By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.

Background and purpose: Paclitaxel is highly efficacious in the treatment of breast, head and neck, non-small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P-glycoprotein (P-gp) at the blood-brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel. Experimental approach: Paclitaxel molecules were conjugated to a brain peptide vector, Angiopep-2, to provide a paclitaxelAngiopep-2 conjugate named ANG1005. We determined the brain uptake capacity, intracellular effects and antitumour properties of ANG1005 in vitro against human tumour cell lines and in vivo in human xenografts. We then determined ANG1005 activity on brain tumours with intracerebral human tumour models in nude mice. Key results: We show by in situ brain perfusion that ANG1005 enters the brain to a greater extent than paclitaxel and bypasses the P-gp. ANG1005 has an antineoplastic potency similar to that of paclitaxel against human cancer cell lines. We also demonstrate that ANG1005 caused a more potent inhibition of human tumour xenografts than paclitaxel. Finally, ANG1005 administration led to a significant increase in the survival of mice with intracerebral implantation of U87 MG glioblastoma cells or NCI-H460 lung carcinoma cells. Conclusions and implications:These results demonstrate the antitumour potential of a new drug, ANG1005, and establish that conjugation of anticancer agents with the Angiopep-2 peptide vector could increase their efficacy in the treatment of brain cancer.

This paper uses patent data to examine the impact of public environmental policy on innovations in environment-related technology. The analysis is conducted using data on an unbalanced panel of 77 countries between 2001 and 2007, drawing upon data obtained from the EPO World Patent Statistical (PATSTAT) database and the World Economic Forum's "Executive Opinion Survey". The results support our hypotheses concerning the positive role of both general innovative capacity and environmental policy stringency on environment-related innovation. A subsequent two-stage model assesses the factors which drive innovation in general and uses the fitted values to estimate environmental innovation. While the analysis is conducted on a smaller sample they confirm the findings of the reduced-form model.

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration-and conformationdependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Background:ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood–brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells.Methods:Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines.Results:Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions.Conclusion:ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.

Mitochondrial biogenesis, which depends on nuclear as well as mitochondrial genes, occurs in response to increased cellular ATP demand. The nuclear transcriptional factors, estrogen‐related receptor α (ERRα) and nuclear respiratory factors 1 and 2, are associated with the coordination of the transcriptional machinery governing mitochondrial biogenesis, whereas coactivators of the peroxisome proliferator‐activated receptor γ coactivator‐1 (PGC‐1) family serve as mediators between the environment and this machinery. In the context of proliferating cells, PGC‐1‐related coactivator (PRC) is a member of the PGC‐1 family, which is known to act in partnership with nuclear respiratory factors, but no functional interference between PRC and ERRα has been described so far. We explored three thyroid cell lines, FTC‐133, XTC.UC1 and RO 82 W‐1, each characterized by a different mitochondrial content, and studied their behavior towards PRC and ERRα in terms of respiratory efficiency. Overexpression of PRC and ERRα led to increased respiratory chain capacity and mitochondrial mass. The inhibition of ERRα decreased cell growth and respiratory chain capacity in all three cell lines. However, the inhibition of PRC and ERRα produced a greater effect in the oxidative cell model, decreasing the mitochondrial mass and the phosphorylating respiration, whereas the nonphosphorylating respiration remained unchanged. We therefore hypothesize that the ERRα–PRC complex plays a role in arresting the cell cycle through the regulation of oxidative phosphorylation in oxidative cells, and through some other pathway in glycolytic cells.

Cardiovascular diseases are often associated with impaired lipid metabolism. Animal models are useful for deciphering the physiological mechanisms underlying these pathologies. However, lipid metabolism is contrasted between species limiting the transposition of findings from animals to human. Hence, we aimed to compare extended lipid profiles of several animal species to bring new insights in animal model selections. Human lipid phenotype was compared with those of 10 animal species. Standard plasma lipids and lipoprotein profiles were obtained by usual methods and lipidomic analysis was conducted by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). As anticipated, we found contrasted lipid profiles between species. Some of them exhibited similar plasma lipids to human (non-human primate, rat, hamster, pig), but only usual lipid profiles of pigs were superimposable with human. LC-HRMS analyses allowed the identification of 106 other molecular species of lipids, common to all samples and belonging to major lipid families. Multivariate analyses clearly showed that hamster and, in a lower extent mouse, exhibited close lipid fingerprints to that of human. Besides, several lipid candidates that were previously reported to study cardiovascular diseases ranged similarly in human and hamster. Hence, hamster appeared to be the best option to study physiological disturbances related to cardiovascular diseases.

BackgroundThe PGC-1 related coactivator (PRC), which shares structural and functional features with PGC-1α, is believed to regulate several metabolic pathways as well as mitochondrial biogenesis. Its involvement in the early programming of cell proliferation suggests the existence of finely regulated crosstalk between mitochondrial functions and the cell cycle status.Methodology/Principal FindingsPRC-regulated pathways were explored in a cell-line model derived from mitochondrial-rich tumours with an essentially oxidative metabolism and specifically high PRC expression. The functional status of mitochondria was compared to the results of microarray analysis under conditions of temporal PRC inhibition. To specify the fine PRC regulation, the expression levels of the genes and proteins involved in the oxidative phosphorylation process were studied by real time quantitative PCR and western blotting. As in earlier studies on PGC-1α, we investigated the role of nitric oxide in PRC-regulated mitochondrial biogenesis and determined its action in the control of the phosphorylation status of the mitogen-activated protein kinase pathway.Conclusion/SignificanceWe found that nitric oxide rapidly influences PRC expression at the transcriptional level. Focusing on mitochondrial energetic metabolism, we observed that PRC differentially controls respiratory chain complexes and coupling efficiency in a time-dependent manner to maintain mitochondrial homeostasis. Our results highlight the key role of PRC in the rapid modulation of metabolic functions in response to the status of the cell cycle.