Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.     

HIV-specific functional antibody responses in breast milk mirror those in plasma and are primarily mediated by IgG antibodies

Despite months of mucosal virus exposure, the majority of breastfed infants born to HIV-infected mothers do not become infected, raising the possibility that immune factors in milk inhibit mucosal transmission of HIV. HIV Envelope (Env)-specific antibodies are present in the milk of HIV-infected mothers, but little is known about their virus-specific functions. In this study, HIV Env-specific antibody binding, autologous and heterologous virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) responses were measured in the milk and plasma of 41 HIV-infected lactating women. Although IgA is the predominant antibody isotype in milk, HIV Env-specific IgG responses were higher in magnitude than HIV Env-specific IgA responses in milk. The concentrations of anti-HIV gp120 IgG in milk and plasma were directly correlated (r = 0.75; P < 0.0001), yet the response in milk was 2 logarithm units lower than in plasma. Similarly, heterologous virus neutralization (r = 0.39; P = 0.010) and ADCC activity (r = 0.64; P < 0.0001) in milk were directly correlated with that in the systemic compartment but were 2 log units lower in magnitude. Autologous neutralization was rarely detected in milk. Milk heterologous virus neutralization titers correlated with HIV gp120 Env-binding IgG responses but not with IgA responses (r = 0.71 and P < 0.0001, and r = 0.17 and P = 0.30). Moreover, IgGs purified from milk and plasma had equal neutralizing potencies against a tier 1 virus (r = 0.65; P < 0.0001), whereas only 1 out of 35 tested non-IgG milk fractions had detectable neutralization. These results suggest that plasma-derived IgG antibodies mediate the majority of the low-level HIV neutralization and ADCC activity in breast milk.     

Involvement of dopamine receptors in binge methamphetamine-induced activation of endoplasmic reticulum and mitochondrial stress pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D(1) receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D(1) and D(2) receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D(1) or D(2) receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58(IPK), in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D(2)-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors.     

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer–dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1 Å) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.     

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.     

Plastic responses to parents and predators lead to divergent shoaling behaviour in sticklebacks

Population divergence in antipredator defence and behaviour occurs rapidly and repeatedly. Genetic differences, phenotypic plasticity or parental effects may all contribute to divergence, but the relative importance of each of these mechanisms remains unknown. We exposed juveniles to parents and predators to measure how induced changes contribute to shoaling behaviour differences between two threespine stickleback species (benthics and limnetics: Gasterosteus spp). We found that limnetics increased shoaling in response to predator attacks, whereas benthics did not alter their behaviour. Care by limnetic fathers led to increased shoaling in both limnetic and benthic offspring. Shoaling helps limnetics avoid trout and avian predation; our results suggest that this adaptive behaviour is the result of a combination of paternal effects, predator-induced plasticity and genetic differences between species. These results suggest that plasticity substantially contributes to the rapid divergence in shoaling behaviour across the post-Pleistocene radiation of sticklebacks.     

Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function

Chronic inflammation in older individuals is thought to contribute to inflammatory, age‐related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross‐sectional study involving 91 healthy male (aged 20–84 years, median 52.4) and 55 female (aged 20–82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age‐related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age‐related diseases.     

Ventilatory phenotypes among four strains of adult rats.

Our purpose in this study was to identify different ventilatory phenotypes among four different strains of rats. We examined 114 rats from three in-house, inbred strains and one outbred strain: Brown Norway (BN; n = 26), Dahl salt-sensitive (n = 24), Fawn-hooded Hypertensive (FHH: n = 27), and outbred Sprague-Dawley rats (SD; n = 37). We measured eupneic (room air) breathing and the ventilatory responses to hypoxia (12% O(2)-88% N(2)), hypercapnia (7% CO(2)), and two levels of submaximal exercise. Primary strain differences were between BN and the other strains. BN rats had a relatively attenuated ventilatory response to CO(2) (P < 0.001), an accentuated ventilatory response to exercise (P < 0.05), and an accentuated ventilatory roll-off during hypoxia (P < 0.05). Ventilation during hypoxia was lower than other strains, but hyperventilation during hypoxia was equal to the other strains (P > 0.05), indicating that the metabolic rate during hypoxia decreased more in BN rats than in other strains. Another strain difference was in the frequency and timing components of augmented breaths, where FHH rats frequently differed from the other strains, and the BN rats had the longest expiratory time of the augmented breaths (probably secondary to the blunted CO(2) sensitivity). These strain differences not only provide insight into physiological mechanisms but also indicate traits (such as CO(2) sensitivity) that are genetically regulated. Finally, the data establish a foundation for physiological genomic studies aimed at elucidating the genetics of these ventilatory control mechanisms.