Main Research Findings

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

2) Treatment with Tβ4 was found to induce angiogenesis in cases of critical limb ischemia.

Selected Data
1) This study conducted by the research team of 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. All other chemicals were analytical grade [1].
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.
Cytotoxicity was evaluated through lactate dehydrogenase (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 [1].
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 [1].
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. 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. 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 [1].
2) This study was conducted by the research team of Shumin et al and utilized human umbilical vein endothelial cells (HUVECs) and 293T/17 cells that were obtained from the American Type Culture Collection for experimental work. Both cell types were maintained in DMEM supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified environment containing 5% CO₂. The 293T/17 cells were used exclusively for the construction of theTβ4 overexpression lentiviral vector, while HUVECs were utilized in downstream functional assays, including MTT viability testing, tube formation, western blotting, and immunofluorescence [2].
For the vector construction, specific Tβ4 sequences were amplified and ligated into a pLJM1 plasmid. Following ligation, the constructs were transformed into competent E. coli cells and cultured on LB medium to allow colony growth. Colonies containing the desired plasmids were selected, and plasmid DNA was extracted. As a negative control, an empty pLJM1 plasmid was also prepared. After obtaining the plasmids, they were co-transfected with packaging vectors psPAX2 and pmD2.G into 293T/17 cells using Lipofectamine 2000. After a brief incubation, the medium was refreshed, and cells were cultured for 48 hours to allow viral particle production. The resulting lentiviral supernatant was harvested, filtered, concentrated, and stored at –80°C for subsequent use in infection experiments.
HUVECs were seeded into culture plates and infected with the Tβ4 overexpression lentivirus. Cells were first exposed to serum-free DMEM to increase infection efficiency and then incubated with the lentiviral solution for six hours. Afterward, the viral medium was replaced with fresh complete medium, and cells were cultured for an additional 48 hours. This ensured robust transgene expression. To further investigate the signaling pathways involved, HUVECs were treated with pharmacological inhibitors. DAPT, an inhibitor of the Notch signaling pathway, and BMS-345541, an inhibitor of NF-κB signaling, were applied to infected cells. After seeding and attachment, HUVECs were exposed to 10 μM DAPT or 1 μM BMS for 48 hours. These concentrations were selected based on prior studies. Treated cells were then subjected to assays designed to probe the functional consequences of Tβ4 expression and pathway inhibition [2].
Cell viability was assessed using an MTT assay. Infected HUVECs were plated into 96-well plates and incubated with an MTT reagent for four hours. Following solubilization with DMSO, absorbance at 570 nm was measured to quantify metabolic activity. Tube formation assays were also performed to assess angiogenic potential. Infected HUVECs were seeded onto ECM gel–coated plates and monitored for capillary-like structure formation. Tube networks were visualized and photographed under a microscope. To evaluate cell migration, wound healing assays were conducted. Confluent HUVEC monolayers were scratched with a pipette tip to generate a vertical wound. Cells were then cultured in serum-free medium, and images were taken at baseline and after 48 hours. Migration into the wound area was analyzed using ImageJ software [2].
Immunofluorescence staining was employed to assess NF-κB/p65 localization. Following infection, HUVECs were fixed, permeabilized, and incubated with anti-p65 antibodies, followed by fluorescent secondary antibodies. Nuclei were counterstained with DAPI, and the samples were examined using a fluorescence microscope. This allowed visualization of pathway activation at the cellular level.
The in vivo portion of the study involved establishing a critical limb ischemia (CLI) model in C57BL/6J mice. Eighty male mice were randomly divided into eight groups, including sham-operated, model, negative control, Tβ4, and combinations of Tβ4 or control with DAPT or BMS treatments. CLI was induced by ligation and excision of the right femoral artery and vein, including the superficial, deep, and saphenous branches. Pain management included buprenorphine administration, and animals were monitored postoperatively. On the seventh day after surgery, animals were euthanized, and gastrocnemius muscles from the ischemic limb were collected for analysis. Lentiviral injections of either control or Tβ4 were administered 14 days before surgery. In groups receiving pharmacological inhibitors, DAPT or BMS was administered daily after model establishment [2].
Western blotting was performed to analyze protein expression in both HUVECs and mouse tissue samples. Cell lysates were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against angiogenesis-related proteins such as Ang2, Tie2, VEGF-A, and signaling pathway markers including Notch intracellular domain (N1ICD), p65, and phosphorylated p65. GAPDH was used as a loading control. Protein bands were detected using chemiluminescence, and expression levels were quantified with image analysis software.
Gene expression analysis was conducted using reverse transcription-quantitative PCR (RT-qPCR). RNA was isolated from cells and mouse tissues, purified, and reverse-transcribed to cDNA. qPCR was then performed to measure relative gene expression, normalized to internal controls, and further analyzed. Immunohistochemistry was employed to evaluate angiogenesis in mouse muscle tissue. Gastrocnemius samples were paraffin-embedded, sectioned, and stained with antibodies against CD31 as a marker of capillaries and α-SMA as a marker of arterioles. The density of positive cells was quantified under a microscope by calculating the ratio of positive to total cells across randomly selected fields [2].
In conclusion, the methods detailed in this study combined in vitro and in vivo approaches to investigate the role of Tβ4 in endothelial function and angiogenesis. The use of lentiviral-mediated Tβ4 overexpression, coupled with pharmacological pathway inhibitors, allowed the researchers to dissect mechanisms involving Notch and NF-κB signaling. Multiple assays including viability, migration, tube formation, western blotting, qPCR, immunofluorescence, and immunohistochemistry were employed to comprehensively evaluate the cellular and molecular effects of Tβ4 in both cultured endothelial cells and a murine model of critical limb ischemia [2].

Discussion
1) 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 [1].

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 [1].
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 [1].
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 [1].
2) Tβ4 has been shown to significantly enhance endothelial cell functions related to angiogenesis, cell viability, and migration, largely through its effects on the Notch/NF-κB signaling pathway. In cultured HUVECs, Tβ4 overexpression was first confirmed by RT-qPCR, and functional assays including MTT, tube formation, and wound healing tests were performed. Compared with negative control cells, Tβ4-treated cells exhibited a notable increase in expression levels of the peptide, which was accompanied by a significant rise in HUVEC viability, angiogenic activity, and migratory ability. These findings suggested that Tβ4 exerts strong pro-angiogenic and pro-survival effects in vitro [2].
To further explore the molecular basis of these effects, the study performed by Shumin et al examined whether Tβ4 influences angiogenesis-related factors and whether the Notch/NF-κB pathway is involved. Both western blotting and RT-qPCR revealed that Tβ4 markedly increased the expression of Ang2, Tie2, and VEGF-A at both transcriptional and translational levels. In addition, Tβ4 upregulated the protein expression of Notch intracellular domain and Notch3, as well as the nuclear expression of NF-κB/p65. These findings indicated that the angiogenic effects of Tβ4 may be mediated by activation of Notch and NF-κB signaling.
To confirm this mechanism, inhibitors of the Notch and NF-κB pathways were applied. Tβ4 alone increased expression of N1ICD and phosphorylated p65, as well as the ratio of p-p65 to total p65, but these effects were reversed when cells were co-treated with DAPT or BMS. The same inhibitory effect was observed for cell viability: the increase in HUVEC viability induced by Tβ4 was blocked when the Notch or NF-κB pathways were inhibited. These results demonstrated that Tβ4’s enhancement of both endothelial viability and pathway-related protein expression is dependent on Notch/NF-κB signaling [2].
The pro-angiogenic and pro-migratory functions of Tβ4 were also confirmed through tube formation and wound healing assays. Tβ4 significantly increased angiogenesis and migration rates in HUVECs, but these effects were diminished following treatment with DAPT or BMS. Similarly, Tβ4’s ability to upregulate angiogenesis-related factors Ang2, Tie2, and VEGF-A was reduced when either inhibitor was applied. Together, these findings further emphasized that the Notch/NF-κB pathway is essential for the actions of Tβ4 on endothelial angiogenesis and migration.
To validate the in vitro results, in vivo experiments were conducted using a mouse model of CLI. In the gastrocnemius tissue of the ischemic hind limb, Tβ4 expression was significantly elevated in the treated group compared with controls. Histological analyses revealed that both capillary density and arteriolar density were increased in the Tβ4 group, whereas these vascular structures were reduced in untreated model and Nc groups. Importantly, the angiogenic effects of Tβ4 in vivo were attenuated when mice were treated with DAPT or BMS, indicating again that the Notch/NF-κB pathway mediates Tβ4’s vascular benefits [2].
The expression of angiogenesis-related proteins in CLI mice was also consistent with the in vitro findings. Tβ4 significantly elevated Ang2, Tie2, and VEGF-A expression at both protein and gene levels, but these effects were reversed with DAPT or BMS treatment. Moreover, Tβ4 enhanced the expression of N1ICD, phosphorylated p65, and the p-p65/p65 ratio, all of which were decreased by pathway inhibition. These results confirmed that the Notch/NF-κB pathway plays a central role in mediating the pro-angiogenic activity of Tβ4 in ischemic tissues [2].
In summary, Tβ4 enhances HUVEC viability, angiogenesis, and migration, and these effects are closely tied to its ability to activate angiogenesis-related factors and the Notch/NF-κB signaling pathway. Inhibition of these pathways diminishes Tβ4’s beneficial actions, underscoring their role in mediating its activity. Furthermore, in a mouse model of critical limb ischemia, Tβ4 increased capillary and arteriolar densities and upregulated angiogenic proteins, again through Notch/NF-κB signaling. Together, these findings provide strong evidence that Tβ4 is a potent pro-angiogenic peptide whose therapeutic potential in ischemic and vascular diseases is mediated by regulation of the Notch/NF-κB pathway [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] 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

[2] Lv S, Cai H, Xu Y, Dai J, Rong X, Zheng L. Thymosin‑β 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF‑κB pathway. Int J Mol Med. 2020;46(4):1347-1358. doi:10.3892/ijmm.2020.4701

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