Apigenin: Molecular Mechanisms and Therapeutic Potential against Cancer Spreading.

Due to its propensity to metastasize, cancer remains one of the leading causes of death worldwide. Thanks in part to their intrinsic low cytotoxicity, the effects of the flavonoid family in the prevention and treatment of various human cancers, both in vitro and in vivo, have received increasing attention in recent years. It is well documented that Apigenin (4',5,7-trihydroxyflavone), among other flavonoids, is able to modulate key signaling molecules involved in the initiation of cancer cell proliferation, invasion, and metastasis, including JAK/STAT, PI3K/Akt/mTOR, MAPK/ERK, NF-κB, and Wnt/ß-catenin pathways, as well as the oncogenic non-coding RNA network. Based on these premises, the aim of this review is to emphasize some of the key events through which Apigenin suppresses cancer proliferation, focusing specifically on its ability to target key molecular pathways involved in angiogenesis, epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cells (CSCs), cell cycle arrest, and cancer cell death.

Redox Biology and Liver Fibrosis.

Hepatic fibrosis is a complex process that develops in chronic liver diseases. Even though the initiation and progression of fibrosis rely on the underlying etiology, mutual mechanisms can be recognized and targeted for therapeutic purposes. Irrespective of the primary cause of liver disease, persistent damage to parenchymal cells triggers the overproduction of reactive species, with the consequent disruption of redox balance. Reactive species are important mediators for the homeostasis of both hepatocytes and non-parenchymal liver cells. Indeed, other than acting as cytotoxic agents, reactive species are able to modulate specific signaling pathways that may be relevant to hepatic fibrogenesis. After a brief introduction to redox biology and the mechanisms of fibrogenesis, this review aims to summarize the current evidence of the involvement of redox-dependent pathways in liver fibrosis and focuses on possible therapeutic targets.

Multi-Faceted Role of Luteolin in Cancer Metastasis: EMT, Angiogenesis, ECM Degradation and Apoptosis.

Luteolin (3',4',5,7-tetrahydroxyflavone), a member of the flavonoid family derived from plants and fruits, shows a wide range of biomedical applications. In fact, due to its anti-inflammatory, antioxidant and immunomodulatory activities, Asian medicine has been using luteolin for centuries to treat several human diseases, including arthritis, rheumatism, hypertension, neurodegenerative disorders and various infections. Of note, luteolin displays many anti-cancer/anti-metastatic properties. Thus, the purpose of this review consists in highlighting the relevant mechanisms by which luteolin inhibits tumor progression in metastasis, i.e., affecting epithelial-mesenchymal transition (EMT), repressing angiogenesis and lysis of extracellular matrix (ECM), as well as inducing apoptosis.

Redox-Dependent Modulation of Human Liver Progenitor Cell Line Fate.

Redox homeostasis is determinant in the modulation of quiescence/self-renewal/differentiation of stem cell lines. The aim of this study consisted of defining the impact of redox modifications on cell fate in a human hepatic progenitor line. To achieve this, the HepaRG cell line, which shows oval ductular bipotent characteristics, was used. The impact of redox status on the balance between self-renewal and differentiation of HepaRG cells was investigated using different methodological approaches. A bioinformatic analysis initially proved that the trans-differentiation of HepaRG toward bipotent progenitors is associated with changes in redox metabolism. We then exposed confluent HepaRG (intermediate differentiation phase) to oxidized (H2O2) or reduced (N-acetylcysteine) extracellular environments, observing that oxidation promotes the acquisition of a mature HepaRG phenotype, while a reduced culture medium stimulates de-differentiation. These results were finally confirmed through pharmacological modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2), a principal modulator of the antioxidant response, in confluent HepaRG. NRF2 inhibition led to intracellular pro-oxidative status and HepaRG differentiation, while its activation was associated with low levels of reactive species and de-differentiation. In conclusion, this study shows that both intra- and extracellular redox balance are crucial in the determination of HepaRG fate. The impact of redox status in the differentiation potential of HepaRG cells is significant on the utilization of this cell line in pre-clinical studies.

Mechanisms of Chemopreventive and Therapeutic Proprieties of Ginger Extracts in Cancer.

Ginger (Zingiber officinale Roscoe, family: Zingiberaceae), originating in South-East Asia, is one of the most used spices and condiments for foods and beverages. It is also used in traditional medicine for many human disorders including fever, gastrointestinal complications, arthritis, rheumatism, hypertension, and various infectious diseases due to its anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties. Intriguingly, many recent studies evidenced the potent chemopreventive characteristics of ginger extracts against different types of cancer. The aim of this work is to review the literature related to the use of ginger extracts as a chemotherapeutic agent and to structure the cellular and molecular mechanisms through which ginger acts in different cancer types. Data summarized from experiments (in vitro or in vivo) and clinical studies, evidenced in this review, show that ginger derivatives perpetrate its anti-tumor action through important mediators, involved in crucial cell processes, such as cell cycle arrest, induction of cancer cell death, misbalance of redox homeostasis, inhibition of cell proliferation, angiogenesis, migration, and dissemination of cancer cells.

Computational analysis of TP53 mutational landscape unveils key prognostic signatures and distinct pathobiological pathways in head and neck squamous cell cancer.

BACKGROUND: Mutations of the tumour-suppressor gene TP53 are the most frequent somatic genomic alterations in head and neck squamous cell carcinoma (HNSCC). However, it is not yet clear whether specific TP53 mutations bear distinct clinical and pathophysiological significance in different HNSCC subgroups. METHODS: A systematic bioinformatics appraisal of TP53 mutations was performed on 415 HNSCC cases available on The Cancer Genome Atlas (TCGA). The following features were analysed and correlated with known clinicopathological variables: mutational profile of TP53, location (within secondary structure and predicted domains of p53 protein) and well-known hotspot mutations. Interactome-genome-transcriptome network analysis highlighted different gene networks. An algorithm was generated to develop a new prognostic classification system based on patients' overall survival. RESULTS: TP53 mutations in HNSCCs exhibited distinct differences in different anatomical sites. The mutational profile of TP53 was an independent prognostic factor in HNSCC. High risk of death mutations, identified by our novel classification algorithm, was an independent prognostic factor in TCGA HNSCC database. Finally, network analysis suggested that distinct p53 molecular pathways exist in a site- and mutation-specific manner. CONCLUSIONS: The mutational profile of TP53 may serve as an independent prognostic factor in HNSCC patients, and is associated with distinctive site-specific biological networks.

Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality.

The chondroitin sulfate proteoglycan 4 ( CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.-Tamburini, E., Dallatomasina, A., Quartararo, J., Cortelazzi, B., Mangieri, D., Lazzaretti, M., Perris, R. Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality.

Neovascularization is a key feature of liver fibrosis progression: anti-angiogenesis as an innovative way of liver fibrosis treatment.

Liver fibrosis affects over 100 million people in the world; it represents a multifactorial, fibro-inflammatory disorder characterized by exacerbated production of extracellular matrix with consequent aberration of hepatic tissue. The aetiology of this disease is very complex and seems to involve a broad spectrum of factors including the lifestyle, environment factors, genes and epigenetic changes. More evidences indicate that angiogenesis, a process consisting in the formation of new blood vessels from pre-existing vessels, plays a crucial role in the progression of liver fibrosis. Central to the pathogenesis of liver fibrosis is the hepatic stellate cells (HSCs) which represent a crossroad among inflammation, fibrosis and angiogenesis. Quiescent HSCs can be stimulated by a host of growth factors, pro-inflammatory mediators produced by damaged resident liver cell types, as well as by hypoxia, contributing to neoangiogenesis, which in turn can be a bridge between acute and chronic inflammation. As matter of fact, studies demonstrated that neutralization of vascular endothelial growth factor as well as other proangiogenic agents can attenuate the progression of liver fibrosis. With this review, our intent is to discuss the cause and the role of angiogenesis in liver fibrosis focusing on the current knowledge about the impact of anti-angiogenetic therapies in this pathology.

Piperine: role in prevention and progression of cancer.

Cancer is among the leading causes of death worldwide. Several pharmacological protocols have been developed in order to block tumor progression often showing partial efficacy and severe counterproductive effects. It is now conceived that a healthy lifestyle coupled with the consumption of certain phytochemicals can play a protective role against tumor development and progression. According to this vision, it has been introduced the concept of "chemoprevention". This term refers to natural agents with the capability to interfere with the tumorigenesis and metastasis, or at least, attenuate the cancer-related symptoms. Piperine (1-Piperoylpiperidine), a main extract of Piper longum and Piper nigrum, is an alkaloid with a long history of medicinal use. In fact, it exhibits a variety of biochemical and pharmaceutical properties, including chemopreventive activities without significant cytotoxic effects on normal cells, at least at doses < of 250 µg/ml. The aim of this review is to discuss the relevant molecular and cellular mechanisms underlying the chemopreventive action of this natural alkaloid.

Paving the path for invasion: The polyedric role of LASP1 in cancer.

Although usually referred to as a structural actin-binding protein, LIM and SH3 domain-containing protein may actually be dynamically involved in the control of a wide spectrum of cellular processes, by virtue of its interaction with several molecular partners. Alongside being ubiquitously expressed in physiological conditions, LIM and SH3 domain-containing protein is overexpressed in a growing number of human cancers, in which it may actively contribute to their aggressiveness by promoting cell proliferation and migration. In view of the recent findings, implicating the protein in cancer progression, we discuss here the most relevant discoveries highlighting the role of this versatile protein in various human tumors. The correlation between LIM and SH3 domain-containing protein expression levels in cancer and the poor outcome and metastatic behavior of tumors denotes the clinical significance of this protein and hints its potential value as a new cancer prognostic or even diagnostic biomarker. This may be decisive not only to optimize existing pharmacological regimes but also to delineate novel, more efficacious therapeutic and/or preventive approaches.

MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line.

Gynaecological leiomyosarcoma (gLMS) represent a heterogeneous group of soft tissue sarcoma, characterized by rare incidence, high aggressiveness and propensity to infiltrate secondary organs, poor prognosis and lethality, because of the lack of biological mechanisms that underlying their progression and effective pharmaceutical treatments. This study was focused on some of the aspects of progression and dissemination of a subtype of gLMS namely vulvar LMS (vLMS). We therefore used a vulvar LMS-derived cell line namely SK-LMS-1, coupled with in vitro and in vivo assays. We observed that SK-LMS-1 cells have a strong invasive capacity in vitro, through the activity of matrix metalloproteinases 2 and 9, while in vivo these cells induce a strong angiogenic response and disseminate to the chick embryo liver. Therefore, we postulate that metalloproteinases are involved in the spreading behaviour of SK-LMS-1. Further investigations are necessary to better understand the molecular and cellular machinery involved in the progression of this malignancy.

Differential structural remodeling of the left-atrial posterior wall in patients affected by mitral regurgitation with or without persistent atrial fibrillation: a morphological and molecular study.

INTRODUCTION: Atrial fibrillation (AF) in mitral regurgitation (MR) is a complex disease where multiple factors may induce left-atrial structural remodeling (SR). We explored the differential SR of the left-atrial posterior wall (LAPW) of patients affected by MR with or without persistent AF, and the expression of key proteins involved in its pathogenesis. METHODS AND RESULTS: Light microscopy of LAPW samples from 27 patients with MR and persistent AF (group 1), 33 with MR in sinus rhythm (group 2), and 15 autopsy controls (group 3) was used to measure myocyte diameter, percentage of myocytolytic myocytes, interstitial fibrosis, and capillary density; RT-PCR and Western blotting were used to assess the mRNA and protein levels of SOD-1, SOD-2, HO-1, calpain, MMP-2, MMP-9, TIMP-1, TIMP-2, and VEGF; immunofluorescence was used to locate these proteins. Myocyte diameter was similar in groups 1 and 2, but larger than controls. Compared to group 2, group 1 had more myocytolytic myocytes (20.8 ± 5.6% vs 14.7 ± 4.5%; P < 0.0001), increased interstitial fibrosis (10.4 ± 5.1% vs 7.5 ± 4.2%; P < 0.05), and decreased capillary density (923 ± 107 No/mm(2) vs 1,040 ± 100 No/mm(2); P < 0.0001). All of the proteins were more expressed in groups 1 and 2 than in controls. The protein and mRNA levels of SOD-1, SOD-2, MMP-2, and MMP-9 were higher in group 1 than in group 2. CONCLUSIONS: The LAPW of MR patients with or without AF shows considerable SR. The former has more severe histopathological changes and higher levels of proteins involved in SR, thereby reaching a threshold beyond which the sinus impulse cannot normally activate atrial myocardium.

Eotaxin/CCL11 in idiopathic retroperitoneal fibrosis.

BACKGROUND: Idiopathic retroperitoneal fibrosis (IRF) is a rare fibro-inflammatory disorder characterized by a periaortic tissue which often encases the ureters causing acute renal failure. IRF histology shows fibrosis and a chronic inflammatory infiltrate with frequent tissue eosinophilia. We assessed a panel of molecules promoting eosinophilia and fibrosis in IRF patients and performed an immunogenetic study. METHODS: Serum levels of eotaxin/CCL11, regulated and normal T-cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-5, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) were measured using a multiplex assay in 24 newly diagnosed, untreated IRF patients and 14 healthy controls. Retroperitoneal biopsies (available in 8/24 patients) were histologically evaluated to assess eosinophil infiltration, whereas mast cells (MCs) were identified by immunohistochemical analysis for human tryptase. Immunohistochemistry for eotaxin/CCL11 and its receptor CCR3 was also performed. Six single nucleotide polymorphisms (SNPs) within the CCL11 gene (rs6505403, rs1860184, rs4795896, rs17735961, rs16969415 and rs17809012) were investigated in 142 IRF patients and 214 healthy controls. RESULTS: Serum levels of eotaxin/CCL11 were higher in IRF patients than in controls (P = 0.009). Eotaxin/CCL11 drives tissue infiltration of eosinophils and MCs, which can promote fibrosis. Eosinophilic infiltration was prominent (>5 cells/hpf) in five (62.5%) cases, and abundant tryptase-positive MCs were found in all cases; notably, MCs were in a degranulating state. Immunohistochemistry showed that CCL11 was highly produced by infiltrating mononuclear cells and that its receptor CCR3 was expressed by infiltrating eosinophils, MCs, lymphocytes and fibroblasts. None of the tested CCL11 SNPs showed disease association, but the TTCCAT haplotype was significantly associated with IRF (P = 0.0005). CONCLUSIONS: These findings suggest that the eotaxin/CCL11-CCR3 axis is active in IRF and may contribute to its pathogenesis; the TTCCAT haplotype within the CCL11 gene is significantly associated with IRF.

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse.

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 α-ß-dystroglycan (DG), α-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/α-ß-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 α-ß-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.

Validation of PDGFRbeta and c-Src tyrosine kinases as tumor/vessel targets in patients with multiple myeloma: preclinical efficacy of the novel, orally available inhibitor dasatinib.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/vessel growth associated with the disease progression. However, target specificity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFRbeta) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFRbeta kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangiogenic factors, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFRbeta/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFRbeta/c-Src TKs in MM, providing a framework for future clinical trials.

Fibrocytes in Chronic Periaortitis: A Novel Mechanism Linking Inflammation and Fibrosis.

OBJECTIVE: Chronic periaortitis (CP) is a rare disease characterized by periaortic and periiliac fibroinflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. This study was undertaken to explore the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. METHODS: We studied 44 patients with newly diagnosed CP and 30 healthy controls. Circulating fibrocytes were identified as Col1+CD45+ cells using flow cytometry. Retroperitoneal tissue biopsy samples from 9 CP patients were stained with anti-type I procollagen, anti-CXCR4, and anti-CD45 antibodies and analyzed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We also characterized T helper polarization in biopsy samples from CP patients and measured serum levels of a panel of cytokines that are hallmarks of T helper responses and capable of influencing fibrocyte differentiation. RESULTS: The frequency of circulating Col1+CD45+ fibrocytes was higher in patients than in controls (P = 0.0371). CD45+proCol1+ and CXCR4+proCol1+ cells were detected in all examined biopsy samples from CP patients. Serum levels of CXCL12 were also higher in CP patients than controls (P = 0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2 cytokines (e.g., interleukin-13 [IL-13] and IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2 polarization; Th2-skewed responses are known to promote fibrocyte differentiation. CONCLUSION: Our findings indicate that fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions. The accumulation of fibrocytes in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.

Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

Mast cells contribute to vasculogenic mimicry in multiple myeloma.

The angiogenic response is amplified during the induction phase of multiple myeloma (MM) and appears to exert a key role in the development of the disease [1]. Thus, inhibitors of angiogenesis have proven therapeutic potential in the treatment of patients with MM. Angiogenesis induced during the development of MM involves the direct production of proangiogenic cytokines by plasma cells within the marrow microenvironment. Mast cells (MCs) contribute to the the composition of the cellular components of the microenvironment in patients with MM, but their role in the pathophysiology of the disease is not clear. In this report, we used electron and confocal microscopy approaches to investigate the participation of MCs in the formation of the vessel wall in biopsy specimens from patients with MM. Results were compared to those obtained from the biopsy material of patients with a benign lesion, namely monoclonal gammopathy of undetermined significance (MGUS). Our results show that patients with MM exhibit typical tryptase-positive MCs, which interact physically with the endothelial cells (ECs) lining the vascular lumina, perhaps as a result of dysregulated vasculogenic development. This evidence highlights the importance of the stromal microenvironment during angiogenesis in the pathophysiology of MM and provides a novel perspective into the complex interplay between stromal and vascular components in the bone marrow microenvironment involved in the induction of hyervascularization in MM.

Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis.

BACKGROUND: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors. METHODS: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. RESULTS: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. CONCLUSIONS: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.

HIF activation and VEGF overexpression are coupled with ZO-1 up-phosphorylation in the brain of dystrophic mdx mouse.

In Duchenne muscular dystrophy (DMD) metabolic and structural alterations of the central nervous system are described. Here, we investigated in the brain of 10 mdx mice and in five control ones, the expression of hypoxia inducible factor-1alpha (HIF-1alpha) and we correlated it with the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) and of the endothelial tight junction proteins zonula occludens-1 (ZO-1) and claudin-1. Results showed an activation of mRNA HIF-1alpha by reverse transcription polymerase chain reaction (RT-PCR) and a strong HIF1-alpha labeling of perivascular glial cells and cortical neurons by immunohistochemistry, in mdx mouse. Moreover, overexpression of VEGF and VEGFR-2, respectively, in neurons and in endothelial cells coupled with changes to endothelial ZO-1 and claudin-1 expression in the latter were detected by immunoblotting and immunohistochemistry, in the mdx brain. Furthermore, by immunoprecipitation, an up-phosphorylation of ZO-1 was demonstrated in mdx endothelial cells in parallel with the reduction in ZO-1 protein content. These data suggest that the activation of HIF-1alpha in the brain of dystrophic mice coupled with VEGF and VEGFR-2 up-regulation and ZO-1 and claudin-1 rearrangement might contribute to both blood-brain barrier opening and increased angiogenesis.