ABSTRACT
Periodontal ligament (PDL) has become an elective source of mesenchymal stem cells (PDLSCs) in dentistry. This research aimed to compare healthy PDLSCs (hPDLSCs) and periodontitis PDLSCs (pPDLSCs) to ascertain any possible functional differences owing to their milieux of origin. Cells were tested in terms of colony-forming unit efficiency; multi differentiating capacity; immunophenotype, stemness, and senescent state were studied by flow cytometry, immunofluorescence, and ß-galactosidase staining; gene expression using RT-PCR. Both hPDLSCs and pPDLSCs were comparable in terms of their immunophenotype and multilineage differentiation capabilities, but pPDLSCs showed a senescent phenotype more frequently. Thus, a selective small molecule inhibitor of DNA methyltransferase (DNMT), RG108, known for its effect on senescence, was used to possibly reverse this phenotype. RG108 did not affect the proliferation and apoptosis of PDLSCs, and it showed little effect on hPDLSCs, while a significant reduction of both p16 and p21 was detected along with an increase of SOX2 and OCT4 in pPDLSCs after treatment at 100 µM RG108. Moreover, the subset of PDLSCs co-expressing OCT4 and p21 decreased, and adipogenic potential increased in pPDLSCs after treatment. pPDLSCs displayed a senescent phenotype that could be reversed, opening new perspectives for the treatment of periodontitis.
ABSTRACT
Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.
Subject(s)
Adipose Tissue , Osteogenesis , Humans , Osteogenesis/genetics , Cell Differentiation/genetics , Adipocytes , Cells, Cultured , Stem CellsABSTRACT
Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.
Subject(s)
Angiomatosis, Bacillary/metabolism , Angiomatosis, Bacillary/microbiology , Bartonella henselae/physiology , Endothelial Cells/metabolism , Mesenchymal Stem Cells/metabolism , Neovascularization, Pathologic/metabolism , Angiomatosis, Bacillary/pathology , Biomarkers , Disease Susceptibility , Host-Pathogen Interactions , HumansABSTRACT
Imiquimod (IMQ) is an immune response modifier clinically used for the treatment of various topical diseases. However, its poor aqueous solubility and skin penetration capability make the topical delivery of IMQ a challenging task. This work aims at developing a nanomedicine-based topical formulation, carrying IMQ to control the scarring process for the treatment of aberrant wounds. For this purpose, IMQ was loaded in ß-cyclodextrin-based nanosponges and dispersed in a hydrogel suitable for dermal application. The formulation was characterized in vitro and compared with IMQ inclusion complexes, with (2-hydroxy)propyl ß-cyclodextrin(HPßCD) and carboxymethyl ß-cyclodextrin (CMßCD) showing enhanced penetration properties. The hydrogel containing IMQ-loaded nanosponges could act as a drug reservoir and guarantee the sustained release of IMQ through the skin. A greater inhibitory effect on fibroblast proliferation was observed for IMQ loaded in nanosponges compared to the other formulations.
ABSTRACT
Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1ß). OPN induction required cell-to-cell contact, mediated at least in part, by ß1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.
Subject(s)
Adaptation, Biological , Cell Communication , Dendritic Cells/metabolism , Mesenchymal Stem Cells/metabolism , Osteogenesis , Osteopontin/biosynthesis , Antigens, CD1/metabolism , Bone Marrow/metabolism , Cell Differentiation , Chemokine CCL5/biosynthesis , Coculture Techniques , Dendritic Cells/immunology , Glycoproteins/metabolism , Humans , Immunohistochemistry , Mesenchymal Stem Cells/cytologyABSTRACT
Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.
Subject(s)
Cell Death/genetics , Cell Respiration/genetics , Mitochondria/genetics , Receptors, Calcitriol/genetics , Cell Cycle/genetics , Cell Death/physiology , Humans , Mitochondria/metabolism , Mitochondria/physiology , Reactive Oxygen Species/metabolism , Receptors, Calcitriol/antagonists & inhibitors , Vitamin D/genetics , Vitamin D/metabolismABSTRACT
BACKGROUND: The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis. OBJECTIVE: The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA. METHODS: It was compared the incubation of the dermis in NaOH (0.06 N) and in the standard culture medium "Dulbecco Modified Eagle Medium" (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA. RESULTS: It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA. CONCLUSIONS: Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.
Subject(s)
Acellular Dermis , DNA/analysis , Extracellular Matrix/chemistry , Dermis/chemistry , Glycerol/chemistry , Humans , Polymerase Chain Reaction/methodsABSTRACT
During their spatial and differentiative progression, keratinocytes face a thermal gradient, from 37 °C in the proliferating basal layer to 32 °C found in skin surface. In our study, we hypothesized that this difference in temperature must be balanced by increasing the heat produced during respiratory activity. We demonstrated that at 33 °C human primary keratinocytes and HaCaT cells raised mitochondrial energy metabolism, but not glycolytic activity. At 33 °C, the increased mitochondrial ATP synthesis was associated with a strong induction of the modulator of the respiratory chain estrogen receptor ß, whereas uncoupling protein 1 expression was not changed. The enhanced mitochondrial oxidative metabolism was accompanied by a remarkable reduction in proliferation. These results suggest that environmental temperature can modulate the energy metabolism and proliferation of human keratinocytes.
ABSTRACT
Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at -80°C and -196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at -80°C and -196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.
ABSTRACT
Viral infections, especially cytomegalovirus (CMV), are a cause of death in burned patients. Aim of this study was to perform an in vitro CMV-infection model comparing fresh and glycerol-treated fibroblasts and keratinocytes. Cells were plated in plates for the two conditions. Each plate was set up with CMV dilutions. Immunofluorescence and real time PCR assays were performed. The assays were negative in both fresh and glycerolized keratinocytes. For fibroblasts, CMV-DNA was positive in both conditions and immunofluorescence test only in fresh cells. Glycerol at 85% confirms its strong virucidal effect as reported also for other viruses.
Subject(s)
Cryoprotective Agents , Cytomegalovirus Infections/virology , Glycerol , Skin Transplantation , Transplants/virology , Cryoprotective Agents/pharmacology , Cryoprotective Agents/toxicity , Cytomegalovirus/drug effects , Cytomegalovirus/genetics , Fibroblasts/metabolism , Fibroblasts/virology , Glycerol/pharmacology , Glycerol/toxicity , Humans , Keratinocytes/metabolism , Keratinocytes/virology , Primary Cell Culture , Skin/drug effects , Skin/metabolism , Skin/virology , Tissue Culture Techniques , Transplantation, HomologousABSTRACT
C/EBPs are a family of B-Zip transcription factors--TFs--involved in the regulation of differentiation in several tissues. The two most studied members--C/EBPα and C/EBPß--play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets--MafB and SOX2--affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation.
Subject(s)
CCAAT-Enhancer-Binding Protein-delta/genetics , Gene Expression Profiling , Keratinocytes/metabolism , Blotting, Western , CCAAT-Enhancer-Binding Protein-delta/metabolism , Cell Differentiation , Cells, Cultured , Humans , Immunohistochemistry , Keratinocytes/cytology , MafB Transcription Factor/genetics , MafB Transcription Factor/metabolism , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Skin/metabolism , Skin/pathology , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tissue Array AnalysisABSTRACT
Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.
Subject(s)
Biocompatible Materials , Keratinocytes , Materials Testing , Skin, Artificial , Basement Membrane/cytology , Basement Membrane/metabolism , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Biocompatible Materials/pharmacology , Cell Adhesion/drug effects , Cell Culture Techniques , Cell Survival/drug effects , Cells, Cultured , Glycerol , Humans , Immunohistochemistry , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Tissue Scaffolds/chemistryABSTRACT
This study evaluated the detection of Human Cytomegalovirus (HCMV)-DNA in donors' skin samples. HCMV-DNA was quantified in 100 skin specimens, including 50 fresh samples and as many corresponding glycerol-preserved specimens by a home-made Real Time PCR. HCMV-DNA was detected in 19/50 (38%) fresh specimens and 23/50 (46%) glycerol-preserved (p = n.s.). Nevertheless, the mere detection of HCMV-DNA does not imply the presence of infectious virions and therefore does not imply a risk of HCMV transmission, as treatment with glycerol is particularly efficacious in inactivating viral particles. Therefore, HCMV serology confirms its pivotal role in the setting of skin grafting.
Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/isolation & purification , Skin Transplantation , Skin/microbiology , Cytomegalovirus/genetics , Dermatologic Surgical Procedures , Humans , Organ Preservation , Tissue DonorsABSTRACT
Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.
Subject(s)
Activins/metabolism , Cell Differentiation/drug effects , Langerhans Cells/cytology , Skin/metabolism , Antigens, CD/biosynthesis , Antigens, CD1/biosynthesis , Cells, Cultured , Dendritic Cells/cytology , Humans , In Vitro Techniques , Keratinocytes/cytology , Lectins, C-Type/biosynthesis , Lipopolysaccharide Receptors/biosynthesis , Mannose-Binding Lectins/biosynthesis , Models, Biological , Monocytes/metabolism , Skin/drug effects , Transforming Growth Factor beta/metabolism , Wound HealingABSTRACT
BACKGROUND: Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. RESULTS: C/EBPdelta, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPdelta as a primary target of DeltaNp63alpha by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPdelta is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the DeltaNp63 promoter. Overexpression of C/EBPdelta leads to alteration in the normal profile of p63 isoforms, with the emergence of DeltaNp63beta and gamma, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPdelta leads to gene expression modifications, in part due to the concomitant repression of DeltaNp63alpha. Finally, C/EBPdelta is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. CONCLUSION: Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets.
Subject(s)
CCAAT-Enhancer-Binding Protein-delta/metabolism , Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Keratinocytes/metabolism , Promoter Regions, Genetic/physiology , Skin/metabolism , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism , CCAAT-Enhancer-Binding Protein-delta/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , Female , Gene Deletion , Gene Expression Profiling , Humans , Keratinocytes/cytology , Oligonucleotide Array Sequence Analysis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Trans-Activators/genetics , Transcription Factors , Tumor Suppressor Proteins/geneticsABSTRACT
Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.
Subject(s)
Activins/metabolism , Cicatrix, Hypertrophic/metabolism , Fibroblasts/metabolism , Follistatin/metabolism , Actins/metabolism , Activin Receptors/metabolism , Adolescent , Adult , Aged , Burns/complications , Burns/metabolism , Cell Proliferation , Cells, Cultured , Cicatrix, Hypertrophic/etiology , Cicatrix, Hypertrophic/pathology , Dendritic Cells/metabolism , Female , Fibroblasts/physiology , Humans , Immunoassay , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Skin/metabolism , Skin/pathology , Transforming Growth Factor beta1/metabolism , Wound Healing/physiologyABSTRACT
p63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63-depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation/genetics , Keratinocytes/metabolism , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism , Cell Differentiation , DNA-Binding Proteins/genetics , Gene Expression Profiling , Humans , Keratinocytes/cytology , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Skin/cytology , Substrate Specificity , Trans-Activators/genetics , Transcription Factors , Tumor Suppressor Proteins/geneticsABSTRACT
p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.
Subject(s)
Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Keratinocytes/metabolism , Trans-Activators/metabolism , Transcriptional Activation/physiology , Tumor Suppressor Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , DNA-Binding Proteins/genetics , Humans , Keratinocytes/cytology , Organogenesis/physiology , Protein Array Analysis/methods , Protein Binding/physiology , Trans-Activators/genetics , Transcription Factors , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that 'take' of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60h after harvesting). 2. The same specimens (stored at 4 and 37 degrees C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12-30h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30h was, therefore, set-up. The data suggested that skin stored at 37 degrees C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 degrees C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 degrees C for 4 days. The PVI of thawed skin samples decreased dramatically within 24h, and had reached 0% within 6 days.