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Limana F.,Centro Cardiologico Monzino Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | Esposito G.,Instituto Dermopatico dellImmacolata IRCCS | D'Arcangelo D.,Instituto Dermopatico dellImmacolata IRCCS | Di Carlo A.,Instituto Dermopatico dellImmacolata IRCCS | And 7 more authors.
PLoS ONE | Year: 2011

Aims: HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts. Methods and Results: Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ~23% reduction in LV volume, a ~48% increase in infarcted wall thickness and a ~14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ~2-fold increase in c-kit+ cell number, a ~13-fold increase in newly formed myocytes and a ~2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20-25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206. Conclusions: HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3. © 2011 Limana et al.


Valorani M.G.,Queen Mary, University of London | Valorani M.G.,BIOS S.p.A | Montelatici E.,Fondazione IRCCS Ca Granda | Germani A.,Fondazione Livio Patrizi | And 12 more authors.
Cell Proliferation | Year: 2012

Objectives: Hypoxia is an important factor in many aspects of stem-cell biology including their viability, proliferation, differentiation and migration. We evaluated whether low oxygen level (2%) affected human adipose tissue mesenchymal stem-cell (hAT-MSC) phenotype, population growth, viability, apoptosis, necrosis and their adipogenic and osteogenic differentiation potential. Materials and methods: hAT-MSCs from four human donors were cultured in growth medium under either normoxic or hypoxic conditions for 7 days and were then transferred to normoxic conditions to study their differentiation potential. Results: Hypoxia enhanced hAT-MSC expansion and viability, whereas expression of mesenchymal markers such as CD90, CD73 and endothelial progenitor cell marker CD34, remained unchanged. We also found that pre-culturing hAT-MSCs under hypoxia resulted in their enhanced ability to differentiate into adipocytes and osteocytes. Conclusions: This protocol could be useful for maximizing hAT-MSC potential to differentiate in vitro into the adipogenic and osteogenic lineages, for use in plastic and reconstructive surgery, and in tissue engineering strategies. © 2012 Blackwell Publishing Ltd.


Valorani M.G.,Queen Mary, University of London | Valorani M.G.,BIOS S.p.A. | Germani A.,Fondazione Livio Patrizi | Otto W.R.,Cancer Research UK | And 14 more authors.
Cell and Tissue Research | Year: 2010

Mesenchymal stem cells (MSCs) are usually cultured under normoxic conditions (21% oxygen). However, in vivo, the physiological "niches" for MSCs have a much lower oxygen tension. Because of their plasticity, stem cells are particularly sensitive to their environments, and oxygen tension is one developmentally important stimulus in stem cell biology and plays a role in the intricate balance between cellular proliferation and commitment towards differentiation. Therefore, we investigated here the effect of hypoxia (2% oxygen) on murine adipose tissue (AT) MSC proliferation and adipogenic differentiation. AT cells were obtained from the omental fat and AT-MSCs were selected for their ability to attach to the plastic dishes, and were grown under normoxic and hypoxic conditions. Prior exposure of MSCs to hypoxia led to a significant reduction of ex vivo expansion time, with significantly increased numbers of Sca-1+ as well as Sca-1+/CD44 +double-positive cells. Under low oxygen culture conditions, the AT-MSC number markedly increased and their adipogenic differentiation potential was reduced. Notably, the hypoxia-mediated inhibition of adipogenic differentiation was reversible: AT-MSCs pre-exposed to hypoxia when switched to normoxic conditions exhibited significantly higher adipogenic differentiation capacity compared to their pre-exposed normoxic-cultured counterparts. Accordingly, the expression of adipocyte-specific genes, peroxisome proliferator activated receptor γ (Pparγ), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) were significantly enhanced in hypoxia pre-exposed AT-MSCs. In conclusion, pre-culturing MSCs under hypoxic culture conditions may represent a strategy to enhance MSC production, enrichment and adipogenic differentiation. © 2010 Springer-Verlag.


Rossini A.,Centro Cardiologico Monzino | Rossini A.,University of Milan | Frati C.,University of Parma | Lagrasta C.,University of Parma | And 18 more authors.
Cardiovascular Research | Year: 2011

AimsBone marrow mesenchymal stromal cell (BMStC) transplantation into the infarcted heart improves left ventricular function and cardiac remodelling. However, it has been suggested that tissue-specific cells may be better for cardiac repair than cells from other sources. The objective of the present work has been the comparison of in vitro and in vivo properties of adult human cardiac stromal cells (CStC) to those of syngeneic BMStC.Methods and resultsAlthough CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC.ConclusionOur findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair. © 2010 The Author.


Malgieri A.,University of Rome Tor Vergata | Kantzari E.,Future Health | Patrizi M.P.,Fondazione Livio Patrizi | Gambardella S.,University of Rome Tor Vergata
International Journal of Clinical and Experimental Medicine | Year: 2010

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in all tissues, as part of the perivascular population. As multipotent cells, MSCs can differentiate into different tissues originating from mesoderm ranging from bone and cartilage, to cardiac muscle. MSCs are an excellent candidate for cell therapy because they are easily accessible, their isolation is straightforward, they can be bio-preserved with minimal loss of potency, and they have shown no adverse reactions to allogeneic versus autologous MSCs transplants. Therefore, MSCs are being explored to regenerate damaged tissue and treat inflammation, resulting from cardiovascular disease and myocardial infarction (MI), brain and spinal cord injury, stroke, diabetes, cartilage and bone injury, Crohn's disease and graft versus host disease (GvHD). Most of the application and clinical trials involve MSCs from bone marrow (BMMSCs). Transplantation of MSCs from bone marrow is considered safe and has been widely tested in clinical trials of cardiovascular, neurological, and immunological disease with encouraging results. There are examples of MSCs utilization in the repair of kidney, muscle and lung. The cells were also found to promote angiogenesis, and were used in chronic skin wound treatment. Recent studies involve also mesenchymal stem cell transplant from umbilical cord (UCMSCt). One of these demonstrate that UCMSCt may improve symptoms and biochemical values in patients with severe refractory systemic lupus erythematosus (SLE), and therefore this source of MSCs need deeper studies and require more attention. However, also if there are 79 registered clinical trial sites for evaluating MSC therapy throughout the world, it is still a long way to go before using these cells as a routinely applied therapy in clinics.


Limana F.,Instituto Of Ricovero E Cura A Carattere Scientifico | Capogrossi M.C.,Instituto Dermopatico dellImmacolata | Germani A.,Fondazione Livio Patrizi
Pharmacology and Therapeutics | Year: 2011

During heart development, the epicardium provides cardiogenic progenitor cells and, together with the myocardium, directs lineage specification and coordinates both myocardial growth and coronary vasculature formation. In the adult heart, the established function of the epicardium is to provide a smooth surface that, together with the pericardium, favors heart movement during contraction and relaxation. Recently, epicardial precursor cells with the ability to differentiate into cardiomyocytes and vascular cells have been identified and the quiescent nature of the adult epicardium has been questioned. Interestingly, the signaling pathways involved in this process appear to be regulated, in the adult heart, by mechanisms similar to those in the embryonic heart. This review will summarize the properties of the embryonic epicardium and will focus on recent advances on the role of the adult epicardium in cardiac regeneration. Specifically, we will present aspects of epicardial cell biology that may be relevant to the development of new therapeutic approaches aimed at inducing heart repair following injury. © 2010 Elsevier Inc. All rights reserved.


Giardina E.,University of Rome Tor Vergata | Stocchi L.,University of Rome Tor Vergata | Cuzzola V.F.,IRCCS Centro Neurolesi Bonino Pulejo | Zampatti S.,University of Rome Tor Vergata | And 6 more authors.
Electrophoresis | Year: 2010

Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA-B*57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA-B*57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence-specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non-infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence-specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Limana F.,Instituto Of Ricovero E Cura A Carattere Scientifico | Bertolami C.,Instituto Dermopatico dellImmacolata | Mangoni A.,Instituto Dermopatico dellImmacolata | Di Carlo A.,Instituto Dermopatico dellImmacolata | And 10 more authors.
Journal of Molecular and Cellular Cardiology | Year: 2010

Stem cells expressing c-kit have been identified in the adult epicardium. In mice, after myocardial infarction, these cells proliferate, migrate to the injury site and differentiate toward myocardial and vascular phenotype. We hypothesized that, acutely after myocardial infarction, pericardial sac integrity and pericardial fluid (PF) may play a role on epicardial cell gene expression, proliferation and differentiation. Microarray analysis indicated that, in the presence of an intact pericardial sac, myocardial infarction modulated 246 genes in epicardial cells most of which were related to cell proliferation, cytoskeletal organization, wound repair and signal transduction. Interestingly, WT1, Tbx18 and RALDH2, notably involved in epicardial embryonic development, were markedly up-regulated. Importantly, coexpression of stem cell antigen c-kit and WT1 and/or Tbx18 was detected by immunohistochemistry in the mouse epicardium during embryogenesis as well as in adult mouse infarcted heart. Injection of human pericardial fluid from patients with acute myocardial ischemia (PFMI) in the pericardial cavity of non-infarcted mouse hearts, enhanced, epicardial cell proliferation and WT1 expression. Further, PFMI supplementation to hypoxic cultured human epicardial c-kit+ cells increased WT1 and Tbx18 mRNA expression. Finally, insulin-like growth factor 1, hepatocyte growth factor and high mobility group box 1 protein, previously involved in cardiac c-kit+ cell proliferation and differentiation, were increased in PFMI compared to the pericardial fluid of non ischemic patients. In conclusion, myocardial infarction reactivates an embryonic program in epicardial c-kit+ cells; soluble factors released in the pericardial fluids following myocardial necrosis may play a role in this process. © 2009 Elsevier Ltd. All rights reserved.


Conte C.,University of Rome Tor Vergata | D'Apice M.R.,University of Rome Tor Vergata | Rinaldi F.,University of Rome Tor Vergata | Gambardella S.,Fondazione Livio Patrizi | And 2 more authors.
BMC Medical Genetics | Year: 2011

Background: Treacher Collins syndrome (TCS) is one of the most severe autosomal dominant congenital disorders of craniofacial development and shows variable phenotypic expression. TCS is extremely rare, occurring with an incidence of 1 in 50.000 live births. The TCS distinguishing characteristics are represented by down slanting palpebral fissures, coloboma of the eyelid, micrognathia, microtia and other deformity of the ears, hypoplastic zygomatic arches, and macrostomia. Conductive hearing loss and cleft palate are often present. TCS results from mutations in the TCOF1 gene located on chromosome 5, which encodes a serine/alanine-rich nucleolar phospho-protein called Treacle. However, alterations in the TCOF1 gene have been implicated in only 81-93% of TCS cases.Methods: In this study, the entire coding regions of the TCOF1 gene, including newly described exons 6A and 16A, were sequenced in 46 unrelated subjects suspected of TCS clinical indication.Results: Fifteen mutations were reported, including twelve novel and three already described in 14 sporadic patients and in 3 familial cases. Moreover, seven novel polymorphisms were also described. Most of the mutations characterised were microdeletions spanning one or more nucleotides, in addition to an insertion of one nucleotide in exon 18 and a stop mutation. The deletions and the insertion described cause a premature termination of translation, resulting in a truncated protein.Conclusion: This study confirms that almost all the TCOF1 pathogenic mutations fall in the coding region and lead to an aberrant protein. © 2011 Conte et al; licensee BioMed Central Ltd.

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