Francesca Cialdai

Inhibition of Nicotinamide Phosphoribosyltransferase: CELLULAR BIOENERGETICS REVEALS A MITOCHONDRIAL INSENSITIVE NAD POOL*

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Pharmacological Effects of Exogenous NAD on Mitochondrial Bioenergetics, DNA Repair, and Apoptosis

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N′-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N′-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

The Impact of Microgravity and Hypergravity on Endothelial Cells

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na+ channels and amplitude of Ca++ currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.

Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g). Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

Quantitative measurement of scattering and extinction spectra of nanoparticles by darkfield microscopy

We demonstrate a versatile concept for the quasi simultaneous and quantitative measurement of light extinction and scattering cross section spectra of nanoparticles in a darkfield microscope. We validate this method by the analysis of an aqueous suspension of gold nanorods and comparison with both numerical simulations and standard spectrophotometry measurements. Our approach holds the promise to allow one to map the principal optical properties of nanoparticles in a biological sample with μm spatial resolution, which is an issue of particular relevance for applications in biomedical optics such as photothermolysis and laser hyperthermia.

In vitro study on the safety of near infrared laser therapy in its potential application as postmastectomy lymphedema treatment

Clinical studies demonstrated the effectiveness of laser therapy in the management of postmastectomy lymphedema, a discomforting disease that can arise after surgery/radiotherapy and gets progressively worse and chronic. However, safety issues restrict the possibility to treat cancer patients with laser therapy, since the effects of laser radiation on cancer cell behavior are not completely known and the possibility of activating postmastectomy residual cancer cells must be considered. This paper reports the results of an in vitro study aimed to investigate the effect of a class IV, dual-wavelength (808 nm and 905 nm), NIR laser system on the behavior of two human breast adenocarcinoma cell lines (namely, MCF7 and MDA-MB361 cell lines), using human dermal fibroblasts as normal control. Cell viability, proliferation, apoptosis, cell cycle and ability to form colonies were analyzed in order to perform a cell-based safety testing of the laser treatment in view of its potential application in the management of postmastectomy lymphedema. The results showed that, limited to the laser source, treatment conditions and experimental models used, laser radiation did not significantly affect the behavior of human breast adenocarcinoma cells, including their clonogenic efficiency. Although these results do not show any significant laser-induced modification of cancer cell behavior, further studies are needed to assess the possibility of safely applying NIR laser therapy for the management of postmastectomy lymphedema.

Irradiation by pulsed Nd:YAG laser induces the production of extracellular matrix molecules by cells of the connective tissues: a tool for tissue repair

Many studies demonstrated that mechanical stress is a key factor for tissue homeostasis, while unloading induce loss of mass and impairment of function. Because of their physiological function, muscle, connective tissue, bone and cartilage dynamically interact with mechanical and gravitational stress, modifying their properties through the continuous modification of their composition. Indeed, it is known that mechanical stress increases the production of extracellular matrix (ECM) components by cells, but the mechanotransduction mechanisms and the optimal loading conditions required for an optimal tissue homeostasis are still unknown. Considering the importance of cell activation and ECM production in tissue regeneration, a proper use of mechanical stimulation could be a powerful tool in tissue repair and tissue engineering. Studies exploring advanced modalities for supplying mechanical stimuli are needed to increase our knowledge on mechanobiology and to develop effective clinical applications. Here we describe the effect of photomechanical stress, supplied by a pulsed Nd:YAG laser on ECM production by cells of connective tissues. Cell morphology, production of ECM molecules (collagens, fibronectin, mucopolysaccharides), cell adhesion and cell energy metabolism have been studied by using immunofluorescence and autofluorescence microscopy. The results show that photomechanical stress induces cytoskeleton remodelling, redistribution of membrane integrins, increase in production of ECM molecules. These results could be of consequence for developing clinical protocols for the treatment of connective tissue dideases by pulsed Nd:YAG laser.

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration

Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes. It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to the environmental conditions.

Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts

ABSTRACT Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.