Thymosin Beta-4

Engineered Tandem Thymosin Peptide Promotes Corneal Wound Healing.

Joseph Nguyen, Sudhir Verma, Vivian T Vuong +3 more

Thymosin beta-4 (TB4) is a small peptide upregulated in injured tissues, playing roles in cell migration, angiogenesis, inflammation, and oxidative stress. Studies show TB4 significantly promotes corneal wound healing after injury, leading to a clinical trial (RGN-259), with full US Food and Drug Administration approval still pending. Current limitations to TB4-based therapies are a short half-life and high peptide synthesis costs, limiting large-scale applications. Here, we engineered a tandem thymosin beta-4 (tTB4) peptide with improved therapeutic potential and scalability for corneal wound repair.

Tβ4-Engineered ADSC Extracellular Vesicles Rescue Cell Senescence Through Separable Microneedle Patches for Diabetic Wound Healing.

Youjun Ding, Jinglin Wang, Jiaye Li +5 more

Microneedles loaded with bioactive substances have demonstrated efficacy in wound healing, while their application in the elderly chronic wounds, aggravated by cellular senescence, is still a significant challenge. Here, a novel therapeutic strategy is presented utilizing Thymosin β4 (Tβ4)-modified adipose-derived stem cell extracellular vesicles (ADSC-EVs) delivered via separable microneedle patches (MN@EVsTβ4). The therapeutic EVsTβ4 are derived from ADSCs that overexpress Tβ4, a factor that reverses cellular senescence. Leveraging the drug-loading and release properties of gelatin methacryloyl and poly(ethylene glycol) diacrylate, EVsTβ4 are encapsulated within the tips of the microneedles. Notably, the soluble hyaluronic acid base layer dissolves rapidly and separates from the tips upon exudate absorption, enabling a sustained release of EVsTβ4. Subsequently, it is demonstrated its ability to mitigate senescence and improve function via the PTEN/PI3K/AKT pathway. Furthermore, MN@EVsTβ4 patches showed significant efficacy in reversing senescence and promoting wound healing in diabetic wound models. Thus, the engineered ADSC-EVs, combined with separable microneedle patches, represent a promising bioengineering strategy for clinical wound management.

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation.

Alexandra Muntiu, Andrea Papait, Federica Vincenzoni +6 more

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SIGNIFICANCE: In this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

In Vitro Study of Thymosin Beta 4 Promoting Transplanted Fat Survival by Regulating Adipose-Derived Stem Cells.

Wandi Li, Yan Yang, Yan Lin +1 more

Autologous fat grafting (AFG) has emerged as a highly sought-after plastic surgery procedure, although its success has been hampered by the uncertain fat survival rate. Current evidence suggests that adipose-derived stem cells (ADSCs) may contribute to fat retention in AFG. In previous studies, it was confirmed that thymosin beta 4 (Tβ4) could enhance fat survival in vivo, although the precise mechanism remains unclear.

Thymosin beta-4 - A potential tool in healing middle ear lesions in adult mammals.

Peter Bako, Balint Lippai, Jazmin Nagy +4 more

Acute tympanic membrane perforations primarily occur due to injury or infection in humans. In acute cases, nearly 80-94 % of the perforations heal spontaneously. In chronic cases, non-surgical treatment becomes significantly limited, and the perforation can be restored only by myringoplasty. In addition to classical grafts such as the fascia or cartilage, promising results have been reported with various biological materials including silk or acellular collagen. However, despite of all the efforts, healing remains insufficient. Consequentially, a need for substances which actively promote tympanic cell migration and proliferation is deemed essential. In our study, we utilized Thymosin beta-4 (TB4), a 43aa peptide possessing many regenerative properties in various organ systems. Our aim was to reveal the impact of externally administered TB4 regarding impairments of the middle ear, particularly the tympanic membrane. We harvested tympanic membranes from adult mice and treated these with TB4 or PBS on both collagen gel matrixes and in the form of floating, ex vivo explants. Cell migration and proliferation was measured, while immunocytochemical analyses were performed to determine cell type and the nature of the targeted molecules. We discovered the peptide affects the behavior of epidermal and epithelial cells of the tympanic membrane in vitro. Moreover, as our initial results imply, it is not the differentiated, yet most likely the local epidermal progenitor cells which are the primary targets of the molecule. Our present results unveil a new, thus far undiscovered field regarding clinical utilization for TB4 in the future.

A Novel Combination Therapy Tβ4/VIP Protects against Hyperglycemia-Induced Changes in Human Corneal Epithelial Cells.

Abdul Shukkur Ebrahim, Thomas W Carion, Thanzeela Ebrahim +7 more

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tβ4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell-substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tβ4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tβ4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tβ4/VIP. These results strongly support the premise that Tβ4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea.

Thymosin beta-4 participate in antibacterial immunity and wound healing in black tiger shrimp, Penaeus monodon.

Changhong Lin, Lihua Qiu, Pengfei Wang +3 more

Thymosin beta-4 (Tβ4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates, which play fundamental roles in innate immune against pathogens and wound healing. In this study, the full-length cDNA of Tβ4 was cloned from Penaeus monodon (designated as PmTβ4), using the technology of rapid amplification of cDNA ends (RACE). The cDNA of PmTβ4 was 1361 bp with an open reading frame (ORF) of 501 bp, which encoding a polypeptide of 166 amino acid. The Quantitative Real-time PCR (qRT-PCR) analysis results showed that PmTβ4 was ubiquitously expressed in all the tested shrimp tissues, with the highest expression level was detected in the hemolymph, while the lowest expression level in the muscle. The expression level of PmTβ4 was significantly up-regulated in hepatopancreas after challenged by Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In vitro antimicrobial test showed that the recombinant protein of PmTβ4 (rPmTβ4) had broad-spectrum of antimicrobial activity, which could inhibit both the growth of gram-negative bacteria and gram-positive bacteria, including Vibrio vulnificus, V. parahaemolyticus, Streptococcus agalactiae, S. aureus and Aeromonas hydrophila. Moreover, rPmTβ4 had a certain binding ability to different bacteria, and this binding ability exhibits a strong dose-dependent effect. In vivo, PmTβ4 could facilitate external bacterial clearance in shrimp, and have beneficial to shrimp survival post V. parahaemolyticus infection. Furthermore, wound-healing assay was carried out to study the role of PmTβ4 in the process of wound healing. The results showed that the PmTβ4 expression was significantly up-regulated by injury treatment, and exerted positive effects to promote wound healing. In addition, PmTβ4 can significantly increase the expression level of superoxide dismutase (SOD) and Catalase (CAT) after injury treatment in shrimp, which would involve in scavenging reactive oxygen species (ROS) caused by the wound. In conclusion, these results indicated that PmTβ4 may play important roles in antibacterial immunity and wound healing in Penaeus monodon.

Thymosin beta 4: A potential novel adjunct treatment for bacterial keratitis.

Gabriel Sosne, Elizabeth A Berger

Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification or scarring, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts.Pseudomonas aeruginosaandStaphylococcus aureusare the two bacteria most commonly associated with this type of infection. Risk factors include patients who are immunocompromised, those who have undergone refractive corneal surgery, and those with prior penetrating keratoplasty, as well as extended wear contact lens users. Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics. Bacterial clearance is of utmost importance yet does not guarantee good visual outcome. Clinicians are often left to rely upon the eye's innate ability to heal itself, as there are limited options beyond antibiotics and corticosteroids for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, and anti-inflammatory drops, do not fully accommodate clinical needs and have many potential harmful complications. To this end, treatments are needed that both regulate the inflammatory response and promote corneal wound healing to resolve visual disturbances and improve quality of life. Thymosin beta 4 is a small, naturally occurring 43-amino-acid protein that promotes wound healing and reduces corneal inflammation and is currently in Phase 3 human clinical trials for dry eye disease. Our previous work has shown that topical Tβ4 as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages) while enhancing bacterial killing and wound healing pathway activation in an experimental model ofP. aeruginosa-induced keratitis. Adjunctive thymosin beta 4 treatment holds novel therapeutic potential to regulate and, optimally, resolve disease pathogenesis in the cornea and perhaps other infectious and immune-based inflammatory disease. We plan to establish the importance of thymosin beta 4 as a therapeutic agent in conjunction with antibiotics with high impact for immediate clinical development.

Thymosin β4 and Actin: Binding Modes, Biological Functions and Clinical Applications.

Yuyuan Ying, Chen Lin, Nana Tao +4 more

Thymosin β4 (Tβ4) is the β-thymosin (Tβs) with the highest expression level in human cells; it makes up roughly 70-80% of all Tβs in the human body. Combining the mechanism and activity studies of Tβ4 in recent years, we provide an overview of the subtle molecular mechanism, pharmacological action, and clinical applications of Tβ4. As a G-actin isolator, Tβ4 inhibits the polymerization of G-actin by binding to the matching site of G-actin in a 1:1 ratio through conformational and spatial effects. Tβ4 can control the threshold concentration of G-actin in the cytoplasm, influence the balance of depolymerization and polymerization of F-actin (also called Tread Milling of F-actin), and subsequently affect cell's various physiological activities, especially motility, development and differentiation. Based on this, Tβ4 is known to have a wide range of effects, including regulation of inflammation and tumor metastasis, promotion of angiogenesis, wound healing, regeneration of hair follicles, promotion of the development of the nervous system, and improving bone formation and tooth growth. Tβ4 therefore has extensive medicinal applications in many fields, and serves to preserve the kidney, liver, heart, brain, intestine, and other organs, as well as hair loss, skin trauma, cornea repairing, and other conditions. In this review, we focus on the mechanism of action and clinical application of Tβ4 for its main biological functions.

Novel Biotherapeutics Targeting Biomolecular and Cellular Approaches in Diabetic Wound Healing.

Suraj Kumar Singh, Shradha Devi Dwivedi, Krishna Yadav +5 more

Wound healing responses play a major role in chronic inflammation, which affects millions of people around the world. One of the daunting tasks of creating a wound-healing drug is finding equilibrium in the inflammatory cascade. In this study, the molecular and cellular mechanisms to regulate wound healing are explained, and recent research is addressed that demonstrates the molecular and cellular events during diabetic wound healing. Moreover, a range of factors or agents that facilitate wound healing have also been investigated as possible targets for successful treatment. It also summarises the various advances in research findings that have revealed promising molecular targets in the fields of therapy and diagnosis of cellular physiology and pathology of wound healing, such as neuropeptides, substance P, T cell immune response cDNA 7, miRNA, and treprostinil growth factors such as fibroblast growth factor, including thymosin beta 4, and immunomodulators as major therapeutic targets.

Thymosin β4 and the anti-fibrotic switch.

Hynda K Kleinman, Veronika Kulik, Allan L Goldstein

Wound healing involves a rapid response to the injury by circulating cells, followed by inflammation with an influx of inflammatory cells that release various factors. Soon after, cellular proliferation begins to replace the damaged cells and extracellular matrix, and then tissue remodeling restores normal tissue function. Various factors can lead to pathological wound healing when excessive and irreversible connective tissue/extracellular matrix deposition occurs, resulting in fibrosis. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. TGFβ is the most well-characterized macrophage derived pro-fibrotic mediator. Other soluble mediators of fibrosis include connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). Thymosin β4 (Tβ4) has shown therapeutic benefit in preventing fibrosis/scarring in various animal models of fibrosis/scarring. The mechanism of action of Tβ4 appears related, in part, to a reduction in the inflammatory response, including a reduction in macrophage infiltration, decreased levels of TGFβ and IL-10, and reduced CTGF activation, resulting in both prevention of fibroblast conversion to myofibroblasts and production of normally aligned collagen fibers. The amino N-terminal end of Tβ4, SDKP (serine-aspartate-lysine-proline), appears to contain the majority of anti-fibrotic activity and has shown excellent efficacy in many animal models of fibrosis, including liver, lung, heart, and kidney fibrosis. Ac-SDKP not only prevents fibrosis but can reverse fibrosis. Unanswered questions and future directions will be presented with regard to therapeutic uses alone and in combination with already approved drugs for fibrosis.

The Role of Tβ4-POP-Ac-SDKP Axis in Organ Fibrosis.

Wei Wang, Wenning Jia, Chunping Zhang

Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin β4 (Tβ4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tβ4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tβ4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tβ4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tβ4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.

Germinal peptide eye drop promotes corneal epithelial and stromal defect healing in rabbit model.

Lijia Zhou, Jieying Guan, Li Wang +2 more

Corneal defect is a common disease in ophthalmology caused by trauma, inflammation, drug toxicity, or surgery. To investigate the effect of germinal peptide eye drop on corneal epithelial and stromal defects after lamellar keratectomy in rabbit model.

In-silico Screening of Phytoconstituents on Wound Healing Targets - Approaches and Current Status.

Vijaya Mandale, Asha Thomas, Ravindra Wavhale +1 more

Over recent years, there has been tremendous research focused on the effective utilization of natural products in wound management. Natural or herbal products contain several phytoconstituents that may act on various stages in wound healing and thereby provide a multi-targeted approach especially in the treatment of chronic wounds. Currently, attempts have been made to screen the phytoconstituents present in herbs on various targets involved in wound healing. This review includes a systematic evaluation of scientific reports by various groups of researchers on the herbals evaluated for wound management, their phytochemical profiling, pre-clinical studies, and molecular modeling studies. Various wound targets discussed include Interleukin-1, Interleukin-6, Tumor necrosis factor-α (TNF-α), Thymosin beta-4 (Tβ-4) that regulate the early inflammatory stage and the novel T cell immune response cDNA 7(TIRC7) that regulates angiogenesis. Also, neuropeptides P and Y act on the inflammatory, migratory, and proliferation phases, and growth factors like vascular endothelial growth factor family (VEGF) and placental growth factor family (PGF) are involved in angiogenesis, while the role of Fibroblast growth factor in tissue remodeling is discussed. As many of the natural products include polyherbal systems, this approach can help in the judicious selection of a combination of herbs that will act on multiple targets in the wound healing process and provide a multi-factorial approach in wound management.

[Research advances on thymosin β4 in promoting wound healing].

Y X Gao, L F Wang, S J Ba +4 more

With the aging of population and the development of social economy, the incidence of chronic wounds is increasing day by day, while the incidence of burns and trauma remains at a high level, making wound repair an increasingly concerned area in clinical practice. Thymosin β4 is a naturally occurring small molecule protein in vivo, which is widely distributed in a variety of body fluids and cells, especially in platelets. Thymosin β4 has biological activities of promoting angiogenesis, anti-inflammation, anti-apoptosis, and anti-fibrosis, and has many important functions in wound repair. Thymosin β4 has been observed to promote the healing of various wounds, such as burns, diabetic ulcers, pressure ulcers. This paper will review the molecular structure, mechanism of wound healing promotion, pharmacokinetics, and clinical application of thymosin β4, aiming to introduce its potential in wound treatment and the shortcomings of current researches.

TMSB4 Overexpression Enhances the Potency of Marrow Mesenchymal Stromal Cells for Myocardial Repair.

Shiyuan Tang, Chengming Fan, Chukwuemeka Daniel Iroegbu +8 more

The actin-sequestering proteins, thymosin beta-4 (Tβ4) and hypoxia-inducible factor (HIF)-1α, are known to be associated with angiogenesis after myocardial infarction (MI). Herein, we aimed to identify the mechanism of HIF-1α induction by Tβ4 and investigate the effects of bone marrow mesenchymal stromal cells (BMMSCs) transfected with the Tβ4 gene (TMSB4) in a rat model of MI.

An Overview on Functional and Structural Properties of Monomeric and Multimeric β-Thymosin in Invertebrates.

Lili Gao, Mujie Huang, Hongkuan Deng +1 more

β-thymosin 4 (Tβ4) is a prototypical actin-monomer sequestering protein that plays an important role in mammalian cells and tissues. In vertebrates, Tβ4 is involved in various physiological and pathophysiological processes, such as angiogenesis, hair follicle and hair regeneration, nervous system development, inflammatory response, wound healing, tumour metastasis, and liver and heart protection. Additionally, thymosin domain-containing protein was discovered in invertebrates and was recently shown to be more homologous to Tβ4. However, the structural and functional properties are more complex and diverse than those of Tβ4. In this review article, we will discuss in detail the structural and functional aspects of β-thymosin in invertebrates.

A first-in-human, randomized, double-blind, single- and multiple-dose, phase I study of recombinant human thymosin β4 in healthy Chinese volunteers.

Xinghe Wang, Long Liu, Lu Qi +8 more

The study evaluated the safety, tolerability, pharmacokinetics (PK) and anti-drug antibody (ADA) of the recombinant human thymosin β4 (NL005) for single and multiple intravenous injections in healthy subjects. Seven cohorts, with 54 healthy subjects, were given a single intravenous dose of NL005 or placebo and were observed for 28 days. The cohorts received ascending doses of either 0.05, 0.25, 0.5, 2.0, 5.0, 12.5 or 25.0 μg/kg in the single-dose trial. A total of 30 healthy subjects were randomly enrolled in the multiple-dose trial, and 3 cohorts (0.5, 2.0 and 5.0 μg/kg) were administered once human thymosin β4 daily for 10 days and observed for 28 days. The adverse events were mild to moderate in intensity. There were no dose-limiting toxicities or serious adverse events. The plasma concentration, maximum peak concentration (Cmax ) and AUC of each dose group increased with the increase in the dose. The tendency of terminal clearance in each dose group was consistent, and there was no obvious accumulation after continuous administration. Thus, the drug can be concluded to be well tolerated and safe in healthy people and suitable for use in a clinical study for the treatment of acute myocardial infarction.

Protection effect of thymosin β4 on ethanol injury in corneal stromal keratocyte.

Jinghua Liu, Chen Guo, Peng Hao +4 more

To investigate the protective effects of thymosin β4 (Tβ4) on ethanol injured human corneal keratocytes (HCKs).

Adjunctive Thymosin Beta-4 Treatment Influences PMN Effector Cell Function during Pseudomonas aeruginosa-Induced Corneal Infection.

Yuxin Wang, Thomas W Carion, Abdul Shukkur Ebrahim +2 more

Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tβ4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tβ4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tβ4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tβ4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tβ4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tβ4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.

Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis.

Yuxin Wang, Thomas W Carion, Abdul Shukkur Ebrahim +2 more

Our previous work has shown that topical thymosin beta 4 (Tβ4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tβ4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tβ4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tβ4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tβ4 alone and adjunctive Tβ4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tβ4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.

Circular RNA PIP5K1A (circPIP5K1A) accelerates endometriosis progression by regulating the miR-153-3p/Thymosin Beta-4 X-Linked (TMSB4X) pathway.

Lin Sun, Yan Wei, Junli Wang

As a common gynecologic disease, endometriosis (EM) poses a threat to the reproductive health of about 10% women globally. Recent studies have revealed that circular RNAs (circRNAs) are deeply implicated in EM pathogenesis. However, the functions of circPIP5K1A in EM have not been studied yet. Our study intended to uncover the molecular mechanism of circPIP5K1A in EM. In this work, gene and protein expressions were determined by RT-qPCR or Western blotting. CCK-8, wound healing, transwell, and flow cytometry assays were conducted to analyze cell viability, migration, invasion, cell cycle, and apoptosis. Additionally, bioinformatics analysis, dual-luciferase reporter assay, as well as RIP assay were performed to investigate the combination between miR-153-3p and circPIP5K1A or TMSB4X. Herein, we found remarkable high circPIP5K1A expression in EM tissues and cells. Silencing of circPIP5K1A suppressed proliferation, restrained cell cycle, increased cell apoptosis, and decreased migration and invasion in EM cells. In addition, miR-153-3p inhibition could abrogate the impacts of circPIP5K1A knockdown on EM progression in vitro. Also, we found that circPIP5K1A regulated TMSB4X level via interaction with miR-153-3p in EM cells. Besides, circPIP5K1A promoted EM progression via TMSB4X. Moreover, TMSB4X could activate the TGF-β signaling in hEM15A cells. To sum up, our study elucidated that circPIP5K1A accelerated EM progression in vitro by activating the TGF-β signaling pathway via the miR-153-3p/TMSB4X axis, providing a potential clinical target for EM treatment.

Purinergic Signaling Involvement in Thymosin β4-mediated Corneal Epithelial Cell Migration.

Heung-Mo Yang, Shin Wook Kang, Jihye Sung +2 more

Purpose: This study aimed to determine the effect of thymosin beta 4 (Tβ4) on human corneal epithelial cell migration and the downstream signaling pathways. Tβ4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tβ4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tβ4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. Methods: Various concentrations of Tβ4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca2+ concentration after treatment with various concentrations of Tβ4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. Results: Tβ4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca2+ influx, and ERK1/2 phosphorylation. Tβ4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tβ4-mediated human corneal epithelial cell migration. Conclusions: These results suggest that Tβ4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca2+ influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tβ4-mediated corneal healing and regeneration.

Thymosin β4 dynamics during chicken enteroid development.

Mohan Acharya, Rohana Liyanage, Anamika Gupta +4 more

The sheared avian intestinal villus-crypts exhibit high tendency to self-repair and develop enteroids in culture. Presuming that this transition process involves differential biomolecular changes, we employed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) to find whether there were differences in the spectral profiles of sheared villi versus the enteroids, assessed in the mass range of 2-18 kDa. The results showed substantial differences in the intensities of the spectral peaks, one particularly corresponding to the mass of 4963 Da, which was significantly low in the sheared villus-crypts compared with the enteroids. Based on our previous results with other avian tissues and further molecular characterization by LC-ESI-IT-TOF-MS, and multiple reaction monitoring (MRM), the peak was identified to be thymosin β4 (Tβ4), a ubiquitously occurring regulatory peptide implicated in wound healing process. The identity of the peptide was further confirmed by immunohistochemistry which showed it to be present in a very low levels in the sheared villi but replete in the enteroids. Since Tβ4 sequesters G-actin preventing its polymerization to F-actin, we compared the changes in F-actin by its immunohistochemical localization that showed no significant differences between the sheared villi and enteroids. We propose that depletion of Tβ4 likely precedes villous reparation process. The possible mechanism for the differences in Tβ4 profile in relation to the healing of the villus-crypts to developing enteroids is discussed.

Thymosin‑β 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF‑κB pathway.

Shumin Lv, Hongwen Cai, Yifei Xu +3 more

Thymosin‑β 4 (Tβ4) has been reported to exert a pro‑angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tβ4 on critical limb ischemia (CLI). The present study aimed therefore to investigate the mechanisms and pro‑angiogenic effects of Tβ4 in CLI mice. Tβ4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF‑κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis‑related factors angiopoietin‑2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α‑smooth muscle actin (α‑SMA) and the Notch/NF‑κB pathways‑related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF‑κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tβ4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF‑κB, and phosphorylated (p)‑p65 in HUVEC. In addition, Tβ4 promoted the expression of CD31, α‑SMA Ang2, tie2, VEGFA, N1ICD and p‑p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tβ4, whereas Tβ4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tβ4 may promote angiogenesis in CLI mice via regulation of Notch/NF‑κB pathways.

Recombinant Human Thymosin Beta-4 Protects against Mouse Coronavirus Infection.

Rui Yu, Yunyun Mao, Kai Li +11 more

Coronaviruses (CoVs) are enveloped and harbor an unusually large (30-32 kb) positive-strand linear RNA genome. Highly pathogenic coronaviruses cause severe acute respiratory syndrome (SARS) (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome (MERS) (MERS-CoV) in humans. The coronavirus mouse hepatitis virus (MHV) infects mice and serves as an ideal model of viral pathogenesis, mainly because experiments can be conducted using animal-biosafety level-2 (A-BSL2) containment. Human thymosin beta-4 (Tβ4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tβ4 regulates actin polymerization and functions as an anti-inflammatory molecule and an antioxidant as well as a promoter of wound healing and angiogenesis. These activities led us to test whether Tβ4 serves to treat coronavirus infections of humans. To test this possibility, here, we established a BALB/c mouse model of coronavirus infection using mouse CoV MHV-A59 to evaluate the potential protective effect of recombinant human Tβ4 (rhTβ4). Such a system can be employed under A-BSL2 containment instead of A-BSL3 that is required to study coronaviruses infectious for humans. We found that rhTβ4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host's immune response, alleviating pathological damage, and promoting repair of the liver. These results will serve as the basis for further application of rhTβ4 to the treatment of human CoV diseases such as COVID-19.

Multiple functions of thymosin β4 in the pearl oyster Pinctada fucata suggest its multiple potential roles in artificial pearl culture.

Lirong Bai, Wenyao He, Sigang Fan +5 more

Thymosin β4 is a multifunctional protein in vertebrates that participates in physiological processes, such as wound healing, immune response, cell proliferation and migration. We assessed the multifarious roles of this small peptide in Pinctada fucata, an oyster commonly used in pearl culture in China. Our results showed that when P. fucata was challenged by bacterial pathogens or LPS, the relative expression level of Pfthymosin β4 mRNA was significantly up-regulated, suggesting its involvement in immune response of the animal. Recombinant Pfthymosin β4 (rPfthymosin β4) was produced and showed in vitro different antibacterial activities against different pathogenic bacteria; the inhibitory effect of rPfthymosin β4 on bacterial growth was relatively stronger in the broth culture than agar culture. The overexpression of Pfthymosin β4 in Escherichia coli BL21(DE3) cells could improve their resistance to Cu2+, Zn2+, Cd2+, and H2O2, suggesting that Pfthymosin β4 is likely involved with antioxidant. rPfthymosin β4 also significantly promoted the proliferation and migration of mouse aortic vascular smooth muscle cells as indicated by MTT assay and cell scratch assay, respectively. In addition, chemically synthesized or recombinant Pfthymosin β4 could transiently increase the circulating total hemocytes counts but down-regulated by RNAi in P. fucata. Taking together above results and previous studies suggested that Pfthymosin β4 is potentially able to promote wound healing through enhancing antibacterial activity and antioxidant capacity, promotion of cell proliferation and migration, and increase of circulating hemocytes in P. fucata due to nucleus implantation injury. Thus, the future of recombinant Pfthymosin β4 should be promising in the culture of pearls in P. fucata.

Thymosin β4-Enhancing Therapeutic Efficacy of Human Adipose-Derived Stem Cells in Mouse Ischemic Hindlimb Model.

Jong-Ho Kim, I-Rang Lim, Chi-Yeon Park +5 more

Thymosin β4 (Tβ4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tβ4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tβ4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tβ4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tβ4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tβ4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tβ4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tβ4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tβ4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.

Thymosin β4 promotes autophagy and repair via HIF-1α stabilization in chronic granulomatous disease.

Giorgia Renga, Vasilis Oikonomou, Silvia Moretti +12 more

Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin β4 (Tβ4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tβ4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tβ4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tβ4.

Ethosomal Gel for Improving Transdermal Delivery of Thymosin β-4.

Xianglei Fu, Yanbin Shi, Hui Wang +5 more

Thymosin β-4(Tβ-4) is a macromolecular protein drug with potential for drug development in wound repair but is limited by the shortcomings of macromolecular protein, such as large volumes, poor membrane permeability, and unstable physicochemical characteristics. Ethosomes could enhance cell membrane fluidity and reduce epidermal membrane density to make macromolecular drugs through the stratum corneum into the deeper layers of the skin easily. Herein, we developed and characterized a novel transdermal delivery vehicle to load macromolecular protein peptides and use Tβ-4 as a model drug wrapped into ethosomes.