TB-500

Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions.

Therapeutic peptides are emerging as promising adjuncts in the management of orthopaedic injuries, grounded in their ability to modulate molecular signaling networks central to cellular medicine. By acting on key pathways such as PI3K/Akt, mTOR, MAPK, TGF-β, and AMPK, peptides exert influence over tissue regeneration, inflammation resolution, and neuromuscular recovery. Wound-healing peptides such as BPC-157, TB-500, and GHK-Cu promote angiogenesis, integrin-mediated extracellular matrix remodeling, and fibroblast activation, whereas growth hormone secretagogues like ipamorelin, CJC-1295, tesamorelin, sermorelin, and AOD-9604 activate IGF-1 signaling and satellite cell repair. Recovery-enhancing agents such as epithalon, delta sleep-inducing peptide, and pinealon target circadian and mitochondrial regulators, and neuroactive peptides like selank, semax, and dihexa enhance brain-derived neurotrophic factor and HGF/c-Met pathways critical to neuroplasticity. Although preclinical studies are promising, there is a current lack of clinical trials. This review integrates current mechanistic insights with orthopaedic relevance, emphasizing safety, efficacy, and future directions for responsible integration into musculoskeletal care.

Thymosin beta 4 as an Alzheimer disease intervention target identified using human brain organoids.

The developmental origin of Alzheimer disease (AD) has been proposed but is arguably debated. Here, we developed cerebral organoids from induced pluripotent stem cells (iPSCs) with mutations in amyloid precursor protein (APP) associated with familial AD (fAD) and analyzed the dynamic changes of cellular states. We found that mature neurons induced in fAD organoids markedly decreased compared to that of health control, accompanied with increased cell senescence and β-amyloid (Aβ) production. Interestingly, the expression level of the gene TMSB4X that encodes thymosin beta 4 (Tβ4) significantly decreased both in fAD organoids' neurons and AD patients' excitatory neurons. Remarkably, the neurodevelopmental deficits and Aβ formation in fAD organoids were rescued by treatment with Tβ4. The beneficial effects of Tβ4 were also revealed in 5xfAD model mice. Thus, this study has identified Tβ4 as a neuroprotective factor that may mitigate altered neurogenesis and AD pathology, highlighting a potential for disease intervention.

Identification of glutamine as a potential therapeutic target in dry eye disease.

Dry eye disease (DED) is a prevalent inflammatory condition significantly impacting quality of life, yet lacks effective pharmacological therapies. Herein, we proposed a novel approach to modulate the inflammation through metabolic remodeling, thus promoting dry eye recovery. Our study demonstrated that co-treatment with mesenchymal stem cells (MSCs) and thymosin beta-4 (Tβ4) yielded the best therapeutic outcome against dry eye, surpassing monotherapy outcomes. In situ metabolomics through matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) revealed increased glutamine levels in cornea following MSC + Tβ4 combined therapy. Inhibition of glutamine reversed the anti-inflammatory, anti-apoptotic, and homeostasis-preserving effects observed with combined therapy, highlighting the critical role of glutamine in dry eye therapy. Clinical cases and rodent model showed elevated expression of glutaminase (GLS1), an upstream enzyme in glutamine metabolism, following dry eye injury. Mechanistic studies indicated that overexpression and inhibition of GLS1 counteracted and enhanced, respectively, the anti-inflammatory effects of combined therapy, underscoring GLS1's pivotal role in regulating glutamine metabolism. Furthermore, single-cell sequencing revealed a distinct subset of pro-inflammatory and pro-fibrotic corneal epithelial cells in the dry eye model, while glutamine treatment downregulated those subclusters, thereby reducing their inflammatory cytokine secretion. In summary, glutamine effectively ameliorated inflammation and the occurrence of apoptosis by downregulating the pro-inflammatory and pro-fibrotic corneal epithelial cells subclusters and the related IκBα/NF-κB signaling. The present study suggests that glutamine metabolism plays a critical, previously unrecognized role in DED and proposes an attractive strategy to enhance glutamine metabolism by inhibiting the enzyme GLS1 and thus alleviating inflammation-driven DED progression.

Safety and Efficacy of Approved and Unapproved Peptide Therapies for Musculoskeletal Injuries and Athletic Performance.

Peptides are short chains of amino acids with a unique pharmacological niche between small-molecule drugs and large proteins. Their use in sports medicine is rapidly expanding, driven by patient demand for accelerated injury recovery and performance enhancement. While numerous peptide drugs have undergone a rigorous approval process that evaluates both safety and efficacy, a parallel "gray market" of unapproved compounds has emerged, operating largely outside of regulatory oversight. Our objective is to present the pharmacological mechanisms, safety profiles, and regulatory status of prominent approved and unapproved peptides marketed direct to patients, including AOD-9604 (anti-obesity drug 9604), BPC-157 (body protection compound 157), CJC-1295, FS-344 (follistatin-344), GHK-Cu (glycyl-L-histidyl-L-lysine copper), ipamorelin, MOTS-C (mitochondrial ORF of the 12S rRNA type-c), sermorelin, SS-31 (elamipretide), tesamorelin (Egrifta), Tβ4 (thymosin beta-4), and TB-500 (thymosin beta-4 fragment). Many unapproved peptides demonstrate favorable tissue repair and metabolic outcomes in animal models, but rigorous human safety data are scarce, and there is potential for serious harm to patients. This narrative review focuses on the utilization of peptides in sports medicine, and alternative treatments that may be considered. We provide a framework to navigate patient discussions about peptides to better facilitate evidence-based practices for musculoskeletal healing and athletic performance. We also discuss the placebo effect as a mediator of peptide efficacy, and how social media amplifies this effect.

Cardioprotection by Thymosin Beta 4.

Treatment with thymosin beta 4 (Tβ4) reduces infarct volume and preserves cardiac function in preclinical models of cardiac ischemic injury. These effects stem in part from decreased infarct size, but additional benefits are likely due to specific antifibrotic and proangiogenic activities. Injected or transgenic Tβ4 increase blood vessel growth in large and small animal models, consistent with Tβ4 converting hibernating myocardium to an actively contractile state following ischemia. Tβ4 and its degradation products have antifibrotic effects in in vitro assays and in animal models of fibrosis not related to cardiac injury. This large number of pleiotropic effects results from Tβ4's many interactions with cellular signaling pathways, particularly indirect regulation of cellular motility and movement via the SRF-MRTF-G-actin transcriptional pathway. Variation in effects and effect sizes in animal models may potentially be due to variable distribution of Tβ4. Preclinical studies of PK/PD relationships and a reliable pharmacodynamic biomarker would facilitate clinical development of Tβ4.

Tβ4-exosome-loaded hemostatic and antibacterial hydrogel to improve vascular regeneration and modulate macrophage polarization for diabetic wound treatment.

Diabetic wounds often exhibit delayed healing due to compromised vascular function and intensified inflammation. In this study, we overexpressed Thymosin β4 (Tβ4) in Adipose-Derived Stem Cells (ADSCs) to produce Exosomes (Exos) rich in Tβ4. We then utilized a dual photopolymerizable hydrogel composed of Hyaluronic Acid Methacryloyl (HAMA) and Poly-L-lysine Methacryloyl (PLMA) for the sustained release of Tβ4-Exos on diabetic wounds. The results showed that Tβ4-Exos could stimulate angiogenesis and collagen synthesis, and mitigate inflammation in diabetic wounds by promoting the polarization of M1-type macrophages and inhibiting that of M2-type macrophages. Furthermore, Tβ4-Exos was found to activate the PI3K/AKT/mTOR/HIF-1a signaling pathway, thereby enhancing vascular proliferation. In summary, the sustained release of Tβ4-Exos in HAMA-PLMA (HP) hydrogel and the management of inflammation through the upregulation of the HIF-1a pathway and modulation of macrophage polarization in vascular proliferation significantly accelerated the healing process of diabetic wounds.

Thymosin β4 Promotes Dermal Healing.

No agent has been identified that significantly accelerates the repair of chronic dermal wounds in humans. Thymosin beta 4 (Tβ4) is a small, abundant, naturally occurring regenerative protein that is found in body fluids and inside cells. It was found to have angiogenic and antiinflammatory activity and to be high in platelets that aggregate at the wound site. Thus we used Tβ4 initially in dermal healing. It has since been shown to have many activities important in tissue protection, repair, and regeneration. Tβ4 increases the rate of dermal healing in various preclinical animal models, including diabetic and aged animals, and is active for burns as well. Tβ4 also accelerated the rate of repair in phase 2 trials with patients having pressure ulcers, stasis ulcers, and epidermolysis bullosa wounds. It is safe and well tolerated and will likely have additional uses in the skin and in injured organs for tissue repair and regeneration.

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

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.

Thymosin β4 and β10 Expression in Human Organs during Development: A Review.

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin β4 and thymosin β10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin β4 and thymosin β10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of β-thymosins in health and disease. The functions of β-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "β-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tβ4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tβ4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

Potential role of thymosin Beta 4 in liver fibrosis.

Liver fibrosis, the main characteristic of chronic liver diseases, is strongly associated with the activation of hepatic stellate cells (HSCs), which are responsible for extracellular matrix production. As such, investigating the effective regulators controlling HSC activation provides important clues for developing therapeutics to inhibit liver fibrosis. Thymosin beta 4 (Tβ4), a major actin-sequestering protein, is known to be involved in various cellular responses. A growing body of evidence suggests that Tβ4 has a potential role in the pathogenesis of liver fibrosis and that it is especially associated with the activation of HSCs. However, it remains unclear whether Tβ4 promotes or suppresses the activation of HSCs. Herein, we review the potential role of Tβ4 in liver fibrosis by describing the effects of exogenous and endogenous Tβ4, and we discuss the possible signaling pathway regulated by Tβ4. Exogenous Tβ4 reduces liver fibrosis by inhibiting the proliferation and migration of HSCs. Tβ4 is expressed endogenously in the activated HSCs, but this endogenous Tβ4 displays opposite effects in HSC activation, either as an activator or an inhibitor. Although the role of Tβ4 has not been established, it is apparent that Tβ4 influences HSC activation, suggesting that Tβ4 is a potential therapeutic target for treating liver diseases.

Thymosin beta 4-like peptides.

Thymosin beta 4 and the eye: the journey from bench to bedside.

Thymosin beta 4 (Tβ4) has important applications in ocular repair and Phase 3 clinical trials using Tβ4 to treat dry eye and neurotrophic keratopathy are currently ongoing. These exciting clinical possibilities for Tβ4 in the eye are the result of seminal basic scientific discoveries and contributions from so many talented investigators. Areas covered: My personal Tβ4 journey began at the NIH in 1998 and propelled my career as a clinician scientist. As a tribute to the amazing individuals who have guided and supported me along with my brilliant colleagues and students who have contributed and collaborated with me over the years, this review will tell the cumulative story of how Tβ4 became a major potential new therapy for corneal wound healing disorders. The journey has been marked by the thrilling exhilaration from fundamental breakthroughs in the laboratory and clinic, combined with the challenging and often harsh realities of submitting grants and obtaining funding. Expert opinion: The electrifying possibility of Tβ4 as a revolutionary novel dry eye therapy is something that could have only been dreamed about just a few years ago. We believe that Tβ4 eyedrops will help many patients suffering from several ocular surface related disorders.

The role of thymosin-β4 in kidney disease.

Therapies that modulate inflammation and fibrosis have the potential to reduce the morbidity and mortality associated with chronic kidney disease (CKD). A promising avenue may be manipulating thymosin-β4, a naturally occurring peptide, which is the major G-actin sequestering protein in mammalian cells and a regulator of inflammation and fibrosis. Thymosin-β4 is already being tested in clinical trials for heart disease and wound healing. This editorial outlines the evidence that thymosin-β4 may also have therapeutic benefit in CKD.

Thymosin Beta 4 Is a Potential Regulator of Hepatic Stellate Cells.

Liver fibrosis, a major characteristic of chronic liver disease, is inappropriate tissue remodeling caused by prolonged parenchymal cell injury and inflammation. During liver injury, hepatic stellate cells (HSCs) undergo transdifferentiation from quiescent HSCs into activated HSCs, which promote the deposition of extracellular matrix proteins, leading to liver fibrosis. Thymosin beta 4 (Tβ4), a major actin-sequestering protein, is the most abundant member of the highly conserved β-thymosin family and controls cell morphogenesis and motility by regulating the dynamics of the actin cytoskeleton. Tβ4 is known to be involved in various cellular responses, including antiinflammation, wound healing, angiogenesis, and cancer progression. Emerging evidence suggests that Tβ4 is expressed in the liver; however, its biological roles are poorly understood. Herein, we introduce liver fibrogenesis and recent findings regarding the function of Tβ4 in various tissues and discuss the potential role of Tβ4 in liver fibrosis with a special focus on the effects of exogenous and endogenous Tβ4. Recent studies have revealed that activated HSCs express Tβ4 in vivo and in vitro. Treatment with the exogenous Tβ4 peptide inhibits the proliferation and migration of activated HSCs and reduces liver fibrosis, indicating it has an antifibrotic action. Meanwhile, the endogenously expressed Tβ4 in activated HSCs is shown to promote HSCs activation. Although the role of Tβ4 has not been elucidated, it is apparent that Tβ4 is associated with HSC activation. Therefore, understanding the potential roles and regulatory mechanisms of Tβ4 in liver fibrosis may provide a novel treatment for patients.

CCN5 suppresses injury-induced vascular restenosis by inhibiting smooth muscle cell proliferation and facilitating endothelial repair via thymosin β4 and Cd9 pathway.

Members of the CCN matricellular protein family are crucial in various biological processes. This study aimed to characterize vascular cell-specific effects of CCN5 on neointimal formation and its role in preventing in-stent restenosis (ISR) after percutaneous coronary intervention (PCI). Stent-implanted porcine coronary artery RNA-seq and mouse injury-induced femoral artery neointima single-cell RNA sequencing were performed. Plasma CCN5 levels were measured by enzyme-linked immunosorbent assay. Endothelial cell (EC)- and vascular smooth muscle cell (VSMC)-specific CCN5 loss-of-function and gain-of-function mice were generated. Mass spectrometry and co-immunoprecipitation were conducted to identify CCN5 interacting proteins. Additionally, CCN5 recombinant protein (CCN5rp)-coated stents were deployed to evaluate its anti-ISR effects in a porcine model. Plasma CCN5 levels were significantly reduced and correlated closely with the degree of restenosis in ISR patients. CCN5 expression was significantly decreased in VSMCs of stent-implanted porcine coronary segments and injured mouse femoral arteries, especially in synthetic VSMCs. In contrast, elevated CCN5 expression was observed in regenerating ECs of injured vessels. Endothelial cell- and VSMC-specific CCN5 deletion mice exhibited exacerbation of injury-induced neointimal hyperplasia, while CCN5 gain-of-function alleviated neointimal formation. Mechanistic studies identified thymosin β4 (Tβ4) as a CCN5 interacting protein in ECs and EC-CCN5 promoted injury repair through Tβ4 cleavage product Ac-SDKP. Also, CCN5rp promoted EC repair to suppress neointimal hyperplasia via interaction with Cd9 extracellular domain. Moreover, implantation with CCN5rp-coated stent significantly increased stent strut coverage with ECs, which suppressed neointimal formation and ultimately alleviated ISR. CCN5 exerts a dual protective effect on ISR by inhibiting VSMC proliferation and facilitating EC repair. CCN5rp-coated stent might be promising in the prevention of ISR after PCI.

Thymosin β4-derived peptides alleviate neuroinflammation and neurite atrophy in both in vitro models and in vivo 5 × FAD mice: A potential therapy for memory improvement in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder defined by neuroinflammation, neurite atrophy, and cognitive decline. This study explored the therapeutic potential of Thymosin β4 (Tβ4)-derived peptides (TB500 and Ac-SDKP) in mitigating AD-related neuropathology. Using the 5 × FAD mouse model and established in vitro AD cell systems, we evaluated the neuroprotective and anti-inflammatory effects of these peptides. In Aβ25-35-treated HT22 cells and primary cortical neurons, TB500 and Ac-SDKP significantly attenuated neurite atrophy, restored cell viability, and modulated the expression of apoptosis-related genes. In BV2 microglia assays, the peptides exhibited robust anti-inflammatory effects, as shown by suppressing lipopolysaccharide (LPS)-induced nitric oxide (NO) production, reducing expression of pro-inflammatory cytokines, and inhibiting M1 microglial polarization. In 5 × FAD mice, TB500 and Ac-SDKP ameliorated cognitive impairments, as evidenced by improved performance in the Morris water maze and novel object recognition tests. Immunohistochemical analyses revealed markedly reduced glial activation and neuronal apoptosis in treated mice. Notably, the peptides restored axonal density in the perirhinal cortex and attenuated β-amyloid (Aβ) plaque-associated dystrophic neurites, though hippocampal Aβ burden remained unchanged. Transcriptomic profiling identified critical regulatory genes, including forkhead box B2 (Foxb2) and olfactory receptor, family 2, subfamily K, member 2 (Or2k2), and linked their neuroprotective effects to the modulation of apoptosis and synaptic plasticity. Collectively, TB500 and Ac-SDKP exert multi-targeted efficacy against AD pathology by enhancing neuronal survival, suppressing neuroinflammation, and promoting axonal regeneration, thereby emerging as promising candidates for AD intervention.

Thymosin beta4 and its posttranslational modifications.

Thymosin beta(4) as well as the other members of the beta-thymosin family are important G-actin sequestering peptides. The chemical properties, the biosynthesis, and posttranslational modifications (PTMs) of these peptides are discussed. During biosynthesis of thymosin beta(4) the initiator methionine is removed and the N-terminus is acetylated. Research on proteomics revealed several acetylated lysine residues and two phosphorylated threonine residues. The enormous number of phosphorylable and acetylable sites in the human proteome raises the question about the biological significance of these PTMs in the context of beta-thymosins. Presently, this question cannot be answered because neither the concentration of these modified beta-thymosins in cells is known nor the consequences of the modifications on the biological function(s) of beta-thymosins have been studied yet. Thymosin beta(4) is also posttranslationally modified by transglutaminase forming covalent bonds with other molecules. Prolyl oligopeptidase generates ac-SDKP from thymosin beta(4). The concentration of C-terminal peptide fragments of thymosin beta(4) is elevated in the blood of patients with rheumatoid arthritis.

Thymosin Beta-4 Induces Mouse Hair Growth.

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.

Thymosin β4 limits inflammation through autophagy.

Thymosin β4 (Tβ4) is a thymic hormone with multiple and different intracellular and extracellular activities affecting wound healing, inflammation, fibrosis and tissue regeneration. As the failure to resolve inflammation leads to uncontrolled inflammatory pathology which underlies many chronic diseases, the endogenous pathway through which Tβ4 may promote inflammation resolution becomes of great interest. In this review, we discuss data highlighting the efficacy of Tβ4 in resolving inflammation by restoring autophagy. The authors provide an overview of the Tβ4's anti-inflammatory properties in several pathologies and provide preliminary evidence on the ability of Tβ4 to resolve inflammation via the promotion of non-canonical autophagy associated with the activation of the DAP kinase anti-inflammatory function. Based on its multitasking activity in various animal studies, including tissue repair and prevention of chronic inflammation, Tβ4 may represent a potential, novel treatment for inflammatory diseases associated with defective autophagy.

Alkaline Phosphatase-Triggered Spatiotemporal Repair of Corneal Injury with TB500 Peptide Hydrogel.

Corneal injury remains a significant clinical challenge due to the limited regenerative capacity of the cornea and the difficulties associated with maintaining drug retention at the injury site. This study presents a novel spatiotemporal repair strategy for corneal wounds, utilizing an alkaline phosphatase (ALP)-triggered, lesion-responsive peptide hydrogel that incorporates TB500, a biologically active peptide with the amino acid sequence LKKTETQ, which has not been previously explored for corneal disease treatment. The hydrogel is designed through enzyme-instructed self-assembly (EISA) of a phosphorylated peptide precursor, Nap-YpYY-TB500, which undergoes site-specific dephosphorylation by elevated ALP levels at the wound site, triggering nanofiber formation and gelation in situ. Among three candidate sequences, Nap-YpYY-TB500 exhibited optimal gelation kinetics, nanostructure, and therapeutic efficacy. In vitro, the hydrogel promoted human corneal epithelial cell (HCEC) migration, proliferation, and tight junction recovery, while also enhancing myofibroblastic differentiation and cytoskeletal reorganization of human corneal stromal fibroblasts (HCSFs). In an alkali burn model, the hydrogel significantly accelerated epithelial regeneration, reduced inflammation, and improved corneal barrier function. Our work represents the first ocular application of TB500 and underscores the potential of enzyme-responsive, self-assembling peptide hydrogel as a localized and sustained delivery system for corneal repair.

Thymosin β4-mediated protective effects in the heart.

Despite recent advances in the treatment of coronary heart disease, a significant number of patients progressively develop heart failure. Reduction of infarct size after acute myocardial infarction and normalization of microvasculature in chronic myocardial ischemia could enhance cardiac survival. Induction of neovascularization using vascular growth factors has emerged as a promising novel approach for cardiac regeneration. Thymosin β4 (Tβ4) might be a promising candidate for the treatment of ischemic heart disease. It has been characterized as a major G-actin-sequestering factor regulating cell motility, migration, and differentiation. During cardiac development, Thymosin β4 seems essential for vascularization of the myocardium. In the adult organism, Thymosin β4 has anti-inflammatory properties, increases myocyte and endothelial cell survival accompanied by differentiation of epicardial progenitor cells. In chronic myocardial ischemia, Tβ4 overexpression enhances micro- and macrovasculature in the ischemic area and thereby improves myocardial function. A comparable effect is seen in diabetic and dyslipidemic pig ischemic hearts, suggesting an attractive therapeutic potential of adeno-associated virus encoding for Tβ4 for patients with ischemic heart disease. Induction of mature micro-vessels is a prerequisite for chronic myocardial ischemia and might be achieved via a long-term overexpression of Thymosin β4.

Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.

Thymosin beta 4 (Tβ4), a secreted 43 amino acid peptide, promotes oligodendrogenesis, and improves neurological outcome in rat models of neurologic injury. We demonstrated that exogenous Tβ4 treatment up-regulated the expression of the miR-200a in vitro in rat brain progenitor cells and in vivo in the peri-infarct area of rats subjected to middle cerebral artery occlusion (MCAO). The up-regulation of miR-200a down-regulated the expression of the following targets in vitro and in vivo models: (i) growth factor receptor-bound protein 2 (Grb2), an adaptor protein involved in epidermal growth factor receptor (EGFR)/Grb2/Ras/MEK/ERK1/c-Jun signaling pathway, which negatively regulates the expression of myelin basic protein (MBP), a marker of mature oligodendrocyte; (ii) ERRFI-1/Mig-6, an endogenous potent kinase inhibitor of EGFR, which resulted in activation/phosphorylation of EGFR; (iii) friend of GATA 2, and phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which are potent inhibitors of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, and resulted in marked activation of AKT; and (iv) transcription factor, p53, which induces pro-apoptotic genes, and possibly reduced apoptosis of the progenitor cells subjected to oxygen glucose deprivation (OGD). Anti-miR-200a transfection reversed all the effects of Tβ4 treatment in vitro. Thus, Tβ4 up-regulated MBP synthesis, and inhibited OGD-induced apoptosis in a novel miR-200a dependent EGFR signaling pathway. Our findings of miR-200a-mediated protection of progenitor cells may provide a new therapeutic importance for the treatment of neurologic injury. Tβ4-induced micro-RNA-200a (miR-200a) regulates EGFR signaling pathways for MBP synthesis and apoptosis: up-regulation of miR-200a after Tβ4 treatment, increases MBP synthesis after targeting Grb2 and thereby inactivating c-Jun from inhibition of MBP synthesis; and also inhibits OGD-mediated apoptosis after targeting EGFR inhibitor (Mig-6), PI3K inhibitors (FOG2 and Pten) and an inducer (p53) of pro-apoptotic genes, for AKT activation and down-regulation of p53. These findings may contribute the therapeutic benefits for stroke and other neuronal diseases associated with demyelination disorders.

Potential role of thymosin beta 4 in the treatment of nonalcoholic fatty liver disease.

As a result of increased prevalence of obesity worldwide, non-alcoholic fatty liver disease (NAFLD) has become one of the most common causes of chronic liver disease. Although most NAFLD cases remain benign, some progress to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Therefore, treatment should be considered for NAFLD patients who are likely to progress to nonalcoholic steatohepatitis (NASH) or fibrosis. Thymosin beta 4 (Tβ4), a G-actin sequestering peptide, regulates actin polymerization in mammalian cells. In addition, studies have reported anti-inflammatory, insulin-sensitizing, and anti-fibrotic effects of Tβ4. However, no research has been done to investigate the effects of Tβ4 on NAFLD. Based on the findings above mentioned, we hypothesize that Tβ4 may represent an effective treatment for NAFLD.

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

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.

Thymosin beta 4 prevents systemic lipopolysaccharide-induced plaque load in middle-age APP/PS1 mice.

Lipopolysaccharide (LPS) produced by the gut during systemic infections and inflammation is thought to contribute to Alzheimer's disease (AD) progression. Since thymosin beta 4 (Tβ4) effectively reduces LPS-induced inflammation in sepsis, we tested its potential to alleviate the impact of LPS in the brain of the APPswePS1dE9 mouse model of AD (APP/PS1) and wildtype (WT) mice. 12.5-month-old male APP/PS1 mice (n = 30) and their WT littermates (n = 29) were tested for baseline food burrowing performance, spatial working memory and exploratory drive in the spontaneous alternation and open-field tests, prior to being challenged with LPS (100ug/kg, i.v.) or its vehicle phosphate buffered saline (PBS). Tβ4 (5 mg/kg, i.v.) or PBS, was administered immediately following and at 2 and 4 h after the PBS or LPS challenge, and then once daily for 6 days (n = 7-8). LPS-induced sickness was assessed though monitoring of changes in body weight and behaviour over a 7-day period. Brains were collected for the determination of amyloid plaque load and reactive gliosis in the hippocampus and cortex. Treatment with Tβ4 alleviated sickness symptoms to a greater extent in APP/PS1 than in WT mice by limiting LPS-induced weight loss and inhibition of food burrowing behaviour. It prevented LPS-induced amyloid burden in APP/PS1 mice but increased astrocytic and microglial proliferation in the hippocampus of LPS-treated WT mice. These data show that Tβ4 can alleviate the adverse effects of systemic LPS in the brain by preventing exacerbation of amyloid deposition in AD mice and by inducing reactive microgliosis in aging WT mice.

Thymosin beta 4 attenuates PrP(106-126)-induced human brain endothelial cells dysfunction.

The blood-brain barrier (BBB) is a highly selective permeability barrier that separates the circulating blood from the brain and extracellular fluid in the central nervous system (CNS). The BBB is formed by cerebral endothelial cells connected by tight junctions. Prion diseases are neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), named PrPSc. Thymosin beta 4 (Tβ4) is an actin-sequestering peptide known to bind monomeric actin and inhibit its polymerization, and it is known to have a neuroprotective effect. However, the effect of Tβ4 on prion disease has not yet been investigated. Therefore, in this study, we investigated the effect of Tβ4 on prion-induced BBB dysfunction in hCMEC/D3 human cerebral endothelial cells. We found that Tβ4 increased the expression of tight junction protein, but reduced the ratio of F-actin to G-actin. Moreover, we showed that Tβ4 significantly improved PrP (106-126)-induced vascular permeability dysfunction in hCMEC/D3 cells. Through human BBB in vitro model, we found that PrP (106-126) could disrupt tight junctions and cytoskeleton arrangement. These results suggest that Tβ4 may play a critical role in barrier stabilization. Furthermore, Tβ4 may prevent neurodegenerative diseases caused by prion-induced BBB dysfunction.

Platelet function and thymosin β4.

Thymosin β4 (Tβ4) is a small, low-molecular-weight peptide ubiquitously expressed in all cells and extracellular fluids. It is a major actin sequestering protein present in the cells. In addition to this, Tβ4 has also been shown to be involved in endothelial cell migration, angiogenesis, corneal wound healing, and stem cell differentiation. It is also released by platelets after activation. The amount of Tβ4 increases at sites of injury and thus suggests an important role of this biopeptide in wound healing. Herein, we provide an overview of the role of Tβ4 in thrombosis and platelet aggregation.

Tβ4-Ac-SDKP pathway: Any relevance for the cardiovascular system?

The last 20 years witnessed the emergence of the thymosin β4 (Tβ4)-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) pathway as a new source of future therapeutic tools to treat cardiovascular and renal diseases. In this review article, we attempted to shed light on the numerous experimental findings pertaining to the many promising cardiovascular therapeutic avenues for Tβ4 and (or) its N-terminal derivative, Ac-SDKP. Specifically, Ac-SDKP is endogenously produced from the 43-amino acid Tβ4 by 2 successive enzymes, meprin α and prolyl oligopeptidase. We also discussed the possible mechanisms involved in the Tβ4-Ac-SDKP-associated cardiovascular biological effects. In infarcted myocardium, Tβ4 and Ac-SDKP facilitate cardiac repair after infarction by promoting endothelial cell migration and myocyte survival. Additionally, Tβ4 and Ac-SDKP have antifibrotic and anti-inflammatory properties in the arteries, heart, lungs, and kidneys, and stimulate both in vitro and in vivo angiogenesis. The effects of Tβ4 can be mediated directly through a putative receptor (Ku80) or via its enzymatically released N-terminal derivative Ac-SDKP. Despite the localization and characterization of Ac-SDKP binding sites in myocardium, more studies are needed to fully identify and clone Ac-SDKP receptors. It remains promising that Ac-SDKP or its degradation-resistant analogs could serve as new therapeutic tools to treat cardiac, vascular, and renal injury and dysfunction to be used alone or in combination with the already established pharmacotherapy for cardiovascular diseases.

Actin-Induced Structure in the Beta-Thymosin Family of Intrinsically Disordered Proteins.

Thymosin β4 (Tβ4) is a 43-amino acid signature motif peptide that defines the beta-thymosin (βT) family of proteins. βTs are intrinsically unstructured in their free states and undergo disorder-to-order transitions in carrying out their biological functions. This property poses challenges in determining their 3D structures, mainly favoring structural studies on the complexes formed between βTs and their interaction partners. One of the βTs' primary binding partners is monomeric actin, a major component of the cytoskeleton in eukaryotic cells. Tβ4's role in this system is to maintain the highly concentrated pool of monomeric actin that can be accessed through profilin by actin filament nucleating machineries. Here, we give an account of the structures of βTs that have been illuminated by nuclear magnetic resonance (NMR) and X-ray crystallography. NMR has been the method of choice for probing regions that have intrinsic conformational preference within the largely disordered βTs in their native states in solution. X-ray crystallography has demonstrated at atomic detail how βTs interact with actin. Detailed analysis of these structures highlights the disorder-to-order transition of Tβ4 in binding to actin and its isoform specificity.

Reconsidering an active role for G-actin in cytoskeletal regulation.

Globular (G)-actin, the actin monomer, assembles into polarized filaments that form networks that can provide structural support, generate force and organize the cell. Many of these structures are highly dynamic and to maintain them, the cell relies on a large reserve of monomers. Classically, the G-actin pool has been thought of as homogenous. However, recent work has shown that actin monomers can exist in distinct groups that can be targeted to specific networks, where they drive and modify filament assembly in ways that can have profound effects on cellular behavior. This Review focuses on the potential factors that could create functionally distinct pools of actin monomers in the cell, including differences between the actin isoforms and the regulation of G-actin by monomer binding proteins, such as profilin and thymosin β4. Owing to difficulties in studying and visualizing G-actin, our knowledge over the precise role that specific actin monomer pools play in regulating cellular actin dynamics remains incomplete. Here, we discuss some of these unanswered questions and also provide a summary of the methodologies currently available for the imaging of G-actin.

Thymosin Beta 4 Protects Hippocampal Neuronal Cells against PrP (106-126) via Neurotrophic Factor Signaling.

Prion protein peptide (PrP) has demonstrated neurotoxicity in brain cells, resulting in the progression of prion diseases with spongiform degenerative, amyloidogenic, and aggregative properties. Thymosin beta 4 (Tβ4) plays a role in the nervous system and may be related to motility, axonal enlargement, differentiation, neurite outgrowth, and proliferation. However, no studies about the effects of Tβ4 on prion disease have been performed yet. In the present study, we investigated the protective effect of Tβ4 against synthetic PrP (106-126) and considered possible mechanisms. Hippocampal neuronal HT22 cells were treated with Tβ4 and PrP (106-126) for 24 h. Tβ4 significantly reversed cell viability and reactive oxidative species (ROS) affected by PrP (106-126). Apoptotic proteins induced by PrP (106-126) were reduced by Tβ4. Interestingly, a balance of neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) and receptors (nerve growth factor receptor p75, tropomyosin related kinase A and B) were competitively maintained by Tβ4 through receptors reacting to PrP (106-126). Our results demonstrate that Tβ4 protects neuronal cells against PrP (106-126) neurotoxicity via the interaction of neurotrophic factors/receptors.

Thymosin β4 reverses phenotypic polarization of glial cells and cognitive impairment via negative regulation of NF-κB signaling axis in APP/PS1 mice.

Thymosin β4 (Tβ4) is the most abundant member of the β-thymosins and plays an important role in the control of actin polymerization in eukaryotic cells. While its effects in multiple organs and diseases are being widely investigated, the safety profile has been established in animals and humans, currently, little is known about its influence on Alzheimer's disease (AD) and the possible mechanisms. Thus, we aimed to evaluate the effects and mechanisms of Tβ4 on glial polarization and cognitive performance in APP/PS1 transgenic mice. Behavior tests were conducted to assess the learning and memory, anxiety and depression in APP/PS1 mice. Thioflavin S staining, Nissl staining, immunohistochemistry/immunofluorescence, ELISA, qRT-PCR, and immunoblotting were performed to explore Aβ accumulation, phenotypic polarization of glial cells, neuronal loss and function, and TLR4/NF-κB axis in APP/PS1 mice. We demonstrated that Tβ4 protein level elevated in all APP/PS1 mice. Over-expression of Tβ4 alone alleviated AD-like phenotypes of APP/PS1 mice, showed less brain Aβ accumulation and more Insulin-degrading enzyme (IDE), reversed phenotypic polarization of microglia and astrocyte to a healthy state, improved neuronal function and cognitive behavior performance, and accidentally displayed antidepressant-like effect. Besides, Tβ4 could downregulate both TLR4/MyD88/NF-κB p65 and p52-dependent inflammatory pathways in the APP/PS1 mice. While combination drug of TLR4 antagonist TAK242 or NF-κB p65 inhibitor PDTC exerted no further effects. These results suggest that Tβ4 may exert its function by regulating both classical and non-canonical NF-κB signaling and is restoring its function as a potential therapeutic target against AD.

Mechanistic study of the Tβ4/SLC7A11 signaling pathway regulating breast cancer evolution.

Thymosin β4 (Tβ4) plays a critical role in breast cancer progression, yet its molecular mechanism remains unclear. In this study, we identified that Tβ4 is significantly upregulated in breast cancer tissues and cell lines, and its high expression correlates with poor clinical outcomes. Functionally, Tβ4 promotes breast cancer cell proliferation, migration, epithelial-mesenchymal transition (EMT), and angiogenesis while inhibiting apoptosis. Mechanistically, Tβ4 directly regulates the expression of SLC7A11, a key cystine/glutamate antiporter, thereby enhancing glutathione biosynthesis and suppressing lipid peroxidation to inhibit ferroptosis. Rescue experiments further demonstrated that silencing SLC7A11 abrogates the oncogenic effects of Tβ4 both in vitro and in vivo. Collectively, these findings uncover a novel Tβ4/SLC7A11 axis that modulates ferroptosis sensitivity and contributes to breast cancer malignancy, offering potential therapeutic implications for targeting ferroptosis resistance.

Plasmacytoid dendritic cells alleviate allergic asthma via airway epithelial cell-dependent thymosin β4 expression.

Plasmacytoid dendritic cells (pDCs) have been previously reported to induce immune tolerance to allergen by inhibiting allergic TH2-cell priming. However, there is limited knowledge on pDC function during the TH2 effector phase of allergic asthma. We sought to investigate the role of pDCs in ongoing allergic asthma following airway sensitization and challenge with house mite dust. Blood dendritic cell antigen 2-diphtheria toxin receptor (BDCA2-DTR) mice were used for pDC depletion. RNA sequencing of sorted mouse lung pDCs, in vitro cell experiments, and in vivo CCR2 blockade or thymosin β4 (Tβ4) supplementation were performed to elucidate the underlying mechanism. pDC depletion during house mite dust challenge enhanced CCR2-dependent inflammatory monocyte-derived cell accumulation, leading to exacerbated TH2-mediated allergic asthma phenotypes. RNA-sequencing analysis revealed that the anti-inflammatory peptide thymosin β4 (Tβ4) was among the most upregulated genes in asthmatic lung pDCs. Airway epithelial cell-derived IL-33 was required for upregulating Tβ4 expression in pDCs, which represented the main source of Tβ4 in asthmatic lungs. Importantly, Tβ4 supplementation reversed the exacerbation of asthmatic phenotypes in BDCA2-DTR mice. Alveolar macrophages were identified as the major source of CCL2 and the target of Tβ4 in asthmatic lungs. Mechanistically, Tβ4 inhibited IL-4/IL-13-induced phosphorylation of Janus kinase 1 and signal transducer and activator of transcription 6 and downstream early growth response 2 expression in macrophages, thereby reducing CCL2 expression and subsequent inflammatory monocyte-derived cell recruitment. Unexpectedly, decreased serum Tβ4 levels were detected in mice and humans with ongoing allergic asthma. This could be due to increased uptake of Tβ4 by alveolar macrophages, which was required for its inhibitory effect on CCL2 expression. Lung pDCs exert anti-inflammatory effects in allergic asthma via expressing Tβ4, which could have therapeutic potential.

Thymosin β as an Actin-binding Protein with a Variety of Functions.

According to current data, the thymosin β family is composed of 20 short (40-44 amino acid) peptides, but in a healthy human body only 2 are expressed - thymosin β4 and β10. Their most characteristic feature is the ability to form a complex with monomeric actin, thereby preventing polymerization into a filamentous form, hence the name Actin-Binding Protein (ABP). These peptides play numerous different functions. Among others, they affect the processes of carcinogenesis, differentiation and angiogenesis, influence metalloproteinase activity and accelerate wound healing. Moreover, significant biological activity has also been displayed by Tβ4 derived peptides: Ac-SDKP, the N-terminal fragment which is involved, inter alia, in stimulating angiogenesis and the inhibition of stem cell proliferation and Tβ4 sulfoxide, an oxidation product of one of the peptide methionine by hydrogen peroxide, which inhibit the development of inflammation. The properties of these peptides have potential applications in cardiovascular medicine, dermatology, ophthalmology and other medical areas.

Thymosin beta-4 is essential for coronary vessel development and promotes neovascularization via adult epicardium.

Ischemic heart disease leading to myocardial infarction causes irreversible cell loss and scarring and is a major cause of morbidity and mortality in humans. Significant effort in the field of cardiovascular medicine has been invested in the search for adult cardiac progenitor cells that may replace damaged muscle cells and/or contribute to new vessel formation (neovascularization) and in the identification of key factors, which may induce such progenitor cells to contribute to myocardial repair and collateral vessel growth. We recently demonstrated that the actin monomer-binding protein, thymosin beta-4 (Tbeta-4), when secreted from the myocardium provides a paracrine stimulus to the cells of the epicardium-derived cells (EPDCs) to promote their inward migration and differentiation into endothelial and smooth muscle cells to form the coronary vasculature. Translating this essential role for Tbeta-4 in coronary vessel development to the adult, we found that treatment of cultured adult explants with Tbeta-4 stimulated extensive outgrowth of epicardin-positive epicardial cells, which, as they migrated away from the explant, differentiated into procollagen type I, SMalphaA, and Flk1-positive cells indicative of fibroblasts, smooth muscle, and endothelial cells; thus releasing the adult epicardium from a quiescent state and restoring pluripotency. The ability of Tbeta-4 to promote coronary vessel development and potentially induce new vasculature in the adult is essential for cardiomyocyte survival and could contribute significantly toward the reported Tbeta4-induced cardioprotection and repair in the adult heart. Tbeta-4 is currently subject to multicenter phase 1 clinical trials for treatment of cardiovascular disease (http://www.regenerx.com), therefore, insight into the repair mechanism(s) induced by Tbeta-4 is an essential step toward harnessing therapeutic survival, migration, and repair properties of the peptide in the context of acute myocardial damage.

Thymosins and muscle regeneration.

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.

TB500/TB1000 and SGF1000: A scientific approach for a better understanding of misbranded and adulterated drugs.

Nowadays, numerous websites attempt to commercialize over the internet various products, regardless of the lack of approval by the EMA or the FDA either for human or veterinary use. These products are often produced after aborted drug development due to insufficient or deleterious biological effects, synthesized based on natural products, or only based on scientific literature. However, the administration of such products is dangerous, considering the lack of official control over the production of these substances and the absence of approval by health authorities. In this short communication, we provide an extensive analysis of three misbranded and adulterated products sold over the internet named TB500, TB1000, and SGF1000. We confirm that the content of TB500/TB1000 products is not systematically consistent with it's former descriptions, but also that SGF1000 is mainly composed of sheep extracellular matrix (ECM) and blood proteins, and the signal corresponding to the purported growth promoters is excessively diluted.

Enhancing fat graft survival: thymosin beta-4 facilitates mitochondrial transfer from ADSCs via tunneling nanotubes by upregulating the Rac/F-actin pathway.

Autologous fat grafting is a widely used technique in plastic and reconstructive surgery, but its efficacy is often limited by the poor survival rate of transplanted adipose tissue. This study aims to enhance the survival of fat grafts by investigating the role of thymosin beta-4 (Tβ4) in facilitating mitochondrial transfer from adipose-derived stem cells (ADSCs) to adipocytes and newly formed blood vessels within the grafts via tunneling nanotubes (TNTs). We demonstrate that Tβ4 upregulates the Rac/F-actin pathway, leading to an increased formation of TNTs and subsequent transfer of mitochondria from ADSCs. This process mitigates oxidative stress, reduces apoptosis, and promotes revascularization, thereby improving the quality and volume retention of fat grafts. Our findings provide a novel mechanistic insight into the enhancement of fat graft survival and suggest that mitochondrial transplantation and Tβ4 are potential therapeutic strategies to improve clinical outcomes in autologous fat transfer procedures.