Federico Alessandro Ruffinatti

Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells

Endothelial progenitor cells could be implicated in the aberrant neoangiogenesis that occurs in bone marrow and spleen in patients with primary myelofibrosis (PMF). However, antivascular endothelial growth factor (VEGF) monotherapy had only a modest and transient effect in these individuals. Recently it was found that VEGF-induced proangiogenic intracellular Ca(2+) oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca(2+) imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca(2+) oscillations are altered in PMF-derived endothelial progenitor cells. We focused on endothelial colony-forming cells (ECFCs), which are the only endothelial progenitor cell subtype capable of forming neovessels both in vivo and in vitro. VEGF triggers repetitive Ca(2+) spikes in both normal ECFCs (N-ECFCs) and ECFCs obtained from PMF patients (PMF-ECFCs). However, the spiking response to VEGF is significantly weaker in PMF-ECFCs. VEGF-elicited Ca(2+) oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca(2+) mobilization and store-operated Ca(2+) entry. However, in most PMF-ECFCs, Ca(2+) oscillations are triggered by a store-independent Ca(2+) entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca(2+) spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca(2+) content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca(2+) oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF.

Interaction of SiO2 nanoparticles with neuronal cells: Ionic mechanisms involved in the perturbation of calcium homeostasis

SiO2 nanoparticles (NPs), in addition to their widespread utilization in consumer goods, are also being engineered for clinical use. They are considered to exert low toxicity both in vivo and in vitro, but the mechanisms involved in the cellular responses activated by these nanoobjects, even at non-toxic doses, have not been characterized in detail. This is of particular relevance for their interaction with the nervous system: silica NPs are good candidates for nanoneuromedicine applications. Here, by using two neuronal cell lines (GT1-7 and GN11 cells), derived from gonadotropin hormone releasing hormone (GnRH) neurons, we describe the mechanisms involved in the perturbation of calcium signaling, a key controller of neuronal function. At the non-toxic dose of 20μgmL(-1), 50nm SiO2 NPs induce long lasting but reversible calcium signals, that in most cases show a complex oscillatory behavior. Using fluorescent NPs, we show that these signals do not depend on NPs internalization, are totally ascribable to calcium influx and are dependent in a complex way from size and surface charge. We provide evidence of the involvement of voltage-dependent and transient receptor potential-vanilloid 4 (TRPV4) channels.

Intracellular Ca 2+ Signals to Reconstruct A Broken Heart: Still A Theoretical Approach?

The infusion of autologous stem cells has recently been put forward as an alternative strategy to regenerate infarcted myocardium and restore the contractile functions of diseased hearts. A growing number of cell types have been probed to induce cardiac repair in several animal models of ischemic myocardium, including human cardiac progenitor cells (hCPCs), human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human endothelial progenitor cells (hEPCs). The enthusiasm raised by pre-clinical studies has been dampened by clinical practice, according to which the extent of cardiac repair by cell based therapy is inadequate with respect to animal models. There is no doubt that regenerative medicine of acute myocardial infarction (AMI) will greatly benefit from the full comprehension of the signal transduction pathways which guide stem cell towards the injury site and their subsequent acquisition of a therapeutically relevant phenotype. The present review will focus on the role that oscillations in intracellular Ca(2+) concentration might play to promote the stem cells-dependent regrowth of ischemic myocardium. We will describe how intracellular Ca(2+) spikes may be manipulated to redirect stem cell fate to the most suitable lineage to restore cardiac vascularisation and contractility.

Calcium signals and FGF-2 induced neurite growth in cultured parasympathetic neurons: spatial localization and mechanisms of activation

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites.

When Neurons Encounter Nanoobjects: Spotlight on Calcium Signalling

Nanosized objects are increasingly present in everyday life and in specialized technological applications. In recent years, as a consequence of concern about their potential adverse effects, intense research effort has led to a better understanding of the physicochemical properties that underlie their biocompatibility or potential toxicity, setting the basis for a rational approach to their use in the different fields of application. Among the functional parameters that can be perturbed by interaction between nanoparticles (NPs) and living structures, calcium homeostasis is one of the key players and has been actively investigated. One of the most relevant biological targets is represented by the nervous system (NS), since it has been shown that these objects can access the NS through several pathways; moreover, engineered nanoparticles are increasingly developed to be used for imaging and drug delivery in the NS. In neurons, calcium homeostasis is tightly regulated through a complex set of mechanisms controlling both calcium increases and recovery to the basal levels, and even minor perturbations can have severe consequences on neuronal viability and function, such as excitability and synaptic transmission. In this review, we will focus on the available knowledge about the effects of NPs on the mechanisms controlling calcium signalling and homeostasis in neurons. We have taken into account the data related to environmental NPs, and, in more detail, studies employing engineered NPs, since their more strictly controlled chemical and physical properties allow a better understanding of the relevant parameters that determine the biological responses they elicit. The literature on this specific subject is all quite recent, and we have based the review on the data present in papers dealing strictly with nanoparticles and calcium signals in neuronal cells; while they presently amount to about 20 papers, and no related review is available, the field is rapidly growing and some relevant information is already available. A few general findings can be summarized: most NPs interfere with neuronal calcium homeostasis by interactions at the plasmamembrane, and not following their internalization; influx from the extracellular medium is the main mechanism involved; the effects are dependent in a complex way from concentration, size and surface properties.

Spatial Wavelet Analysis of Calcium Oscillations in Developing Neurons

Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs) along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone) or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the expression of specific mechanisms controlling calcium influx/efflux.

VEGF-induced intracellular Ca2+ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells

Endothelial colony forming cells (ECFCs) represent a population of truly endothelial precursors that promote the angiogenic switch in solid tumors, such as breast cancer (BC). The intracellular Ca2+ toolkit, which drives the pro-angiogenic response to VEGF, is remodelled in tumor-associated ECFCs such that they are seemingly insensitive to this growth factor. This feature could underlie the relative failure of anti-VEGF therapies in cancer patients. Herein, we investigated whether and how VEGF uses Ca2+ signalling to control angiogenesis in BC-derived ECFCs (BC-ECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and in vitro tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca2+ oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca2+ oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER) and maintained by store-operated Ca2+ entry (SOCE). However, InsP3-dependent Ca2+ release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca2+ levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca2+ response to VEGF was seemingly due to the reduction in ER Ca2+ concentration, which prevents VEGF from triggering robust intracellular Ca2+ oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and in vitro tubulogenesis. These findings demonstrate for the first time that BC-ECFCs are insensitive to VEGF, which might explain at cellular and molecular levels the failure of anti-VEGF therapies in BC patients, and hint at SOCE as a novel molecular target for this disease.

Calcium signals induced by FGF-2 in parasympathetic neurons: role of second messenger pathways.

Basic Fibroblast Growth Factor, or FGF-2, has been shown to promote neuronal survival and neurite outgrowth in dissociated neurons from the embryonic chick ciliary ganglion; in these effects the three main signal transduction pathways downstream the activated FGFR receptor, i.e. the MAPK, the PI3-K and the PLCγ ones, are differentially involved. While it has been shown that FGF-2 can elicit long lasting elevations in intracellular calcium concentration, [Ca(2+)](i), the role of the three pathways in this process has not been elucidated. Here we show, by means of pharmacological inhibitors, that all three are involved, at a different extent, in the generation of the [Ca(2+)](i) increase induced by FGF-2; in particular, inhibition of the PLCγ pathway, in addition to reducing the number of responsive cells, induces, in a significant population of cells, basal calcium oscillations in the absence of the growth factor and interferes with calcium signals elicited by depolarization. We propose that this complex behaviour can be due to a perturbation in PIP(2) levels at the plasmamembrane.

Susceptibility of different mouse strains to oxaliplatin peripheral neurotoxicity: Phenotypic and genotypic insights

Peripheral neurotoxicity is one of the most distressing side effects of oxaliplatin therapy for cancer. Indeed, most patients that received oxaliplatin experience acute and/or chronic severe sensory peripheral neuropathy. However, despite similar co-morbidities, cancer stage, demographics and treatment schedule, patients develop oxaliplatin-induced peripheral neurotoxicity with remarkably different severity. This suggests individual genetic variability, which might be used to glean the mechanistic insights into oxaliplatin neurotoxicity. We characterized the susceptibility of different mice strains to oxaliplatin neurotoxicity investigating the phenotypic features of neuropathy and gene expression profiles in dorsal root ganglia of six genetically different mice strains (Balb-c, C57BL6, DBA/2J, AJ, FVB and CD1) exposed to the same oxaliplatin schedule. Differential gene expression in dorsal root ganglia from each mice strain were assayed using a genome-wide expression analysis and selected genes were validated by RT-PCR analysis. The demonstration of consistent differences in the phenotypic response to oxaliplatin across different strains is interesting to allow the selection of the appropriate strain based on the pre-defined read-out parameters. Further investigation of the correlation between gene expression changes and oxaliplatin-induced neurotoxicity phenotype in each strain will be useful to deeper investigate the molecular mechanisms of oxaliplatin neurotoxicity.

SiO 2 nanoparticles modulate the electrical activity of neuroendocrine cells without exerting genomic effects

Engineered silica nanoparticles (NPs) have attracted increasing interest in several applications, and particularly in the field of nanomedicine, thanks to the high biocompatibility of this material. For their optimal and controlled use, the understanding of the mechanisms elicited by their interaction with the biological target is a prerequisite, especially when dealing with cells particularly vulnerable to environmental stimuli like neurons. Here we have combined different electrophysiological approaches (both at the single cell and at the population level) with a genomic screening in order to analyze, in GT1-7 neuroendocrine cells, the impact of SiO2 NPs (50 ± 3 nm in diameter) on electrical activity and gene expression, providing a detailed analysis of the impact of a nanoparticle on neuronal excitability. We find that 20 µg mL−1 NPs induce depolarization of the membrane potential, with a modulation of the firing of action potentials. Recordings of electrical activity with multielectrode arrays provide further evidence that the NPs evoke a temporary increase in firing frequency, without affecting the functional behavior on a time scale of hours. Finally, NPs incubation up to 24 hours does not induce any change in gene expression.