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Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.
Mesothelium is the simple squamous epithelium covering all abdominal organs and the coeloms in which those organs reside. While the structural characteristics of this cell type were documented a century ago, its potential in development, disease, and wound healing is only now becoming apparent. In the embryo, mesothelia provide vasculogenic cells for the developing heart, lungs, and gut. Furthermore, adult mesothelial cells can be reactivated using thymosin β4 and mobilized to aid in tissue repair. Despite their positive role in development and repair, mesothelia are also susceptible to adhesion and tumor formation. With knowledge that the mesothelium is an important mediator of tissue repair as well as disease, it will be important to identify other factors like thymosin β4 that have the ability to potentiate these cells. Future use of chemical and genetic agents in conjunction with mesothelial cells will lead to enhanced therapeutic potential and mitigation of deleterious outcomes.
© 2012 New York Academy of Sciences.
Inflammation and angiogenesis are inevitable in vivo responses to biomaterial implants. Continuous progress has been made in biomaterial design to improve tissue interactions with an implant by either reducing inflammation or promoting angiogenesis. However, it has become increasingly clear that the physiological processes of inflammation and angiogenesis are interconnected through various molecular mechanisms. Hence, there is an unmet need for engineering functional tissues by simultaneous activation of pro-angiogenic and anti-inflammatory responses to biomaterial implants. In this work, the modulus and fibrinogen adsorption of porous scaffolds were tuned to meet the requirements (i.e., ~100 kPa and ~10 nm, respectively), for soft tissue regeneration by employing tyrosine-derived combinatorial polymers with polyethylene glycol crosslinkers. Two types of functional peptides (i.e., pro-angiogenic laminin-derived C16 and anti-inflammatory thymosin β4-derived Ac-SDKP) were loaded in porous scaffolds through collagen gel embedding so that peptides were released in a controlled fashion, mimicking degradation of the extracellular matrix. The results from (1) in vitro coculture of human umbilical vein endothelial cells and human blood-derived macrophages and (2) in vivo subcutaneous implantation revealed the directly proportional relationship between angiogenic activities (i.e., tubulogenesis and perfusion capacity) and inflammatory activities (i.e., phagocytosis and F4/80 expression) upon treatment with either type of peptide. Interestingly, cotreatment with both types of peptides upregulated the angiogenic responses, while downregulating the inflammatory responses. Also, anti-inflammatory Ac-SDKP peptides reduced production of pro-inflammatory cytokines (i.e., interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor alpha) even when treated in combination with pro-angiogenic C16 peptides. In addition to independent regulation of angiogenesis and inflammation, this study suggests a promising approach to improve soft tissue regeneration (e.g., blood vessel and heart muscle) when inflammatory diseases (e.g., ischemic tissue fibrosis and atherosclerosis) limit the regeneration process.
Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin β4 (Tβ4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tβ4-induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tβ4-stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tβ4 promoted proliferation, migration and trans-differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell-based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm-setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields.
Copyright © 2012 John Wiley & Sons, Ltd.
The pathogenesis of impaired healing within pressure ulcers remains poorly characterized and rarely examined. We describe the results of a pilot study that applies matrix-assisted laser desorption/ionization imaging mass spectrometry technology for direct tissue analysis to evaluate proteomic signatures ranging from 2 to 20 kDa and phospholipids from 300-1,200 Da in focal regions within the wound microenvironment. Distinguishing molecular differences were apparent between upper vs. lower regions of ulcers and further contrasted against adjacent dermis and epidermal margins using protein profiles, ion density maps, principal component analysis and significant analysis of microarrays. Several proteins previously uncharacterized in pressure ulcers, the α-defensins (human neutrophil peptide [HNP]-1, -2, -3), are potential markers indicating whether the wound status is improving or being prolonged in a deleterious, chronic state. Thymosin β4 appears to be a favorable protein marker showing higher relative levels in adjacent dermis and maturing areas of the wound bed. Lipidomic examination revealed the presence of major lipid classes: glycerophosphocholines, glycerophosphoglycerols, glycerophosphoinositols, and triacylglycerols. Our pilot data examined from either a global perspective using proteomic or lipidomic signatures or as individual distributions reveal that imaging mass spectrometry technology can be effectively used for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic wounds.
2011 by the Wound Healing Society.
Acute myocardial infarction is still one of the leading causes of death in the industrial nations. Even after successful revascularization, myocardial ischemia results in a loss of cardiomyocytes and scar formation. Embryonic EPCs (eEPCs), retroinfused into the ischemic region of the pig heart, provided rapid paracrine benefit to acute and chronic ischemia in a PI-3K/Akt-dependent manner. In a model of acute myocardial ischemia, infarct size and loss of regional myocardial function decreased after eEPC application, unless cell pre-treatment with thymosin beta4 shRNA was performed. Thymosin beta4 peptide retroinfusion mimicked the eEPC-derived improvement of infarct size and myocardial function. In chronic ischemia (rabbit model), eEPCs retroinfused into the ischemic hindlimb enhanced capillary density, collateral growth, and perfusion. Therapeutic neovascularization was absent when thymosin beta4 shRNA was introduced into eEPCs before application. In conclusion, eEPCs are capable of acute and chronic ischemia protection in a thymosin beta4 dependent manner.
Substantial evidence demonstrates a link of increased plasminogen activator inhibitor-1 (PAI-1) and glomerulosclerosis and kidney fibrosis, providing a novel therapeutic option for prevention and treatment of chronic kidney diseases. Several mechanisms contributing to increased PAI-1 will be addressed, including classic key profibrotic factors such as the renin-angiotensin-system (RAS) and transforming growth factor-beta (TGF-b???and novel molecules identified by proteomic analysis, such as thymosin- b4. The fibrotic sequelae caused by increased PAI-1 in kidney depend not only on its classic inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA), but also its influence on cell migration.
BACKGROUND - Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection.
METHODS AND RESULTS - In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tbeta4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10(6) cells) or cells transfected with Tbeta4 shRNA when indicated or 15 mg Tbeta4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32+/-4% to 90+/-2% after eEPC application, an effect sensitive to shRNA transfection compared with Tbeta4 (45+/-7%). In vivo, infarct size decreased with eEPC application (38+/-4% versus 54+/-4% of area at risk; P<0.01), an effect abolished by Tbeta4 shRNA (62+/-3%). Segmental subendocardial shortening improved after eEPC treatment (22+/-3% versus -3+/-4% of control area) unless Tbeta4 shRNA was transfected (-6+/-4%). Retroinfusion of Tbeta4 mimicked eEPC application (infarct size, 37+/-3%; segmental endocardial shortening, 34+/-7%). Myeloperoxidase activity (3323+/-388 U/mg in controls) was decreased by eEPCs (1996+/-546 U/mg) or Tbeta4 alone (1455+/-197 U/mg) but not Tbeta4 shRNA-treated eEPCs (5449+/-829 U/mg).
CONCLUSIONS - Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tbeta4.
PURPOSE - Prothymosin-alpha and ERp57 were previously identified as markers for gastric metaplasia in a mouse model of Helicobacter-induced gastric metaplasia and neoplasia. In this paper we assess whether the expression of these putative biomarkers in humans is correlated with gastric metaplasia and adenocarcinoma and clinical outcomes.
METHODS - Eight tissue microarrays, containing 749 paraffin-embedded tissue cores from 164 gastric cancer patients, were stained for prothymosin-alpha and ERp57 by horseradish peroxidase immunohistochemical techniques. The proportion of stained cells per core was quantitated using the Ariol SL-50 automated image analysis system.
RESULTS - Prothymosin-alpha stained a significantly higher percentage of nuclei in cancer and metastases compared with normal gastric mucosa. ERp57 staining was significantly decreased in cancer and metastases compared with both normal gastric mucosa and metaplasias. ERp57 expression also correlated with greater depth of tumor invasion and advanced stage of disease. Kaplan-Meier survival analysis determined that tumors with the highest quartile of ERp57 expression were statistically associated with longer postoperative survival. A Cox proportional hazard analysis showed that maintenance of ERp57 expression was associated with longer postoperative survival.
CONCLUSIONS - These results suggest that although prothymosin-alpha is overexpressed in gastric adenocarcinoma, it is not associated with alterations in survival. In contrast, loss of ERp57 expression correlated with more aggressive disease and could provide useful prognostic information for gastric cancer patients.
Protein expression profiles linked to sclerosis in the 5/6 nephrectomy (Nx) rat model of focal segmental glomerulosclerosis were investigated. Sections of control glomeruli from normal baseline Nx tissue and nonsclerotic and sclerotic glomeruli from 12 wk after 5/6 Nx were isolated by laser capture microdissection. Protein profiles were acquired directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Classification accuracy was 99.2% for distinguishing normal versus sclerotic glomeruli and 96.7 and 97.8% for nonsclerotic versus normal and sclerotic glomeruli, respectively. The proteomic pattern of the nonsclerotic glomeruli was more similar to sclerotic than normal glomeruli (P < 0.0001). Thymosin beta4, a protein with relevant interactions with plasminogen activator inhibitor-1, angiogenesis, and wound healing, was identified as a key differentially expressed protein. Thymosin beta4 immunostaining was increased in sclerotic glomeruli, predominantly in endothelial cells. Downregulation of thymosin beta4 by RNAi in cultured glomerular endothelial cells decreased angiotensin II-induced plasminogen activator inhibitor-1 expression. In conclusion, proteomic patterns can accurately distinguish normal versus nonsclerotic versus sclerotic glomeruli. The closely related proteomic patterns of nonsclerotic and sclerotic glomeruli suggest early activation of prosclerotic mechanisms even in seemingly intact glomeruli. Thymosin beta4 is a marker of such early events and may even contribute to sclerosis.