Lucia Petrelli

Evaluation of gold nanoparticles toxicity towards human endothelial cells under static and flow conditions.

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).

Decellularized human skeletal muscle as biologic scaffold for reconstructive surgery

Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits) and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.

Isolation of human adrenocortical aldosterone-producing cells by a novel immunomagnetic beads method.

We detected intense CD56 immunostaining in the zona glomerulosa (ZG) and medulla of the normal human adrenal gland and therefore identified CD56, the neural cell adhesion molecule, as a membrane antigen specific for the ZG, aldosterone-producing adenoma (APA), and chromaffin cells. The APA and pheochromocytoma cells, which are histogenetically derived from the ZG and medulla, respectively, also showed intense CD56 immunostaining. Based on these findings we developed a strategy for isolating cells from the ZG and APA using CD56 immunobinding to magnetic beads. Morphology, gene expression studies, and aldosterone measurement confirmed that CD56 positive (+) cells were ZG and APA cells. Analysis of CD56+ cells under light and phase contrast microscopy evidenced that these cells formed clumps, as the ZG cells usually do; with electron microscopy they showed multiple features typical of a steroidogenic phenotype. Expression levels of the CD56 and the aldosterone synthase (CYP11B2) gene were markedly higher in CD56+ cells than CD56- cells (+1600 and +2100% increase, respectively). Moreover, aldosterone secretion was higher (+1380%) from CD56+ cells than from CD56- cells. Hence, this novel methodology allows isolation of a pure population of ZG and APA cells exhibiting multiple characteristics of the aldosterone-producing cells.

An easy-to-handle microfluidic device suitable for immunohistochemical procedures in mammalian cells grown under flow conditions

Microfluidics, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

Neurotrophin-3 upregulates NGF receptors in a central nervous system glial cell line

The low-affinity nerve growth factor (NGF) receptor (LNGFR) binds the neurotrophins NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) with similar affinities. Here we report on the ability of NT-3 to regulate the expression of the LNGFR in C6 glioma cells. LNGFR-like immunoreactivity (LNGFR-IR) was examined in C6 cells treated for 16 h with NT-3 and exposed to the antibody 192-IgG followed by immunoglobulins conjugated with colloidal gold by means of ultrastructural morphometric analysis. Untreated C6 cells exhibited some positive LNGFR-IR, while C6 cells treated with NT-3 displayed significantly increased (2.3 fold) LNGFR-IR. The increase in LNGFR protein was accompanied by a greater quantity of LNGFR mRNA in NT-3-treated cells. Thus, LNGFR can be upregulated by the structurally related neurotrophin NT-3.

The infrapatellar adipose body: A histotopographic study

The infrapatellar fat pad (IFP) can be regarded as a peculiar form of fibro-adipose tissue localized close to the synovial membrane and articular cartilage. The aims of the present study were to analyze the microscopic anatomy of the IFP through histological and ultrastructural methods, comparing it with that of the subcutaneous tissue of the abdomen and of the knee. Ten specimens of IFP were sampled from bodies of the Donation Program of the University of Padua without a history of osteoarthritis. The IFP consisted of white adipose tissue, of lobular type, with lobules delimited by thin connective septa. The IFP lobule areas were smaller (p < 0.05) and the interlobular septa were thicker (p > 0.05) than those of subcutaneous tissues of the abdomen, whereas the IFP lobule areas were larger (p < 0.05) and the interlobular septa were thinner than those of the subcutaneous tissue of the knee (p < 0.05). The IFP adipocytes present a mean area of 3,708 ± 976 µm2 with a large intercellular space, whereas the mean area of the abdominal tissues was greater (6,082 ± 628 µm2; p < 0.05). At scanning electron microscopy the IFP adipocytes were covered by thick fibrillary sheaths, creating a basket around the adipocytes. The structural characteristics of the IFP (lobular aspect of the adipose tissue, thickness of the septa with scarce elastic fibers) could act as a plastic portion aimed at the absorption of pressure variation during knee articular activity. The extensive distribution of nerves suggests a possible role of the IFP as a mechanoreceptor, corresponding to a tridimensional connective mesh working in the proprioceptive regulation of the activity of the knee joint.

Characterization and growth-dependent regulation of the nerve growth factor receptor gp140trk in rat C6 glioma cells.

The glioma cell line C6 was used to study the expression and growth-dependent regulation of the nerve growth factor (NGF) tyrosine kinase receptor gp140trk, which is the mature protein product of the trk proto-oncogene. Chemical cross-linking of 125I-NGF to C6 cells, followed by immunoprecipitation with polyclonal anti-NGF antibodies and separation by polyacrylamide gel electrophoresis, revealed the presence of 90-95 and 150 kDa species. Immunocytochemical staining of C6 cells with antibodies directed against either the low-affinity NGF receptor gp75NGFR or trk proto-oncogene products demonstrated a heterogeneous cellular distribution of both antigens. Brief treatment of C6 cells with NGF led to the tyrosine phosphorylation of 80, 110 and 140 kDa protein species, as detected on anti-phosphotyrosine Western blots. Similar molecular weight species were found with anti-Trk antibodies in the NGF-treated cells. Intracellular localization of Trk-like immunoreactivity in C6 cells released from a growth-arrested state indicated an initial immunostaining of the nuclear periphery, progressing to cytoplasmic vesicles and finally to the plasma membrane. These observations at the light microscopic level were confirmed using immunoelectron microscopy with the same anti-Trk antibodies, and showed clearly the trafficking of Trk-like immunostained particles from the endoplasmic reticulum to the plasmalemma. The cellular localization of trk gene products also appeared to depend on their glycosylation state. Such growth-dependent expression of NGF receptors on glial cells may be important in controlling autocrine regulatory processes of glia to NGF, which these cells produce.

Behavioral and morpho-functional correlates of brain aging: a preclinical study with phosphatidylserine.

In the aging brain, changes in membrane lipid composition or content have been related to reduction of membrane fluidity, loss of enzymatic activities, and decreased efficiency of signal transduction and transport mechanisms (Schroeder, 1984). This has led to the assumption that administration of endogenously occurring phospholipids may preserve both structural and functional integrity of central nervous system membranes and prevent, or restore, neuronal deficits that occur with aging or age-related neurodegenerative disorders. Furthermore, exogenous phospholipids may participate in phospholipid metabolism, yielding biologically active intermediates in response to physiopathological phenomena (Toffano and Bruni, 1980; Bruni, 1988).

Endothelin‐1 Drives Epithelial‐Mesenchymal Transition in Hypertensive Nephroangiosclerosis

Background Tubulointerstitial fibrosis, the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT). Endothelin‐1 (ET‐1) activates EMT in cancer cells, but it is not known whether it drives EMT in the kidney. We therefore tested the hypothesis that tubulointerstitial fibrosis involves EMT driven by ET‐1. Methods and Results Transgenic TG[mRen2]27 (TGRen2) rats developing fulminant angiotensin II–dependent hypertension with prominent cardiovascular and renal damage were submitted to drug treatments targeted to ET‐1 and/or angiotensin II receptor or left untreated (controls). Expressional changes of E‐cadherin and α‐smooth muscle actin (αSMA) were examined as markers of renal EMT. In human kidney HK‐2 proximal tubular cells expressing the ETB receptor subtype, the effects of ET‐1 with or without ET‐1 antagonists were also investigated. The occurrence of renal fibrosis was associated with EMT in control TGRen2 rats, as evidenced by decreased E‐cadherin and increased αSMA expression. Irbesartan and the mixed ET‐1 receptor antagonist bosentan prevented these changes in a blood pressure–independent fashion (P < 0.001 for both versus controls). In HK‐2 cells ET‐1 blunted E‐cadherin expression, increased αSMA expression (both P < 0.01), collagen synthesis, and metalloproteinase activity (P < 0.005, all versus untreated cells). All changes were prevented by the selective ETB receptor antagonist BQ‐788. Evidence for involvement of the Rho‐kinase signaling pathway and dephosphorylation of Yes‐associated protein in EMT was also found. Conclusions In angiotensin II–dependent hypertension, ET‐1 acting via ETB receptors and the Rho‐kinase and Yes‐associated protein induces EMT and thereby renal fibrosis.

Expression of the endocannabinoid receptors in human fascial tissue.

Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.