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Background "Trousseau's Sign" was the first demonstration of a close relationship between cancer and thrombosis. Currently, venous thromboembolism (VTE) is 5-6 times more likely to occur in cancer patients, while there is a greater risk of cancer diagnoses following thromboses. In considering novel players, factor VIII (FVIII), an essential coagulation co-factor with emerging extra-coagulative functions, has been identified as an independent VTE risk factor in cancer, however, the basis of this increase is unknown. Objective To investigate the possible direct expression and secretion of FVIII by cancer cells. Methods Bladder cancer, with a high VTE-risk, and normal bladder tissue and epithelium, were used to investigate FVIII. Factor VIII protein and secretion were examined in bladder cancer cell lines. Expanding to other cancers, the Cancer Cell line Encyclopedia (CCLE) database was used to analyze FVIII, Tissue Factor (TF), FV, FVII, FIX, FX and von Willebrand factor (vWF) mRNA in 811 cell lines subdivided according to origin. Factor VIII protein synthesis, secretion and bioactivity were investigated in a profile of cancer cell lines of differing origins. Results and conclusions While expressed in the normal bladder epithelium, FVIII mRNA and protein were higher in matched bladder neoplasms, with synthesis and secretion of bioactive FVIII evident in bladder cancer cells. This can be extended to other cancer cell lines, with a pattern reflecting the tumor origin, and which is independent of vWF and other relevant players in the coagulation cascade. Herein, evidence is provided of a possible independent role for FVIII in cancer-related pathophysiology.

Amajor challenge in the development of a gene therapy for hemophilia A is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help to optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets- 1/Est-2 combination, while Ets-2 alone was ineffective. Moreover, Ets-1/Ets- 2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of green fluorescent protein (GFP) or FVIII cassettes driven by the shortened promoters, led to GFP expression mainly in endothelial cells in the liver and to longterm FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in hemophilia A gene therapy.

Here we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)–secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

Gene expression regulation is the result of complex interactions between transcriptional and post-transcriptional controls, resulting in cell-type-specific gene expression patterns that are determined by the developmental and differentiation stage of pathophysiological conditions. Understanding the complexity of gene expression regulatory networks is fundamental to gene therapy, an approach which has the potential to treat and cure inherited disorders by delivering the correct gene to patient specific cells or tissues by means of both viral and non-viral vectors. Besides the issues of biosafety, in recent years efforts have focused on achieving a robust and sustained transgene expression, which attains a phenotypic correction in several diseases, while avoiding transgene-related adverse effects, such as overexpression-associated cytotoxicity and/or immune responses to the transgene. In this sense, the use of cell-type-specific promoters and microRNA target sequences (miRTs) in gene transfer expression cassettes have allowed for a restricted expression after gene transfer in several studies. This review will focus on the use of transcriptional and post-transcriptional regulation to achieve a highly specific and safe transgene expression, as well as their application in ex vivo and in vivo gene therapeutic approaches.

Summary We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy., Graphical Abstract, Highlights • Endothelial cells were differentiated from iPSCs obtained from hemophilic donors • LV transduced-hemophilic endothelial cells secreted FVIII in vitro and in vivo, Follenzi and colleagues show that endothelial cells can be obtained starting from blood CD34+ cells of both healthy and hemophilic donors passing through iPSC generation. During the differentiation process the cells can be genetically modified by LV to express FVIII; corrected endothelial cells secrete FVIII in vitro and ameliorate the hemophilic phenotype in a mouse model of the disease.

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA., Graphical abstract, BOECs from HA patients and healthy donors were isolated, expanded, and LV-FVIII-transduced under GMP-like procedures. Amelioration of bleeding phenotype in a preclinical mouse model was reached by implantation of FVIII gene-corrected hemophilic BOECs injected in a pre-vascularized Cell Pouch reaching a long-term engraftment with a safe genomic integration profile.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants’ causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.

HemAcure: Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A

Background We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. Objectives To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. Methods Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte-derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site-directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA-mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti-FVIII antibodies and FVIII antigen, by ELISA. Results HA-MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti-FVIII antibodies. Conclusions Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.

Background & aims: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. Methods: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. Results: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor β (TRβ). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRβ axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. Conclusions: Collectively, these findings suggest that reactivation of the T3/TRβ axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRβ, could be useful tools in HCC therapy. Lay summary: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Kupffer cells (KC) play major roles in immunity and tissue injury or repair. Because recapitulation of KC biology and function within liver will allow superior insights into their functional repertoire, we studied the efficacy of the cell transplantation approach for this purpose. Mouse KC were isolated from donor livers, characterized, and transplanted into syngeneic recipients. To promote cell engraftment through impairments in native KC, recipients were preconditioned with gadolinium chloride. The targeting, fate, and functionality of transplanted cells were evaluated. The findings indicated that transplanted KC engrafted and survived in recipient livers throughout the study period of 3 months. Transplanted KC expressed macrophage functions, including phagocytosis and cytokine expression, with or without genetic modifications using lentiviral vectors. This permitted studies of whether transplanted KC could affect outcomes in the context of acetaminophen hepatotoxicity or hepatic ischemia-reperfusion injury. Transplanted KC exerted beneficial effects in these injury settings. The benefits resulted from cytoprotective factors including vascular endothelial growth factor. In conclusion, transplanted adult KC were successfully targeted and engrafted in the liver with retention of innate immune and tissue repair functions over the long term. This will provide excellent opportunities to address critical aspects in the biogenesis, fate, and function of KC within their native liver microenvironment and to develop the cell and gene therapy potential of KC transplantation.

Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration., Graphical Abstract, Highlights • Fibroblasts from two Sanfilippo C patients were reprogrammed to obtain iPSCs • iPSCs were successfully differentiated to neural cells that mimic the disease • Networks of patients’ neurons show altered activity and connectivity • Early functional phenotypes are prevented in gene-corrected patients’ neurons, iPSC technology has been used to model dozens of diseases. In this article, Raya, Grinberg, and colleagues take a step forward and show that this technology can also be used to model pre-symptomatic stages of human diseases. In particular, they used it to generate neurons from Sanfilippo C patients and found functional alterations in how these neurons interacted with one another, before the neurons themselves had any obvious alteration.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e. miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA., SCOPUS: ar.j, info:eu-repo/semantics/published

// Maria Maddalena Angioni 1, * , Kevin Bellofatto 2, * , Simone Merlin 2 , Silvia Menegon 3 , Andrea Perra 1 , Annalisa Petrelli 3 , Pia Sulas 1 , Silvia Giordano 3 , Amedeo Columbano 1 , Antonia Follenzi 2 1 Department of Biomedical Sciences, University of Cagliari, 09124 Cagliari, Italy 2 Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy 3 Department of Oncology, University of Torino School of Medicine, Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Italy * These authors have contributed equally to this work Correspondence to: Antonia Follenzi, email: antonia.follenzi@med.uniupo.it Amedeo Columbano, email: columbano@unica.it Silvia Giordano, email: silvia.giordano@unito.it Keywords: resistant hepatocyte, CD90.1, CD24, HCC cell lines, immortalized non-tumorigenic cells Received: March 03, 2016 Accepted: January 03, 2017 Published: February 01, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo . On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development.

To better understand cellular basis of hemophilia, cell types capable of producing FVIII need to be identified. We determined whether bone marrow (BM)–derived cells would produce cells capable of synthesizing and releasing FVIII by transplanting healthy mouse BM into hemophilia A mice. To track donor-derived cells, we used genetic reporters. Use of multiple coagulation assays demonstrated whether FVIII produced by discrete cell populations would correct hemophilia A. We found that animals receiving healthy BM cells survived bleeding challenge with correction of hemophilia, although donor BM-derived hepatocytes or endothelial cells were extremely rare, and these cells did not account for therapeutic benefits. By contrast, donor BM-derived mononuclear and mesenchymal stromal cells were more abundant and expressed FVIII mRNA as well as FVIII protein. Moreover, injection of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from BM, or of healthy BM-derived mesenchymal stromal cells, protected hemophilia A mice from bleeding challenge with appearance of FVIII in blood. Therefore, BM transplantation corrected hemophilia A through donor-derived mononuclear cells and mesenchymal stromal cells. These insights into FVIII synthesis and production in alternative cell types will advance studies of pathophysiological mechanisms and therapeutic development in hemophilia A.

Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi's sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways. © 2011 Japanese Cancer Association.

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo, de novo osteoclastogenesis is observed but it is currently unknown whether, besides increased osteoclast differentiation from undifferentiated precursors, other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study, an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment, the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally, chromosomal painting identified nuclei from female DCs, previously injected into a male recipient, among the nuclei of giant cells at sites of osteolysis. Finally, osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r−/− mice, which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether, our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases.

Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form.

EphrinB transmembrane ligands and their cognate EphB receptor tyrosine kinases regulate vascular development through bidirectional cell-to-cell signaling, but little is known about the role of EphrinB during postnatal vascular remodeling. We report that EphrinB is a critical mediator of postnatal pericyte-to-endothelial cell assembly into vascular structures. This function is dependent upon extracellular matrix-supported cell-to-cell contact, engagement of EphrinB by EphB receptors expressed on another cell, and Src-dependent phosphorylation of the intracytoplasmic domain of EphrinB. Phosphorylated EphrinB marks angiogenic blood vessels in the developing and hypoxic retina, the wounded skin, and tumor tissue, and is detected at contact points between endothelial cells and pericytes. Furthermore, inhibition ofEphrinB activity prevents proper assembly of pericytes and endothelial cells into vascular structures. These results reveal a role for EphrinB signaling in orchestrating pericyte/endothelial cell assembly, and suggest that therapeutic targeting of EphrinB may prove useful for disrupting angiogenesis when it contributes to disease.

The c-MET proto-oncogene, encoding the p190 hepatocyte growth factor tyrosine kinase receptor, can acquire oncogenic potential by multiple mechanisms, such as gene rearrangement, amplification and overexpression, point mutation, and ectopic expression, all resulting in its constitutive activation. Hepatocyte growth factor receptor truncated forms are generated by post-translational cleavage: p140 and p130 lack the kinase domain and are inactive. Their C-terminal remnant fragments are generally undetectable in normal cells, but a membrane-associated truncated form is recognized by anti-C-terminus antibodies in some human tumors, suggesting that a hepatocyte growth factor receptor lacking the ectodomain, but retaining the transmembrane and intracellular domains (Met-EC-), could acquire oncogenic properties. Herein we show that NIH-3T3 cells transduced with MET-EC- expressed a membrane-associated constitutively tyrosine-phosphorylated 60-kDa protein and, similarly to NIH-3T3 cells expressing the cytosolic oncoprotein Tpr-Met, showed activated extracellular regulated kinase 1/2 mitogen-activated protein kinase and Akt downstream transducers. Compared to control NIH-3T3 cells, NIH-3T3-Met-EC- cells grew faster and showed anchorage-independent growth and invasive properties in all aspects similar to cells expressing the transforming TPR-MET. Nude female mice injected subcutaneously with NIH-3T3-Met-EC- cells developed visible tumors, displaying the typical morphology of carcinomas with polygonal cells, in contrast to sarcomas with spindle-shaped cells induced by the injection of NIH-3T3-Tpr-Met cells. It is suggested that the different subcellular localization of the oncoproteins, more than differences in signal transduction, could be responsible for the tumor phenotype. All together, these data show that deletion of the ectodomain activates the hepatocyte growth factor receptor and its downstream signaling pathways, unleashing its transforming, invasive, and tumorigenic potential.

Multicellular networks based on the IEEE 802.16 standard appear to be very promising candidates to provide end users with broadband wireless access. However, they also pose interesting challenges in terms of radio resource management, where several design choices are not specified in the standard, intentionally left open to implementors. For this reason, we focus in this article on scheduling and resource allocation, and investigate how they could operate in a cross-layer fashion. In particular, we describe the principles of joint scheduling and resource allocation for IEEE 802.16 operating in AMC mode, and discuss the critical role played by physical layer considerations, especially intercell interference estimation and channel state awareness, in the obtained performance. This leads to identifying key open issues and possible general solutions

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Background & Aims Ischemia-reperfusion (IR) of liver results in hepatocytes (HP) and sinusoidal endothelial cells (LSEC) irreversible damage. Ischemic preconditioning protects IR damage upon adenosine A2a receptor (A2aR) stimulation. Understanding the phenotypic changes that underlie hepatocellular damage and protection is critical to optimize strategies against IR. Methods The proteome of HP and LSEC, isolated from sham or IR exposed mice, receiving or not the A2aR agonist CGS21680 (0.5mg/kg b.w.), was analyzed by 2-D DIGE/MALDI-TOF. Results We identified 64 proteins involved in cytoprotection, regeneration, energy metabolism and response to oxidative stress; among them, 34 were associated with IR injury and A2aR protection. The main pathways, downregulated by IR and upregulated by CGS21680 in HP and LSEC, were related to carbohydrate, protein and lipid supply and metabolism. In LSEC, IR reduced stress response enzymes that were instead upregulated by CGS21680 treatment. Functional validation experiments confirmed the metabolic involvement and showed that inhibition of pyruvate kinase, 3-chetoacylCoA thiolase, and arginase reduced the protection by CGS21680 of in vitro hypoxia-reoxygenation injury, whereas their metabolic products induced liver cell protection. Moreover, LSEC, but not HP, were sensitive to H 2 O 2 -induced oxidative damage and CGS21680 protected against this effect. Conclusions IR and A2aR stimulation produces pathological and protected liver cell phenotypes, respectively characterized by down- and upregulation of proteins involved in the response to O 2 and nutrients deprivation during ischemia, oxidative stress, and reactivation of aerobic energy synthesis at reperfusion. This provides novel insights into IR hepatocellular damage and protection, and suggests additional therapeutic options.