Thymosin Beta-4

Abstract

Over the past decades, the escalating global burden of kidney disease has underscored an urgent need for innovative therapeutic strategies. Thymosin β4 (Tβ4), a highly conserved 43-amino-acid peptide encoded by the X-linked TMSB4x gene, is the predominant β-thymosin in mammalian cells and a multifunctional regulator of cellular homeostasis. Once considered mainly an actin-sequestering molecule, Tβ4 and its N-terminal metabolite N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) now emerge as dynamic mediators of renal injury and repair. In this review, we synthesize current evidence on the Tβ4-Ac-SDKP axis. We map intra- and extracellular mechanisms and relevant signaling pathways, delineate cell-type and spatial expression across glomerular and tubular compartments, and critically evaluate its renoprotective efficacy-including cytoprotection, anti-inflammatory and antifibrotic actions-across models of acute and chronic kidney injury. To reconcile disparate findings, we propose conceptual frameworks that consider bidirectional effects on fibrosis and model-dependent mechanisms. Finally, translational opportunities are appraised with attention to pharmacokinetics, peptide stability and delivery strategies. Key challenges moving forward include validating efficacy in additional clinically relevant models, overcoming peptide instability and completing comprehensive safety assessments.

Abstract

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.

Abstract

Acute ischemic injury causes impairment of blood brain barrier (BBB) permeability and is considered as secondary insult in the brain after traumatic brain injury (TBI). The mechanisms underlying these events are incomprehensible and therefore therapeutic opportunities are limited. Although drugs have been showing some promise in TBI outcome, the restoration of BBB damage remain elusive. Thymosin β4 (Tβ4) is a secreted 43 amino acid peptide showed beneficial outcome in cerebral ischemia or TBI, however, it's role in hypoxia-induced BBB damage remains elusive. We hypothesize that Tβ4 protect hypoxia-induced BBB disruption via Sphingosine 1-phosphate receptor 1 (S1PR1) modulation. In the current study, we investigated the beneficial effects of Tβ4 in hypoxia induced gene expression of several tight junction proteins, S1PR1, endothelial cell permeability and tight junction dynamics in human brain microvascular endothelial cells (hBMVECs), one of the important cell types in the BBB integrity. The data suggests that pretreatment with Tβ4 reversed the hypoxia-induced damage of BBB components in hBMVECs. Furthermore, results identify S1PR1, a possible target for Tβ4. Inhibition of S1PR1 showed that Tβ4 failed to offer protection. Together, data provided evidence that S1PR1 is pivotal and Tβ4 can serve as a protective agent in BBB integrity and may offer a promising therapeutic target. In conclusion, we propose that depletion of S1PR1signaling is vital in hypoxia-induced BBB pathophysiology and Tβ4 may be tested as a potential treatment modality and warrant further investigation.

Abstract

Mast cells (MCs) under stress conditions contribute to the development of irritable bowel syndrome (IBS), yet their precise mechanisms in IBS remain unclear.

Abstract

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.

Abstract

Despite advancements in primary percutaneous coronary intervention (PCI), cardiac dysfunction remains a challenge in patients with ST-segment elevation myocardial infarction (STEMI). Although thymosin beta 4 has shown cardioprotective effects in preclinical MI models, its impact on chronic cardiac functional recovery post ischemia/reperfusion (I/R), especially in STEMI, warrants further investigation. This study aims to explore the therapeutic potential of recombinant human thymosin beta 4 (rhTB4) in both murine models subjected to I/R and in subjects with STEMI post-PCI.

Abstract

Ovarian cancer is a gynecologic malignancy with high mortality and poor prognosis. Chemoresistance is a key cause of ovarian cancer recurrence and metastasis. It has been found that some bioactive peptides can inhibit the growth and metastasis of cancer cells and promote cell apoptosis, thus exerting anti-cancer effects. Tβ4-17 is a small polypeptide that we selected using ITRAQ technology, and its precursor protein is thymosin β4. This study mainly investigated its effect in combination with cisplatin (DDP) on the proliferation, migration and apoptosis of ovarian cancer resistant cells and related molecular mechanisms. Our results showed that Tβ4-17 peptide combined with DDP significantly inhibited the proliferation and migration of drug resistance cells in ovarian cancer, promoted apoptosis, and increased the chemo-sensitivity of ovarian cancer cells to DDP. In addition, qRT-PCR and Western blot showed that NF-κB was significantly highly expressed in DDP-resistant cells of ovarian cancer. After application of NF-κB inhibitors and activators, Western blot, CCK8, EDU fluorescence proliferation assay, and cell scratch assay showed that Tβ4-17 peptide down-regulated NF-κB p65 protein expression and inhibited cell proliferation and migration. In conclusion, our study demonstrates that Tβ4-17 peptide enhances the sensitivity of ovarian cancer cells to DDP by down-regulating NF-κB expression.

Abstract

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.

Abstract

Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer's disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.

Abstract

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.

Abstract

Accelerating angiogenesis, neurogenesis, and in situ stem cell recruitment at the site of bone defects is critical for bone regenerative repair. Bone marrow mesenchymal stem cell (BMSC) exosomes are cell-free therapeutic agents with bone-enhancing effects. Thymosin β4 (Tβ4) is a short peptide known for its key role in tissue repair and angiogenesis. In this study, we successfully developed a multifunctional injectable Exo@Tβ4/HAMA hydrogel platform by grafting Tβ4 onto methylmalonic anhydride-modified hyaluronic acid (HAMA) via photo-cross-linking and then encapsulating BMSC-derived exosomes. In vitro results demonstrated that the Exo@Tβ4/HAMA hydrogel exhibited improved mechanical properties, favorable biocompatibility, and the ability to significantly recruit BMSCs. Additionally, it showed superior vasculogenic effects on HUVECs and osteogenic differentiation potentials on BMSCs. In vivo studies revealed that the hydrogel successfully promoted both neurogenesis, angiogenesis, and new bone formation. It also facilitated osteogenesis through the ERK1/2-dependent RUNX2 signaling pathway. Our results suggest that this hydrogel platform exerts a robust multisystemic regulatory effect, fostering rat bone repair through the synergistic promotion of in situ stem cell recruitment, angiogenesis, neurogenesis, and osteogenesis. As a simple-to-prepare and multifunctional integrated bone graft, this hydrogel platform holds a significant promise in establishing a conducive microenvironment for optimal bone healing.

Abstract

Although a myocardial infarction occurs roughly every minute in the U.S. alone, medical research has yet to unlock the key to fully enabling post-hypoxic myocardial regeneration. Thymosin beta-4 (TB4), a short, secreted peptide, was shown to possess a beneficial impact regarding myocardial cell survival, coronary re-growth and progenitor cell activation following myocardial infarction in adult mammals. It equally reduces scarring, however, the precise mechanisms through which the peptide assists this phenomenon have not been properly elucidated. Accordingly, the primary aim of our study was to identify novel molecular contributors responsible for the positive impact of TB4 during the remodeling processes of the infarcted heart. We performed miRNA profiling on adult mice hearts following permanent coronary ligation with or without systemic TB4 injection and searched for targets and novel mechanisms through which TB4 may mitigate pathological scarring in the heart. Our results revealed a significant increase in miR139-5p expression and identified ROCK1 as a potential target protein aligned. Real-time PCR, Western blot and immunostaining on adult mouse hearts and human cardiac cells revealed the peptide indirectly or directly modulates ROCK1 protein levels both in vivo and in vitro. We equally discovered TB4 may reverse or inhibit fibroblast/myofibroblast transformation and the potential downstream mechanisms by which TB4 alters cellular responses through ROCK1 are cell type specific. Given the beneficial effects of ROCK1 inhibition in various cardiac pathologies, we propose a potential utilization for TB4 as a ROCK1 inhibitor in the future.

Abstract

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.

Abstract

Available models currently adopted for preclinical studies in the cardiovascular field either fail to recapitulate human cardiac physiology or are extremely expensive and time-consuming. Translational research would greatly benefit from the development of novel models that reflect the native mature phenotype of the human heart while being cost and time effective. Living myocardial slices (LMSs) have emerged as a novel, powerful ex vivo tool for translational research. Although the number of studies adopting LMSs is rapidly increasing, this model remains largely under-characterized. In this study, we make use of LMSs and compare them to a murine model to deliver the cardioprotective factor zinc finger E box-binding homeobox 2 (ZEB2), a transcription factor known to exert cardioprotective effects after ischemic injury and promote the secretion of pro-angiogenetic factors thymosin beta-4 (TMSB4) and prothymosin alpha (PTMA). Our data show that viral-mediated delivery of these factors induced similar cardiomyocyte gene expression changes in LMS and mouse models. We also show that the delivery of these pro-angiogenic factors enhances an angiogenic response in both models, indicating that LMSs are a suitable alternative to mice for studying the effects of gene transfer in various cardiac cell types.

Abstract

Although peptides have been shown to have biological functions in neurodegenerative diseases, their role in Parkinson's disease has been understudied. A previous study by our group, which used a 6-hydroxydopamine zebrafish model, suggested that nine intracellular peptides may play a part in this condition. In this context, our aim is to better understand the role of five of these nine peptides. The selection of peptides was made based on their precursor proteins, which are fatty acid binding protein 7, mitochondrial ribosomal protein S36, MARCKS-related protein 1-B, excitatory amino acid transporter 2 and thymosin beta-4. The peptides were chemically synthesized in solid phase and characterized by high-performance liquid chromatography and mass spectrometry. Circular dichroism was performed to determine the secondary structure of each peptide, which showed that all five peptides maintain a random structure in the aqueous solutions that were studied. Two molecules show a helical profile in trifluoroethanol, a known structuring agent. Cell viability by the MTT assay indicates that all five peptides are not cytotoxic in all concentrations tested in both mouse and human cell lines. Behavioral assay using a 6-OHDA zebrafish larvae model suggest that all peptides help in the recovery of motor function with 24 h treatment at two concentrations. Three peptides showed a complete recovery from the 6-OHDA-induced motor impairment. Further studies are needed to better understand the mechanism of action of these peptides and whether they are truly a potential ally against Parkinson's disease.

Abstract

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.

Abstract

Plasma protein alterations may occur in patients with acute myocardial infarction (AMI). In this study, we investigated the plasma proteomics of patients with first-onset AMI to identify a novel diagnostic target for myocardial infarction.