Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7β-hydroxycholesterol (7βOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24 h, which was preceded by early lipid accumulation (6 h) and lysosomal membrane permeabilization (6−12 h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7βOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7βOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.     

Control of lipopolysaccharide biosynthesis by FtsH-mediated proteolysis of LpxC is conserved in enterobacteria but not in all gram-negative bacteria

Despite the essential function of lipopolysaccharides (LPS) in Gram-negative bacteria, it is largely unknown how the exact amount of this molecule in the outer membrane is controlled. The first committed step in LPS biosynthesis is catalyzed by the LpxC enzyme. In Escherichia coli, the cellular concentration of LpxC is adjusted by the only essential protease in this organism, the membrane-anchored metalloprotease FtsH. Turnover of E. coli LpxC requires a length- and sequence-specific C-terminal degradation signal. LpxC proteins from Salmonella, Yersinia, and Vibrio species carry similar C-terminal ends and, like the E. coli enzyme, were degraded by FtsH. Although LpxC proteins are highly conserved in Gram-negative bacteria, there are striking differences in their C termini. The Aquifex aeolicus enzyme, which is devoid of the C-terminal extension, was stable in E. coli, whereas LpxC from the alphaproteobacteria Agrobacterium tumefaciens and Rhodobacter capsulatus was degraded by the Lon protease. Proteolysis of the A. tumefaciens protein required the C-terminal end of LpxC. High stability of Pseudomonas aeruginosa LpxC in E. coli and P. aeruginosa suggested that Pseudomonas uses a proteolysis-independent strategy to control its LPS content. The differences in LpxC turnover along with previously reported differences in susceptibility against antimicrobial compounds have important implications for the potential of LpxC as a drug target.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.     

Vascular endothelial growth factors and receptors: anti-angiogenic therapy in the treatment of cancer

Vascular endothelial growth factors (VEGFs) are critical regulators of vascular and lymphatic function during development, in health and in disease. There are five mammalian VEGF ligands and three VEGF receptor tyrosine kinases. In addition, several VEGF co-receptors that lack intrinsic catalytic activity, but that indirectly modulate the responsiveness to VEGF contribute to the final biological effect. This review describes the molecular features of VEGFs, VEGFRs and co-receptors with focus on their role in the treatment of cancer.     

Conformational transformation and selection of synthetic prion strains

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.     

Cultural transmission of tool use by Indo-Pacific bottlenose dolphins (Tursiops sp.) provides access to a novel foraging niche

Culturally transmitted tool use has important ecological and evolutionary consequences and has been proposed as a significant driver of human evolution. Such evidence is still scarce in other animals. In cetaceans, tool use has been inferred using indirect evidence in one population of Indo-Pacific bottlenose dolphins (Tursiops sp.), where particular dolphins (‘spongers’) use marine sponges during foraging. To date, evidence of whether this foraging tactic actually provides access to novel food items is lacking. We used fatty acid (FA) signature analysis to identify dietary differences between spongers and non-spongers, analysing data from 11 spongers and 27 non-spongers from two different study sites. Both univariate and multivariate analyses revealed significant differences in FA profiles between spongers and non-spongers between and within study sites. Moreover, FA profiles differed significantly between spongers and non-spongers foraging within the same deep channel habitat, whereas the profiles of non-spongers from deep channel and shallow habitats at this site could not be distinguished. Our results indicate that sponge use by bottlenose dolphins is linked to significant differences in diet. It appears that cultural transmission of tool use in dolphins, as in humans, allows the exploitation of an otherwise unused niche.