Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury.

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Role of parnaparin in atherosclerosis.

Atherosclerosis is characterized by a proliferation of vascular smooth muscle cells (VSMCs) and their migration to the intima, which induces thickening of the intima itself, but the mechanism remains poorly understood. Low molecular weight heparin (LMWH) inhibits the proliferation of VSMCs. Previous studies have shown that a LMWH, parnaparin (PNP), acts on the processes of atherogenesis and atheroprogression in experimental animal models. The aim of this study was to investigate the involvement of oxidative stress, inflammation and VSMCs in the regulation of vascular wall homeostasis. We also considered the possibility of restoring vascular pathological changes using PNP treatment. In order to evaluate vascular remodelling in this study we have analysed the morphological changes in aortas of an animal model of atherosclerosis, apolipoprotein E-deficient mice (ApoE-/-) fed with a normal or a western diet without treatment or treated with PNP. We also analysed, by immunohistochemistry, the expression of proteins linked to atherogenesis and atheroprogression - an enzyme involved in oxidative stress, iNOS, examples of inflammatory mediators, such as tumour necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1 and IL-6), and markers of VSMC changes, in particular plasminogen activator inhibitor-1 and thrombospondin-1 (PAI-1 and TSP-1). Our results could suggest that PNP downregulates VSMC proliferation and migration, mediated by PAI-1 and TSP-1, and reduces inflammation and oxidative stress in vessels. These data suggested that LMWH, in particular PNP, could be a theoretically practical tool in the prevention of atherosclerotic vascular modification.

Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications.

PURPOSE: Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS: Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS: Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS: These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.

Regenerative potential of the Bichat fat pad determined by the quantification of multilineage differentiating stress enduring cells.

Published studies regarding Bichat fat pad focused, quite exclusively, on the implant of this adipose depot for different facial portions reconstruction. The regenerative components of Bichat fat pad were poorly investigated. The present study aimed to describe by an ultrastructural approach the Bichat fat pad, providing novel data at the ultrastructural and cellular level. This data sets improve the knowledge about the usefulness of the Bichat fat pad in regenerative and reconstructive surgery. Bichat fat pads were harvested form eight patients subjected to maxillofacial, dental and aesthetic surgeries. Biopsies were used for the isolation of mesenchymal cell compartment and for ultrastructural analysis. Respectively, Bichat fat pads were either digested and placed in culture for the characterization of mesenchymal stem cells (MSCs) or, were fixed in glutaraldehyde 2% and processed for transmission or scanning electron microscopy. Collected data showed very interesting features regarding the cellular composition of the Bichat fat pad and, in particular, experiments aimed to characterized the MSCs showed the presence of a sub-population of MSCs characterized by the expression of specific markers that allow to classify them as multilineage differentiating stress enduring cells.  This data set allows to collect novel information about regenerative potential of Bichat fat pad that could explain the success of its employment in reconstructive and regenerative medicine.

5-Azacytidine makes human preadipocytes able to differentiate into mesoderm-derived cell lineages.

In the present study we have evaluated whether (i) 5-azacytidine (AZA), a well-known demethylating agent, could be able to modify the phenotype of human preadipocytes and (ii) the modified cells could possess multilineage differentiation potential. Human preadipocytes at the 3rd passage were treated for 48 or 96 h with 10 µM AZA and then expanded up to passage 5. Stem cell markers, such as OCT-4, Nanog, and Sox2, were upregulated after 96 h of treatment with the demethylating treatment. Further, decreases in the expression of genes, such as adipose differentiation-related protein, characterizing the preadipocytes were noted. Our data showed that AZA-treated preadipocytes differentiated into cell lineages derived from mesoderm. Indeed, after incubation with inductive media for 3 weeks, osteblast-, chondrocyte-, and myoblast-like cells were detected in the cultures. Interestingly, both upregulation of stem cell markers and differentiation potential were maintained by the treated cultures expanded until the 5th passage. Taken together, our results suggest that AZA, without the use of transduction methods, convert preadipocytes to a less differentiated state that can be induced, under suitable stimuli, to the formation of mesoderm-derived cell lineages.

Exploring epidermal growth factor receptor (EGFR) inhibitor features: the role of fused dioxygenated rings on the quinazoline scaffold.

A number of dioxolane, dioxane, and dioxepine quinazoline derivatives have been synthetized and evaluated as EGFR inhibitors. Their cytotoxic activity has been tested against two cell lines overexpressing and not expressing EGFR. Most derivatives were able to counteract EGF-induced EGFR phosphorylation, and their potency was comparable to the reference compound PD153035. The size of the fused dioxygenated ring was crucial for the biological activity, the dioxane derivatives being the most promising class of this series.

Differential availability/processing of decorin precursor in arterial and venous smooth muscle cells.

The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of M(r) 33-226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury.