Gabriel Sosne

Biological activities of thymosin β4 defined by active sites in short peptide sequences

Thymosin ß4, a small ubiquitous protein containing 43 aa, has structure/function activity via its actin‐binding domain and numerous biological affects on cells. Since it is the major actin‐sequestering molecule in eukaryotic cells and is found essentially in all cells and body fluids, thymosin ß4 has the potential for significant roles in tissue development, maintenance, repair, and pathology. Several active sites with unique functions have been identified, including the amino‐terminal site containing 4 aa (Ac‐SDKP) that generally blocks inflammation and reduces fibrosis. Another active site at the amino terminus contains 15 aa, including Ac‐SDKP, and promotes cell survival and blocks apoptosis, while a short sequence containing LKKTETQ, the central actin‐binding domain (aa 17–23) plus 1 additional amino acid (Q), promotes angiogenesis, wound healing, and cell migration. Several additional biological activities have been identified but not yet localized in the molecule, including its antimicrobial activity, the induction of various genes (including laminin‐5, MMPs, TGF ß, zyxin, terminal deoxynucleotidyl transferase, and angiogenesis‐related proteins), and the ability to activate ILK/PINCH/Akt, and other signaling molecules important in both apoptosis and inflammatory pathways. This review details these important physiologically and pathologically active sites and their potential therapeutic uses.—Sosne, G., Qiu, P., Goldstein, A L., Wheater. M. Biological activities of thymosin β4 defined by active sites in short peptide sequences. FASEB J. 24, 2144–2151 (2010). www.fasebj.org

Thymosin β4: a multi-functional regenerative peptide. Basic properties and clinical applications.

Introduction: Thymosin β4, a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin β4, is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin β4 binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue. Thymosin β4 also decreases the number of myofibroblasts in wounds, resulting in decreased scar formation and fibrosis Areas covered: This article will cover the many thymosin β4 activities that directly affect the repair and regeneration cascade with emphasis on its therapeutic uses and potential. Our approach has been to evaluate the basic biology of the molecule as well as its potential for clinical applications in the skin, eye, heart and brain. Expert opinion: The considerable advances in our understanding of the functional biology and mechanisms of action of thymosin β4 have provided the scientific foundation for ongoing and projected clinical trials in the treatment of dermal wounds, corneal injuries and in the regeneration and repair of heart and CNS tissue following ischemic insults and trauma.

Thymosin β4 inhibits TNF-α-induced NF-κB activation, IL-8 expression, and the sensitizing effects by its partners PINCH-1 and ILK

The mechanisms by which thymosin β 4 (Tβ4) regulates the inflammatory response to injury are poorly understood. Previously, we demonstrated that ectopic Tβ4 treatment inhibits injury‐induced proin‐flammatory cytokine and chemokine production. We have also shown that Tβ4 suppresses TNF‐α‐mediated NF‐κB activation. Herein, we present novel evidence that Tβ4 directly targets the NF‐κB RelA/p65 subunit. We find that enforced expression of Tβ4 interferes with TNF‐α‐mediated NF‐κB activation, as well as downstream IL‐8 gene transcription. These activities are independent of the G‐actin‐binding properties of Tβ4. Tβ4 blocks RelA/p65 nuclear translocation and targeting to the cognate kB site in the proximal region of the IL‐8 gene promoter. Tβ4 also inhibits the sensitizing effects of its intracellular binding partners, PINCH‐1 and ILK, on NF‐κB activity after TNF‐α stimulation. The identification of a functional regulatory role by Tβ4 and the focal adhesion proteins PINCH‐1 and ILK on NF‐κB activity in this study opens a new window for scientific exploration of how Tβ4 modulates inflammation. In addition, the results of this study serve as a foundation for developing Tβ4 as a new anti‐inflammatory therapy.—Qiu, P., Kurpakus Wheater, M., Qiu, Y., Sosne, G. Thymosin β4 inhibits TNF‐α‐induced NF‐κB activation, IL‐8 expression, and the sensitizing effects by its partners PINCH‐1 and ILK FASEB J. 25, 1815‐1826 (2011). www.fasebj.org

Thymosin β4 promotes human conjunctival epithelial cell migration

Purpose. In this study the effects of thymosin ß4 (Tß4) on migration and production of laminin-5 in the human conjunctival cell line HC0597 was analyzed. Methods. Boyden chamber assays assessed the ability of Tß4 to stimulate in vitro cell migration. Control or Tß4-treated cells were processed for immunofluorescence microscopy using antibodies to vinculin or laminin-5. Cell lysates were processed for Western blot and densitometric analysis using antibodies to laminin-5 a3 or ?2 chains. Results. Tß4 stimulated migration in a dose-dependent manner. Focal adhesions present in Tß4-treated cells were smaller and more rounded compared to the “streaks” characteristic of controls. Western blot analysis and densitometry revealed that Tß4-treated cells expressed more laminin-5 a3 and ?2 chain protein. Conclusions. Tß4 stimulates in vitro conjunctival epithelial cell migration, and results in altered focal adhesion formation and increased extracellular laminin-5 deposition. The increased migration may be correlated with increased production of laminin-5.

Treatment of chronic nonhealing neurotrophic corneal epithelial defects with thymosin β4

Neurotrophic corneal defects are difficult to heal and all too often lead to scarring and vision loss. Medical management is often of limited success. We describe the results of nine patients (ages 37–84) with chronic nonhealing neurotrophic corneal epithelial defects who were treated with thymosin beta 4 (Tβ4) sterile eye drops for 28 or 49 days with a follow‐up period of 30 days. Those with geographic defects (six patients) showed dramatic healing without clinically significant neovascularization. Stromal thinning was observed in one patient. Three patients with punctate epithelial defects did not have a demonstrable change in their clinical findings. Reduced ocular irritation was reported by all patients soon after treatment initiation. Results from these compassionate use cases indicate that Tβ4 may provide a novel, topical approach to wound healing in chronic nonhealing neurotrophic corneal ulcers.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration.

Studies from our laboratory provide substantial evidence that thymosin beta 4, (Tβ4), an actin‐sequestering protein, promotes corneal wound healing through its ability to stimulate epithelial cell migration. Matrix metalloproteinases (MMPs), which are expressed in a wide variety of tissues including the cornea, also play a key role in epithelial cell migration and wound healing. In this study we investigated the role of MMPs in Tβ4‐stimulated corneal epithelial cell migration. In Boyden chamber assays, XG076, an inhibitor of the conversion of pro‐ to active MMPs, had no effect on epithelial cell migration stimulated by exogenous activated MMP‐1. However, in in vitro migration assays where the activation of pro‐MMPs was blocked, XG076 significantly inhibited cell migration and wound healing in the presence or absence of Tβ4. GM6001, a broad‐spectrum inhibitor of active MMPs and selective MMP inhibitors, also suppressed Tβ4‐stimulated cell migration. Tβ4 upregulated MMP‐1 gene and protein expression in primary human corneal epithelial cells and in transformed human corneal epithelial cells following scrape wounding. From these results we conclude that MMP catalytic activity is necessary for Tβ4 promotion of epithelial cell migration. These novel findings are the first to demonstrate a functional link between the two. J. Cell. Physiol. 212: 165–173, 2007.

Thymosin β4 and corneal wound healing: visions of the future

Persistent corneal epithelial defects and inflammation within the central cornea can directly distort visual acuity and may lead to permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents such as corticosteroids would result in improved clinical outcome and would represent a major advance in the field. Despite much progress in the areas of corneal wound healing research, clinically available pharmacological therapies that can promote repair and limit the visual complications from persistent corneal wounds are severely limited and remains a major deficiency in the field. Prior studies from our laboratory have demonstrated the potent wound healing and anti‐inflammatory effects of thymosin β4 (Tβ4; Tβ4) in numerous models of corneal injury. We are studying the mechanisms by which Tβ4 suppresses inflammation and promotes repair. Herein, we discuss some of our new basic scientific directions that may lead to the use of Tβ4 as a novel corneal wound healing and anti‐inflammatory therapy.

Thymosin β-4 and the Eye

Abstract:  The cornea epithelium responds to injury by synthesizing several cytokines, growth factors, and tissue remodeling molecules. Proinflammatory cytokines have been implicated in the inflammation that follows corneal epithelial injury and cytokine‐mediated processes play a significant role in corneal epithelial wound healing. Poorly regulated corneal inflammatory reactions that occur after injury can retard healing. In turn, persistent corneal epithelial defects and inflammation may lead to ocular morbidity and permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents, such as corticosteroids, would result in improved clinical outcome and would represent a major advance in the field. Evidence is mounting to support the idea that thymosin β‐4 (Tβ‐4) has multiple, seemingly diverse, cellular functions. In the cornea, as in other tissues, Tβ‐4 promotes cell migration and wound healing, has anti‐inflammatory properties, and suppresses apoptosis. Prior studies from our laboratory have demonstrated the potent wound healing and anti‐inflammatory effects of Tβ‐4 in numerous models of corneal injury. Recently, we demonstrated that Tβ‐4 suppresses the activation of the transcription factor, nuclear factor‐kappa b (NF‐κB) in TNF‐α‐stimulated cells. TNF‐α initiates cell signaling pathways that converge on the activation of NF‐κB, thus both are known mediators of the inflammatory process. These results have important clinical implications for the potential role of Tβ‐4 as a corneal anti‐inflammatory and wound‐healing agent.

Treatment of chronic nonhealing neurotrophic corneal epithelial defects with thymosin beta 4.

Neurotrophic keratopathy is a degenerative disease of the corneal epithelium and stroma that results from impaired corneal innervation. Reduced corneal sensitivity is responsible for producing recurring or chronic epithelial defects that may lead to subsequent ulceration and/or perforation. It is most frequently associated with topical medications, long-standing diabetes mellitus, herpes zoster ophthalmicus (HZO), herpes simplex keratitis, neurologic disease, or localized trauma. Conventional treatments include prophylactic topical antibiotic drops or ointment, frequent nonpreserved ocular lubricants, patching, and bandage contact lenses. In recalcitrant cases, oral doxycycline, autologous serum, and the surgical application of an amniotic membrane, tarsorrhaphy, or a conjunctival flap are used alone or in combination. Successful modulation healing in these patients is erratic at best and vexing for both the patient and ophthalmologist. The potent wound healing and anti-inflammatory effects of thymosin beta 4, a naturally occurring, 43–amino acid, G-actin– sequestering molecule, has been demonstrated in numerous animal and cellular models of corneal injury. We sought to evaluate thymosin beta 4 in a human disorder that did not have an infectious component or one in which stem cell dysfunction or conjunctival disruption was extensive. A preliminary unpublished evaluation of thymosin beta 4 in diabetic corneal defects was encouraging. Here we describe the treatment results of 4 patients with chronic neurotrophic corneal epithelial defects who were treated under a Food and Drug Administration investigational new drug compassionate use protocol (approved by the Wayne State University Human Investigation Committee) with a sterile, single-dose, nonpreserved, ophthalmic formulation of thymosin beta 4 eye drops supplied by RegeneRx Biopharmaceuticals, Inc (Rockville, Maryland).