lncRNA cytoskeleton regulator reduces non­small cell lung cancer radiosensitivity by downregulating miRNA­206 and activating prothymosin α.

The present study aimed to explore the role of the long noncoding RNA cytoskeleton regulator (CYTOR) in non­small cell lung cancer (NSCLC) radiosensitivity by manipulating the microRNA (miR)­206/prothymosin α (PTMA) axis. First, 58 pairs of NSCLC and paracancerous tissues, normal human lung epithelial cells and NSCLC cells were collected to analyze CYTOR expression and the relationship between CYTOR and NSCLC prognosis. Subsequently, CYTOR expression in radioresistant cells was assessed. Radioresistant cells with low CYTOR expression and parental cells with high CYTOR expression were established. Functional assays were then performed to assess changes in cell radiosensitivity after irradiation treatment. Subsequently, the downstream mechanism of CYTOR was explored. The binding interactions between CYTOR and miR­206 and between miR­206 and PTMA were predicted and certified. Xenograft transplantation was applied to confirm the role of CYTOR in the radiosensitivity of NSCLC. CYTOR was overexpressed in NSCLC and was associated with poor prognosis. CYTOR was further upregulated in NSCLC cells with radioresistance. CYTOR knockdown enhanced the radiosensitivity of NSCLC cells, while overexpression of CYTOR led to the opposite result. Mechanistically, CYTOR specifically bound to miR­206 and silencing CYTOR promoted miR­206 to enhance the radiosensitivity of NSCLC cells. PTMA is a target of miR­206 and silencing CYTOR inhibited PTMA expression via miR­206, thus promoting radiosensitivity of NSCLC cells. CYTOR knockdown also enhanced NSCLC cell radiosensitivity in vivo. CYTOR was highly expressed in NSCLC, while silencing CYTOR potentiated NSCLC cell radiosensitivity by upregulating miR­206 and suppressing PTMA. The present study preliminarily revealed the role of CYTOR in radiotherapy sensitivity of NSCLC and provided a novel potential target for the clinical treatment of NSCLC.

Thymosin ß4 increases cardiac cell proliferation, cell engraftment, and the reparative potency of human induced-pluripotent stem cell-derived cardiomyocytes in a porcine model of acute myocardial infarction.

Rationale: Previous studies have shown that human embryonic stem cell-derived cardiomyocytes improved myocardial recovery when administered to infarcted pig and non-human primate hearts. However, the engraftment of intramyocardially delivered cells is poor and the effectiveness of clinically relevant doses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in large animal models of myocardial injury remains unknown. Here, we determined whether thymosin ß4 (Tb4) could improve the engraftment and reparative potency of transplanted hiPSC-CMs in a porcine model of myocardial infarction (MI). Methods: Tb4 was delivered from injected gelatin microspheres, which extended the duration of Tb4 administration for up to two weeks in vitro. After MI induction, pigs were randomly distributed into 4 treatment groups: the MI Group was injected with basal medium; the Tb4 Group received gelatin microspheres carrying Tb4; the CM Group was treated with 1.2 × 108 hiPSC-CMs; and the Tb4+CM Group received both the Tb4 microspheres and hiPSC-CMs. Myocardial recovery was assessed by cardiac magnetic resonance imaging (MRI), arrhythmogenesis was monitored with implanted loop recorders, and tumorigenesis was evaluated via whole-body MRI. Results: In vitro, 600 ng/mL of Tb4 protected cultured hiPSC-CMs from hypoxic damage by upregulating AKT activity and BcL-XL and promoted hiPSC-CM and hiPSC-EC proliferation. In infarcted pig hearts, hiPSC-CM transplantation alone had a minimal effect on myocardial recovery, but co-treatment with Tb4 significantly enhanced hiPSC-CM engraftment, induced vasculogenesis and the proliferation of cardiomyocytes and endothelial cells, improved left ventricular systolic function, and reduced infarct size. hiPSC-CM implantation did not increase incidence of ventricular arrhythmia and did not induce tumorigenesis in the immunosuppressed pigs. Conclusions: Co-treatment with Tb4-microspheres and hiPSC-CMs was safe and enhanced the reparative potency of hiPSC-CMs for myocardial repair in a large-animal model of MI.

PTEN/AKT upregulation of TMSB10 contributes to lung cancer cell growth and predicts poor survival of the patients.

PTEN/AKT signaling cascade is frequently activated in various cancers, including lung cancer. The downstream effector of this signaling cascade is poorly understood. ß-Thymosin 10 (TMSB10) functions as an oncogene or tumor suppressors in cancers, whereas its significance in lung cancer remains unknown. In this study, we showed that the activation of PTEN/AKT signaling promoted the expression of TMSB10. Based on the TCGA database, TMSB10 was upregulated in lung cancer tissues and its overexpression was correlated with poor prognosis of lung cancer patients. Functional experiments demonstrated that TMSB10 knockdown suppressed, while its overexpression promoted the proliferation, growth, and migration of lung cancer cells. Apoptosis and epithelial-mesenchymal transition were also regulated by TMSB10. We therefore suggest that TMSB10 is a novel oncogene for lung cancer. Targeting TMSB10 may benefit lung cancer patients with activated PTEN/AKT signaling.

Progress on the Function and Application of Thymosin ß4.

Thymosin ß4 (Tß4) is a multifunctional and widely distributed peptide that plays a pivotal role in several physiological and pathological processes in the body, namely, increasing angiogenesis and proliferation and inhibiting apoptosis and inflammation. Moreover, Tß4 is effectively utilized for several indications in animal experiments or clinical trials, such as myocardial infarction and myocardial ischemia-reperfusion injury, xerophthalmia, liver and renal fibrosis, ulcerative colitis and colon cancer, and skin trauma. Recent studies have reported the potential application of Tß4 and its underlying mechanisms. The present study reveals the progress regarding functions and applications of Tß4.

Thymosin α1 protects from CTLA-4 intestinal immunopathology.

The advent of immune checkpoint inhibitors has represented a major boost in cancer therapy, but safety concerns are increasingly being recognized. Indeed, although beneficial at the tumor site, unlocking a safeguard mechanism of the immune response may trigger autoimmune-like effects at the periphery, thus making the safety of immune checkpoint inhibitors a research priority. Herein, we demonstrate that thymosin α1 (Tα1), an endogenous peptide with immunomodulatory activities, can protect mice from intestinal toxicity in a murine model of immune checkpoint inhibitor-induced colitis. Specifically, Tα1 efficiently prevented immune adverse pathology in the gut by promoting the indoleamine 2,3-dioxygenase (IDO) 1-dependent tolerogenic immune pathway. Notably, Tα1 did not induce IDO1 in the tumor microenvironment, but rather modulated the infiltration of T-cell subsets by inverting the ratio between CD8+ and Treg cells, an effect that may depend on Tα1 ability to regulate the differentiation and chemokine expression profile of DCs. Thus, through distinct mechanisms that are contingent upon the context, Tα1 represents a plausible candidate to improve the safety/efficacy profile of immune checkpoint inhibitors.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions.

Prothymosin alpha (ProTα) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProTα is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as "alarmins". Antibodies and immunochemical techniques for ProTα have played a leading role in the investigation of the biological role of ProTα, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProTα (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Thymosin ß4-mediated protective effects in the heart.

INTRODUCTION: Despite recent advances in the treatment of coronary heart disease, a significant number of patients progressively develop heart failure. Reduction of infarct size after acute myocardial infarction and normalization of microvasculature in chronic myocardial ischemia could enhance cardiac survival. AREAS COVERED: Induction of neovascularization using vascular growth factors has emerged as a promising novel approach for cardiac regeneration. Thymosin ß4 (Tß4) might be a promising candidate for the treatment of ischemic heart disease. It has been characterized as a major G-actin-sequestering factor regulating cell motility, migration, and differentiation. During cardiac development, Thymosin ß4 seems essential for vascularization of the myocardium. In the adult organism, Thymosin ß4 has anti-inflammatory properties, increases myocyte and endothelial cell survival accompanied by differentiation of epicardial progenitor cells. In chronic myocardial ischemia, Tß4 overexpression enhances micro- and macrovasculature in the ischemic area and thereby improves myocardial function. A comparable effect is seen in diabetic and dyslipidemic pig ischemic hearts, suggesting an attractive therapeutic potential of adeno-associated virus encoding for Tß4 for patients with ischemic heart disease. EXPERT OPINION: Induction of mature micro-vessels is a prerequisite for chronic myocardial ischemia and might be achieved via a long-term overexpression of Thymosin ß4.

Intrinsic, Functional, and Structural Properties of ß-Thymosins and ß-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

ß-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/ß-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling.

Phosphorylation of Prothymosin α. An Approach to Its Biological Significance.

Prothymosin α (ProTα), the precursor of the thymosin α1 and thymosin α11, is a 109-111 amino acids protein widely distributed in the mammalian tissues that is essential for the cell proliferation and survival through its implication on chromatin remodeling and in the proapoptotic activity. ProTα is phosphorylated at Thr residues by the M2 isoenzyme of the pyruvate kinase in a process that is dependent on the cell proliferation activity, which constitutes a novel dual functionality of this enzyme. The Thr residues phosphorylated are apparently dependent on the carcinogenic transformation of the cells. Thus, in normal lymphocytes residues Thr11 or Thr12 are phosphorylated in addition to a Thr7 residue, while in tumor cells Thr7 is the only residue phosphorylated. Phosphorylation of ProTα seems to be related to its antiapoptotic activity, although other possibilities cannot be discarded.

Thymosin Beta 4 Is a Potential Regulator of Hepatic Stellate Cells.

Liver fibrosis, a major characteristic of chronic liver disease, is inappropriate tissue remodeling caused by prolonged parenchymal cell injury and inflammation. During liver injury, hepatic stellate cells (HSCs) undergo transdifferentiation from quiescent HSCs into activated HSCs, which promote the deposition of extracellular matrix proteins, leading to liver fibrosis. Thymosin beta 4 (Tß4), a major actin-sequestering protein, is the most abundant member of the highly conserved ß-thymosin family and controls cell morphogenesis and motility by regulating the dynamics of the actin cytoskeleton. Tß4 is known to be involved in various cellular responses, including antiinflammation, wound healing, angiogenesis, and cancer progression. Emerging evidence suggests that Tß4 is expressed in the liver; however, its biological roles are poorly understood. Herein, we introduce liver fibrogenesis and recent findings regarding the function of Tß4 in various tissues and discuss the potential role of Tß4 in liver fibrosis with a special focus on the effects of exogenous and endogenous Tß4. Recent studies have revealed that activated HSCs express Tß4 in vivo and in vitro. Treatment with the exogenous Tß4 peptide inhibits the proliferation and migration of activated HSCs and reduces liver fibrosis, indicating it has an antifibrotic action. Meanwhile, the endogenously expressed Tß4 in activated HSCs is shown to promote HSCs activation. Although the role of Tß4 has not been elucidated, it is apparent that Tß4 is associated with HSC activation. Therefore, understanding the potential roles and regulatory mechanisms of Tß4 in liver fibrosis may provide a novel treatment for patients.

Primary Mechanisms of Thymosin ß4 Repair Activity in Dry Eye Disorders and Other Tissue Injuries.

Dry eye disorders are becoming more common due to many causes, including an aging population, increased pollution, and postrefractive surgery. Current treatments include artificial tears; gels; lubricants; tear duct plugs; and anti-inflammatory agents such as steroids, doxycycline, and cyclosporine. For more severe forms of the disease, serum tears and scleral contact lenses are employed. Despite these therapies, successful resolution of the problem is limited because none of these treatments fully addresses the underlying causes of dry eye to promote ocular surface repair. Thymosin ß4 (Tß4), a small, naturally occurring protein, promotes complete and faster corneal healing than saline alone or prescription agents (doxycycline and cyclosporine) in various animal models of eye injury. In human trials, it improves both the signs and symptoms of moderate to severe dry eye with effects lasting beyond the treatment period. This review will cover the multiple activities of Tß4 on cell migration, inflammation, apoptosis, cytoprotection, and gene expression with a focus on mechanisms of cell migration, including laminin-332 synthesis and degradation, that account for this paradigm-shifting potential new treatment for dry eye disorders. We will also speculate on additional mechanisms that might promote eye repair based on data from other tissue injury models. Such studies provide the rationale for use of Tß4 in other types of eye disorders beyond dry eye. Finally, we will identify the gaps in our knowledge and propose future research avenues.

Thymosin-ß4-mediated therapeutic neovascularization: role of the PI3K/AKT pathway.

OBJECTIVES: Thymosin ß4 (Tß4) is known to have pro-angogenic abilities in vitro and in vivo, and its cardioprotective effect is PI3/AKT-dependent. Tß4-induced vessel formation requires transcriptional activation via the MRTF/SRF pathway. However, the relevance of PI3/AKT signaling for Tß4-induced angiogenesis remains unclear. Here, we analyzed the PI3K/AKT cascade after Tß4 transduction in models of chronic hindlimb ischemia. METHODS: Tube formation assays of endothelial cells transfected with Tß4 ± AKT-dn or PI3Kα/Rho inhibition were performed. In mice, rAAV.Tß4 was injected (intramuscular [i.m.]) 14 days before femoral artery ligation. In addition, either rAAV.AKT-dn was co-applied or Rho/PI3K/AKT pathways were inhibited. Capillary density and hindlimb perfusion were obtained. In rabbits, chronic ischemia was induced by femoral artery excision and subsequent i.m. injection of rAAV.Tß4 ± rAAV.AKT-dn. Analyses of capillary density, collateral formation and perfusion were performed. RESULTS: Tß4-induced ring formation was blunted by inhibiting the Rho-kinase (ROCK) or the PI3K/AKT pathway. In vivo, Tß4 transduction induced angiogenesis and perfusion, an effect abrogated by inhibition of Rho-signaling, or PI3Kα/AKT. In the rabbit model, inhibition of AKT in the lower limb not only abolished angiogenesis but also collateral formation. CONCLUSION: Tß4 requires PI3Kα/AKT pathway signaling for induction of therapeutic neovascularization in ischemic limb disease.

Thymosin ß4 attenuates microcirculatory and hemodynamic destabilization in sepsis.

OBJECTIVE: The actin polymerization regulator Thymosin ß4 (Tß4) has been shown to be involved in angiogenesis, wound healing, cell survival and anti-inflammatory responses. We have previously shown that Tß4 is capable of recruiting pericytes, thus stabilizing the endothelial barrier function. Here, we analyzed whether treatment with Tß4 is able to reduce the pericytes loss in lipopolysaccharides (LPS)-induced sepsis and to improve the hemodynamic function and survival in C57BL/6 mice. METHODS: Fourteen days before LPS injection, the mice were injected with an adeno-associated virus carrying the Tß4 (rAAV.Tß4) or LacZ gene (rAAV.LacZ). A sepsis-severity score was assessed, and non-invasive hemodynamic and permeability measurements were performed. Heart and muscle samples were analyzed for PECAM-1(+) capillaries and NG2(+)pericytes. RESULTS: At 36 h, there was a decrease of sepsis severity score in rAAV.Tß4-treated animals as compared to rAAV.LacZ-treated control. rAAV.Tß4-treated animals displayed lower perivascular leakage and higher blood pressure compared to control. Of note, the rAAV.Tß4 group showed a higher pericyte count in heart and peripheral muscle samples. Finally, Tß4-treatment reduced mortality compared to control. CONCLUSION: The data indicate a preventive role of Tß4 in septic hypercirculation and highlight Tß4 as a potential therapeutic target in severe sepsis.

Oncogenic c-Myc and prothymosin-alpha protect hepatocellular carcinoma cells against sorafenib-induced apoptosis.

Prothymosin alpha (PTMA) is overexpressed in various human tumors, including hepatocellular carcinoma (HCC). The significance of PTMA overexpression and its underlying mechanism remain unclear. We show here that silencing PTMA sensitizes HCC cells to the kinase inhibitor sorafenib. In contrast, ectopic expression of PTMA induces cell resistance to the drug. While inhibitors targeting JNK, ERK or PI3K reduce PTMA expression, only ERK activation is suppressed by sorafenib. In addition, inhibition of ERK produces a dramatic decrease in both endogenous PTMA level and promoter activation. Ectopic expression of active MKK1/2 considerably induces PTMA expression. We also identify a sorafenib-responsive segment lying 1000-1500-bp upstream of the PTMA transcription start site and observe that it is controlled by c-Myc and ERK. Mutation in the PTMA promoter at the predicted c-Myc binding site and silencing of c-Myc both abrogate sorafenib's effect on PTMA transcription. We also find that silencing PTMA potentiates Bax translocation to mitochondria in response to sorafenib and this is associated with increased cytochrome c release from mitochondria and enhanced caspase-9 activation. These results indicate that PTMA is positively regulated by the oncoprotein c-Myc and protects HCC cells against sorafenib-induced cell death, thus identifying PTMA as a new target for chemotherapy against HCC.

Depletion of thymosin ß4 promotes the proliferation, migration, and activation of human hepatic stellate cells.

BACKGROUND & AIMS: It has recently been reported that thymosin beta-4 (Tß4) has anti-fibrogenic effects in human hepatic stellate cells (HSCs) in vitro, but the mechanisms underlying these effects remain unclear. The aim of this study was to investigate the roles of Tß4 in the proliferation, migration, and activation of HSCs. METHODS: Enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and western blot assays were utilized to determine the expression levels of Tß4 in serum, liver tissues, and LX-2 cells. Tß4 was depleted in LX-2 cells using small interfering RNAs (siRNAs). Cell proliferation was analyzed using cell counting kit-8 (CCK-8) viability assays, and cell migration was investigated using wound-healing and transwell migration assays. RESULTS: The expression of Tß4 was significantly reduced during the progression of liver fibrosis. The depletion of Tß4 significantly promoted the proliferation and migration of LX-2 cells via the activation of the PI3K/Akt signaling pathway. The pro-migratory and pro-proliferative effects of Tß4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tß4 depletion was also associated with the activation of HSCs via the enhanced expression of α-smooth muscle actin (α-SMA) and vimentin. CONCLUSIONS: Our results suggest that Tß4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tß4 may be an effective target in the treatment of liver fibrosis.

Thymosin ß4 increases the potency of transplanted mesenchymal stem cells for myocardial repair.

BACKGROUND: Thymosin ß4 (Tß4) has been shown to enhance the survival of cultured cardiomyocytes. Here, we investigated whether the cytoprotective effects of Tß4 can increase the effectiveness of transplanted swine mesenchymal stem cells (sMSCs) for cardiac repair in a rat model of myocardial infarction (MI). METHODS AND RESULTS: Under hypoxic conditions, cellular damage (lactate dehydrogenase leakage), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelingc cells), and caspase-8 activity were significantly lower, whereas B-cell lymphoma-extra large protein expression was significantly higher, in sMSCs cultured with Tß4 (1 µg/mL) than in sMSCs cultured without Tß4, and Tß4 also increased sMSC proliferation. For in vivo experiments, animals were treated with basal medium (MI: n=6), a fibrin patch (Patch: n=6), a patch containing sMSCs (sMSC: n=9), or a patch containing sMSCs and Tß4 (sMSC/Tß4: n=11); Tß4 was encapsulated in gelatin microspheres to extend Tß4 delivery. Four weeks after treatment, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly better (P<0.05) in sMSC/Tß4 animals (left-ventricular ejection fraction=51.7 ± 1.1%; fractional shortening=26.7 ± 0.7%) than in animals from MI (39 ± 3%; 19.5 ± 1.7%) and Patch (43 ± 1.4%; 21.6 ± 0.9%) groups. Histological assessment of infarct wall thickness was significantly higher (P<0.05) in sMSC/Tß4 animals (50%, [45%, 80%]) than in animals from MI (25%, [20%, 25%]) group. Measurements in sMSC (left-ventricular ejection fraction=45 ± 2.6%; fractional shortening=22.9 ± 1.6%; TH = 43% [25%, 45%]), Patch, and MI animals were similar. Tß4 administration also significantly increased vascular growth, the retention/survival of the transplanted sMSCs, and the recruitment of endogenous c-Kit(+) progenitor cells to the infarcted region. CONCLUSIONS: Extended-release Tß4 administration improves the retention, survival, and regenerative potency of transplanted sMSCs after myocardial injury.

The expression and function of thymosin beta 10 in tooth germ development.

This study presents the expression pattern and functions of thymosin beta 10 (Tbeta10), a Tbeta4 homologue during the development of mouse lower first molars. An in situ signal of Tbeta10 was detected on embryonic day 10.5 (E10.5)-E15.5 mainly in dental mesenchymal cells as well as in dental epithelial cells, while Tbeta4 was expressed in dental epithelial cells. In the late bell stage, preodontoblasts with strong Tbeta10 expression and preameloblasts with strong Tbeta4 expression exhibited face-to-face localization, suggesting that an intimate cell-cell interaction might exist between preodontoblasts and preameloblasts to form dentin and enamel matrices. A strong Tbeta10 signal was found in odontoblasts in the lateral side of the dental pulp and in Hertwig’s epithelial root sheath, thus suggesting that Tbeta10 participates in the formation of the outline of the tooth root. An inhibition assay using Tbeta10-siRNA in E11.0 mandibles showed significant growth inhibition in the tooth germ. The Tbeta10-siRNA-treated E15.0 tooth germ also showed significant developmental arrest. The number of Ki67-positive cells significantly decreased in the Tbeta10-siRNA-treated mandibles. The cellular proliferative activity was also significantly suppressed in Tb10-siRNA-treated cultured mouse dental pulpal and epithelial cells. These results indicate that developmental arrest of the tooth germ might be caused by a reduction in cell proliferative activity. The stage-specific temporal and spatial expression pattern of Tbeta10 in the developing tooth germ is indicative of multiple functions of Tbeta10 in the developmental course from initiation to root formation of the tooth germ.

The role of immunohistochemistry in the study of the newborn kidney.

The identification of the different cell types involved in human nephrogenesis, when solely based on morphology, may lead to errors in its interpretation, given the complexity of the histological picture of the fetal and of the newborn kidney. In this study, the most recent works utilizing immunohistochemistry for the identification of the multiple cell types involved in human nephrogenesis are reviewed. The role of WT1, MUC1, Thymosin beta 10, Thymosin beta 4, CD10 and CD44 in the different phases of glomerulogenesis and of tubulogenesis is here described, with particular emphasis on their expression in the early phases of nephrogenesis. On the basis of our data, immunohistochemistry appears to be a useful tool in the study of human nephrogenesis, giving new data on the different steps of the differentiation of metanephric mesenchyme towards the multiple cell types characterizing the mature human kidney. Moreover, allowing a better knowledge of the protein products involved in the generation of new nephrons, immunohistochemistry could open new perspectives in the field of renal regenerating medicine, evidencing the factors able to prolong nephrogenesis after birth, helping us to reach our goal: allowing newborn kidneys to restore their nephron endowment, escaping susceptibility to hypertension and renal disease in adulthood.

In vitro and in vivo pro-angiogenic effects of thymosin-ß4-derived peptides.

Thymosin-ß4 (Tß4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tß4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tß4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tß4.