Plasticity of human dedifferentiated adipocytes toward endothelial cells
2014; Elsevier BV; Volume: 43; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2014.10.003
ISSN1873-2399
AutoresAntonella Poloni, Giulia Maurizi, Sara Anastasi, Eleonora Mondini, Domenico Mattiucci, Giancarlo Discepoli, Fabiola Tiberi, Stefania Mancini, Stefano Partelli, Angela Maurizi, Saverio Cinti, Attilio Olivieri, Pietro Leoni,
Tópico(s)Zebrafish Biomedical Research Applications
Resumo•Dedifferentiated adipocytes represent a plastic stem cell population•Cells grew in specific medium were able to differentiate into endothelial-like structures•After endothelial induction cells expressed specific molecular and functional markers•After reseeding of endothelial-like cells into basal medium, they returned undifferentiated MSCs•The cells obtained showed normal karyotype The process of cellular differentiation in terminally differentiated cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Our previous study showed that mature adipocytes represent an alternative source of mesenchymal stem cells. Here, results showed the capacity of mature adipocytes to differentiate into endothelial-like cells, using the ability of these cells to revert into an immature phase without any relievable chromosomal alterations. Mature adipocytes were isolated from human omental and subcutaneous fat and were dedifferentiated in vitro. The resulting cells were subcultivated for endothelial differentiation and were analyzed for their expression of specific genes and proteins. Endothelial-like cells were harvested from the differentiation medium and were traditionally cultured to evaluate the endothelial markers and the karyotype. Cells cultured in specific medium formed tube-like structures and expressed several endothelial marker genes and proteins. The endothelial-like cells expressed significantly higher levels of vascular endothelium growth factor receptor 2, vascular endothelial cadherin, Von Willebrand factor, and CD133 than the untreated cells. These cells were positively stained for CD31 and vascular endothelial cadherin, markers of mature endothelial cells. Moreover, the low-density lipoprotein–uptake assay demonstrated a functionally endothelial differentiation of these cells. When these cells were harvested and reseeded in basal medium, they lost the endothelial markers and reacquired the typical mesenchymal stem cell markers and the ability to expand in a short time period. Moreover, karyotype analysis showed that these cells reverted into an immature phase without any karyotype alterations. In conclusion, the results showed that adipocytes exhibited a great plasticity toward the endothelial lineage, suggesting their possible use in cell therapy applications for vascular disease. The process of cellular differentiation in terminally differentiated cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Our previous study showed that mature adipocytes represent an alternative source of mesenchymal stem cells. Here, results showed the capacity of mature adipocytes to differentiate into endothelial-like cells, using the ability of these cells to revert into an immature phase without any relievable chromosomal alterations. Mature adipocytes were isolated from human omental and subcutaneous fat and were dedifferentiated in vitro. The resulting cells were subcultivated for endothelial differentiation and were analyzed for their expression of specific genes and proteins. Endothelial-like cells were harvested from the differentiation medium and were traditionally cultured to evaluate the endothelial markers and the karyotype. Cells cultured in specific medium formed tube-like structures and expressed several endothelial marker genes and proteins. The endothelial-like cells expressed significantly higher levels of vascular endothelium growth factor receptor 2, vascular endothelial cadherin, Von Willebrand factor, and CD133 than the untreated cells. These cells were positively stained for CD31 and vascular endothelial cadherin, markers of mature endothelial cells. Moreover, the low-density lipoprotein–uptake assay demonstrated a functionally endothelial differentiation of these cells. When these cells were harvested and reseeded in basal medium, they lost the endothelial markers and reacquired the typical mesenchymal stem cell markers and the ability to expand in a short time period. Moreover, karyotype analysis showed that these cells reverted into an immature phase without any karyotype alterations. In conclusion, the results showed that adipocytes exhibited a great plasticity toward the endothelial lineage, suggesting their possible use in cell therapy applications for vascular disease. Endothelial progenitor cells (EPCs) have been described as a rare population of nonhematopoietic cells that reside in the bone marrow and support the integrity of the vascular endothelium [1Asahara T. Murohara T. 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Several studies have demonstrated that mesenchymal stem cells (MSCs) from various tissues have the potential to differentiate into other cell types, suggesting that precommitted and committed cells have plasticity in cell fate determination [18Oki Y. Watanabe S. Endo T. Kano K. Mature adipocyte-derived dedifferentiated fat cells can trans-differentiate into osteoblasts in vitro and in vivo only by all-trans retinoic acid.Cell Struct Funct. 2008; 33: 211-222Crossref PubMed Scopus (62) Google Scholar]. Mesenchymal stem cells can be isolated from a variety of adult tissues and organs, and adipose tissue is abundant and easily accessible at most ages. Mature adipocytes are numerous in adipose tissue, and they are easily isolated without painful procedures or donor site injuries. We previously demonstrated that mature adipocytes isolated from fat tissue can dedifferentiate into MSCs using an easy in vitro dedifferentiation strategy and a small amount of adipose tissue. Thus, white mature adipocytes gave rise to dedifferentiated cells when they lose their fat in culture. During this process, gene reprogramming events occur, leading to changes in the epigenetic status of the cells, which allow them to acquire morphologic and functional stem cell properties [19Poloni A. Maurizi G. Leoni P. et al.Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.Stem Cells. 2012; 30: 965-974Crossref PubMed Scopus (97) Google Scholar]. Therefore, dedifferentiated adipocytes may be an attractive source of stem cells for regenerative medicine and other cell-based therapies. The aim of the present study was to examine whether dedifferentiated fat cells could be induced into the endothelial lineage, using the ability of these cells to revert into an immature phase without any relievable karyotype alterations. In accordance with the guidelines of the local ethical committee (300/DG), omental and subcutaneous fat tissue (5–10 g) were obtained from patients (n = 10 + 10, 53–81 years old) at the time of abdominal surgery. The patients were not obese and were undergoing surgery for pancreatic diseases that were localized and had not enlarged to the omental and subcutaneous fat tissues. Adipose tissue was promptly washed with Dulbecco's modified Eagle's medium (DMEM; Biological Industries, Milan, Italy), and the visible blood vessels were removed. The samples were minced into smaller pieces and treated with 3 mg/mL type I collagenase (Gibco, Milan, Italy) at 37°C for 2 hours. To obtain isolated mature adipocytes free of stromal-vascular elements, after collagenase digestion, the disrupted tissues were filtered through a 200-μm nylon sieve. The filtered cells were washed four times with DMEM and centrifuged at 250 g for 5 min. Only the floating top layer was collected after each centrifugation step, which allowed for the isolation of a pure fraction of floating adipocytes and a pellet containing the stromal vascular fraction (SVF) cells. After the last centrifugation step, the fatty layer was transferred to an inverted 25-cm2 cell culture flask that was completely filled with DMEM supplemented with 20% fetal bovine serum (Stem Cell Technologies, Vancouver, Canada), and the cells were seeded for ceiling culture, where the bottom of the flask is on top [5Rafii S. Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nat Med. 2003; 9: 702-712Crossref PubMed Scopus (1434) Google Scholar]. Only mature adipocytes free of any detectable contamination of stromal-vascular elements were allowed to adhere to the top layer of the flask in a 5% CO2 incubator (37°C), and the cultures were monitored daily for cell attachment. When the cells lost their spherical shape, they were trypsinized and subcultivated at an initial seeding density of 2 × 103 cells/cm2. When the cells reached confluence, the culture supernatants of the dedifferentiated adipocytes were collected and frozen at −20°C. Multiplex human cytokine, chemokine, and growth factor detection assays (BioPlex, BioRad, Milan, Italy) were utilized to measure the levels of interleukin (IL) 6, IL-8, basic fibroblast growth factor (FGF-β), basic granulocyte colony-stimulating factor (G-CSF), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and vascular endothelium growth factor (VEGF) in the culture supernatants. Matrigel (BD Biosciences, Milan, Italy) was added to a 24-well plate (200 μL well−1) and allowed to solidify for 30 min at 37°C. Cells (0.1 × 106) were suspended in 500 μL of DMEM and were placed on top of the Matrigel. After 24-hour incubation, the formation of cord- or tube-like networks was examined and recorded using a phase contrast microscope. Early outgrowth cells were obtained after 7 days in culture and were analyzed for the expression of several endothelial markers. The cells were harvested and studied by flow cytometry, real-time polymerase chain reaction (PCR), and immunocytochemistry. Huvec cell line and SVF-derived mesenchymal stem cell (cultured in Matrigel) were analyzed in all the experiments as positive controls. The dedifferentiated adipocytes were characterized by flow cytometry before and after the endothelial differentiation procedure. To harvest the cells from Matrigel, a step of collagenase digestion was performed. The cells were stained with fluorescein isothiocyanate–, phycoerythrin- or PerCP-conjugated antibodies against CD31 (platelet endothelial cell adhesion molecule), CD34 (hematopoietic progenitor cell antigen, BD Biosciences), CD45 (leukocyte common antigen, BD Biosciences), CD90 (Thy-1, BD Pharmingen) and CD73 (ecto-5′-nucleotidase). We used fluorescein isothiocyanate, phycoerythrin (Dako, Glostrup, Denmark), and PerCP (Becton Dickinson Pharmingen, Milan, Italy) negative isotypes as control antibodies. The cells were incubated with the primary antibodies at 4°C for 30 min. Thereafter, cell fluorescence was evaluated by flow cytometry using a FACSCalibur instrument (Becton Dickinson Pharmingen). The data were analyzed using the CellQuest software (Milan, Italy). To harvest the cells from Matrigel, a step of collagenase digestion was performed. Total RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN, Milan, Italy) according to the manufacturer's instructions. The purity of the RNA was confirmed by determining the 260 nm/280 nm absorbance ratio (>1.8). For each sample, 1 μL of total RNA was reverse-transcribed in a 20 μL reaction containing 5 × reaction buffer (Invitrogen, Milan, Italy), 100 mmol/L dNTPs (Biotech, Milan, Italy), 50 mmol/L MgCl2 (Promega, Milan, Italy), 3 μg/μL random hexamers (Invitrogen), 100 mmol/L dithiothreitol (DTT; Invitrogen), 40 U/μL RNase inhibitor (Takara, Shiga, Japan), and 200 U/μL Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen). The reactions proceeded for 10 min at 70°C, 10 min at 20°C, 45 min at 42°C, and 3 min at 99°C. Real-time PCR analysis of the dedifferentiated adipocytes and SVF-derived MSCs were performed to quantify the expression of CD133 (Prominin 1; NM_006017.2; 172 bp), VEGF receptor 2 (VEGFR-2; NM_002253.2; 131 bp), Von Willebrand factor (VWF; NM_000552.3; 111 bp), and CD144 (vascular endothelial-cadherin [VE-cadherin]; NM_001795.3; 152 bp). The primers for CD133, VEGFR-2, VWF, and CD144 were designed using the Primer3 software (http://frodo.wi.mit.edu/primer3), and the primer pairs were designed to span at least one exon-intron junction to exclude any possibility of genomic DNA amplification. The transcript levels of these genes were normalized to the expression of the constitutive gene GAPDH (NM_002046.4, 185 bp) and an endogenous calibrator (Huvec), following analysis with the 2−ΔΔCt method [20Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-Delta Delta Ct method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar]. The SYBR Select Master Mix (Life Technologies, Monza, Italy) and specific primers were optimized for the ABI 7500 Fast system [Applied Biosystems, Foster City, CA], and reactions were performed in a 20 μL mixture containing 10 μL of MasterMix, 10 μmol/L of each primer, and 2 μL of cDNA template. After 20 sec at 50°C and 10 min at 95°C, the samples were incubated for 40 cycles of denaturation for 15 sec at 95°C and annealing/extension for 1 min at 60°C. The MeltCurve Stage was performed using one cycle each at 95°C for 15 sec, 60°C for 1 min, 95°C for 30 sec, and 60°C for 15 sec. Moreover, the PCR products were separated by electrophoresis in a 2% agarose gel stained with ethidium bromide and were visualized using an ultraviolet illuminator. To harvest the cells from Matrigel, a step of collagenase digestion was performed. Cells were attached in slides for immunocytochemistry by cytocentrifuge and were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-X. We used 3% hydrogen peroxide to inactivate the endogenous peroxidase, followed by incubation with 10% normal goat serum (Vector Laboratories, Burlingame, CA) to reduce the nonspecific staining. Then, the cells were incubated for 1 hour at room temperature with a polyclonal rabbit anti–vascular endothelial cadherin (anti-VE-cadherin; 1:200; Abcam, Cambridge, United Kingdom) primary antibody, according to the Avidin Biotin Complex method. The biotinylated horse radish peroxidase (HRP) -conjugated secondary antibody was a goat-antirabbit immunoglobulin G (Vector Laboratories, Burlingame, CA). Immunocytochemical reactions were performed using the Vector's Vectastain ABC Kit (Vector Laboratories) and Sigma Fast 3,3′-diaminobenzidine as a substrate (Sigma, St. Louis, MO). The sections were counterstained with hematoxylin. After the differentiation process the endothelial-like cells (ECs) were tested for the presence of scavenger receptor to assess the binding low-density lipoprotein (LDL) capacity of these cells. Cells harvested from Matrigel were attached in slides for immunocytochemistry by cytocentrifuge and were fixed in cold methanol. Purified acetylated LDL (Ac-LDL) labeled with the fluorescent probe 1,1′-dioctadecy 1-3,3,3′3′-tetramethyl-indocarbocyanine perchlorate (DiI) was purchased from Molecular Probes (Life Technologies). Cells grown on glass coverslips were preincubated at 37°C in a serum-free medium containing 0.5% bovine serum album for 24 hours. Thereafter, the cells were directly incubated in basal medium containing 10 μg/mL DiI-Ac-LDL for 4 hours at 37°C. The medium was then removed and the cells were washed once with probe-free medium for 10 min and three times with phosphate-buffered saline. Cells were observed by fluorescence microscopy. The cells harvested from Matrigel by collagenase digestion were reseeded in basal medium and, at this time, the cytogenetic evaluation was performed. The cells were harvested from a confluent flask and were then transferred to petri dishes with a glass slide. The cells were expanded in 2 mL of Chang medium (Irvine Scientific, Santa Ana, CA); after 24–48 hours, the cells were exposed to 200 μL of 10 μg/μL colchicine (Karyomax Colcemid, Life technologies, Monza, Italy) for 1.5–2 hours and to 0.075 mol/L KCl for 25 min. The slides were fixed in a methanol-acetic acid solution (3:1), subjected to quinacrine fluorescence quenching (QFQ) banding and then examined using a Zeiss Axioplan 2 (Carl Zeiss, Gottingen, Germany) epifluorescence microscope. Images of the metaphase chromosome spreads were captured and analyzed using the PSI MacKtype 4.5 software (Bury St Edmunds, UK), which was run on a Macintosh computer. The karyotypes were described following the recommendations of the 2005 International System for Human Cytogenetic Nomenclature [21Shaffer L.G. Tommerup N. An International System for Human Cytogenetic Nomenclature. S. Karger AG, Basel, Switzerland2005Google Scholar]. The data are presented as the mean ± standard deviations and were analyzed using Student's t test. The differences were considered statistically significant when p < 0.05. Ceiling cultures were performed using mature adipocytes isolated from omental and subcutaneous fat tissues. Beginning around day 5–6 of culture, the cytoplasm of the ceiling culture began to spread. The adipocytes then changed to a fibroblast-like morphology. Upon culturing, they lost a considerable amount of lipid, the nuclei became more centralized, and the cells became elongated in shape. When the culture medium was changed and the flasks were inverted on day 8–10 of the ceiling culture, we obtained dedifferentiated cells that had entered in a proliferative phase. When the cells reached the confluence of the flask, they were harvested and used for endothelial differentiation (Fig. 1). Dedifferentiated adipocyte-culture supernatants were analyzed and compared with those of mature adipocytes and SVF-derived MSCs using a multiplex detection assay for different factors. The results were divided into four groups: value <50 pg/mL (−), 50 pg/mL < value < 500 pg/mL (+), 500 pg/mL < value < 5,000 pg/mL (++), and value > 5,000 pg/mL (+++). The samples showed high levels of β-FGF (+), G-CSF (+), TNF-α (+) and even higher values for IL-6 (++), IL-8 (++), IFN-γ (++), and VEGF (++). Notably, VEGF, one of the most important proangiogenic factors, was expressed without significant difference between dedifferentiated adipocytes and SVF-derived MSCs, suggesting similar endothelial differentiation ability of these two cell populations. Data demonstrated that adipocytes secreted similar levels of these specific factors before and after the dedifferentiation process, indicating their potential plasticity properties (Table 1).Table 1Angiogenic growth factors produced by dedifferentiated adipocytesDedifferentiated adipocytes (Pg/mL)Adipocytes (Pg/mL)SVF-MSC (Pg/mL)β-FGF+ (77)− (44)+ (81)G-CSF+ (81)+ (84)+ (93)TNF-α+ (344)+ (89)+ (381)IL-6++ (2800)++ (1368)++ (3669)IL-8++ (979)++ (2938)++ (2211)IFN-γ++ (558)+ (139)++ (655)VEGF++ (2787)+ (316)++ (3130) Open table in a new tab The dedifferentiated cells were suspended in DMEM and were cultured on top of Matrigel. Just after 24 hours, the dedifferentiated cells began to change their spindle-shaped morphologies, forming a tube-like network. Only cells cultured in Matrigel formed these structures, whereas cells in basal medium did not show any shape alterations. In 1 week, the cells cultured in the semisolid medium achieved a cord-like morphology. The endothelial-like cells formed tube structures with a variable tube length (30–70 μm) and area (40–100 μm2). Moreover, each endothelial-like structure expressed 5–10 branch points. The SVF-derived MSC acquired the same shape as dedifferentiated adipocytes in Matrigel in the same time of culture (Fig. 2). Both dedifferentiated adipocytes and SVF-derived MSCs, after Matrigel culture, expressed the endothelial lineage-specific marker CD31 (92% ± 7%) at their cellular surface and were also positively stained for CD34 (83% ± 5%), CD73 (87% ± 5%), and CD90 (95% ± 4%); additionally, these cells were negative for CD45 (Fig. 3). Notably, dedifferentiated adipocytes only cultured in basal medium were CD31-negative stained, whereas Huvec cells were CD31-positive stained and were used as positive control. The resulting ECs were harvested from Matrigel and reseeded in basal medium. Immunophenotype analysis of these cells was performed within 24 hours after seeding in basal medium to exclude any possibility that results obtained were representative of residual MSCs present in Matrigel culture. The data showed that cells in this phase lost their endothelial features and reacquired typical MSC markers. These cells were positive for CD90, CD44, CD73, and CD29 and were negative for CD31, CD34, and CD45. These cells were also able to expand in a short time period (2 ± 0.3 logs in three passages of culture in 35 days; Fig. 4). The expression of genes associated with endothelial differentiation was investigated using real-time PCR to determine whether the dedifferentiated adipocytes cultured in specific medium have properties of endothelial cells. Endothelial differentiation involves a series of transcriptional events, including the functions of VEGFR-2 and VE-cadherin. It appears that VEGFR-2 mediates almost all of the known cellular responses to VEGF, which is an important signaling protein involved in both vasculogenesis and angiogenesis. Vascular endothelial cadherin plays an important role in endothelial cell biology by controlling cohesion, the organization of the intercellular junction, and the maintenance of newly formed vessels. Moreover, VWF is an important marker of the endothelium; this protein binds to collagen when it is exposed in endothelial cells owing to blood vessel damage. CD133 is a glycoprotein that is expressed in endothelial progenitor cells. The results showed the positive activation of the endothelial specific markers. Quantitative data demonstrated a significantly higher expression of VEGFR-2, VWF, CD133, and VE-cadherin (CD144) in the ECs compared with the untreated, dedifferentiated cells (p < 0.05). The same data were obtained with SVF-derived MSCs. The Huvec cell line was used as a positive control and endogenous calibrator for the quantitative assay (Fig. 5). Immunocytochemical analysis was performed to confirm the molecular data. The differentiated cells expressed VE-cadherin in the cytoplasm and around the cellular surface, showing that these cells can undergo terminal endothelial differentiation in vitro. Notably, the negative staining for this marker in the untreated cells is in line with this hypothesis (Fig. 6). Results showed a higher capacity of both endothelial-like cells derived from dedifferentiated adipocytes and SVF-derived cells to bind DiI-Ac-LDL, demonstrating the expression of scavenger receptor after the differentiation process (Fig. 7). At the end of the cultures, the cells obtained from the dedifferentiated adipocytes and SVF-derived MSCs were harvested from the Matrigel by a step of collagenase digestion and were subcultivated in basal medium. The karyotypes of these cells were analyzed. A median of 50 metaphases (range 16–75) was analyzed for each sample at approximately the 350–450 band level. In all of the investigated cells, no clonal abnormalities, structural abnormalities, or other abnormalities, including the loss of the same chromosome in three or more metaphases or the gain of the same chromosome in two or more metaphases, were found (Fig. 8). The present study provides structural and molecular evidence that dedifferentiated adipocytes have the ability to undergo the mesenchymal-endothelial transition, thus highlighting the plasticity of these cells. The resulting endothelial cells were tested for their proliferation potential in vitro and were characterized for endothelial specific markers. We investigated the phenotype of the cells cultured in the presence of endothelial growth supplements and after differentiation. Moreover, we analyzed the karyotype of the tested cells to study their biological safety and ensured that these cells could be used in clinical applications. Adipose tissue has recently been reported as an important reservoir of stem cells that can be potentially used in medicine [22Shen J.F. Sugawara A. Yamashita J. Ogura H. Sato S. Dedifferentiated fat cells: An alternative source of adult multipotent cells from the adipose tissues.Int J Oral Sci. 2011; 3: 117-124Crossref PubMed Scopus (68) Google Scholar]. Our group has also shown that adult mature adipocytes can reversibly change their phenotype and transform into new cells with different morphologies and physiologies in vitro; thus, they can essentially reprogram their genomes [19Poloni A. Maurizi G. Leoni P. et al.Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.Stem Cells. 2012; 30: 965-974Crossref PubMed Scopus (97) Google Scholar]. Previously, we have shown that isolated mature adipocytes express stem-cell genes and reprogramming genes, suggesting a potential use of this committed population as stem cells [23Dodson M.V. Fernyhough M.E. Mature adipocytes: Are there still novel things that we can learn from them?.Tissue Cell. 2008; 40: 307-308Crossref PubMed Scopus (15) Google Scholar]. In particular, when maintained in culture, mature adipocytes undergo a process of dedifferentiation [24Poloni A. Maurizi G. Rosini V. et al.Selection of CD271(+) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow.Cytotherapy. 2009; 11: 153-162Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Adipocytes lost their morphology and lineage gene expression profile, acquiring the structural and molecular features of bone marrow-derived MSCs. Thus, human dedifferentiated adipocytes hold potential promise for therapeutic applications in regenerative medicine because they are an easily accessible source of stem cells with great plasticity. Endothelial progenitor cells have been isolated from human blood and may contribute to the repair of damaged endothelium and the formation of new blood vessels [25Liu J.W. Dunoyer-Geindre S. Serre-Beinier V. et al.Characterization of endothelial-like cells derived from human mesenchymal stem cells.J Thromb Haemost. 2007; 5: 826-834Crossref PubMed Scopus (100) Google Scholar]. A major obstacle in the therapeutic use of EPCs is the limited number of cells that can be obtained from blood. There are several advantages to working with adipocytes and dedifferentiated adipocytes when studying endothelial differentiation or as a potential source of multipotent cells. These cells are readily available in large quantities; thus, there is less need for propagation to generate sufficient cell numbers. Another advantage of the dedifferentiated adipocytes is the simple method used for endothelial differentiation. Here, we showed an easy technique to obtain endothelial-like cells from mature adipocytes, which may contribute to the repair of damaged endothelium and the formation of new blood vessels. Dedifferentiated adipocytes are able to differentiate into endothelial-like cells when cultivated in specific semisolid medium, resulting in the formation of a network of cord- or tube-like structures. In our results, the dedifferentiated adipocytes were initially negatively stained for CD34, CD31, and CD45 and were positive for CD90 and CD73, identifying them as mesenchymal stem cells. After specific treatment, we detected the expression of all these markers, with the exception of CD45, suggesting that the dedifferentiated cells may serve as precursor cells to several types of stromal progenitor cell populations. Thus, they rapidly acquired an endothelial phenotype and expressed specific markers on their cellular surface, including CD31 and CD34. Moreover, data obtained in this study showed that endothelial lineage genes, VEGFR-2, VWF, CD133, and CD144, were expressed at higher levels in these cells than in the untreated cells. Our immunocytochemistry results indicate that adipocytes can undergo terminal endothelial differentiation in vitro, as shown by the expression of VE-cadherin. Indeed, VE-cadherin is required for the formation of vasculature and is expressed specifically in endothelial cells. It is indispensable for proper vascular development and the maintenance of newly formed vessels. Moreover, the cellular uptake of DiI-Ac-LDL has been used as one of the characteristics of ECs. Here, we showed the expression of scavenger receptors in the treated cells, giving a further evidence of dedifferentiated adipocytes' endothelial differentiation process. 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On the contrary, a recent study demonstrated that overexpression of VEGF in adipose tissue, resulting in increased adipose vascularity, may even protect against diet-induced obesity and insulin resistance [32Elias I. Franckhauser S. Ferre T. et al.Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance.Diabetes. 2012; 61: 1801-1813Crossref PubMed Scopus (222) Google Scholar]. This would, in part, challenge previous studies that showed that angiogenesis inhibitors have promise in preventing obesity [33Cao Y. Adipose tissue angiogenesis as a therapeutic target for obesity and metabolic diseases.Nat Rev Drug Discov. 2010; 9: 107-115Crossref PubMed Scopus (303) Google Scholar]. In this study, we provided evidence that cells derived from human mature adipocytes are able to undergo endothelial differentiation following a simple specific treatment. Thus, our data support the close relationship between adipocytes and endothelial cells that was previously suggested by Planat-Bernard et al. [34Planat-Benard V. Silvestre J.S. Cousin B. et al.Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives.Circulation. 2004; 109: 656-663Crossref PubMed Scopus (1184) Google Scholar] and Jumabay et al. [35Jumabay M. Abdmaulen R. Urs S. et al.Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.J Mol Cell Cardiol. 2012; 53: 790-800Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar] and highlight the concept that adipose lineage cells represent a suitable new cell source for therapeutic angiogenesis for the treatment of ischemic disease and cardiovascular regeneration. In line with these results, previous data suggested that white and brown adipocytes share the same precursor cells, endothelial cells, and the existence of a shared precursor is consistent with data supporting the physiologic reversible transdifferentiating properties of the adipocytes in the adipose organ [36Barbatelli G. Murano I. Madsen L. et al.The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.Am J Physiol Endocrinol Metab. 2010; 298: E1244-1253Crossref PubMed Scopus (532) Google Scholar]. Moreover, these data support an endothelial origin of adipocytes, suggesting a model for how adipogenesis and angiogenesis are coordinated during adipose tissue expansion. These data gave new perspectives on adipose tissue plasticity; these findings are particularly important because of the availability of adipose tissue and of the capacity of these cells to expand in vitro. Cells with a phenotype similar to our endothelial-like cells were obtained through the differentiation of bone marrow derived-MSCs (MSCEs) [25Liu J.W. Dunoyer-Geindre S. Serre-Beinier V. et al.Characterization of endothelial-like cells derived from human mesenchymal stem cells.J Thromb Haemost. 2007; 5: 826-834Crossref PubMed Scopus (100) Google Scholar], and this differentiation was accompanied by an increase in endothelial marker proteins. Subcutaneous implantation of MSCEs in collagen plugs into nonobese diabetic severe combined immunodeficient mice resulted in the formation of functional blood vessels that incorporated the MSCEs. The process of cellular differentiation in terminally differentiated mammalian cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Here, in addition to characterizing the endothelial-like cells obtained from adipocytes, we also reported on the ability of these cells to revert into an immature phase without any karyotype alteration. Instead, the endothelial-like cells that were harvested from the specific medium and cultivated in general proliferative medium lost their lineage markers and reacquired the noncommitted status of dedifferentiated adipocytes. These differentiation/transdifferentiation processes may represent the manifestation of morphologic, molecular, and functional changes that occur in the adipocytes when exposed to specific microenvironments, suggesting the great plasticity of thes2e cells. Moreover, in line with these results, the dedifferentiated fat cells displayed long telomeres (11.5 ± 0.7 kb) [37Poloni A. Maurizi G. Serrani F. et al.Molecular and functional characterization of human bone marrow adipocytes.Exp Hematol. 2013; 41: 558-566Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar]. Thus, telomere length and the absence of karyotype alterations demonstrated that these cells were not susceptible to malignant transformation. Normal karyotypes were preserved throughout expansion and endothelial differentiation, suggesting the safety of the adipocytes. In conclusion, our results indicate that adipose cells could be an alternative, more accessible resource for cell therapy and regenerative medicine. These data demonstrated that adipocytes exhibited a great plasticity toward different lineages, and this potency could be directed toward the endothelial lineage. This work was supported by grants from Associazione Italiana Contro le Leucemie, Linfomi e Mielomi , and Sezione di Ancona-ONLUS . No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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