Search Results (24)

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. Full article

Background/Objectives: Tumor cell migration depends on the actin cytoskeleton modified by actin-binding proteins (ABPs). Overexpression of cofilin or thymosin beta4 (Tß4) has been correlated with an increase or decrease in their migratory activity, respectively. Methods: Immunostaining of tumor cells and transfection with EGFP-tagged cofilin or bicistronic vectors leading to independent expression of EGFP and Tß4. Determination of cell migration by transwell or agarose drop assay. Results: We modulated by transfection the intracellular concentrations of cofilin and Tß4 of two colon (3LNLN and EB3) and one breast carcinoma (MDA-MB-231) cell line and analyzed their migratory activity. Increasing wild-type cofilin did not alter their migratory activity, whereas the constitutively active S3A–cofilin mutant elevated migration. Transfection leading to an up- or downregulation of Tß4 showed that MDA-MB-231 and 3LNLN cells responded with a decrease or increase in migration, respectively. Exposure of MDA-MB-231 and 3LNLN cells to increasing concentrations of extracellular Tβ4 (or His-tagged Tß4) induced a biphasic response of migration, being highest around 0.24 µM and decreased at higher extracellular Tß4. Immunostaining of 3LNLN cells exposed to 0.24 µM extracellular His-tagged Tß4 with anti-His antibody indicated its uptake co-localizing with integrin-linked kinase at cell attachment points. Furthermore, the exposure to 0.24 µM His-tagged Tß4 led to increased phosphorylation of AKT1/2 and secretion of matrix metalloproteases. These effects and tumor cell migration were abrogated after exposure of 3LNLN cells to 2.8 µM His-Tß4, also inducing apoptosis in a number of cells. Conclusions: Tumor cell migration can be inhibited by high extracellular Tß4. Full article

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. Full article

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tβ4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell–substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tβ4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tβ4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tβ4/VIP. These results strongly support the premise that Tβ4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea. Full article

Liver fibrosis is the most common feature of liver disease, and activated hepatic stellate cells (HSCs) are the main contributors to liver fibrosis. Thus, finding key targets that modulate HSC activation is important to prevent liver fibrosis. Previously, we showed that thymosin β4 (Tβ4) influenced HSC activation by interacting with the Hedgehog pathway in vitro. Herein, we generated Tβ4 conditional knockout (Tβ4-flox) mice to investigate in vivo functions of Tβ4 in liver fibrosis. To selectively delete Tβ4 in activated HSCs, double-transgenic (DTG) mice were generated by mating Tβ4-flox mice with α-smooth muscle actin (α-Sma)-Cre-ER^T2 mice, and these mice were administered carbon tetrachloride (CCl₄) or underwent bile duct ligation to induce liver fibrosis. Tβ4 was selectively suppressed in the activated HSCs of DTG mouse liver, and this reduction attenuated liver injury, including fibrosis, in both fibrotic models by repressing Hedgehog (Hh) signaling. In addition, the re-expression of Tβ4 by an adeno-associated virus reversed the effect of HSC-specific Tβ4 deletion and led to liver fibrosis with Hh activation in CCl₄-exposed mice treated with tamoxifen. In conclusion, our results demonstrate that Tβ4 is a crucial regulator of HSC activation, suggesting it as a novel therapeutic target for curing liver fibrosis. Full article

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. Full article

Wound healing responses play a major role in chronic inflammation, which affects millions of people around the world. One of the daunting tasks of creating a wound-healing drug is finding equilibrium in the inflammatory cascade. In this study, the molecular and cellular mechanisms to regulate wound healing are explained, and recent research is addressed that demonstrates the molecular and cellular events during diabetic wound healing. Moreover, a range of factors or agents that facilitate wound healing have also been investigated as possible targets for successful treatment. It also summarises the various advances in research findings that have revealed promising molecular targets in the fields of therapy and diagnosis of cellular physiology and pathology of wound healing, such as neuropeptides, substance P, T cell immune response cDNA 7, miRNA, and treprostinil growth factors such as fibroblast growth factor, including thymosin beta 4, and immunomodulators as major therapeutic targets. Full article

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. At present, the mortality rate of the deadly disease is still very high, while the existing treatments only delay the progression of the disease and improve the quality of life of patients. Lung cancer (LC) is the most fatal disease in the world. In recent years, IPF has been considered to be an independent risk factor for the development of LC. The incidence of lung cancer is increased in the patients with IPF and the mortality is also significantly increased in the patients inflicted with the two diseases. In this study, we evaluated an animal model of pulmonary fibrosis complicated with LC by implanting LC cells orthotopically into the lungs of mice several days after bleomycin induction of the pulmonary fibrosis in the same mice. In vivo studies with the model showed that exogenous recombinant human thymosin beta 4 (exo-rhTβ4) alleviated the impairment of lung function and severity of damage of the alveolar structure by the pulmonary fibrosis and inhibited the proliferation of LC tumor growth. In addition, in vitro studies showed that exo-rhTβ4 inhibited the proliferation and migration of A549 and Mlg cells. Furthermore, our results also showed that rhTβ4 could effectively inhibit the JAK2-STAT3 signaling pathway and this might exert an anti-IPF-LC effect. The establishment of the IPF-LC animal model will be helpful for the development of drugs for the treatment of IPF-LC. Exogenous rhTβ4 can be potentially used for the treatment of IPF and LC. Full article

Risk signals are characteristic of many common inflammatory diseases and can function to activate nucleotide-binding oligomerization (NLR) family pyrin domain-containing 3 (NLRP3), the innate immune signal receptor in cytoplasm. The NLRP3 inflammasome plays an important role in the development of liver fibrosis. Activated NLRP3 nucleates the assembly of inflammasomes, leading to the secretion of interleukin (IL)-1β and IL-18, the activation of caspase-1, and the initiation of the inflammatory process. Therefore, it is essential to inhibit the activation of the NLRP3 inflammasome, which plays a vital role in the immune response and in initiating inflammation. RAW 264.7 and LX-2 cells were primed with lipopolysaccharide (LPS) for 4 h and subsequently stimulated for 30 min with 5 mM of adenosine 5′-triphosphate (ATP) to activate the NLRP3 inflammasome. Thymosin beta 4 (Tβ4) was supplemented to RAW264.7 and LX-2 cells 30 min before ATP was added. As a result, we investigated the effects of Tβ4 on the NLRP3 inflammasome. Tβ4 prevented LPS-induced NLRP3 priming by inhibiting NF-kB and JNK/p38 MAPK expression and the LPS and ATP-induced production of reactive oxygen species. Moreover, Tβ4 induced autophagy by controlling autophagy markers (LC3A/B and p62) through the inhibition of the PI3K/AKT/mTOR pathway. LPS combined with ATP significantly increased thee protein expression of inflammatory mediators and NLRP3 inflammasome markers. These events were remarkably suppressed by Tβ4. In conclusion, Tβ4 attenuated NLRP3 inflammasomes by inhibiting NLRP3 inflammasome-related proteins (NLRP3, ASC, IL-1β, and caspase-1). Our results indicate that Tβ4 attenuated the NLRP3 inflammasome through multiple signaling pathway regulations in macrophage and hepatic stellate cells. Therefore, based on the above findings, it is hypothesized that Tβ4 could be a potential inflammatory therapeutic agent targeting the NLRP3 inflammasome in hepatic fibrosis regulation. Full article

The thymus is the most sensitive organ under various pathophysiological conditions, such as aging, starvation, and infection. As a key stromal cell for T cell development, it is well-known that thymic epithelial cells (TECs) play an important role in the thymus response to the external environment. Thymosin beta 15 (Tβ15) is a G-actin binding protein secreted by TECs, it plays an important role in maintaining the dynamic balance of actin, angiogenesis, axonal formation, and wound healing, but the relationship between Tβ15 and TECs is not clear yet. Here, we show the impact of Tβ15 on the TEC’s spatial development, as well as the T-cell differentiation and thymic output. As a result, TEC is the main effector cell of Tβ15 in the thymus. Tβ15 OX inhibits the chemotaxis of TECs to the medulla and subsequently blocks the positive selection of thymocytes from CD3⁺TCRβ⁺CD4⁺CD8⁺ double positive cells to CD3⁺TCRβ⁺CD4⁺CD8⁻ single-positive (CD4SP) cells. Tβ15-knockdown accelerates the reticular differentiation of astral TECs and medullary TECs. Importantly, mice implanted with Tβ15-knockdown iTECs show high thymic output but low peripheral T cell maturity and activity. In a word, our results explain the role of Tβ15 on the differentiation and function of TECs and provide a new perspective for understanding the process of thymus development and degeneration. Full article

Alzheimer’s disease (AD) represents the most frequent type of dementia in elderly people. There are two major forms of the disease: sporadic (SAD)—whose causes are not completely understood—and familial (FAD)—with clear autosomal dominant inheritance. The two main hallmarks of AD are extracellular deposits of amyloid-beta (Aβ) peptide and intracellular deposits of the hyperphosphorylated form of the tau protein (P-tau). An ever-growing body of research supports the infectious hypothesis of sporadic forms of AD. Indeed, it has been documented that some pathogens, such as herpesviruses and certain bacterial species, are commonly present in AD patients, prompting recent clinical research to focus on the characterization of antimicrobial peptides (AMPs) in this pathology. The literature also demonstrates that Aβ can be considered itself as an AMP; thus, representing a type of innate immune defense peptide that protects the host against a variety of pathogens. Beyond Aβ, other proteins with antimicrobial activity, such as lactoferrin, defensins, cystatins, thymosin β4, LL37, histatin 1, and statherin have been shown to be involved in AD. Here, we summarized and discussed these findings and explored the diagnostic and therapeutic potential of AMPs in AD. Full article

Thymosin β4 (Tβ4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tβ4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tβ4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tβ4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron²⁺ and iron³⁺ binding regions along the peptide and show that the presence of Tβ4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tβ4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tβ4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tβ4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tβ4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies. Full article

Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tβ4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tβ4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tβ4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tβ4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tβ4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tβ4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study. Full article

Our previous work has shown that topical thymosin beta 4 (Tβ4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tβ4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tβ4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tβ4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tβ4 alone and adjunctive Tβ4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tβ4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study. Full article

Our dream of defeating the processes of aging has occupied the curious and has challenged scientists globally for hundreds of years. The history is long, and sadly, the solution is still elusive. Our endeavors to reverse the magnitude of damaging cellular and molecular alterations resulted in only a few, yet significant advancements. Furthermore, as our lifespan increases, physicians are facing more mind-bending questions in their routine practice than ever before. Although the ultimate goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. As our initial approach to enhance the endogenous restorative capacity by delivering exogenous progenitor cells appears limited, we propose, utilizing small molecules critical during embryonic development may prove to be a powerful tool to increase regeneration and to reverse the processes associated with aging. In this review, we introduce Thymosin beta-4, a 43aa secreted peptide fulfilling our hopes and capable of numerous regenerative achievements via systemic administration in the heart. Observing the broad capacity of this small, secreted peptide, we believe it is not the only molecule which nature conceals to our benefit. Hence, the discovery and postnatal administration of developmentally relevant agents along with other approaches may result in reversing the aging process. Full article