Marzia Di Chio

Endothelial progenitor cells in patients with essential hypertension.

Objective(s) The eventual role of blood pressure on the endothelial progenitor cell (EPC) has rarely been evaluated and data collected so far relate to patients with co-existing coronary heart disease. Methods We have studied the number and functional activity of EPC as well as the number of EPC endothelial colony-forming units (CFU) in a carefully selected group of 36 patients with essential hypertension and 24 normotensive control subjects. Results In patients with essential hypertension, the EPC number was not statistically different from that found in control subjects (mean ± SD, essential hypertension 58 ± 29, controls 53 ± 20; EPC/high power field). CFU per well were not statistically different in patients with essential hypertension compared with normotensive controls (mean ± SD, patients with essential hypertension 2.4 ± 2.6, normotensive controls 3 ± 3.3 CFU/well). In essential hypertension patients, the EPC number was inversely correlated with both total (R = 0.635, P < 0.0001) and low-density lipoprotein (LDL)-cholesterol (R = 0.486, P < 0.05). Neither the EPC number nor the EPC CFU were correlated with age, systolic blood pressure, diastolic blood pressure, body mass index, lipoprotein(a), high-sensitivity C-reactive protein or homocysteine. Conclusions The present study shows that essential hypertension is not characterized by the altered number or functional activity of EPC. Plasma total and LDL-cholesterol are independent predictors of reduced numbers of circulating EPC in essential hypertension patients. The absence of any correlation between the characteristics of EPC and several markers predictive of cardiovascular damage merits further investigation.

Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

The modulation of BDNF expression and signalling dissects the antidepressant from the reinforcing properties of ketamine: Effects of single infusion vs. chronic self-administration in rats.

Ketamine is a drug of abuse with a unique profile, which besides its inherent mechanism of action as a non-competitive antagonist of the NMDA glutamate receptor, displays both antidepressant and reinforcing properties. The major aim of our study was to find a molecular signature of ketamine that may help in discriminating between its reinforcing and antidepressant effects. To this end, we focused our attention on BDNF, a neurotrophin that has been shown to play a role in both antidepressant and reinforcing properties of several drugs. Rats were exposed to self-administer intravenous (IV) ketamine (S/A) for 43 days or to receive a single IV ketamine 0.5mg/kg, or vehicle infusion. Although the dose we employed is lower than that reported by the literature, it however yields Cmax values that correspond to those achieved in humans after antidepressant treatment. Our results show that while the single infusion of ketamine increased the neurotrophin expression in the hippocampus while reducing it in the ventral striatum, a feature shared with other antidepressants, the repeated self-administration reduced mBDNF expression and its downstream signalling in both ventral striatum and hippocampus. Further, we here show that phosphorylation of Akt is oppositely regulated by ketamine, pointing to this pathway as central to the different actions of the drug. Taken together, we here point to BDNF and its downstream signalling pathway as a finely tuned mechanism whose modulation might subserve the different features of ketamine.

Meninges harbor cells expressing neural precursor markers during development and adulthood.

Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

Sex-specific eNOS activity and function in human endothelial cells

Clinical and epidemiological data show that biological sex is one of the major determinants for the development and progression of cardiovascular disease (CVD). Impaired endothelial function, characterized by an imbalance in endothelial Nitric Oxide Synthase (eNOS) activity, precedes and accelerates the development of CVD. However, whether there is any sexual dimorphism in eNOS activity and function in endothelial cells (ECs) is still unknown. Here, by independently studying human male and female ECs, we found that female ECs expressed higher eNOS mRNA and protein levels both in vitro and ex vivo. The increased eNOS expression was associated to higher enzymatic activity and nitric oxide production. Pharmacological and genetic inhibition of eNOS affected migratory properties only in female ECs. In vitro angiogenesis experiments confirmed that sprouting mostly relied on eNOS-dependent migration in female ECs. At variance, capillary outgrowth from male ECs was independent of eNOS activity but required cell proliferation. In this study, we found sex-specific differences in the EC expression, activity, and function of eNOS. This intrinsic sexual dimorphism of ECs should be further evaluated to achieve more effective and precise strategies for the prevention and therapy of diseases associated to an impaired endothelial function such as CVD and pathological angiogenesis.

Nicotine-induced phosphorylation of phosphorylated cyclic AMP response element-binding protein (pCREB) in hippocampal neurons is potentiated by agrin

The scope of this study was to test whether increased levels of the extracellular matrix molecule (ECM) agrin might enhance nicotine effects on those molecular mechanisms that initiate neuroadaptative processes in the hippocampus, a key brain area for learning and memory. We studied the effects of repetitive applications of neuronal agrin to primary hippocampal cell culture on nicotine-induced phosphorylated cyclic AMP response element-binding protein (pCREB) expression, a marker of neuroadaptation, by using immunofluorescence-based assessment of pCREB-positive neurons. We also tested agrin effects on nicotine-induced expression of a marker of metabolic activation, the immediate early gene c-fos. Agrin was shown to significantly enhance nicotine-induced pCREB, but not c-fos, expression. By using Western blotting analysis, cumulative agrin has been shown to increase nicotine-induced pCREB phosphorylation. These analyses, however, showed that inhibition of the CaMKII pathway blocked general pCREB phosphorylation, whereas inhibition of the MAPK pathway potentiated the synergistic effect of cumulative agrin and nicotine. These findings suggest that increasing the concentration of an ECM molecule, i.e. agrin, may enhance nicotine effects on pCREB and that both MAPK and CaMKII signalling may play a regulatory role.

All muscarinic acetylcholine receptors (M 1 -M 5 ) are expressed in murine brain microvascular endothelium

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

Polymyositis in solid organ transplant recipients receiving tacrolimus.

Tacrolimus, also known as FK506, is an immunosuppressive agent widely used for the prevention of acute allograft rejection in organ transplantation and for the treatment of immunological diseases. This study reports two male patients who underwent solid organ transplantation (liver and kidney). After transplant, the patients received continuous immunosuppressive therapy with oral tacrolimus and later presented clinical manifestations and laboratory signs of myopathy. Muscle biopsies of both patients clearly documented an inflammatory myopathy with the histological features of polymyositis including CD8+ T cells which invaded healthy muscle fibers and expressed granzyme B and perforin, many CD68+ macrophages and MHC class I antigen upregulation on the surface of most fibers. Because of the temporal association while receiving tacrolimus and since other possible causes for myopathy were excluded, the most likely cause of polymyositis in our patients was tacrolimus toxicity. We suggest that patients on tacrolimus should be carefully monitored for serum CK levels and clinical signs of muscle disease.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.

The metaplastic effects of NMDA receptors blockade on reactivation of instrumental memories in rats

HIGHLIGHTSMK‐801 given 24h before instrumental memory retrieval affected reconsolidation.Changes of Zif268 and rpS6P in key brain areas indicated reconsolidation inhibition.MK‐801 given 24h before also increased glutamate receptors in a metaplastic fashion.‘Metaplastic’ MK‐801 might facilitated extinction rather than inhibited reconsolidation. ABSTRACT Metaplasticity, defined as the plasticity of synaptic plasticity, could affect learning and memory at different neural levels. It was hypothesized that metaplasticity changes on glutamate receptors may affect memory destabilization, promoting or preventing reconsolidation. We investigated the metaplastic effect of NMDA channel blocker MK‐801 on sucrose instrumental memory reconsolidation in a behavioural rat model associated to the assessment of molecular markers of metaplasticity, memory retrieval, destabilization and reconsolidation. Following instrumental conditioning and forced abstinence, rats were intraperitoneally treated with MK‐801 or vehicle 24h before the exposure to memory retrieval or not‐retrieval. Separate groups were tested for in‐vivo extinction of responding (24h and 7d after reactivation) or ex‐vivo assessment of transcription factor Zif268 and ribosomal protein rpS6 phosphorylation in nucleus accumbens (NAc) and amygdala (Amy). MK‐801 significantly inhibited instrumental responding at extinction test, suggesting reconsolidation blockade of instrumental memory. The decrease of Zif268 and phosphorylated‐rpS6 levels in NAc and Amy in MK‐801/Retrieval vs. Vehicle/Retrieval group supported the behavioural findings. An increase of GluN2B, GluA1 and mGluR5 in NAc, and GluN2B in Amy, 24h after MK‐801 indicated the trigger of associated metaplastic changes. Our findings show that metaplastic changes induced by NMDA receptors blockade affected sucrose instrumental memory retrieval as shown by both behavioural and molecular changes. We hypothesize that these findings however suggested a switch to extinction rather than a reconsolidation.