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Biochimica et biophysica acta (1)
Biological Procedures Online (1)
Guarnieri, Carlo (2)
Agnetti, Giulio (1)
Basile, Ilaria (1)
Bezstarosti, Karel (1)
Bonafé, Francesca (1)
Caldarera, Claudio M. (1)
Caldarera, Claudio Marcello (1)
Capitani, Ombretta (1)
Dekkers, Dick H.W. (1)
Gamberini, Chiara (1)
Giordano, Emanuele (1)
Lamers, Jos M.J. (1)
Muscari, Claudio (1)
Valgimigli, Simond (1)
Van Eyk, Jennifer E. (1)
Verhoeven, Adrie J.M. (1)
Year of Publication
Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and α-B-crystallin
Dekkers, Dick H.W.
Verhoeven, Adrie J.M.
Caldarera, Claudio M.
Van Eyk, Jennifer E.
Lamers, Jos M.J.
Biochimica et biophysica acta
We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as α-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and α-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.
Endothelin; Hypertrophy; Proteomics; Desmin; Mitochondria
Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage
Caldarera, Claudio Marcello
Biological Procedures Online
The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.
Blood Cells; Bone Marrow Cells; Cell Proliferation; Cell Survival; Mesenchymal Stem Cells; Physiologic Neovascularization
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