Main Research Findings

1) Treatment with Tβ4 has been shown to improve overall functioning and potency of diabetic endothelial cells by enhancing hiPSC-EC viability and reducing endothelin-1 production.

2) Tβ4 was shown to reduce endothelial cell dysfunction induced by prion proteins in brain tissue.

Selected Data
1) Researchers Su et al investigated the biological role of Tβ4 in diabetic human induced pluripotent stem cells–derived endothelial cells (hiPSC-ECs) both in vitro and in vivo. Two previously established hiPSC lines, DP2C8iPS and DP3C6iPS, reprogrammed from dermal fibroblasts of patients with type 2 diabetes mellitus (T2DM), served as the cell source. These cells were maintained in a feeder-free culture system and differentiated into endothelial cells (ECs) using a protocol optimized in earlier studies. Endothelial cells expressing the markers CD31 and CD144 were isolated via fluorescence-activated cell sorting (FACS), expanded, and cultured in EGM2-MV medium supplemented with B27, VEGF165, and SB-431542. This system provided a reliable platform for examining how Tβ4 influences diabetic endothelial cell function [1].
To assess the proliferative capacity of these diabetic hiPSC-ECs, researchers calculated population doubling time during the first week following sorting. Cells were grown in endothelial growth medium supplemented either with or without recombinant Tβ4 protein, with media refreshed every two days. On day seven, cells were harvested and counted to determine proliferation rates. Endothelial function was further assessed by tube formation assays. In these experiments, cells were seeded on Matrigel and incubated for 24 hours. The resulting capillary-like networks were quantified by analyzing nodes, junctions, branches, and branch length using ImageJ software.
The study also explored whether Tβ4 provided cytoprotective effects under stress conditions. hiPSC-ECs were exposed to hypoxia for 24 hours in basal medium with or without Tβ4 supplementation. Lactate dehydrogenase (LDH) release into the culture supernatant was measured as an indicator of membrane damage and cytotoxicity, while a DNA fragmentation factor ELISA detected DNA damage. To probe underlying mechanisms, pharmacological inhibitors of AKT (MK-2206) and Bcl-XL (A-1155463) were applied before Tβ4 treatment. LDH release was then measured to determine whether Tβ4’s protective effects depended on these signaling pathways [1].
The researchers also measured endothelial secretory activity and inflammatory phenotype. Supernatants from diabetic hiPSC-EC cultures were analyzed by Western blot for endothelin-1, a vasoconstrictor, and MMP-1, a marker of senescence-associated secretory phenotype (SASP). ICAM-1, an adhesion molecule linked to endothelial inflammation, was also evaluated by Western blot, normalized against GAPDH. Senescence itself was assessed two weeks after sorting through β-galactosidase staining, with intensity quantified by ImageJ. Western blot analysis was further applied to detect protein levels of p21, p53, and acetylated p53, all markers of cellular aging and stress response [1].
Mitochondrial function was evaluated by measuring membrane potential with JC-1 dye. Cells were stained and imaged, with red fluorescence intensity calculated per cell. Additionally, mitochondrial serine hydroxymethyltransferase (mSHMT) protein levels were determined by Western blot. Together, these assays provided insight into how Tβ4 influences diabetic endothelial bioenergetics and senescence. Western blotting was also used to study key signaling proteins involved in Tβ4’s effects. Total protein was extracted from hiPSC-ECs, quantified, and separated by SDS-PAGE before transfer to nitrocellulose membranes. Blots were probed with antibodies against GAPDH, phosphorylated AKT, total AKT, and Bcl-XL, followed by chemiluminescent detection. Expression levels were normalized to GAPDH and reported as relative percentages.
For in vivo experiments, Tβ4 was delivered using gelatin microspheres. Microspheres were generated by emulsifying gelatin in olive oil, cooling, and crosslinking with glutaraldehyde. After neutralization, washing, and drying, microspheres were loaded with Tβ4 by incubation with peptide solution. These served as a sustained-release system for local delivery. The therapeutic potential of Tβ4 was tested in a diabetic mouse model of hind-limb ischemia (HLI). Male KK.Cg-Ay mice, a diabetic strain, were screened for glucose intolerance by intraperitoneal glucose tolerance testing. Following confirmation of diabetes, mice underwent femoral artery ligation under anesthesia to induce limb ischemia. Animals were randomly divided into groups: untreated control, EC transplantation alone, defined as DP2-EC or DP3-EC, Tβ4-treated EC transplantation defined as DP2-EC+Tβ4 or DP3-EC+Tβ4, Tβ4-loaded microspheres alone, or basal medium injection. Endothelial cells for transplantation were pretreated with Tβ4 in vitro before injection. Treatments were administered intramuscularly three days after surgery at multiple sites around the ligated artery [1].
Limb perfusion recovery was monitored using laser Doppler imaging, with blood flow in the ischemic limb normalized to the contralateral limb. To assess neovascularization, immunohistochemistry was performed on limb tissue sections. Human CD31 staining identified transplanted cells, while staining for total CD31 and smooth muscle actin quantified vessel and arteriole density. These analyses allowed the researchers to evaluate whether Tβ4 enhanced vascular regeneration in ischemic diabetic limbs.
In summary, this study employed a comprehensive set of in vitro and in vivo experiments to examine the effects of Tβ4 on diabetic hiPSC-ECs. Assays addressed proliferation, angiogenesis, cytoprotection, inflammation, senescence, mitochondrial health, and signaling pathways. The in vivo HLI model extended these findings by testing whether Tβ4-treated cells or Tβ4 delivery systems could enhance vascular repair in diabetic ischemic tissue. Collectively, the methodology provides a robust framework to evaluate Tβ4 as a potential therapeutic agent for diabetic vascular complications [1].
2) This study conducted by Song et al employed a series of biochemical, cellular, and imaging techniques to investigate the role of Tβ4 in human cerebral endothelial cells (hCMEC/D3), particularly under conditions of prion protein (PrP) fragment exposure. The reagents included commercially sourced Tβ4, Tβ4-specific small interfering RNA (siRNA), and antibodies for proteins of interest such as ZO-1, occludin, Tβ4, and β-actin. Secondary antibodies conjugated with horseradish peroxidase were also used. The pathogenic prion peptide fragment PrP (106–126), which mimics the properties of infectious PrPSc, along with a scrambled control peptide, was synthesized with a purity of 80%. These peptides were dissolved in dimethyl sulfoxide at 10 mM and stored at –72 °C [2].
For cell culture, the hCMEC/D3 line was grown in EBM-2 medium supplemented with fetal bovine serum, penicillin–streptomycin, hydrocortisone, ascorbic acid, lipid concentrate, HEPES, and basic fibroblast growth factor. Cells were maintained at 37 °C in 5% CO₂. Treatments included 200 μM PrP (106–126) with or without Tβ4 at 0.5 μg/mL or Tβ4 siRNA. For gene silencing, Tβ4 siRNA or scrambled siRNA was transfected into confluent cells using DharmaFECT 1 reagent, following a 24-hour starvation period. The siRNA concentration used was 50 nM. Cell viability was assessed using the MTT assay. Cells were seeded into 96-well plates, exposed to the relevant treatments, and then incubated with MTT. This assay relies on mitochondrial enzymes converting MTT into a colored formazan product, which was quantified at 570 nm using spectrophotometry [2].
Cytotoxicity was evaluated through LDH release assays. LDH levels in culture supernatants served as a marker of cell damage. The LDH content was measured spectrophotometrically at 490 nm using a detection kit. For protein expression analysis, immunoblotting was performed. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with skim milk, membranes were incubated with primary antibodies overnight, followed by secondary antibodies. Detection was carried out with chemiluminescent substrate, and results were analyzed using imaging software.
The study also measured the ratio of filamentous actin (F-actin) to globular actin (G-actin) using a commercial assay kit. Cellular proteins were extracted, and actin fractions were isolated according to the kit instructions. Protein concentrations were quantified using the Bradford assay, and F-actin and G-actin contents were determined by immunoblotting. The F/G actin ratio was then calculated relative to controls. Phalloidin staining was conducted to visualize actin filaments. Cells were seeded in 96-well plates, treated with PrP and/or Tβ4, fixed, permeabilized, and blocked. They were then stained with fluorescently labeled phalloidin and Hoechst dye. Images were captured by fluorescence microscopy. For immunofluorescence assays, cells were fixed and permeabilized after treatment, then incubated overnight with antibodies against tight junction proteins ZO-1 and occludin. Fluorescently labeled secondary antibodies were applied, along with nuclear staining. Images were again obtained using fluorescence microscopy [2].
Finally, in vitro vascular permeability was assessed using a commercial trans-well assay. Cells were seeded on collagen-coated inserts and grown to confluence. Following treatment with Tβ4 and PrP, FITC-dextran was added to the upper chamber. The degree of permeability was determined by measuring fluorescence in the medium of the lower chamber. Together, these methods provided an experimental framework to determine how Tβ4 influences endothelial cell viability, cytotoxicity, cytoskeletal organization, tight junction integrity, and barrier function in the context of prion peptide challenge [2].

Discussion
1) This study performed by Su et al examined the effects of Tβ4 under both in vitro and in vivo conditions. The researchers first focused on Tβ4’s ability to activate AKT signaling and upregulate Bcl-XL, a key anti-apoptotic protein. A dose-dependent experiment was performed in which dia-hiPSC-ECs were exposed to increasing concentrations of Tβ4 including 300, 600, and 1000 ng/mL. Western blot analysis revealed that both 600 and 1000 ng/mL significantly enhanced AKT activity, while 300 and 600 ng/mL increased Bcl-XL expression. Based on these findings, 600 ng/mL was chosen as the optimal concentration for subsequent experiments [1].
To test whether Tβ4 could enhance angiogenic potential, tube formation assays were conducted using DP2-ECs and DP3-ECs cultured on Matrigel. Compared with untreated controls, Tβ4-treated cells formed more extensive tubular networks, with significantly greater numbers of nodes, junctions, and total branching length. Although there was a trend toward increased branch numbers, the difference was not statistically significant. These results indicated that Tβ4 improves the angiogenic potential of diabetic endothelial cells in vitro.
Next, the effect of Tβ4 on cell proliferation and senescence was evaluated. Both DP2-ECs and DP3-ECs treated with 600 ng/mL Tβ4 exhibited shorter doubling times, demonstrating enhanced proliferative capacity. Western blot analysis further showed increased expression of cyclin D2, a key regulator of cell cycle progression. At the same time, senescence was reduced, as indicated by decreased β-galactosidase staining in both cell lines following Tβ4 treatment. Interestingly, protein levels of p21, p53, and acetylated p53 did not change significantly, suggesting that Tβ4’s effects on proliferation and senescence were not mediated by these pathways [1].
The researchers also investigated whether Tβ4 reduced the secretion of molecules associated with endothelial dysfunction and senescence. Western blot analysis of culture supernatants showed that endothelin-1, a vasoconstrictor, and MMP-1, a marker of the SASP, were both significantly decreased in Tβ4-treated cells. This reduction suggested improved cellular viability and diminished senescence-associated signaling. However, not all dysfunction markers were affected. ICAM-1, an adhesion molecule linked to endothelial inflammation, remained unchanged after Tβ4 treatment. Similarly, neither mSHMT expression nor mitochondrial membrane potential was improved, indicating that Tβ4 did not influence these particular mitochondrial parameters in diabetic endothelial cells [1].
The cytoprotective effects of Tβ4 were further tested under hypoxic conditions. Dia-hiPSC-ECs exposed to low oxygen levels for 24 hours showed significant cellular damage, as measured by LDH leakage and DNA fragmentation. Treatment with Tβ4 at both 600 and 1000 ng/mL significantly reduced LDH release and DNA damage, demonstrating its protective effect. Moreover, Tβ4 enhanced AKT activity and Bcl-XL protein expression during hypoxia. Importantly, when inhibitors of AKT or Bcl-XL were applied, the protective effects of Tβ4 were completely abolished. These findings indicated that Tβ4 confers cytoprotection primarily by activating AKT signaling and upregulating Bcl-XL.
To assess the therapeutic potential of Tβ4-treated endothelial cells in vivo, a diabetic mouse model of HLI was employed. Mice received intramuscular injections of either untreated dia-hiPSC-ECs, Tβ4-treated dia-hiPSC-ECs combined with gelatin microspheres, Tβ4-loaded microspheres alone, or control medium. Blood perfusion recovery was monitored using laser Doppler imaging. Perfusion was markedly improved in groups treated with Tβ4-preconditioned endothelial cells delivered with microspheres. Specifically, perfusion in the DP2-EC+Tβ4-microsphere and DP3-EC+Tβ4-microsphere groups reached over 65–70%, compared to much lower recovery rates in control, untreated EC, or microsphere-only groups. Although Tβ4 microspheres alone modestly improved perfusion, they were significantly less effective than Tβ4-treated endothelial cell groups [1].
Histological analysis supported these findings. Cryosections stained for CD31, which labels both human and mouse endothelial cells, showed significantly higher vessel densities in ischemic limbs treated with Tβ4-preconditioned ECs compared to all control groups. Vessel density in the Tβ4-only group was also improved relative to controls but remained lower than in the Tβ4+EC groups. Interestingly, arteriole density did not differ significantly among groups, suggesting that Tβ4’s primary benefit was in capillary formation rather than arteriole development. Further immunostaining using human-specific CD31 demonstrated that transplanted dia-hiPSC-ECs integrated into ischemic tissues, contributing directly to capillary and, in some cases, arteriole formation. These results confirmed that transplanted diabetic endothelial cells can survive, integrate, and support neovascularization in ischemic limbs, and that preconditioning them with Tβ4 further enhances these regenerative properties [1].
In summary, Tβ4 at 600 ng/mL was found to be an optimal dose for improving endothelial cell viability, proliferation, angiogenesis, and resistance to hypoxic damage in vitro. These effects were largely mediated through the activation of AKT and the upregulation of Bcl-XL. Tβ4 also reduced markers of senescence and dysfunction such as endothelin-1 and MMP-1, though it did not affect ICAM-1 expression or mitochondrial parameters. In vivo, Tβ4-treated diabetic endothelial cells significantly improved perfusion recovery and vessel density in a diabetic mouse model of hind-limb ischemia, demonstrating enhanced angiogenic potential. Together, these findings suggest that Tβ4 is a promising therapeutic strategy for improving the function of diabetic endothelial cells and enhancing vascular repair in ischemic disease [1].
2) This study investigated the protective effects of Tβ4 on human cerebral endothelial cells (hCMEC/D3) exposed to the toxic prion protein fragment PrP (106–126). The experiments focused on cell viability, cytoskeletal regulation, tight junction protein expression, and vascular permeability.
The first set of experiments assessed whether Tβ4 could protect hCMEC/D3 cells from PrP (106–126)-induced toxicity. Cell viability was measured using both MTT and LDH assays. PrP (106–126) reduced cell viability in a dose-dependent manner, with concentrations ranging from 50 to 400 μM, while a scrambled control peptide showed no toxicity. At 200 μM, PrP (106–126) decreased viability to approximately 60% after 24 hours, so this dose was chosen for subsequent experiments. Tβ4 itself, when administered at concentrations from 0.01 to 0.5 μg/mL, did not induce cell death. However, pretreatment with Tβ4 at 0.1 or 0.5 μg/mL significantly restored viability in PrP (106–126)-treated cells in a dose-dependent fashion. LDH assays confirmed that these doses of Tβ4 also reduced cytotoxicity caused by PrP (106–126). Based on these results, 0.5 μg/mL Tβ4 and 200 μM PrP (106–126) were selected as the working concentrations for later studies [2].

Figure 1: Changes in cell viability in cells treated with PrP (106-126) and Tβ4-treated cells.
The researchers next explored the relationship between Tβ4 and actin cytoskeleton regulation, since Tβ4 is known to bind G-actin and prevent its polymerization into F-actin. Treatment with PrP (106–126) increased the F-actin to G-actin ratio in a dose-dependent manner, suggesting disrupted cytoskeletal balance. In contrast, treatment with Tβ4 alone decreased this ratio in a dose-dependent way. Importantly, Tβ4 reversed the PrP (106–126)-induced elevation of the F-actin to G-actin ratio, indicating that it helps maintain cytoskeletal homeostasis. These findings were supported by phalloidin staining, which showed that cells treated with both Tβ4 and PrP (106–126) exhibited reduced F-actin distribution compared to cells treated with PrP (106–126) alone. Together, these results highlight the role of Tβ4 in regulating cytoskeletal integrity in prion-challenged endothelial cells [2].
To further examine the contribution of endogenous Tβ4, cells were transfected with Tβ4 siRNA. Silencing Tβ4 expression reduced cell viability, especially when combined with PrP (106–126), compared to cells treated with the prion peptide alone. Conversely, adding exogenous Tβ4 rescued viability even under Tβ4 knockdown conditions. Protein expression assays confirmed that Tβ4 siRNA and PrP (106–126) both decreased Tβ4 levels, but exogenous Tβ4 restored its expression. Examination of the F-actin to G-actin ratio revealed that Tβ4 knockdown exaggerated the increase caused by PrP (106–126), further disrupting cytoskeletal homeostasis. Supplementation with external Tβ4 reversed this imbalance, reducing the F-actin to G-actin ratio and reinforcing the importance of Tβ4 in maintaining normal cytoskeletal regulation.
The study then evaluated the impact of Tβ4 on tight junction (TJ) proteins, which are essential for blood-brain barrier (BBB) integrity. Treatment with PrP (106–126) alone markedly reduced expression of the tight junction proteins ZO-1 and occludin. Knockdown of Tβ4 using siRNA further decreased these protein levels in PrP (106–126)-treated cells. However, supplementation with exogenous Tβ4 reversed this reduction, restoring ZO-1 and occludin expression. These findings suggest that Tβ4 plays a protective role in maintaining TJ protein levels under prion-induced stress [2].
Finally, the researchers tested whether Tβ4 could mitigate PrP (106–126)-induced increases in endothelial permeability, a hallmark of BBB disruption. An in vitro vascular permeability assay demonstrated that PrP (106–126) significantly increased permeability in hCMEC/D3 cultures. Cells treated with Tβ4 siRNA also exhibited increased permeability compared to controls, indicating that endogenous Tβ4 contributes to barrier stability. Importantly, exogenous Tβ4 treatment counteracted the heightened permeability observed in cells exposed to both Tβ4 siRNA and PrP (106–126). These results confirm that Tβ4 helps preserve BBB integrity by protecting tight junction proteins and reducing abnormal vascular permeability.
In summary, this study demonstrated that Tβ4 protects cerebral endothelial cells from PrP (106–126)-induced damage by multiple mechanisms. It enhances cell viability, reduces cytotoxicity, and stabilizes the actin cytoskeleton by maintaining the balance between F-actin and G-actin. Both endogenous and exogenous Tβ4 play critical roles in this regulation. Furthermore, Tβ4 preserves the expression of tight junction proteins ZO-1 and occludin, thereby preventing disruption of the endothelial barrier. Functional assays confirmed that Tβ4 reduces prion-induced increases in vascular permeability, reinforcing its protective effects on BBB integrity. Collectively, these findings highlight the therapeutic potential of Tβ4 in mitigating endothelial dysfunction and maintaining barrier stability in the face of prion-related toxicity [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Su L, Kong X, Loo S, et al. Thymosin beta-4 improves endothelial function and reparative potency of diabetic endothelial cells differentiated from patient induced pluripotent stem cells. Stem Cell Res Ther. 2022;13(1):13. Published 2022 Jan 10. doi:10.1186/s13287-021-02687-x

[2] Song K, Han HJ, Kim S, Kwon J. Thymosin beta 4 attenuates PrP(106-126)-induced human brain endothelial cells dysfunction. Eur J Pharmacol. 2020;869:172891. doi:10.1016/j.ejphar.2019.172891

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