Giulia Cantini

Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue

Adipose tissue is a dynamic endocrine organ with a central role in metabolism regulation. Functional differences in adipose tissue seem associated with the regional distribution of fat depots, in particular in subcutaneous and visceral omental pads. Here, we report for the first time the isolation of human adipose‐derived adult stem cells from visceral omental and subcutaneous fat (V‐ASCs and S‐ASCs, respectively) from the same subject. Immunophenotyping shows that plastic culturing selects homogeneous cell populations of V‐ASCs and S‐ASCs from the corresponding stromal vascular fractions (SVFs), sharing typical markers of mesenchymal stem cells. Electron microscopy and electrophysiological and real‐time RT‐PCR analyses confirm the mesenchymal stem nature of both V‐ASCs and S‐ASCs, while no significant differences in a limited pattern of cytokine/chemokine expression can be detected. Similar to S‐ASCs, V‐ASCs can differentiate in vitro toward adipogenic, osteogenic, chondrogenic, muscular, and neuronal lineages, as demonstrated by histochemical, immunofluorescence, real‐time RT‐PCR, and electrophysiological analyses, suggesting the multipotency of such adult stem cells. Our data demonstrate that both visceral and subcutaneous adipose tissues are a source of pluripotent stem cells with multigermline potential. However, the visceral rather than the subcutaneous ASC could represent a more appropriate in vitro cell model for investigating the molecular mechanisms implicated in the pathophysiology of metabolic disorders such as obesity.—Baglioni, S., Francalanci, M., Squecco, R., Lombardi, A., Cantini, G., Angeli, R., Gelmini, S., Guasti, D., Benvenuti, S., Annunziato, F., Bani, D., Liotta, F., Francini, F., Perigli, G., Serio, M., Luconi, M. Characterization of human adult stem‐cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 23, 3494–3505 (2009). www.fasebj.org

Functional Differences in Visceral and Subcutaneous Fat Pads Originate from Differences in the Adipose Stem Cell

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K+-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.

A New Mechanism Involving ERK Contributes to Rosiglitazone Inhibition of Tumor Necrosis Factor-α and Interferon-γ Inflammatory Effects in Human Endothelial Cells

Objective—Microvascular endothelium is one of the main targets of the inflammatory response. On specific activation, endothelial cells recruit Th1-lymphocytes at the inflammatory site. We investigated the intracellular signaling mediating tumor necrosis factor (TNF)-α and interferon (IFN)-γ inflammatory response in human microvascular endothelial cells (HMEC-1) and the interfering effects of the peroxisome-proliferator-activated-receptor (PPARγ) agonist, rosiglitazone (RGZ). Methods and Results—TNFα and IFNγ, mainly when combined, stimulate IFNγ-inducible protein of 10 kDa (IP10) and fractalkine production evaluated by ELISA and TaqMan analyses. This effect is not only mediated by activation of the NFkB and Stat1 classic pathways, but also involves a rapid increase in phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) as measured by Western blot. RGZ interferes with TNFα and IFNγ stimulation of IP10, fractalkine, and adhesion molecule through a novel rapid mechanism which involves the blocking of ERK activation. Conclusions—Our findings shed new light on the mechanisms underlying the inflammatory response of microvascular endothelium and on the possible therapeutic use of RGZ in vasculopathies involving Th1-responses.

Peroxisome proliferator-activated receptor gamma (PPARγ): Is the genomic activity the only answer?

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily of transcription factors, widely expressed in the organism, including adipose, vascular and immune cells. Besides the well-known role in lipid/glycidic homeostasis, PPARgamma has also recently emerged as a key regulator of inflammatory and immune responses. Besides the natural ligands, more potent synthetic agonists of PPARgamma have been developed, including thiazolidinediones (TZDs), currently used in type 2 diabetes treatment, which also exert anti-inflammatory and anti-neoplastic effects. PPARgamma mechanism of action has focused considerable attention over the years. This receptor was initially shown to act on gene expression through a direct transcription and an indirect transrepression activity, mainly associated with metabolic and anti-inflammatory effects. Different post-translational modifications of the receptor can modulate PPARgamma activity. More recently, rapid nongenomic activity of TZDs affecting post-translation modifications of extranuclear proteins involved in cell signaling, has been reported. In particular, PPARgamma can physically interact with protein kinases resulting in a compartment specific recruitment and activity modulation of these enzymes. Among them, ERK can be positively/negatively regulated by PPARgamma ligands, as in endothelial cells, where TZDs exert anti-inflammatory effects through a novel mechanism involving a rapid inhibition of ERK1/2 phosphorylation/activation. Finally, some of the TZD anti-tumor effects seem to be PPARgamma-independent, raising the possibility that alternative receptors can act through extranuclear nongenomic pathways. In conclusion, different mechanisms of action of PPARgamma seem to coexist in an interacting functional network in the cell, concurring in mediating both pharmacological and natural ligand effects.

Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa

BACKGROUND Protein tyrosine phosphorylation is one of the main processes associated with sperm activation. Although this process and its targets have been well characterized, only few tyrosine kinases have been identified so far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying sperm activation. METHODS AND RESULTS Specific anti-Src antibodies, against different epitopes of the protein, identified a single band of approximately 70 kDa relating to a protein which is mainly localized in the post-acrosomal region of the head, neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies against Src phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation during sperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phosphorylation. Moreover, this inhibitor also blocked the progesterone-induced acrosome reaction and interfered with the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motility and hyperactivation resulted from incubation with SU6656. CONCLUSIONS We identified a novel Src isoform in human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation and acrosome reaction.

Rosiglitazone Inhibits Adrenocortical Cancer Cell Proliferation by Interfering with the IGF-IR Intracellular Signaling

Rosiglitazone (RGZ), a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR)-γ, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R) of human adrenocortical carcinoma (ACC) and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR). We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K)-Akt, and extracellular signal-regulated kinase (ERK1/2) cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.

Role of a-kinase anchoring proteins (AKAPs) in reproduction

Rapid spatio-temporal organized intracellular signaling is a pivotal mechanism for regulation of functions in many cells, in particular in the female and male gametes, in which functional regulation through rapid increases in protein content is not possible since the mechanisms of transcription/translation are somehow frozen due to meiosis block or DNA condensation respectively. A kinase-anchoring proteins (AKAPs) represent a functional conserved family of signal-organizing scaffolding proteins, which due to the specific subcellular distribution and focally compartmentalized cyclic-AMP-dependent protein kinase (PKA) and other enzymes, assuring the coordination of cAMP-responsive events and their integration with other intracellular signals. This review summarizes the actual knowledge on AKAP structure and functions, taking into particular account the role of different AKAPs in regulating reproductive functions such as gametogenesis. Evidence for sperm specific AKAP isoforms and their initiated signaling cascades in mature sperm and the role of this focally activated super-molecular signaling complex in motility are discussed in details with particular emphasis on putative relations between AKAP structural and functional alterations and defects in sperm motility.

Rosiglitazone impairs proliferation of human adrenocortical cancer: preclinical study in a xenograft mouse model

Adrenocortical carcinoma (ACC) is a rare aggressive tumor with a poor prognosis. The lack of a specific and effective medical treatment is due to the poor knowledge of the mechanisms underlying tumor growth. Research on potential drugs able to specifically interfere with tumor proliferation is essential to develop more efficacious therapies. We evaluated for the first time the in vivo effect of rosiglitazone (RGZ), an anti-diabetic drug with in vitro anti-tumor properties, on ACC proliferation in a xenograft model obtained by s.c. injection of human ACC H295R cells in athymic mice. When the tumor size reached 5 mm, animals were allocated to 5 mg/kg RGZ- or water-treated groups. Tumor volume was measured twice a week. A significant reduction of tumor growth in RGZ versus control (control) group was observed and was already maximal following 17 day treatment (1-T/C=75.4% (43.7-93.8%)). After 31 days of treatment, mice were killed and tumor analyzed. Tumor histological evaluation revealed characteristics of invasiveness, richness in small vessels and mitotic figures in control group, while RGZ group tumors presented non infiltrating borders, few vessels, and many apoptotic bodies. Tumor immunohistochemistry showed that Ki-67 was reduced in RGZ versus control group. Quantitative real-time RT-PCR demonstrated a significant reduction in the expression of angiogenic (VEGF), vascular (CD31), proliferation (BMI-1), and anti-apoptotic (Bcl-2) genes in RGZ versus control group tumors. The same inhibitory effects were confirmed in in vitro RGZ-treated H295R. Our findings support and expand the role of RGZ in controlling ACC proliferation and angiogenesis in vivo and in vitro.

Impact on Left Ventricular Function and Remodeling and on 1-Year Outcome in Patients With Left Bundle Branch Block After Transcatheter Aortic Valve Implantation.

Conflicting results have been reported about the prognostic impact of left bundle branch block (LBBB) after transcatheter aortic valve implantation (TAVI). The aim of this study was to evaluate the impact of LBBB after TAVI on left ventricular (LV) function and remodeling and on 1-year outcomes. Of 101 TAVI patients, 9 were excluded. All complications were evaluated according to the Valve Academic Research Consortium 2 definition. Of 92 patients, 34 developed LBBB without more advanced myocardial damage or inflammation biomarkers in comparison with patients without LBBB. The only predictor of new LBBB was larger baseline LV end-diastolic volume. LBBB plus advanced atrioventricular block was strongly correlated with permanent pacemaker implantation (p <0.0001). Patients with LBBB had a higher rate of permanent pacemaker implantation at 30 days (59% vs 19%, p <0.0001) and less recovery of LV systolic function and a trend toward a lower rate of LV reverse remodeling at 1 year. The development of acute kidney injury and the logistic European System for Cardiac Operative Risk Evaluation score were associated with poor outcomes (all-cause mortality and heart failure) (hazard ratio 6.86, 95% confidence interval 2.51 to 18.74, p <0.0001, and hazard ratio 1.04, 95% confidence interval 1.01 to 1.08, p = 0.021, respectively), but not LBBB. In conclusion, after TAVI, 37% of patients developed new LBBB without more advanced myocardial damage or inflammation biomarkers. LBBB was associated with a higher rate of permanent pacemaker implantation, which negatively affected the recovery of LV systolic function. The development of acute kidney injury, rather than LBBB, increases the 1-year risk for mortality and hospitalization for heart failure.

Acrosome reaction is impaired in spermatozoa of obese men: a preliminary study

OBJECTIVE To compare spontaneous (Sp-AR) and P-induced acrosome reaction (AR) in spermatozoa of obese and lean subjects. SETTING Bariatric unit at a university hospital. DESIGN Prospective, observational study. PATIENT(S) Twenty-three obese (mean±SD body mass index [BMI], 44.3±5.9 kg/m2) and 25 age-matched lean (BMI, 24.2±3.0 kg/m2) subjects. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Spontaneous and P-induced AR in spermatozoa of obese and lean subjects. RESULT(S) A statistically significant difference was found between obese and lean cohorts in total T and calculated free T, E2, glycated hemoglobin, and high-density lipoproteins, whereas among the routine semen parameters analyzed, only immotile sperm percentage and ejaculate volume differed significantly. Spermatozoa of obese (n=13) vs. lean men (n=19) showed a higher Sp-AR (17.9%±7.2% vs. 8.3%±4.2%), which resulted in a reduced ability to respond to P evaluated as the AR-after-P-challenge parameter (3.5%±3.2% vs. 17.6%±9.2%). Multivariate analysis adjusted for age revealed a significant correlation between BMI, waist, E2, and glycated hemoglobin with both Sp-AR (age-adjusted r=0.654, r=0.711, r=0.369, and r=0.644, respectively) and AR-after-P-challenge (age-adjusted r=-0.570, r=-0.635, r=-0.507, and r=-0.563, respectively). A significant difference in sperm cholesterol content was reported between obese and lean men (29.8±19.5 vs. 19.1±14.6 ng/μg of proteins). CONCLUSION(S) Sperm AR is impaired in obese men, showing reduced response to P and elevated Sp-AR, associated with altered circulating levels of E2 and sperm cholesterol content.