Thymosin beta 4 induces hair growth via stem cell migration and differentiation.

Thymosin beta 4 is a small 43-amino-acid molecule that has multiple biological activities, including promotion of cell migration angiogenesis, cell survival, protease production, and wound healing. We have found that thymosin beta 4 promotes hair growth in various rat and mice models including a transgenic thymosin beta 4 overexpressing mouse. We have also determined the mechanism by which thymosin beta 4 acts to promote hair growth by examining its effects on follicle stem cell growth, migration, differentiation, and protease production.

Inhibition of VEGF receptors significantly impairs mammary cancer growth in C3(1)/Tag transgenic mice through antiangiogenic and non-antiangiogenic mechanisms.

Cancer growth and progression is often critically influenced by the production of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. VEGF produced by tumor cells stimulates endothelial cell growth through the binding and activation of the KDR/Flk-1 receptor (VEGFR-2) on endothelial cells. Recently, some human breast cancer epithelial cells have been shown to express VEGF receptors, suggesting a potential autocrine-mediated growth stimulation of a subset of cancers by VEGF. We demonstrate that mammary tumors in the C3(1)/Tag transgenic model express VEGF and VEGF receptors and tumor growth is stimulated by this autocrine mechanism. GW654652, an indazolylpyrimidine, is a VEGFRs tyrosine kinase inhibitor that dramatically reduces both angiogenesis and tumor cell growth in this model, as demonstrated using both in vitro and in vivo assays. GW654652 significantly decreased cell proliferation and induced apoptosis in human umbilical vein endothelial cells and M6 mammary tumor cells derived from C3(1)/Tag (Tag: simian virus 40 T-antigen) transgenic mice. A 75% reduction in VEGF-induced angiogenesis was observed with GW654652 using the chick chorioallantoic membrane assay, whereas GW654652 produced an approximately 85% reduction in angiogenesis as assessed by the Matrigel plug assay. A profound inhibitory effect on tumor growth in the C3(1)/Tag transgenic model of human breast cancer was observed with oral administration of GW654652 as measured by delayed tumor onset, decreased multiplicity, reduced tumor volume, and extended animal survival. The antitumor effects of GW654652 were associated with reduced tumor vascularization and no apparent toxicity. Tumor growth, however, rapidly advanced following cessation of treatment. This is the first demonstration that a VEGF receptor inhibitor, GW654652, has a strong inhibitory effect on angiogenesis and tumor progression in a transgenic model of mammary cancer, suggesting that this is a useful approach for preclinical testing of such agents.

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

The actin binding site on thymosin beta4 promotes angiogenesis.

Thymosin beta4 is a ubiquitous 43 amino acid, 5 kDa polypeptide that is an important mediator of cell proliferation, migration, and differentiation. It is the most abundant member of the beta-thymosin family in mammalian tissue and is regarded as the main G-actin sequestering peptide. Thymosin beta4 is angiogenic and can promote endothelial cell migration and adhesion, tubule formation, aortic ring sprouting, and angiogenesis. It also accelerates wound healing and reduces inflammation when applied in dermal wound-healing assays. Using naturally occurring thymosin beta4, proteolytic fragments, and synthetic peptides, we find that a seven amino acid actin binding motif of thymosin beta4 is essential for its angiogenic activity. Migration assays with human umbilical vein endothelial cells and vessel sprouting assays using chick aortic arches show that thymosin beta4 and the actin-binding motif of the peptide display near-identical activity at ~50 nM, whereas peptides lacking any portion of the actin motif were inactive. Furthermore, adhesion to thymosin beta4 was blocked by this seven amino acid peptide demonstrating it as the major thymosin beta4 cell binding site on the molecule. The adhesion and sprouting activity of thymosin beta4 was inhibited with the addition of 5-50 nM soluble actin. These results demonstrate that the actin binding motif of thymosin beta4 is an essential site for its angiogenic activity.

Thymosin beta4 increases hair growth by activation of hair follicle stem cells.

Thymosin beta4, a 43-amino acid polypeptide that is an important mediator of cell migration and differentiation, also promotes angiogenesis and wound healing. Here, we report that thymosin beta4 stimulates hair growth in normal rats and mice. A specific subset of hair follicular keratinocytes in mouse skin expresses thymosin beta4 in a highly coordinated manner during the hair growth cycle. These keratinocytes originate in the hair follicle bulge region, a niche for skin stem cells. Rat vibrissa follicle clonogenic keratinocytes, closely related, if not identical, to the bulge-residing stem cells, were isolated and their migration and differentiation increased in the presence of nanomolar concentrations of thymosin beta4. Expression and secretion of the extracellular matrix-degrading enzyme matrix metalloproteinase-2 were increased by thymosin beta4. Thus, thymosin beta4 accelerates hair growth, in part, due to its effect on critical events in the active phase of the hair follicle cycle, including promoting the migration of stem cells and their immediate progeny to the base of the follicle, differentiation, and extracellular matrix remodeling.

Role of the extracellular matrix in morphogenesis.

The extracellular matrix is a complex, dynamic and critical component of all tissues. It functions as a scaffold for tissue morphogenesis, provides cues for cell proliferation and differentiation, promotes the maintenance of differentiated tissues and enhances the repair response after injury. Various amounts and types of collagens, adhesion molecules, proteoglycans, growth factors and cytokines or chemokines are present in the tissue- and temporal-specific extracellular matrices. Tissue morphogenesis is mediated by multiple extracellular matrix components and by multiple active sites on some of these components. Biologically active extracellular matrix components may have use in tissue repair, regeneration and engineering, and in programming stem cells for tissue replacement.

Animal studies with thymosin beta, a multifunctional tissue repair and regeneration peptide.

Studies in various animal models of disease and repair with thymosin beta(4) (Tbeta(4)), the major actin-sequestering molecule in mammalian cells, have provided the scientific foundation for the ongoing dermal, corneal, and cardiac wound repair multicenter clinical trials. Tbeta(4) has of multiple biological activities, which include down-regulation of inflammatory chemokines and cytokines, and promotion of cell migration, blood vessel formation, cell survival, and stem cell maturation. All of these activities contribute to the multiple wound healing properties that have been observed in animal studies. This paper reviews and discusses the topical and systemic uses of Tbeta(4) in various animal models that demonstrate its potential for clinical use.

Over-expression of thymosin beta 4 promotes abnormal tooth development and stimulation of hair growth.

Thymosin beta 4 has multi-functional roles in cell physiology. It accelerates wound healing, hair growth and angiogenesis, and increases laminin-5 expression in corneal epithelium. Furthermore, thymosin beta 4 stimulates tumor growth and metastasis by induction of cell migration and vascular endothelial growth factor-mediated angiogenesis. Using a construct on the skin-specific keratin-5 promoter, we have developed thymosin beta 4 over-expressing transgenic mice to further study its functional roles. Thymosin beta 4 in adult skin and in embryonic stages of the transgenic mouse was analyzed by both Western blot and immunohistochemistry. The over-expression of thymosin beta 4 was observed especially around hair follicles and in the teeth in the transgenic mice. We examined the phenotype of the thymosin beta 4 over-expressing mice. Hair growth was accelerated. In addition, the transgenic mice had abnormally-shaped white teeth and dull incisors. We found that the expression of laminin-5 was up-regulated in the skin of the transgenic mice. We conclude that thymosin beta 4 has an important physiological role in hair growth and in tooth development.

Angiogenic activity of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT).

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719-2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None of the homologous sequences from the laminin alpha2, alpha3, alpha4, or alpha5 chains was as active as AG73 in promoting sprouting formation. AG73 also mediated angiogenesis in the chick chorioallantonic membrane (CAM) assay. Using subcutaneously injected Cultrex BME supplemented with AG73, we observed a large angiogenic response. Furthermore, AG73-conjugated to a chitosan membrane promoted a strong angiogenic response in the CAM assay. These results indicate that the AG73 peptide is a potent syndecan-binding angiogenesis stimulator and may be useful for therapeutic application to treat ischemic injuries.

Integrin-dependent cell behavior on ECM peptide-conjugated chitosan membranes.

Extracellular matrix (ECM) plays an important role in tissue regeneration by promoting cell adhesion, migration, proliferation, and differentiation. ECM mimetics are of importance for tissue engineering because of their functions as scaffolds for cells. Previously, we developed bioactive laminin-derived peptide-conjugated chitosan membranes and demonstrated their cell- and peptide-type specific functions. Here, we conjugated twelve integrin-binding peptides derived from ECM proteins onto chitosan membranes and examined biological activity. Seven peptide-chitosan membranes promoted human foreskin fibroblast attachment. Additionally, FIB1 (YAVTGRGDSPAS; from fibronectin), A99 (AGTFALRGDNPQG; from laminin alpha1 chain), EF1zz (ATLQLQEGRLHFXFDLGKGR, X = Nle; from laminin alpha1 chain), and 531 (GEFYFDLRLKGDKY; from collagen alpha1 (IV) chain) conjugated chitosan membranes promoted integrin-dependent cell adhesion. Various integrins, including alphav, beta1, and beta3, were involved in the cell adhesion to the peptide-chitosan membranes. Further, only the FIB1- and A99-chitosan membranes promoted neurite outgrowth with PC12 rat pheochromocytoma cells. These data demonstrate that peptide-chitosan membranes can regulate specific integrin-mediated cell responses and are useful constructs as ECM mimetics.

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair.

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.

Complex extracellular matrices promote tissue-specific stem cell differentiation.

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.

Heparanase regulates murine hair growth.

Heparanase is an endoglycosidase that cleaves heparan sulfate, the main polysaccharide component of the extracellular matrix. Heparan sulfate moieties are responsible for the extracellular matrix barrier function, as well as for sequestration of heparin-binding growth factors in the extracellular matrix. Degradation of heparan sulfate by heparanase enables cell movement through extracellular barriers and releases growth factors from extracellular matrix depots, making them bioavailable. Here, we demonstrate a highly coordinated temporospatial pattern of heparanase expression and enzymatic activity during hair follicle cycling. This pattern paralleled the route and timing of follicular stem cell progeny migration and reconstitution of the lower part of the follicle, which is a prerequisite for hair shaft formation. By monitoring in vivo activation of luciferase reporter gene driven by heparanase promoter, we observed activation of heparanase gene transcription at a specific stage of the hair cycle. Heparanase was produced by rat vibrissa bulge keratinocytes, closely related to a follicular stem cell population. Heparanase contributed to the ability of the bulge-derived keratinocytes to migrate through the extracellular matrix barrier in vitro. In heparanase-overexpressing transgenic mice, increased levels of heparanase enhanced active hair growth and enabled faster hair recovery after chemotherapy-induced alopecia. Collectively, our results identify heparanase as an important regulator of hair growth and suggest that cellular mechanisms of its action involve facilitation of follicular stem cell progeny migration and release of extracellular matrix-resident, heparin-bound growth factors, thus regulating hair cycle.

Thymosin beta 4 and a synthetic peptide containing its actin-binding domain promote dermal wound repair in db/db diabetic mice and in aged mice.

Impaired wound healing is a problem for immobilized patients, diabetics, and the elderly. Thymosin beta 4 has previously been found to promote dermal and corneal repair in normal rats. Here we report that thymosin beta 4 was also active in accelerating wound repair in full-thickness dermal wounds in both db/db diabetic and aged mice. We found that thymosin beta 4 in either phosphate-buffered saline or a hydrogel formulation is active in promoting dermal wound repair in normal rats. In diabetic mice, where healing is delayed, we found that wound contracture and collagen deposition were significantly increased in the mice treated with thymosin beta 4 in either phosphate buffered saline solution or a hydrogel formulation. No difference was observed in keratinocyte migration, with all of the diabetic animals showing almost complete coverage of the wound at 8 days. Wound healing in 26-month-old (aged) animals was significantly delayed. Thymosin beta 4 accelerated wound healing in these aged mice, with increases in keratinocyte migration, wound contracture, and collagen deposition. The hydrogel formulation generally showed similar wound healing activity with thymosin beta 4 in PBS. The actin-binding domain of thymosin beta 4 duplicated in a seven-amino acid synthetic peptide, LKKTETQ, was able to promote repair in the aged animals comparable to that observed with the parent molecule. These studies show that thymosin beta 4 is active for wound repair in models of impaired healing and may have efficacy in chronic wounds in humans.