The S1P mimetic fingolimod phosphate regulates mitochondrial oxidative stress in neuronal cells.

Fingolimod is one of the few oral drugs available for the treatment of multiple sclerosis (MS), a chronic, inflammatory, demyelinating and neurodegenerative disease. The mechanism of action proposed for this drug is based in the phosphorylation of the molecule to produce its active metabolite fingolimod phosphate (FP) which, in turns, through its interaction with S1P receptors, triggers the functional sequestration of T lymphocytes in lymphoid nodes. On the other hand, part if not most of the damage produced in MS and other neurological disorders seem to be mediated by reactive oxygen species (ROS), and mitochondria is one of the main sources of ROS. In the present work, we have evaluated the anti-oxidant profile of FP in a model of mitochondrial oxidative damage induced by menadione (Vitk3) on neuronal cultures. We provide evidence that incubation of neuronal cells with FP alleviates the Vitk3-induced toxicity, due to a decrease in mitochondrial ROS production. It also decreases regulated cell death triggered by imbalance in oxidative stress (restore values of advanced oxidation protein products and total thiol levels). Also restores mitochondrial function (cytochrome c oxidase activity, mitochondrial membrane potential and oxygen consumption rate) and morphology. Furthermore, increases the expression and activity of protective factors (increases Nrf2, HO1 and Trx2 expression and GST and NQO1 activity), being some of these effects modulated by its interaction with the S1P receptor. FP seems to increase mitochondrial stability and restore mitochondrial dynamics under conditions of oxidative stress, making this drug a potential candidate for the treatment of neurodegenerative diseases other than MS.

IGF-II promotes neuroprotection and neuroplasticity recovery in a long-lasting model of oxidative damage induced by glucocorticoids.

Insulin-like growth factor-II (IGF-II) is a naturally occurring hormone that exerts neurotrophic and neuroprotective properties in a wide range of neurodegenerative diseases and ageing. Accumulating evidence suggests that the effects of IGF-II in the brain may be explained by its binding to the specific transmembrane receptor, IGFII/M6P receptor (IGF-IIR). However, relatively little is known regarding the role of IGF-II through IGF-IIR in neuroprotection. Here, using adult cortical neuronal cultures, we investigated whether IGF-II exhibits long-term antioxidant effects and neuroprotection at the synaptic level after oxidative damage induced by high and transient levels of corticosterone (CORT). Furthermore, the involvement of the IGF-IIR was also studied to elucidate its role in the neuroprotective actions of IGF-II. We found that neurons treated with IGF-II after CORT incubation showed reduced oxidative stress damage and recovered antioxidant status (normalized total antioxidant status, lipid hydroperoxides and NAD(P) H:quinone oxidoreductase activity). Similar results were obtained when mitochondria function was analysed (cytochrome c oxidase activity, mitochondrial membrane potential and subcellular mitochondrial distribution). Furthermore, neuronal impairment and degeneration were also assessed (synaptophysin and PSD-95 expression, presynaptic function and FluoroJade B® stain). IGF-II was also able to recover the long-lasting neuronal cell damage. Finally, the effects of IGF-II were not blocked by an IGF-IR antagonist, suggesting the involvement of IGF-IIR. Altogether these results suggest that, in or model, IGF-II through IGF-IIR is able to revert the oxidative damage induced by CORT. In accordance with the neuroprotective role of the IGF-II/IGF-IIR reported in our study, pharmacotherapy approaches targeting this pathway may be useful for the treatment of diseases associated with cognitive deficits (i.e., neurodegenerative disorders, depression, etc.).

Abnormal phenotype of in vitro dermal fibroblasts from patients with Pseudoxanthoma elasticum (PXE).

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha2, alpha5, alphav, whereas beta1 subunit as well as alpha2beta1 and alpha5beta1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery.

Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients.

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts.

In human dermal fibroblasts, brought to quiescence (G0) by serum starvation, the S phase peaked 24 h and G2/M phases 36 h after serum re-addition. Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h. Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater. S-Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter. Spermidine/spermine N1-acetyltransferase mRNA and activity reached the highest values at 36-48 h. Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine. We discuss how this comprehensive study may help to understand the involvement of polyamines in the control of cell proliferation.

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts.

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

Hyaluronan uptake by adult human skin fibroblasts in vitro.

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

Connective tissue in skin biopsies from patients suffering systemic sclerosis.

Little information is available on elastin during systemic sclerosis since biochemical and morphological data have primarily focused on collagen and glycosaminoglycan alterations of connective tissues in this pathological process. We performed ultrastructural, morphometric, biochemical and in situ hybridisation analyses on skin biopsies from patients affected by scleroderma and from site and age-matched control subjects. Affected skin revealed alterations in the distribution and organisation of collagen bundles and fibrils together with zonal increase of the microfibrillar component. Elastic fibres were significantly more numerous than in control skin, were more frequently vacuolated and characterised by electron-dense inclusions; moreover, morphometric analysis provided evidence for a significant increase of the percentage of both collagen bundles and elastin fibres in the measured tissue, compared to normal skin. Biochemical analysis seemed to confirm the increased elastin content per unit of dried weight tissue in sclerodermic skin. Differences observed among patients were only partially associated with disease duration and/or to severity of clinical manifestations. The abnormal amount of elastic fibres observed in skin biopsies from patients, and data from in situ hybridisation suggest the presence of a deregulation of the whole extracellular matrix that might be related to the role of cytokines such as TGF-beta, which has already been suggested to be involved in systemic sclerosis and in enhanced collagen and elastin production.

[Interpretations of uptake defects in hepatic scintiscanning]. / Interpretazione dei difetti di captazione nella scintigrafia epatica.

The absence of precise parameters for the interpretation of defective uptake means that personal experience is of great assistance in the examination of scintiscans. Classification of uptake defects in 208 cases was carried out by two experts in accordance with the following parameters: 1) irregular uptake or distinct defects; 2) central or peripheral defects; 3) increased size of the liver picture; 4) uptake by the spleen. Irregular uptake or central or peripheral uptake defects (with or without spleen uptake in each case) were observed as categories. The series as a whole showed that peripheral defects are associated with a greater frequency of false positives, and diagnosis must therefore be checked, spleen uptake is an accurate pointer to diffuse chronic hepatopathy, and central defects are indicative of substitutive pathology.

Comparison of ex vivo and in vitro human fibroblast ageing models.

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.

[Topical treatment of some dystrophic and inflammatory lesions of the skin and soft tissues]. / Trattamento topico di alcune lesioni distrofiche e flogistiche della cute e dei tessuti molli.

An investigation has been carried out on lysozyme cleasing antiphlogistic granulogenic activity when topically applied to various lesions such as varicous ulcers, bed sores, herpetic infections or actinic ulcerative processes. 38 patients who had undergone surgery and were affected with these lesions, were treated with lysozyme ointment applied twice daily for 15 days. Parameters for evaluating the therapeutic effect of the drug were, besides the typical subjective symptomatology, planimetry of the lesions, hyperemia and perilesional erythema. At the end of the investigation it was observed that lysozyme applications had favourable restorative effects and, in the case of herpetic lesions, complete resolution was achieved. Tolerance was satisfactory, although in 2 cases treatment had to be precautionarily discontinued having the subject complained of reacutization of pruritus.

Ultrastructural and morphometrical evaluations on normal human dermal connective tissue--the influence of age, sex and body region.

In order to give detailed structural and quantitative evaluations for some of the most important dermal constituents such as collagen, elastic fibres and mesenchymal cells, and for the non-structured extracellular matrix, we performed ultrastructural investigations on dermal biopsies from 50 healthy caucasian subjects aged from 6 fetal months to 83 years. Striking changes were observed, mainly in the perinatal period, for collagen, elastin and mesenchymal cells and, after 50 years of age, for collagen and elastin. Only slight or negligible differences were noted between males and females and in skin specimens taken from different parts of the body but similarly exposed to environmental factors (i.e. UV radiation). Modifications of the non-structured extracellular matrix appeared to be the consequence of changes affecting the other components. The results, therefore, emphasize the importance of the ageing factor in connective tissue metabolism and give further information on both qualitative and quantitative characteristics of normal human dermis.

Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis.

Ultrathin sections from the dermis of five normal subjects and from 10 patients suffering from pseudoxanthoma elasticum (PXE) were analyzed by immunoelectron microscopy with the aim of identifying and localizing proteins associated with the mineral precipitates within the altered elastic fibers. Serial sections were processed by indirect immunogold cytochemistry using primary antibodies against human fibronectin, vitronectin, bone sialoprotein, alkaline phosphatase, osteonectin, and osteopontin. In the latter two cases, antibodies against synthetic peptides were also used. The results indicate that normal elastic fibers contained osteopontin, and that this protein was associated with the apparently normal elastin as well as with the needle-shaped mineral precipitates in the elastic fibers of patients. On the contrary, significant amounts of vitronectin, alkaline phosphatase and, less, of bone sialoprotein were associated with the polymorphous mineral precipitates inside the elastic fibers. Large amounts of osteonectin and fibronectin, together with vitronectin, were localized on the microfilament aggregates, which were often associated with altered elastic fibers in PXE dermis and were never visualized in the dermis of control subjects. The results seem to indicate once more that PXE is a complex disorder of the fibroblast synthetic control. Elastic fiber mineralization might be considered a secondary event, which could depend on the abnormal synthesis and accumulation within the elastic fibers of proteins that are normally involved in mineralization processes.

Morphological analysis of knee synovial membrane biopsies from a randomized controlled clinical study comparing the effects of sodium hyaluronate (Hyalgan) and methylprednisolone acetate (Depomedrol) in osteoarthritis.

OBJECTIVE: The study was part of a randomized open-label clinical trial designed to evaluate the effects of intra-articular injections of hyaluronan (Hyalgan) (HY) in osteoarthritis (OA) of the human knee. Data were compared with those obtained after treatment with methylprednisolone acetate (Depomedrol) (MP). METHODS: Synovial membranes from patients with OA of the knee, primary or secondary to a traumatic event and classified according to the American College of Rheumatology criteria, were examined by arthroscopy and by light and electron microscopy before and 6 months after local injection of HY (2 ml of 500-730 000 MW hyaluronan, 10 mg/ml in saline, one injection per week for 5 weeks) or MP (1 ml of methylprednisolone acetate, 40 mg/ml, one injection per week for 3 weeks). RESULTS: Arthroscopy revealed a significant decrease in inflammatory score after both treatments. Histology showed that HY treatment was effective (P< or =0.05) in reducing the number and aggregation of lining synoviocytes, as well as the number and calibre of the vessels. MP treatment significantly reduced the number of mast cells in primary OA. Both treatments tended to decrease the number of hypertrophic and to increase the number of fibroblast-like lining cells, to decrease the numbers of macrophages, lymphocytes, mast cells and adipocytes, and to decrease oedema, especially in primary OA, and to increase the number of fibroblasts and the amount of collagen. These phenomena were evident throughout the thickness of the synovial tissue. CONCLUSION: At least in the medium term, both HY and MP modified a number of structural variables of the synovial membrane of the osteoarthritic human knee towards the appearance of that of normal synovium. The effect was more evident in primary OA than in OA secondary to a traumatic event. This is the first evidence that local hyaluronan injections modify the structural organization of the human knee synovium in OA.

Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE).

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

Cell behavior and cell-matrix interactions of human palmar aponeurotic cells in vitro.

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren's affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren's aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha 2, alpha 1, and alpha 5 integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren's patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren's patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren's contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren's disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia.

BACKGROUND: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta-thalassemia. OBJECTIVE AND METHODS: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. RESULTS: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta-thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta-thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. CONCLUSION: Data indicate that patients with beta-thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.