Introduction of Thymosin β4
Thymosin, initially isolated from the calf thymus by Goldstein and White, is a vital lymphocyte growth factor. Within the thymosin family, categorized into α, β, and γ types based on their isoelectric points, thymosin β (Tβ) occupies a range between 5.0-7.0. Among the β-thymosin variants, including Tβ4, Tβ10, and Tβ15, Tβ4 stands out as the most prevalent, constituting 70%–80% of β-thymosin in the human body. Thymosin β4 (Tβ4) is a small protein encoded by the TMSB4X gene, located on the X chromosome in humans. Its distribution spans various tissues, with notable concentrations in the thymus, spleen, and peritoneal macrophages, while exhibiting high expression levels in the brain, liver, kidney, testis, myocardium, platelets, and leukocytes.
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Biological Function of Tβ4
Tβ4's Role in Angiogenesis
Thymosin β4 (Tβ4) emerges as a pivotal promoter of angiogenesis, fostering endothelial progenitor cell (EPC) vitality and orchestrating cell proliferation, migration, and capillary-like structure formation. Notably, it upregulates the expression of vascular endothelial growth factor (VEGF), a key paracrine factor secreted by progenitor cells, thereby enhancing angiogenesis. This effect is evident in studies where Tβ4-primed EPCs, upon transplantation into infarcted rat hearts, significantly elevate VEGF expression compared to EPC transplantation alone. Additionally, in murine hindlimb ischemia models, the combination of Tβ4 with human adipose-derived stem cells triggers endothelial differentiation and elevates angiogenic factors like angiopoietin-1 and von Willebrand factor, fostering blood perfusion and collateral formation.
Impact of Tβ4 on Cell Proliferation and the Cell Cycle
Tβ4 profoundly influences the cell cycle and proliferation dynamics. Knocking out Tβ4 in intestinal epithelial cells dampens cell proliferation, disrupts the cell cycle with decreased G0/G1 population, increased polyploid cells, and compromised DNA replication due to DNA damage. Moreover, Tβ4 administration, such as N-acetyl-erythritosyl-lysyl proline infusion, facilitates neurogenesis in the hippocampus. It also enhances proliferation in mesenchymal stem cells (MSCs), particularly adipose-derived ones, mediated by interleukin-8 (IL-8). Furthermore, Tβ4 fosters oligodendrocyte progenitor cell (OPC) proliferation and maturation into myelinating oligodendrocytes, and stimulates adult rat cardiac progenitor cell proliferation and differentiation into various cardiac cell types. Its effects extend to vascular endothelial cells, guarding against post-ischemic cardiac dysfunction.
Tβ4's Anti-apoptotic Effects
Tβ4 exhibits notable anti-apoptotic properties across diverse cellular contexts. It curtails tubular epithelial cell apoptosis by suppressing the transforming growth factor (TGF)-β pathway in chronic renal tubular interstitial fibrosis in rats. Additionally, it mitigates nucleus pulposus cell apoptosis, retards cellular aging, and fosters proliferation. Moreover, Tβ4 diminishes EPC apoptosis induced by serum depletion, downregulating apoptosis-related proteins like caspase-3 and caspase-9. It also safeguards human corneal epithelial cells from ethanol-induced mitochondrial disruption and caspase-mediated apoptosis, indicating potential antiapoptotic prowess.
Tβ4 and Signaling Pathways
Thymosin β4 (Tβ4) exerts a multifaceted influence on cytokine secretion and governs diverse signaling cascades, thereby modulating inflammatory responses, tissue repair, and various cellular processes. It mitigates inflammatory damage by regulating pathways such as NF-κB and Toll-like receptor pathways, thus curbing the release of cytokines like TNF-α and IL-1 receptor-associated kinases. During tissue repair, Tβ4 orchestrates pathways including PI3K/Akt/eNOS, Notch, and TGF-β, among others, to promote healing and regeneration. Additionally, it regulates the Wnt pathway to foster hair follicle generation and attenuates fibrosis via TGFβ/Smad signaling.
Fig. 1 Tβ4 regulates various signaling pathways (Xing Y., et al. 2021).
PI3K/Akt/eNOS Pathway
The PI3K/Akt/eNOS pathway, pivotal in microangiogenesis, cell migration, survival, and angiogenesis, serves as a prime target of Tβ4's actions. Tβ4's administration spurs EPC proliferation, migration, and adhesion via this pathway, bolstering local mobilization of EPCs and participating in angiogenesis. Studies in rats with cerebral ischemia/reperfusion show heightened Akt phosphorylation and eNOS expression in response to Tβ4 treatment, fostering blood vessel regeneration and neurological recovery. Moreover, in ischemic limb diseases, Tβ4 promotes angiogenesis through PI3K/AKT signaling, enhancing myocyte survival, coronary vessel growth, and curbing inflammation in murine and porcine models.
Notch Pathway
Tβ4's influence extends to the Notch signaling pathway, pivotal in various cellular functions including angiogenesis and cell proliferation. It stimulates angiogenesis in endothelial cells via Notch signaling, accelerating lumen formation and angiogenic efficacy. In liver fibrosis models, Tβ4 inhibits hepatic stellate cell activation and fibrosis by dampening Notch signaling. Additionally, Tβ4 augments endothelial cell viability, angiogenesis, and migration while upregulating key angiogenic factors in critical limb ischemia models. Notably, endothelial-specific Notch1 knockdown impedes neovascularization post-hindlimb ischemia, implicating Notch's involvement in Tβ4-induced angiogenesis.
TGFβ/Smad Pathway
Tβ4's anti-fibrotic actions are evident through its modulation of the TGFβ/Smad pathway. It suppresses TGFβ1-mediated fibrosis by reducing TGFβR II, Smad2, and Smad3 expression in hepatic tissues, alleviating cholestatic liver fibrosis. Similarly, Tβ4 treatment mitigates neuroinflammation and enhances neuroprotection post-traumatic brain injury by inhibiting the TGFβ1/NF-κB pathway.
Wnt Signaling Pathway
Tβ4's regulatory role extends to the Wnt signaling pathway, crucial for cell proliferation, differentiation, and hair follicle morphogenesis. By stimulating Wnt ligands and promoting β-catenin accumulation, Tβ4 accelerates hair follicle growth and limb regeneration in animal models. Moreover, it counters Ang II-induced myocardial hypertrophy by modulating the Wnt pathway, thereby safeguarding against cardiac dysfunction.
Apoptosis Pathway
Tβ4's anti-apoptotic prowess involves modulating the apoptosis pathway, chiefly by upregulating anti-apoptotic proteins and suppressing the Bax/BCL2 ratio. In various cell types, Tβ4 treatment mitigates apoptosis induced by ethanol exposure, FasL-mediated activation, or antitumor drugs, underscoring its cytoprotective potential across diverse cellular contexts.
Applications of Tβ4
Thymosin β4 (Tβ4) showcases diverse therapeutic applications owing to its potent biological activity and anti-inflammatory properties. Its multifaceted effects span various injuries and diseases, including corneal injury, ulcerative colitis, liver fibrosis, renal fibrosis, and myocardial infarction. Tβ4 mitigates tissue fibrosis, promotes angiogenesis, tissue repair, and regeneration, and reduces scar formation. Notably, it accelerates wound healing, aids in myocardial infarction recovery, promotes hair growth, and alleviates inflammation.
In myocardial infarction, Tβ4 reduces infarct size and enhances cardiac function by preserving ischemic myocardium and activating vascular or cardiac progenitor cells. Clinical trials confirm its safety and efficacy in protecting and repairing the heart post-infarction, reducing scar volume, and improving cardiac function. Additionally, Tβ4 attenuates rejection in heart transplantation, enhances mesenchymal stem cell therapy efficacy, and prolongs heart regeneration potential.
For corneal injury and dry eye syndrome, Tβ4 accelerates corneal re-epithelialization, dampens inflammation, and promotes wound repair, thereby improving corneal transparency and alleviating xerophthalmia symptoms. Its therapeutic efficacy extends to promoting skin wound healing by stimulating cell migration and angiogenesis, regulating cytokine balance, and reducing oxidative damage.
In liver-related conditions, Tβ4 ameliorates acute liver injury and inhibits fibrosis progression by suppressing oxidative stress, inflammation, and HSC activation. Moreover, it promotes hair growth, alleviates renal fibrosis, and exhibits potential in treating ulcerative colitis and colon cancer by modulating immune responses and reducing inflammation.
Overall, Tβ4's wide-ranging therapeutic effects underscore its potential as a promising drug candidate for various medical conditions, offering hope for improved clinical outcomes and enhanced patient well-being.
References
- Xing Y., et al. Progress on the function and application of thymosin β4. Frontiers in Endocrinology. 2021, 12: 767785.
- Kim J., Jung Y. Potential role of thymosin beta 4 in liver fibrosis. International Journal of Molecular Sciences. 2015, 16(5): 10624-35.
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