Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. A new c-kit + /CD34 + /CD45 − cell population of stromal bone producing cells (SBP/DPSCs) was selected, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44 + /RUNX-2 + ), still capable of selfrenewing, and then in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue, which is markedly positive for several bone antibodies. This tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone-containing osteocytes.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.

No abstract

␤ adrenoceptor (␤AR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, ␤1AR activates the conventional Gs͞ cAMP pathway, whereas ␤2AR sequentially activates both the Gs and Gi pathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by ␤2AR in neonatal cardiac myocytes, while having little or no effect on ␤1AR signaling. Although ␤2AR activation leads to an increase in cAMP production, the cAMP generated does not have access to the protein kinase A-dependent signaling pathways by which the ␤1AR regulates the contraction rate. However, this restricted access is lost in the presence of PDE4 inhibitors or after ablation of PDE4D. These results not only suggest that PDE4D is an integral component of the ␤2AR signaling complex, but also underscore the critical role of subcellular cAMP regulation in the complex control of receptor signaling. They also illustrate a mechanism for fine-tuned ␤AR subtype signaling specificity and intensity in the cardiac system. cAMP ͉ heart ͉ knockout

BackgroundMuscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.ResultsWe addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.ConclusionsCeramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.

Background-cGMP phosphodiesterase type 5 protein is upregulated in myocardial hypertrophy. However, it has never been ascertained whether phosphodiesterase type 5 inhibition exerts an antiremodeling effect in nonischemic heart disease in humans. We explored the cardioreparative properties of a selective phosphodiesterase type 5 inhibitor, sildenafil, in a model of diabetic cardiomyopathy. Methods and Results-Fifty-nine diabetic men (60.3Ϯ7.4 years) with cardiac magnetic resonance imaging consistent with nonischemic, nonfailing diabetic cardiomyopathy (reduced circumferential strain [], Ϫ12.6Ϯ3.1%; increased left ventricular [LV] torsion [], 18.4Ϯ4.6°; and increased ratio of LV mass to volume, 2.1Ϯ0.5 g/mL) were randomized to receive sildenafil or placebo (100 mg/d). At baseline, the metabolic indices were correlated with torsion, strain, N-terminal pro-B-type natriuretic peptide, vascular endothelial growth factor, monocyte chemotactic protein-1, and blood pressure. After 3 months, sildenafil produced a significant improvement compared with placebo in LV torsion (⌬: sildenafil, Ϫ3.89Ϯ3.11°versus placebo, 2.13Ϯ2.35°; PϽ0.001) and strain (⌬: sildenafil, Ϫ3.30Ϯ1.86% versus placebo, 1.22Ϯ1.84%; PϽ0.001). Sildenafil-induced improvement of LV contraction was accompanied by consistent changes in chamber geometry and performance, with a 6.5Ϯ11% improvement in mass-to-volume ratio over placebo (Pϭ0.021). Monocyte chemotactic protein-1 and transforming growth factor-␤ were the only markers affected by active treatment (⌬monocyte chemotactic protein-1: Ϫ75.30Ϯ159.28 pg/mL, Pϭ0.032; ⌬transforming growth factor-␤: 5.26Ϯ9.67 ng/mL, Pϭ0.009). No changes were found in endothelial function, afterload, or metabolism. Conclusions-The early features of diabetic cardiomyopathy are LV concentric hypertrophy associated with altered myocardial contraction dynamics. Chronic phosphodiesterase type 5 inhibition, at this stage, has an antiremodeling effect, resulting in improved cardiac kinetics and circulating markers. This effect is independent of any other vasodilatory or endothelial effects and is apparently exerted through a direct intramyocardial action. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00692237. (Circulation. 2012;125:2323-2333.) Key Words: cardiac magnetic resonance imaging Ⅲ diabetes mellitus type 2 Ⅲ diabetic diastolic heart failure Ⅲ fibrosis Ⅲ phosphodiesterase inhibitors heart failure I nhibition of phosphodiesterase type 5 (PDE5) exerts a relaxant effect on the smooth muscle cells of the trabecular structures of the corpora cavernosa, resulting in improved erections. More recently, PDE5 inhibitors have been claimed to offer cardioprotective effects. In vitro and in vivo studies in mice have shown that cGMP and its downstream protein kinase G are signals common to most pathways activated in cardiac hypertrophy. 1 Low levels of PDE5 protein and cGMP-mediated activation of protein kinase G have been found in unstimulated hearts. 2 However, in isolated mouse cardiomyocytes tr...

Aim To parse out the impact of advanced ageing and disuse on skeletal muscle function, we utilized both in vivo and in vitro techniques to comprehensively assess upper- and lower-limb muscle contractile properties in 8 young (YG; 25±6yrs) and 8 oldest-old mobile (OM; 87±5yrs) and 8 immobile (OI; 88±4yrs) women. Methods In vivo, maximal voluntary contraction (MVC), electrically evoked resting twitch force (RT), and physiological cross sectional area (PCSA) of the quadriceps and elbow flexors was assessed. Muscle biopsies of the vastus lateralis and biceps brachii facilitated the in vitro assessment of single fibre specific tension (Po). Results In vivo, compared to the young, both the OM and OI exhibited a more pronounced loss of MVC in the lower-limb (OM (−60%) and OI (−75%)) than the upper-limb (OM=−51%; OI=−47%). Taking into account the reduction in muscle PCSA (OM=−10%; OI=−18%), only evident in the lower-limb, by calculating voluntary muscle specific force, the lower-limb of the OI (−40%) was more compromised than the OM (−13%). However, in vivo, RT in both upper- and lower-limbs (~9.8 N·m·cm−2) and Po (~123 mN·mm−2), assessed in vitro, implies preserved intrinsic contractile function in all muscles of the oldest-old and were well correlated (r=0.81). Conclusion These findings suggest that in the oldest-old neither advanced ageing nor disuse, per se, impact intrinsic skeletal muscle function, as assessed in vitro. However, in vivo, muscle function is attenuated by age and exacerbated by disuse, implicating factors other than skeletal muscle, such as neuromuscular control, in this diminution of function.

To determine the properties of the cAMP-specific, rolipramsensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93-and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90 -87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.The high affinity, cAMP-specific phosphodiesterases [type 4 according to the nomenclature proposed by Beavo et al. (1994)] are a class of enzymes with similar kinetic properties that are inhibited by the antidepressant rolipram and structurally related compounds. Although the presence of these forms has long been recognized, their distinctive properties are becoming evident only recently (Conti et al., 1995b). Early attempts to purify these forms have been hampered by their low abundance and instability (Conti and Swinnen, 1990). The molecular mass attributed to this group of enzymes ranges between 29 and 89 kDa (Conti and Swinnen, 1990). The definition of th...