Serous cutaneous glands substructural organization in anurans as a model of peptide/protein aggregation

A combined Transmission Electron Microscope (TEM) and Fourier Transformation (FT) analysis has been performed on the secretory granules storing active peptides/ proteins in cutaneous glands of n. 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM analysis revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns that depends on the degree of subunit clustering. Since the ultrastructural features of these biological samples were found to be suitable for FT analysis, this variety of recurrent patterns has been plotted into a range of FT frequency spectra. Through this quantitative approach we found that the variable granule substructure can be reduced to a main mechanism of peptides/proteins aggregation

The neuron-astrocyte-microglia triad in a rat model of chronic cerebral hypoperfusion: Protective effect of dipyridamole

Chronic cerebral hypoperfusion during aging may cause progressive neurodegeneration as ischemic conditions persist. Proper functioning of the interplay between neurons and glia is fundamental for the functional organization of the brain. The aim of our research was to study the pathophysiological mechanisms, and particularly the derangement of the interplay between neurons and astrocytes-microglia with the formation of triads, in a model of chronic cerebral hypoperfusion induced by the 2-vessel occlusion (2VO) in adult Wistar rats (n=15). The protective effect of dipyridamole given during the early phases after 2VO (4 mg/kg/day i.v., the first 7 days after 2VO) was verified (n=15). Sham-operated rats (n=15) were used as controls. Immunofluorescent triple staining of neurons (NeuN), astrocytes (GFAP) and microglia (IBA1) was performed 90 days after 2VO. We found significantly higher amount of ectopic neurons, neuronal debris and apoptotic neurons in CA1 Str. Radiatum and Str. Pyramidale of 2VO rats. In CA1 Str. Radiatum of 2VO rats the amount of astrocytes (cells/mm2) did not increase. In some instances several astrocytes surrounded ectopic neurons and formed a micro scar around them. Astrocyte branches could infiltrate the cell body of ectopic neurons, and, together with activated microglia cells formed the triads. In the triad, significantly more numerous in CA1 Str. Radiatum of 2VO than in sham rats, astrocytes and microglia cooperated in the phagocytosis of ectopic neurons. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Dypiridamole significantly reverted all the above described events. The protective effect of chronic administration of dipyridamole might be a consequence of its vasodilatory, antioxidant and anti-inflammatory role during the early phases after 2VO

Astrocyte clasmatodendrosis affects clearance mechanisms of Aß-fibrils in the hippocampus of aged rats

Aging is frequently accompanied by a low-grade pro-inflammatory condition which is considered a prodrome of Alzheimer Disease (AD). Indeed a common event of aging and AD is the deposition of beta amyloid (Aß) fibrils in the central nervous system, that has been associated to cognitive decline also in normal aging. Identifying traits of amyloid aggregates are the irreversibility of their molecular interactions, the ability to propagate from one cell to another and resist to clearance mechanisms [1]. We previously reported that, in aged rats, astrocyte clasmatodendrosis promote neuron to neuron propagation of Aß-fibrils and, therefore, their prion like spread. Clasmatodendrosis is a phenomenon described in astrocytes that consists in the loss of their distal processes. It’s known that astrocytes constitutively remove Aß-fibrils from neurolemma with their cytoplasmic processes and, in pathological conditions, this activity may cause their necrosis. Moreover, they regulate microglial activity in the central nervous system. Although microglial cells are involved in Aß-fibril clearance, they have also been associated to neuronal cell death in Alzheimer Disease. In this work we found that clasmatodendrosis is associated with a decrease of astrocyte activity of Aß fibril clearance in the pyramidal layer and affects microglial phagocytic activation in the hippocampus of aged rats. To discriminate immunofluorescence signals from autofluorescence typical of amyloid aggregates on confocal acquisitions, we designed a specific method of linear unmixing. Moreover, multiphoton microscopy analyses were implemented with an innovative method of fluorescence lifetime analysis (FLIM/Phasor), suitable to discriminate multiple fluorescences. On the whole our data suggest that clasmatodendrosis consistently affects clearance mechanisms of Aß fibril in the central nervous system and foreshadow new strategies in the development of therapeutical protocols against AD

Relaxin prevents cardiac fibroblast-myofibroblast transition via Notch-1-mediated Inhibition of TGF-β/Smad3 signaling

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells

Sphingosine 1-phosphate receptor 1 is required for MMP-2 function in bone marrow mesenchymal stromal cells: implications for cytoskeleton assembly and proliferation

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs' ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair