Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1? (PGC-1?) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1? activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1? activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1?'s target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.
Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.
The aquaporins (AQPs) are a family of 13 small hydrophobic integral transmembrane water channel proteins involved in transcellular and transepithelial water movement, transport of fluid and cell migration.
The mdx mouse, the most widely used animal model of Duchenne muscular dystrophy (DMD), develops a seriously impaired blood-brain barrier (BBB). As glucocorticoids are used clinically to delay the progression of DMD, we evaluated the effects of chronic treatment with ?-methyl-prednisolone (PDN) on the expression of structural proteins and markers in the brain and skeletal muscle of the mdx mouse. We analyzed the immunocytochemical and biochemical expression of four BBB markers, including endothelial ZO-1 and occludin, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71, the dystrophin-associated proteins (DAPs), and aquaporin-4 (AQP4) and ?-? dystroglycan (DG) in the brain. We evaluated the BBB integrity of mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP). The expression of DAPs was also assessed in gastrocnemius muscles and correlated with utrophin expression, and laminin content was measured in the muscle and brain. PDN treatment induced a significant increase in the mRNA and protein content of the BBB markers; a reduction in the phosphorylation of occludin in the brain and of AQP4/? DG in both tissues; an increase of Dp71 protein content; and an increase of both mRNA and protein levels of the AQP4/?-? DG complex. The latter was associated with enhanced laminin and utrophin in the muscle. The HRP assay demonstrated functional restoration of the BBB in the PDN-treated mdx mice. Specifically, mdx mice showed extensive perivascular labeling due to escape of the marker, while HRP was exclusively intravascular in the PDN-treated mice and the controls. These data illustrate for the first time that PDN reverses the BBB alterations in the mdx mouse and re-establishes the proper expression and phosphorylation of ?-DG in both the BBB and skeletal muscle. Further, PDN partially protects against muscle damage. The reduction in AQP4 and occludin phosphorylation, coupled with their anchoring to glial and endothelial membranes in PDN-treated mice, suggests that the drug may target the glial and endothelial cells. Our results suggest a novel mechanism for PDN action on cerebral and muscular function, restoring the link between DAPs and the extracellular matrix, most likely through protein kinase inactivation.
Angiogenesis plays a key role in several rheumatic diseases, including rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic sclerosis, systemic lupus erythematosus, and vasculitides. An imbalance between angiogenic inducers and inhibitors seems to be a critical factor in pathogenesis of these diseases. Macrophages promote angiogenesis during rheumatoid arthritis. In addition, macrophages can produce a variety of pro-angiogenic factors that have been associated with the angiogenic response occurring during other rheumatic diseases. Lastly, macrophages could be a target in the treatment of rheumatoid arthritis and other rheumatic diseases. Nevertheless, further studies are needed to better elucidate the exact role of macrophage in angiogenesis in these diseases.
No literature data are available concerning the expression of aquaporin-4 in primary central nervous system lymphomas and the relationship between aquaporin-4 expression and the morphological characteristics of blood vessels. Here, we have investigated this relationship in 24 human diffuse large B-cell primary central nervous system lymphomas by means of immunocytochemistry and confocal laser microscopy. Results have shown that: (i) a high aquaporin-4 expression correlated with a high Ki-67 index and aquaporin-4 marked tumour and endothelial cells in cytoplasm and plasma membranes, while aquaporin-4 expression was low in tumour areas with a low Ki-67 index where few tumour cells were positive to aquaporin-4, and endothelial cells showed aquaporin-4 expression on their abluminal side. (ii) Different type of cells participated in vessels formation: CD20(+) tumour cells and factor VIII(+) endothelial cells; aquaporin-4(+) tumour cells and CD31(+) endothelial cells; CD20(+) and aquaporin-4(+) tumour cells; glial fibrillary acidid protein(+) endothelial cells surrounded by glial fibrillary acidic protein(+) tumour cells. Overall, these data suggest the importance of aquaporin-4 in primary central nervous system lymphomas due to its involvement in cerebral oedema formation and resolution and tumour cell migratory activity, and have documented that tumour microvasculature in lymphomas is extremely heterogeneous, confirming the importance of neoangiogenesis in the pathogenesis of lymphomas.
In this study, the extent of angiogenesis, evaluated as microvascular volume density, immunoreactivity of tumour cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human primary melanoma specimens. Results showed that Epo/EpoR expression correlate with angiogenesis and tumour thickness. These findings suggest that Epo is secreted by tumour cells and it affects vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner, playing an important role in melanoma angiogenesis.
In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs ?-?-dystroglycan (DG), ?-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/?-?-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of ?-?-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane.
Previous studies have shown that increased vascularity is associated with haematogenous metastasis and poor prognosis in gastric cancer. The role of mast cells in gastric cancer angiogenesis has not been clarified completely. In this study, we correlated microvascular density and tryptase- and chymase-positive mast cells with histopathological type in gastric cancer. Specimens of primary gastric adenocarcinomas obtained from 30 patients who had undergone curative gastrectomy were investigated immunohistochemically by using anti-CD31 antibody to stain endothelial cells and anti-tryptase and anti-chymase antibodies to stain mast cells. The results showed that stage IV gastric carcinoma has a higher degree of vascularization than other stages and that both tryptase- and chymase-positive mast cells increase in parallel with malignancy grade even if the density of chymase-positive mast cells was significantly lower than the density of tryptase-positive mast cells and is highly correlated with the extent of angiogenesis. This study has demonstrated that mast cell density correlates with angiogenesis and progression of patients with gastric carcinoma. Understanding the mechanisms of gastric cancer angiogenesis provides a basis for a rational approach to the development of an antiangiogenic therapy in patients with this malignancy.
In this study, the extent of angiogenesis, evaluated as microvascular density, and the immunoreactivity of tumor cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human glioma specimens, and the effect of anti-Epo antibody on glioma-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) has been investigated. Results show that: (1) Epo/EpoR expression correlates with angiogenesis, (2) in the CAM assay, tumor bioptic specimens induce a strong angiogenic response, comparable to that induced by VEGF, and (3) an anti-Epo antibody co-administered with tumor bioptic specimens significantly inhibits the angiogenic response. These findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by glioma tumor cells and it affects glioma vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner. Moreover, as demonstrated by in vivo experiments, Epo is responsible for the strong angiogenic response induced by human glioma bioptic specimens, because an anti-Epo antibody is able to significantly inhibit this response.
Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkins lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.
Brain tumour oedema is coupled with blood-brain barrier damage and alteration in water flow. Aquaporin-4 (AQP4) is involved in the development and resolution of brain oedema, and it is strongly upregulated in glioblastoma multiforme (GBM). Here, we evaluated AQP4 expression and content in GBM and correlated with VEGF-VEGFR-2 expression. In the relapse after chemotherapy and radiotherapy, AQP4 content reduced in parallel with VEGF-VEGFR-2, as compared with primary tumours, and in the peripheral areas of relapsed tumours AQP4 mimicked normal findings of perivascular rearrangement. After immunogold electron microscopy, gold particles were attached on the glial membrane facing the perivascular side, likewise AQP4 gold labelling of the vessels of the control areas. In primary tumours the peripheral vessels appeared faintly marked by AQP4, while the perivascular tumour cells showed a strong expression. The vasculature of the inner tumour areas was unlabelled by AQP4, while tumour cells were labelled, in both primary and relapsing tumours. Relapsed tumours after radiotherapy alone showed slight AQP4 reduction and perivascular restoring in the peripheral areas of the tumour. These data indicate that in GBM chemotherapy and radiotherapy induce a down-regulation in AQP4 expression restoring its perivascular rearrangement suggesting its potential role in the resolution of brain oedema.
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