Emilio Clementi

Acid sphingomyelinase activity triggers microparticle release from glial cells.

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL‐1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP‐induced shedding and IL‐1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL‐1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL‐1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

TNF-α downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Defective Mitochondrial Biogenesis A Hallmark of the High Cardiovascular Risk in the Metabolic Syndrome

The metabolic syndrome is a group of risk factors of metabolic origin that are accompanied by increased risk for type 2 diabetes mellitus and cardiovascular disease. These risk factors include atherogenic dyslipidemia, elevated blood pressure and plasma glucose, and a prothrombotic and proinflammatory state. The condition is progressive and is exacerbated by physical inactivity, advancing age, hormonal imbalance, and genetic predisposition. The metabolic syndrome is a particularly challenging clinical condition because its complex molecular basis is still largely undefined. Impaired cell metabolism has, however, been suggested as a relevant pathophysiological process underlying several clinical features of the syndrome. In particular, defective oxidative metabolism seems to be involved in visceral fat gain and in the development of insulin resistance in skeletal muscle. This suggests that mitochondrial function may be impaired in the metabolic syndrome and, thus, in the consequent cardiovascular disease. We have recently found that mitochondrial biogenesis and function are enhanced by nitric oxide in various cell types and tissues, including cardiac muscle. Increasing evidence suggests that this mediator acts as a metabolic sensor in cardiomyocytes. This implies that a defective production of nitric oxide might be linked to dysfunction of the cardiomyocyte metabolism. Here we summarize some recent findings and propose a hypothesis for the high cardiovascular risk linked to the metabolic syndrome.

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events

Macropinocytosis, a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes, has been studied mostly in relation to antigen presentation. Early membrane traffic events occurring in this process are, however, largely unknown. Using human dendritic cells we show that a marked increase in the rate of macropinocytosis occurs a few minutes after application of two markers (small latex beads or dextran), depends on a slow intracellular Ca2+ concentration ([Ca2+]i) rise that precedes the PI3K-dependent step, and is preceded and accompanied by exocytosis of enlargeosomes compensating in part for the macropinocytic plasma membrane internalization. Unexpectedly, macropinosomes themselves, which share markers with endosomes, undergo Ca2+-dependent exocytosis so that, after ∼20 minutes of continuous bead or dextran uptake, an equilibrium is reached preventing cells from overloading themselves with the organelles. Large [Ca2+]i increases induced by ionomycin trigger rapid (<1 minute) exocytic regurgitation of all macropinosomes, whereas endosomes remain apparently unaffected. We conclude that, in dendritic cells, the rate of macropinocytosis is not constant but increases in a regulated fashion, as previously shown in other cell types. Moreover, macropinosomes are not simple containers that funnel cargo to an endocytic pathway, but unique organelles, distinct from endosomes by their competence for regulated exocytosis and other membrane properties.

Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (α-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

Inflammatory and alternatively activated human macrophages attract vessel-associated stem cells, relying on separate HMGB1- and MMP-9-dependent pathways.

Inflammatory macrophages recruited at the site of damaged muscles progressively acquire an alternative activation profile. Inflammatory (M1) and alternatively activated (M2) macrophages exert various and even opposite functions. M1 cells amplify tissue damage, and M2 cells dispose of necrotic fibers and deliver survival signals to myogenic precursors, finally supporting healing. A critical step in muscle healing is the recruitment of myogenic stem cells, including vessel‐associated stem cells (mesoangioblasts), which have been demonstrated to home to damaged skeletal muscle selectively and preferentially. Little information is available about the signals involved and the role played by infiltrating macrophages. Here, we report that the polarization of macrophages dramatically skews the secretion of high mobility group box 1 (HMGB1), TNF‐α, vascular endothelial growth factor, and metalloproteinase 9 (MMP‐9), molecules involved in the regulation of cell diapedesis and migration. All polarized macrophage populations were strikingly effective at inducing mesoangioblast migration. By means of specific inhibitors, we verified that the recruitment of mesoangioblasts requires the secretion of HMGB1 and TNF‐α by M1 cells and of MMP‐9 by M2 cells. Together, these data demonstrate a feature, unrecognized previously, of macrophages: their ability to attract stem cells, which is conserved throughout their polarization. Moreover, they open the possibility of novel strategies, aimed at interfering selectively with signals that recruit blood‐derived stem cells toward pro‐ or anti‐inflammatory macrophages.

HER2 Expression in Breast Cancer Cells Is Downregulated Upon Active Targeting by Antibody-Engineered Multifunctional Nanoparticles in Mice

Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.

Assessing the in vivo targeting efficiency of multifunctional nanoconstructs bearing antibody-derived ligands

A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their in vivo localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct.

Endothelial Nitric Oxide Synthase Activation by Tumor Necrosis Factor α Through Neutral Sphingomyelinase 2, Sphingosine Kinase 1, and Sphingosine 1 Phosphate Receptors: A Novel Pathway Relevant to the Pathophysiology of Endothelium

Objective—Tumor necrosis factor α (TNF-α), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. Methods and Results—In human endothelial cells, we found that eNOS activation by TNF-α is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-α. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P1 and S1P3, either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-α via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-α. Conclusions—This study establishes a previously undescribed link among TNF-α, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-α.