A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations

Portal hypertension (PH) is a common complication and a leading cause of death in patients with chronic liver diseases. PH is underlined by structural and functional derangement of liver sinusoid vessels and its fenestrated endothelium. Because in most clinical settings PH is accompanied by parenchymal injury, it has been difficult to determine the precise role of microvascular perturbations in causing PH. Reasoning that Vascular Endothelial Growth Factor (VEGF) is required to maintain functional integrity of the hepatic microcirculation, we developed a transgenic mouse system for a liver-specific-, reversible VEGF inhibition. The system is based on conditional induction and de-induction of a VEGF decoy receptor that sequesters VEGF and preclude signaling. VEGF blockade results in sinusoidal endothelial cells (SECs) fenestrations closure and in accumulation and transformation of the normally quiescent hepatic stellate cells, i.e. provoking the two processes underlying sinusoidal capillarization. Importantly, sinusoidal capillarization was sufficient to cause PH and its typical sequela, ascites, splenomegaly and venous collateralization without inflicting parenchymal damage or fibrosis. Remarkably, these dramatic phenotypes were fully reversed within few days from lifting-off VEGF blockade and resultant re-opening of SECs' fenestrations. This study not only uncovered an indispensible role for VEGF in maintaining structure and function of mature SECs, but also highlights the vasculo-centric nature of PH pathogenesis. Unprecedented ability to rescue PH and its secondary manifestations via manipulating a single vascular factor may also be harnessed for examining the potential utility of de-capillarization treatment modalities.     

Two Distinct States of the HAMP Domain from Sensory Rhodopsin Transducer Observed in Unbiased Molecular Dynamics Simulations

HAMP domain is a ubiquitous module of bacterial and archaeal two-component signaling systems. Considerable progress has been made recently in studies of its structure and conformational changes. However, the mechanism of signal transduction through the HAMP domain is not clear. It remains a question whether all the HAMPs have the same mechanism of action and what are the differences between the domains from different protein families. Here, we present the results of unbiased molecular dynamics simulations of the HAMP domain from the archaeal phototaxis signal transducer NpHtrII. Two distinct conformational states of the HAMP domain are observed, that differ in relative position of the helices AS1 and AS2. The longitudinal shift is roughly equal to a half of an α-helix turn, although sometimes it reaches one full turn. The states are closely related to the position of bulky hydrophobic aminoacids at the HAMP domain core. The observed features are in good agreement with recent experimental results and allow us to propose that the states detected in the simulations are the resting state and the signaling state of the NpHtrII HAMP domain. To the best of our knowledge, this is the first observation of the same HAMP domain in different conformations. The simulations also underline the difference between AMBER ff99-SB-ILDN and CHARMM22-CMAP forcefields, as the former favors the resting state and the latter favors the signaling state.     

Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O₂. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.     

Bleomycin Exerts Ambivalent Antitumor Immune Effect by Triggering Both Immunogenic Cell Death and Proliferation of Regulatory T Cells

Bleomycin (BLM) is an anticancer drug currently used for the treatment of testis cancer and Hodgkin lymphoma. This drug triggers cancer cell death via its capacity to generate radical oxygen species (ROS). However, the putative contribution of anticancer immune responses to the efficacy of BLM has not been evaluated. We make here the observation that BLM induces immunogenic cell death. In particular, BLM is able to induce ROS-mediated reticulum stress and autophagy, which result in the surface exposure of chaperones, including calreticulin and ERp57, and liberation of HMBG1 and ATP. BLM induces anti-tumor immunity which relies on calreticulin, CD8⁺ T cells and interferon-γ. We also find that, in addition to its capacity to trigger immunogenic cell death, BLM induces expansion of Foxp3+ regulatory T (Treg) cells via its capacity to induce transforming growth factor beta (TGFβ) secretion by tumor cells. Accordingly, Treg cells or TGFβ depletion dramatically potentiates the antitumor effect of BLM. We conclude that BLM induces both anti-tumor CD8⁺ T cell response and a counteracting Treg proliferation. In the future, TGFβ or Treg inhibition during BLM treatment could greatly enhance BLM anti-tumor efficacy.     

Modified Hammerhead Ribozymes as Potential Therapeutics

Abstract

Hammerhead ribozymes were modified in the 2′-position by flouro-, amino-, deoxy-, O-methyl- and methoxyethoxy-groups to stabilize against degradations. They were tested for their ability to cut specifically oncogenic N-ras RNA in vitro. Exogenous delivery by lipofection as well as viral vector mediated transfection showed comparable results in reducing N-ras mRNA.     

ASARM peptides: PHEX-dependent and -independent regulation of serum phosphate

Increased acidic serine aspartate-rich MEPE-associated motif (ASARM) peptides cause mineralization defects in X-linked hypophosphatemic rickets mice (HYP) and "directly" inhibit renal phosphate uptake in vitro. However, ASARM peptides also bind to phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and are a physiological substrate for this bone-expressed, phosphate-regulating enzyme. We therefore tested the hypothesis that circulating ASARM peptides also "indirectly" contribute to a bone-renal PHEX-dependent hypophosphatemia in normal mice. Male mice (n = 5; 12 wk) were fed for 8 wk with a normal phosphorus and vitamin D(3) diet (1% P(i) diet) or a reduced phosphorus and vitamin D(3) diet (0.1% P(i) diet). For the final 4 wk, transplantation of mini-osmotic pumps supplied a continuous infusion of either ASARM peptide (5 mg·day(-1)·kg(-1)) or vehicle. HYP, autosomal recessive hypophosphatemic rickets (ARHR), and normal mice (no pumps or ASARM infusion; 0.4% P(i) diet) were used in a separate experiment designed to measure and compare circulating ASARM peptides in disease and health. ASARM treatment decreased serum phosphate concentration and renal phosphate cotransporter (NPT2A) mRNA with the 1% P(i) diet. This was accompanied by a twofold increase in serum ASARM and 1,25-dihydroxy vitamin D(3) [1,25 (OH)(2)D(3)] levels without changes in parathyroid hormone. For both diets, ASARM-treated mice showed significant increases in serum fibroblast growth factor 23 (FGF23; +50%) and reduced serum osteocalcin (-30%) and osteopontin (-25%). Circulating ASARM peptides showed a significant inverse correlation with serum P(i) and a significant positive correlation with fractional excretion of phosphate. We conclude that constitutive overexpression of ASARM peptides plays a "component" PHEX-independent part in the HYP and ARHR hypophosphatemia. In contrast, with wild-type mice, ASARM peptides likely play a bone PHEX-dependent role in renal phosphate regulation and FGF23 expression. They may also coordinate FGF23 expression by competitively modulating PHEX/DMP1 interactions and thus bone-renal mineral regulation.     

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the ‘S genome’ originated from Nicotiana sylvestris and the ‘T genome’ derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.