain/awt333. Epub 2013 Dec 18.

Thymosin β 4 gene silencing decreases stemness and invasiveness in glioblastoma

Wirsching HG1, Krishnan S, Florea AM, Frei K, Krayenbühl N, Hasenbach K, Reifenberger G, Weller M, Tabatabai G.

Author information

  • 11 Department of Neurology, Laboratory of Molecular Neuro-Oncology, University Hospital Zurich, Zurich, Switzerland.

Abstract

Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres.

A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFβ and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.

J Neurol Sci. 2014 Oct 15;345(1-2):61-7. doi: 10.1016/j.jns.2014.07.006. Epub 2014 Jul 10.

A dose-response study of thymosin β4 for the treatment of acute stroke

Morris DC1, Cui Y2, Cheung WL3, Lu M4, Zhang L5, Zhang ZG6, Chopp M7.

Author information

  • 1Department of Emergency Medicine, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: morris@neuro.hfh.edu.

  • 2Department of Neurology, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: yisheng@neuro.hfh.edu.

  • 3Department of Emergency Medicine, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: wcheung1@hfhs.org.

  • 4Department of Public Health Sciences, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: mlu1@hfhs.org.

  • 5Department of Neurology, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: lzhang@neuro.hfh.edu.

  • 6Department of Neurology, Henry Ford Health Systems, Detroit, MI 48202, USA. Electronic address: zhazh@neuro.hfh.edu.

  • 7Department of Neurology, Henry Ford Health Systems, Detroit, MI 48202, USA; Department of Physics, Oakland University, Rochester, MI 48309, USA. Electronic address: michael.chopp@gmail.com.

Abstract

BACKGROUND:

Thymosin β4 (Tβ4) is a 5K actin binding peptide. Tβ4 improves neurological outcome in a rat model of embolic stroke and research is now focused on optimizing its dose for clinical trials. The purpose of this study was to perform a dose-response study of Tβ4 to determine the optimal dose of neurological improvement in a rat model of embolic stroke.

METHODS:

Male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were divided into 4 groups of 10 animals/group: control, 2, 12 and 18mg/kg. Tβ4 was administered intraperitoneally 24h after MCAo and then every 3days for 4 additional doses in a randomized controlled fashion. Neurological tests were performed after MCAo and before treatment and up to 8weeks after treatment. The rats were sacrificed 56days after MCAo and lesion volumes measured. Generalized estimating equation was used to compare the treatment effect on long term functional recovery at day 56. A quartic regression model was used for an optimal dose determination.

RESULTS:

Tβ4 significantly improved neurological outcome at dose of 2 and 12mg/kg at day 14 and extended to day 56 (p-values <0.05). The higher dose of 18mg/kg did not show significant improvement. The estimated optimal dose of 3.75mg/kg would provide optimal neurological improvement.

CONCLUSIONS:

This study shown that Tβ4 significantly improved the long term neurological functional recovery at day 56 after MCAo with an optimal dose of 3.75mg/kg. These results provide preclinical data for human clinical trials.

 

J Orthop Res. 2014 Oct;32(10):1277-82. doi: 10.1002/jor.22686. Epub 2014 Jul 8.

Thymosin β4 administration enhances fracture healing in mice

Brady RD1, Grills BL, Schuijers JA, Ward AR, Tonkin BA, Walsh NC, McDonald SJ.

Author information

  • 1Department of Human Biosciences, La Trobe University, Victoria, Australia.

Abstract

Thymosin β4 (Tβ4 ) is a regenerative peptide that we hypothesized would promote healing of fractured bone. Mice received a bilateral fibular osteotomy and were given i.p. injections of either Tβ4 (6 mg/kg) or saline. Calluses from saline- and Tβ4 -treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro-computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tβ4 -treated calluses had a 41% increase in peak force to failure (p < 0.01) and were approximately 25% stiffer (p < 0.05) than saline-treated controls. µCT analysis at 21 days post-fracture showed that the fractional volume of new mineralized tissue and new highly mineralized tissue were respectively 18% and 26% greater in calluses from Tβ4 -treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post-fracture, Tβ4 -treated calluses were almost 23% smaller (p < 0.05), had nearly 47% less old cortical bone (p < 0.05) and had a 31% increase in new trabecular bone area/total callus area fraction compared with controls (p < 0.05). Our finding of enhanced biomechanical properties of fractures in mice treated with Tβ4 provides novel evidence of the therapeutic potential of this peptide for treating bone fractures.

 

Am J Physiol Heart Circ Physiol. 2014 Sep 1;307(5):H741-51. doi: 10.1152/ajpheart.00129.2014. Epub 2014 Jul 11.

Thymosin-β4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction

Peng H1, Xu J1, Yang XP1, Dai X1, Peterson EL2, Carretero OA1, Rhaleb NE3.

Author information

  • 1Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan;

  • 2Department of Public Health Sciences, Henry Ford Hospital, Detroit, Michigan; and.

  • 3Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan; Department of Physiology, Wayne State University, Detroit, Michigan nrhaleb1@hfhs.org.

Abstract

Thymosin-β4 (Tβ4) promotes cell survival, angiogenesis, and tissue regeneration and reduces inflammation. Cardiac rupture after myocardial infarction (MI) is mainly the consequence of excessive regional inflammation, whereas cardiac dysfunction after MI results from a massive cardiomyocyte loss and cardiac fibrosis. It is possible that Tβ4 reduces the incidence of cardiac rupture post-MI via anti-inflammatory actions and that it decreases adverse cardiac remodeling and improves cardiac function by promoting cardiac cell survival and cardiac repair. C57BL/6 mice were subjected to MI and treated with either vehicle or Tβ4 (1.6 mg·kg(-1)·day(-1) ip via osmotic minipump) for 7 days or 5 wk. Mice were assessed for 1) cardiac remodeling and function by echocardiography; 2) inflammatory cell infiltration, capillary density, myocyte apoptosis, and interstitial collagen fraction histopathologically; 3) gelatinolytic activity by in situ zymography; and 4) expression of ICAM-1 and p53 by immunoblot analysis. Tβ4 reduced cardiac rupture that was associated with a decrease in the numbers of infiltrating inflammatory cells and apoptotic myocytes, a decrease in gelatinolytic activity and ICAM-1 and p53 expression, and an increase in the numbers of CD31-positive cells. Five-week treatment with Tβ4 ameliorated left ventricular dilation, improved cardiac function, markedly reduced interstitial collagen fraction, and increased capillary density. In a murine model of acute MI, Tβ4 not only decreased mortality rate as a result of cardiac rupture but also significantly improved cardiac function after MI. Thus, the use of Tβ4 could be explored as an alternative therapy in preventing cardiac rupture and restoring cardiac function in patients with MI.

 

Neuropharmacology. 2014 Oct;85:408-16. doi: 10.1016/j.neuropharm.2014.06.004. Epub 2014 Jun 14.

Beneficial effects of thymosin β4 on spinal cord injury in the rat

Cheng P1, Kuang F1, Zhang H1, Ju G2, Wang J3.

Author information

  • 1Institute of Neurosciences, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, China.

  • 2Institute of Neurosciences, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, China. Electronic address: jugong@fmmu.edu.cn.

  • 3Institute of Neurosciences, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, China. Electronic address: jwangfm@fmmu.edu.cn.

Abstract

Thymosin β4 (Tβ4) has many physiological functions that are highly relevant to spinal cord injury (SCI), including neuronal survival, anti-inflammation, wound repair promotion, and angiogenesis. The present study investigated the therapeutic value of Tβ4 in SCI, with a focus on its neuroprotective, anti-inflammatory, and vasculoprotective properties. Tβ4 or a saline control was administered by intraperitoneal injection 30 min, 3 days, or 5 days after SCI with mild compression in rat. Locomotor recovery was tested with the Basso-Beattie-Bresnahan scale and a footprint analysis. All behavioral assessments were markedly improved with Tβ4 treatment. Histological examination at 7 days post injury showed that the numbers of surviving neurons and oligodendrocytes were significantly increased in Tβ4-treated animals compared to saline-treated controls. Levels of myelin basic protein, a marker of mature oligodendrocytes, in Tβ4-treated rats were 57.8% greater than those in saline-treated controls. The expression of ED1, a marker of activated microglia/macrophages, was reduced by 36.9% in the Tβ4-treated group compared to that of the saline-treated group. Tβ4 treatment after SCI was also associated with a significant decrease in pro-inflammatory cytokine gene expression and a significant increase in the mRNA levels of IL-10 compared to the control. Moreover, the size of lesion cavity delineated by astrocyte scar in the injured spinal cord was markedly reduced in Tβ4-treated animals compared to saline-treated controls. Given the known safety of Tβ4 in clinical trials and its beneficial effects on SCI recovery, the results of this study suggested that Tβ4 is a good candidate for SCI treatment in humans.

 

J Biol Chem. 2014 Jul 11;289(28):19508-18. doi: 10.1074/jbc.M113.529966. Epub 2014 May 14.

Thymosin β4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway

Santra M1, Zhang ZG1, Yang J2, Santra S1, Santra S1, Chopp M3, Morris DC4.

Author information

  • 1From the Departments of Neurology.

  • 2Public Health Sciences, and.

  • 3From the Departments of Neurology, the Department of Physics, Oakland University, Rochester, Michigan 48309.

  • 4Emergency Medicine, Henry Ford Health Systems, Detroit, Michigan 48202 and morris@neuro.hfh.edu.

Abstract

Thymosin β4 (Tβ4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tβ4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tβ4 on the TLR pathway. Cells were grown in the presence of Tβ4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tβ4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tβ4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tβ4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tβ4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tβ4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.

 

Eur J Histochem. 2014 Mar 11;58(1):2242. doi: 10.4081/ejh.2014.2242.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma

Theunissen W1, Fanni D, Nemolato S, Di Felice E, Cabras T, Gerosa C, Van Eyken P, Messana I, Castagnola M, Faa G.

Author information

  • 1. wesley.theunissen@gmail.com.

Abstract

Thymosin beta 4 (Tβ4) and thymosin beta 10 (Tβ10) are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tβ4 and Tβ10 in hepatocellular carcinoma (HCC). To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tβ4 and Tβ10.

Immunoreactivity for Tβ4 and Tβ10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tβ4 reactivity was detected in 7/23 cases (30%) and Tβ10 reactivity in 22/23 (97%) cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tβ10 showed a strong homogeneous expression, was Tβ4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tβ10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tβ10 in HCC progression.

 

Immunobiology. 2014 Jun;219(6):425-31. doi: 10.1016/j.imbio.2014.02.001. Epub 2014 Feb 14.

Thymosin β4 reduces IL-17-producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice

Conte E1, Iemmolo M2, Fagone E2, Gili E2, Fruciano M2, Genovese T3, Esposito E3, Cuzzocrea S3, Vancheri C2.

Author information

  • 1Department of Clinical and Molecular Biomedicine, University of Catania, Italy. Electronic address: econte@unict.it.

  • 2Department of Clinical and Molecular Biomedicine, University of Catania, Italy.

  • 3Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy.

Abstract

Thymosin β4 (Tβ4) is a highly conserved peptide with immunomodulatory properties. In this research we investigated the effects of Tβ4 on the bleomycin-induced lung damage in CD-1 mice and the changes in the number of IL-17-producing cells as well as the IL-17 expression in the lung. Male CD-1 mice were treated with bleomycin (1mg/kg) in the absence or the presence of Tβ4 (6mg/kg delivered intra-peritoneally on the day of bleomycin treatment and for 2 additional doses). After sacrifice one week later, lung histology, measurement of collagen content of the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, evaluation of IL17-producing cells in the blood as well as RT-PCR and IHC in the lung tissue were performed. As expected, bleomycin-induced inflammation and lung damage were substantially reduced by Tβ4 treatment in CD-1 mice, as shown by the significant reduction of (i) leukocytes in BALF, (ii) histological evidence of the lung damage, and (iii) total collagen content in the lung. Importantly, the bleomycin-induced increase in the number of IL17-producing cells in the blood was significantly blocked by Tβ4. Accordingly, IHC and RT-PCR results demonstrated that Tβ4 substantially inhibited bleomycin-induced IL-17 over-expression in the lung tissue. This is the first report showing that a decreased amount of IL17-producing cells and inhibited IL-17 expression in the lung with Tβ4 treatment correlate with its anti-inflammatory and anti-fibrotic effects.

 

Tuberculosis (Edinb). 2014 May;94(3):323-31. doi: 10.1016/j.tube.2014.01.003. Epub 2014 Feb 4.

Over-expression of thymosin β4 in granulomatous lung tissue with active pulmonary tuberculosis

Kang YJ1, Jo JO1, Ock MS1, Yoo YB2, Chun BK3, Oak CH4, Cha HJ5.

Author information

  • 1Department of Parasitology and Genetics, Kosin University College of Medicine, Busan, Republic of Korea.

  • 2Department of Biomedical Laboratory Science, Konyang University, Daejon, Republic of Korea.

  • 3Department of Pathology, Kosin University College of Medicine, Busan, Republic of Korea.

  • 4Department of Internal Medicine, Kosin University College of Medicine, Busan, Republic of Korea. Electronic address: oaks70@daum.net.

  • 5Department of Parasitology and Genetics, Kosin University College of Medicine, Busan, Republic of Korea; Institute for Medical Science, Kosin University College of Medicine, Busan, Republic of Korea. Electronic address: hcha@kosin.ac.kr.

Abstract

Recent studies have shown that thymosin β4 (Tβ4) stimulates angiogenesis by inducing vascular endothelial growth factor (VEGF) expression and stabilizing hypoxia inducible factor-1α (HIF-1α) protein. Pulmonary tuberculosis (TB), a type of granulomatous disease, is accompanied by intense angiogenesis and VEGF levels have been reported to be elevated in serum or tissue inflamed by pulmonary tuberculosis. We investigated the expression of Tβ4 in granulomatous lung tissues at various stages of active pulmonary tuberculosis, and we also examined the expression patterns of VEGF and HIF-1α to compare their Tβ4 expression patterns in patients' tissues and in the tissue microarray of TB patients. Tβ4 was highly expressed in both granulomas and surrounding lymphocytes in nascent granulomatous lung tissue, but was expressed only surrounding tissues of necrotic or caseous necrotic regions. The expression pattern of HIF-1α was similar to that of Tβ4. VEGF was expressed in both granulomas and blood vessels surrounding granulomas. The expression pattern of VEGF co-localized with CD31 (platelet endothelial cell adhesion molecule, PECAM-1), a blood endothelial cell marker, and partially co-localized with Tβ4. However, the expression of Tβ4 did not co-localize with alveolar macrophages. Stained alveolar macrophages were present surrounding regions of granuloma highly expressing Tβ4. We also analyzed mRNA expression in the sputum of 10 normal and 19 pulmonary TB patients. Expression of Tβ4 was significantly higher in patients with pulmonary tuberculosis than in normal controls. These data suggest that Tβ4 is highly expressed in granulomatous lung tissue with active pulmonary TB and is associated with HIF-1α- and VEGF-mediated inflammation and angiogenesis. Furthermore, the expression of Tβ4 in the sputum of pulmonary tuberculosis patients can be used as a potential marker for diagnosis.

 

Ann N Y Acad Sci. 2012 Oct;1269:110-6. doi: 10.1111/j.1749-6632.2012.06651.x.

Treatment of neurological injury with thymosin β4

Morris DC1, Zhang ZG, Zhang J, Xiong Y, Zhang L, Chopp M.

Author information

  • 1Department of Emergency Medicine, Henry Ford Health Sciences Center, Detroit, Michigan, USA. Dmorris4@hfhs.org

Abstract

Neurorestorative therapy targets multiple types of parenchymal cells in the intact tissue of injured brain tissue to increase neurogenesis, angiogenesis, oligodendrogenesis, and axonal remodeling during recovery from neurological injury. In our laboratory, we tested thymosin β4 (Tβ4) as a neurorestorative agent to treat models of neurological injury. This review discusses our results demonstrating that Tβ4 improves neurological functional outcome in a rat model of embolic stroke, a mouse model of multiple sclerosis, and a rat model of traumatic brain injury. Tβ4 is a pleiotropic peptide exhibiting many actions in several different types of tissues. One mechanism associated with improvement of neurological improvement from Tβ4 treatment is oligodendrogenesis involving the differentiation of oligodendrocyte progenitor cells to mature myelin-secreting oligodendrocytes. Moreover, our preclinical data provide a basis for movement of Tβ4 into clinical trials for treatment of these devastating neurological diseases and injuries.

 

Ann N Y Acad Sci. 2012 Oct;1269:84-91. doi: 10.1111/j.1749-6632.2012.06752.x.

Thymosin β4 and cardiac protection: implication in inflammation and fibrosis

Gupta S1, Kumar S, Sopko N, Qin Y, Wei C, Kim IK.

Author information

  • 1Texas A & M Health Science Center, College of Medicine, Department of Medicine, Scott and White, Central Texas Veterans Health Care System, Temple, Texas, USA. sgupta@medicine.tamhsc.edu

Abstract

Thymosin beta 4 (Tβ4) is a ubiquitous protein with diverse biological functions. The effecter molecules targeted by Tβ4 in cardiac protection remain unknown. We summarize previously published work showing that treatment with Tβ4 in the myocardial infarction setting improves cardiac function by activating Akt phosphorylation, promoting the ILK-Pinch-Parvin complex, and suppressing NF-κB and collagen synthesis. In the presence of Wortmannin, Tβ4 showed minimal cardiac protection. In vitro findings revealed that pretreatment with Tβ4 resulted in reduction of intracellular ROS in the cardiac fibroblasts and was associated with increased expression of antioxidant enzymes, reduction of Bax/Bcl(2) ratio, and attenuation of profibrotic genes. Silencing of Cu/Zn-SOD, catalase, and Bcl(2) genes abrogated the protective effect of Tβ4. Our findings suggest that Tβ4 improves cardiac function by enhancing Akt and ILK activation and suppressing NF-κB activity and collagen synthesis. Furthermore, Tβ4 selectively upregulates catalase, Cu/Zn-SOD, and Bcl(2), thereby protecting cardiac fibroblasts from H(2)O(2) -induced oxidative damage. Further studies are warranted to elucidate the signaling pathway(s) involved in the cardiac protection afforded by Tβ4.

 

Ann N Y Acad Sci. 2012 Oct;1269:16-25. doi: 10.1111/j.1749-6632.2012.06718.x.

Controlled delivery of thymosin β4 for tissue engineering and cardiac regenerative medicine

Chiu LL1, Reis LA, Radisic M.

Author information

  • 1Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada.

Abstract

Thymosin β4 (Tβ4) is a peptide with multiple biological functions. Here, we focus on the role of Tβ4 in vascularization, and review our studies of the controlled delivery of Tβ4 through its incorporation in biomaterials. Tβ4 promotes vascularization through VEGF induction and AcSDKP-induced migration and differentiation of endothelial cells. We developed a collagen-chitosan hydrogel for the controlled release of Tβ4 over 28 days. In vitro, the Tβ4-encapsulated hydrogel increased migration of endothelial cells and tube formation from epicardial explants that were cultivated on top of the hydrogel, compared to Tβ4-free hydrogel and soluble Tβ4 in the culture medium. In vivo, subcutaneously injected Tβ4-containing collagen-chitosan hydrogel in rats led to enhanced vascularization compared to Tβ4-free hydrogel and collagen hydrogel with Tβ4. Furthermore, the injection of the Tβ4-encapsulated hydrogel in the infarct region improved angiogenesis, reduced tissue loss, and retained left ventricular wall thickness after myocardial infarction in rats.

 

Crit Rev Eukaryot Gene Expr. 2012;22(2):109-16.

Thymosin β4: a potential molecular target for tumor therapy

Xiao Y1, Chen Y, Wen J, Yan W, Zhou K, Cai W.

Author information

  • 1Shanghai Institute of Pediatric Research, Shanghai, China.

Abstract

Thymosin β4 (Tβ4), a 5 kDa protein, has been demonstrated to play an important role in a variety of biological activities, such as actin sequestering, cellular motility, migration, inflammation, and damage repair. Recently, several novel findings provided compelling evidence that Tβ4 played a key role in facilitating tumor metastasis and angiogenesis. It has been found that Tβ4 expressed increasingly in a number of metastatic tumors, which was associated with an increased expression of a known angiogenic factor, vascular endothelial growth factor. Thus, Tβ4 provided a potential target of opportunity for cancer management, especially for cancer metastasis therapy.

 

 

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Vitam Horm. 2011;87:277-90. doi: 10.1016/B978-0-12-386015-6.00032-9.

Thymosins and muscle regeneration

Hara T.

Author information

  • Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Abstract

Thymosins are a family of highly conserved small peptides originally isolated from calf thymus. One representative member of the family is thymosin-β₄ (Tβ₄), a major G-actin-sequestering peptide present in many tissues. In the last decade, various studies have uncovered several important functions for Tβ₄ related to the regeneration of injured tissues including skin and heart. In particular, Tβ₄ promotes endothelial cell migration via the activation of Akt2 kinase at the leading edge of the cell. In the case of skeletal muscle injury, increased levels of Tβ₄ are produced by muscle fibers and surrounding immune cells. Satellite cell-derived myoblasts and myocytes are chemoattracted by Tβ₄, which facilitates skeletal muscle regeneration. Recently, it was reported that Tβ₄ interacts physically with F₁-F₀ ATP synthase on the plasma membrane to increase the local concentration of ATP, which stimulates the P2X₄ purinergic receptor to elicit a migratory response from endothelial cells. Thus, it is clear that Tβ₄ is an important chemotactic factor involved in stem/progenitor cell-mediated tissue regeneration.

 

Ann N Y Acad Sci. 2010 Apr;1194:223-9. doi: 10.1111/j.1749-6632.2010.05474.x.

A randomized, placebo-controlled, single and multiple dose study of intravenous thymosin beta4 in healthy volunteers

Ruff D1, Crockford D, Girardi G, Zhang Y.

Author information

Abstract

Synthetic thymosin beta 4 (Tbeta4) may have a potential use in promoting myocardial cell survival during acute myocardial infarction. Four cohorts, with 10 healthy subjects each, were given a single intravenous dose of placebo or synthetic Tbeta4. Cohorts received ascending doses of either 42, 140, 420, or 1260 mg. Following safety review, subjects were given the same dose regimen daily for 14 days. Safety evaluations, incidence of Treatment-Emergent Adverse Events, and pharmacokinetic parameters were evaluated. Adverse events were infrequent, and mild or moderate in intensity. There were no dose limiting toxicities or serious adverse events. Pharmacokinetic profile for single dose showed a dose proportional response, and an increasing half-life with increasing dose. Synthetic Tbeta4 given intravenously as a single dose or in multiple daily doses for 14 days over a dose range of 42-1260 mg was well tolerated with no evidence of dose limiting toxicity. Further development for use in cardiac ischemia should be considered.

 

Ann N Y Acad Sci. 2010 Apr;1194:179-89. doi: 10.1111/j.1749-6632.2010.05492.x.

Thymosin beta4: structure, function, and biological properties supporting current and future clinical applications

Crockford D1, Turjman N, Allan C, Angel J.

Author information

Abstract

Published studies have described a number of physiological properties and cellular functions of thymosin beta4 (Tbeta4), the major G-actin-sequestering molecule in mammalian cells.

  • Those activities include the promotion of cell migration, blood vessel formation, cell survival, stem cell differentiation, the modulation of cytokines, chemokines, and specific proteases, the upregulation of matrix molecules and gene expression, and the downregulation of a major nuclear transcription factor.

 

Such properties have provided the scientific rationale for a number of ongoing and planned dermal, corneal, cardiac clinical trials evaluating the tissue protective, regenerative and repair potential of Tbeta4, and direction for future clinical applications in the treatment of diseases of the central nervous system, lung inflammatory disease, and sepsis. A special emphasis is placed on the development of Tbeta4 in the treatment of patients with ST elevation myocardial infarction in combination with percutaneous coronary intervention.

 

Ann N Y Acad Sci. 2010 Apr;1194:125-9. doi: 10.1111/j.1749-6632.2010.05470.x.

Thymosin beta4 in multiple myeloma: friend or foe

Caers J1, Otjacques E, Hose D, Klein B, Vanderkerken K.

Author information

  • 1Department of Hematology, CHU University of Liège, Liège, Belgium.

Abstract

Multiple myeloma (MM) is a malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow (BM). Because of the known involvement of thymosin beta4 (Tbeta4) in metastasis of tumor cells, we examined the expression and role of Tbeta4 in MM disease. In a large patient population, we demonstrated that Tbeta4 expression was significantly lower in myeloma cells compared to normal plasma cells and that patients with a high Tbeta4 expression had a longer event free and overall survival. The decreased Tbeta4 expression was also found in the murine 5TMM model. To study its function, we overexpressed the Tbeta4 gene in 5T33MMvt cells by lentiviral transduction. These cells demonstrated a decreased proliferative capability and an increased sensitivity to apoptosis. Mice injected with Tbeta4-overexpressing cells showed a prolonged survival compared to mice injected with controls. In contrast to its role in solid tumors, we found a decreased expression in myeloma cells compared to their normal counterpart and studies with overexpression of the Tbeta4 gene indicated a tumor suppressive function of Tbeta4 in myeloma development.

 

Ann N Y Acad Sci. 2010 Apr;1194:81-6. doi: 10.1111/j.1749-6632.2010.05479.x.

Animal studies with thymosin beta, a multifunctional tissue repair and regeneration peptide

Philp D1, Kleinman HK.

Author information

  • 1NIH, NIDCR, Bethesda, Maryland, USA.

Abstract

Studies in various animal models of disease and repair with thymosin beta(4) (Tbeta(4)), the major actin-sequestering molecule in mammalian cells, have provided the scientific foundation for the ongoing dermal, corneal, and cardiac wound repair multicenter clinical trials. Tbeta(4) has of multiple biological activities, which include down-regulation of inflammatory chemokines and cytokines, and promotion of cell migration, blood vessel formation, cell survival, and stem cell maturation. All of these activities contribute to the multiple wound healing properties that have been observed in animal studies. This paper reviews and discusses the topical and systemic uses of Tbeta(4) in various animal models that demonstrate its potential for clinical use.

 

Neuroreport. 2005 Oct 17;16(15):1629-33.

Expression of thymosin beta4 mRNA by activated microglia in the denervated hippocampus

Dong JH1, Ying GX, Liu X, Wang WY, Wang Y, Ni ZM, Zhou CF.

Author information

  • 1Key Laboratory of Neurobiology, Shanghai Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences.

Abstract

Thymosin beta4 is a major actin-sequestering molecule. Here, we report a prominent upregulation of thymosin beta4 in the hippocampus following entorhinal deafferentation. Northern blotting displayed a transient increase of thymosin beta4 mRNA in the deafferented hippocampus by 1.8, 2.3, 1.3 and 1.1-fold of controls, respectively, at 1, 3, 7 and 15 days post-lesion. In-situ hybridization confirmed that the induction of thymosin beta4 mRNA specifically occurred in the entorhinally denervated zones of the hippocampus. The double labeling of in-situ hybridization for thymosin beta4 mRNA with isolectin B4 cytochemistry showed that isolectin B4-positive microglial cells are responsible for deafferentation-induced thymosin beta4 mRNA expression. The results suggest that thymosin beta4 may participate in the process of microglial activation, which is the earliest event in lesion-induced plasticity.

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