Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.The survival of metazoan organisms is dependent upon their ability to efficiently generate energy through the process of mitochondrial oxidative phosphorylation in which reducing equivalents, derived from the oxidation of acetyl CoA in the tricarboxylic acid cycle, are transferred from NADH and FADH 2 to the electron transport chain and ultimately to O 2 , a process which produces an electrochemical gradient that is used to synthesize ATP (1). Although oxidative phosphorylation is more efficient than glycolysis in generating ATP, it carries the inherent risk of generating reactive oxygen species (ROS) 2 as a result of electrons prematurely reacting with O 2 at respiratory complex I or complex III. Transient, low level ROS production is utilized for signal transduction in metazoan cells, but prolonged elevations of ROS result in the oxidation of protein, lipid, and nucleic acid leading to cell dysfunction or death.O 2 delivery and utilization must, therefore, be precisely regulated to maintain energy and redox homeostasis.Hypoxia-inducible factor 1 (HIF-1) plays a key role in the regulation of oxygen homeostasis (2, 3). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1␤ subunit and an O 2 -regulated HIF-1␣ subunit (4). Under aerobic conditions, HIF-1␣ is hydroxylated on proline residue 402 and/or 564 by prolyl hydroxylase 2 a dioxygenase that utilizes O 2 and ␣-ketoglutarate as co-substrates with ascorbate as co-factor in a reaction that generates succinate and CO 2 as side products (5-8). Under hypoxic conditions the rate of hydroxylation declines, either as a result of inadequate substrate (O 2 ) or as a result of hypoxia-induced mitochondrial ROS production, which may oxidize Fe(II) in the catalytic center of the hydroxylase (9, 10). Hydroxylated HIF-1␣ is bound by the von HippelLindau protein, which recruits a ubiquitin protein ligase complex that targets HIF-1␣ for proteasomal degradation (11)(12)(13)(14).HIF-1 regulates the transcription of hundreds of genes in response to hypoxia (15, 16), including the EPO (17) and VEGF (18) genes that encode proteins required for erythropoiesis and angiogenesis, respectively, which serve to increase O 2 delivery. In addition, HIF-1 controls a series of molecular mechanisms designed to maintain energy and redox homeostasis. First, HIF-1 coordinates a switch in the composition of cytochrome c oxidase (mitochondrial electron-transp...

Background— Stromal cell–derived factor-1 (SDF-1) is a chemokine considered to play an important role in the trafficking of hematopoietic stem cells. Given the close relationship between hematopoietic stem cells and endothelial progenitor cells (EPCs), we investigated the effect of SDF-1 on EPC-mediated vasculogenesis. Methods and Results— Flow cytometric analysis demonstrated expression of CXCR4, the receptor of SDF-1, by 66±3% of EPCs after 7 days in culture. In vitro modified Boyden chamber assay showed a dose-dependent EPC migration toward SDF-1 (control versus 10 ng/mL SDF-1 versus 100 ng/mL SDF-1, 24±2 versus 71±3 versus 140±6 cells/mm 2 ; P <0.0001). SDF-1 attenuated EPC apoptosis (control versus SDF-1, 27±1 versus 7±1%; P <0.0001). To investigate the effect of SDF-1 in vivo, we locally injected SDF-1 into athymic ischemic hindlimb muscle of nude mice combined with human EPC transplantation to determine whether SDF-1 augmented EPC-induced vasculogenesis. Fluorescence microscopic examination disclosed increased local accumulation of fluorescence-labeled EPCs in ischemic muscle in the SDF-1 treatment group (control versus SDF-1=241±25 versus 445±24 cells/mm 2 , P <0.0001). At day 28 after treatment, ischemic tissue perfusion was improved in the SDF-1 group and capillary density was also increased. (control versus SDF-1, 355±26 versus 551±30 cells/mm 2 ; P <0.0001). Conclusion— These findings indicate that locally delivered SDF-1 augments vasculogenesis and subsequently contributes to ischemic neovascularization in vivo by augmenting EPC recruitment in ischemic tissues.

The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmnϪ/Ϫ). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) ␥-subunit to the adult -subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH-evoked cardio-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia-inducible factor-1 (HIF-1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF-1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF-1 and if so whether it contributes to cardio-respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild-type (WT) and heterozygous (HET) mice partially deficient in HIF-1α, the O 2 regulated subunit of the HIF-1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h −1 , 8 h day −1 ) or to 10 days of 21% O 2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long-term facilitation (sLTF) of the chemoreceptor activity in CIH-exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH-exposed HET mice. Analysis of cardio-respiratory responses in CIH-exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH-exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH-induced increases in ROS but also CIH-evoked HIF-1α up-regulation in WT mice. These results indicate that: (a) HIF-1 activation is critical for eliciting CIH-induced carotid body-mediated cardio-respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF-1 and ROS.