The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1α

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1α in respiratory-deficient RCC4-ρ0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O ₂ ⁻ in combination with NO to destabilize HIF-1α was corroborated in RCC4-p0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1α required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O ₂ ⁻ blocked HIF-1α degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O ₂ ⁻ coformation triggers a calcium increase that activates calpain to degrade HIF-1α, independently of the proteasome. Received 14 August 2007; received after revision 4 October 2007; accepted 22 October 2007     

Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.     

Mitochondrial activity: a possible determinant of anoxic injury in renal medulla

In brain, heart and kidney, cell work in the absence of oxygen has been thought to precipitate anoxic damage by increasing the rate of depletion of cellular energy stores. In the medullary thick ascending limb of isolated perfused rat kidneys, however, reduction of ATP synthesis by a variety of mitochondrial or metabolic inhibitors caused ATP depletion comparable to that produced by oxygen deprivation but did not reproduce the lesions of anoxia. In these cells, unrestrained mitochondrial activity may be an important source of anoxic injury.     

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

Uteroglobin: a novel cytokine?

Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, multifunctional protein secreted by the mucosal epithelial of virtually all mammals. It is present in the blood and in other body fluids including urine. An antigen immunoreactive to UG antibody is also detectable in the mucosal epithelia of all vertebrates. UG-binding proteins (putative receptor), expressed on several normal and cancer cell types, have been characterized. The human UG gene is mapped to chromosome 11q12.2 13.1, a region that is frequently rearranged or deleted in many cancers. The generation of UG knockout mice revealed that disruption of this gene causes: (i) severe renal disease due to an abnormal deposition of fibronectin and collagen in the glomeruli; (ii) predisposition to a high incidence of malignancies; and (iii) a lack of polychlorinated biphenyl binding and increased oxygen toxicity in the lungs. The mechanism(s) of UG action is likely to be even more complex as it also functions via a putative receptor-mediated pathway that has not yet been clearly defined. Molecular characterization of the UG receptor and signal transduction via this receptor pathway may show that this protein belongs to a novel cytokine/chemokine family.     

Mitochondrial activity: A possible determinant of anoxic injury in renal medulla

Summary In brain¹, heart² and kidney³, cell work in the absence of oxygen has been thought to precipitate anoxic damage by increasing the rate of depletion of cellular energy stores. In the medullary thick ascending limb of isolated perfused rat kidneys, however, reduction of ATP synthesis by a variety of mitochondrial or metabolic inhibitors caused ATP depletion comparable to that produced by oxygen deprivation but did not reproduce the lesions of anoxia. In these cells, unrestrained mitochondrial activity may be an important source of anoxic injury.     

The response of human skeletal muscle tissue to hypoxia

Hypoxia refers to environmental or clinical settings that potentially threaten tissue oxygen homeostasis. One unique aspect of skeletal muscle is that, in addition to hypoxia, oxygen balance in this tissue may be further compromised when exercise is superimposed on hypoxia. This review focuses on the cellular and molecular responses of human skeletal muscle to acute and chronic hypoxia, with emphasis on physical exercise and training. Based on published work, it is suggested that hypoxia does not appear to promote angiogenesis or to greatly alter oxidative enzymes in skeletal muscle at rest. Although the HIF-1 pathway in skeletal muscle is still poorly documented, emerging evidence suggests that muscle HIF-1 signaling is only activated to a minor degree by hypoxia. On the other hand, combining hypoxia with exercise appears to improve some aspects of muscle O₂ transport and/or metabolism.     

The increase of thiobarbituric acid reacting substances in rats with experimental chronic hypoxia

Summary Feeding under conditions of reduced oxygen supply prompted an increase in serum, arterial and brain tissue levels of thiobarbituric acid (TBA)-reacting substances. These observations indicated the possibility that hypoxia might be one of the factors predisposing to the accumulation of lipid peroxide.     

Adenosine is a sensitive oxygen sensor in the heart

Cardiac adenosine is formed both by an oxygen-sensitive (AMP----adenosine) and by an oxygen-insensitive (S-adenosylhomocysteine----adenosine) pathway. The phasic adenosine release during beta-adrenergic stimulation with isoproterenol is closely linked to coronary venous PO2 (isolated heart) and can be almost fully prevented when diastolic aortic pressure is maintained constant (heart in situ). During pressure autoregulation the transmural gradient of free adenosine is only increased when the autoregulatory reserve is exhausted. The critical PO2 below which adenosine formation is enhanced was found to be 3 mm Hg (isolated cardiomyocytes). Collectively, these data indicate that the formation of adenosine is not primarily coupled to the energy expenditure of the heart but to the supply/demand ratio for oxygen.     

Apolipoprotein M affecting lipid metabolism or just catching a ride with lipoproteins in the circulation?

Apolipoprotein M (apoM) is a novel apolipoprotein found mainly in high-density lipoproteins (HDL). Its function is yet to be defined. ApoM (25 kDa) has a typical lipocalin ?-barrel fold and a hydrophobic pocket. Retinoids bind apoM but with low affinity and may not be the natural ligands. ApoM retains its signal peptide, which serves as a hydrophobic anchor to the lipoproteins. This prevents apoM from being lost in the urine. Approximately 5% of HDL carries an apoM molecule. ApoM in plasma (1 μM) correlates strongly with both low-density lipoprotein (LDL) and HDL cholesterol, suggesting a link to cholesterol metabolism. However, in casecontrol studies, apoM levels in patients with coronary heart disease (CHD) and controls were similar, suggesting apoM levels not to affect the risk for CHD in humans. Experiments in transgenic mice suggested apoM to have antiatherogenic properties; possible mechanisms include increased formation of pre-? HDL, enhanced cholesterol mobilization from foam cells, and increased antioxidant properties. Received 28 November 2008; received after revision 15 December 2008; accepted 16 December 2008     

Defining the resistance to oxygen transfer in tissue hypoxia

Studies of O2 supply in freshly isolated adult mammalian cells provide new insight into the factors that limit mitochondrial oxygenation in vivo. Of particular importance, mitochondria are present at high densities and often in apparent clusters, both of which contribute to local O2 gradients under hypoxic conditions. Current evidence indicates that the mitochondrial distribution is a component of the differentiated phenotype of adult mammalian cells and that specific motors and anchoring mechanisms are present to allow redistribution in response to developmental, physiological and pathological challenges. To compare the importance of resistance to O2 transfer under different conditions and at different sites along the supply path in vivo, a simple mathematical expression of relative resistance to O2 supply is introduced. Under various pathophysiological conditions, this resistance increases in specific regions of the pulmonary, circulatory or cellular supply path and results in O2 deficiency in the mitochondria. Regardless of cause, the relative resistance increases dramatically in the vicinity of mitochondrial clusters during hypoxia.     

Molecular basis of autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common monogenetic diseases in humans. The discovery that mutations in the PKD1 and PKD2 genes are responsible for ADPKD has sparked extensive research efforts into the physiological and pathogenetic role of polycystin-1 and polycystin-2, the proteins encoded by these two genes. While polycystin-1 may mediate the contact among cells or between cells and the extracellular matrix, a lot of evidence suggests that polycystin-2 represents an endoplasmic reticulum-bound cation channel. Cyst development has been compared to the growth of benign tumors and this view is highlighted by the model that a somatic mutation in addition to the germline mutation is responsible for cystogenesis (two-hit model of cyst formation). Since in vitro polycystin-1 and polycystin-2 interact through their COOH termini, the two proteins possibly act in a common pathway, which controls the width of renal tubules. The loss of one protein may lead to a disruption of this pathway and to the uncontrolled expansion of tubules. Our increasing knowledge of the molecular events in ADPKD has also started to be useful in designing novel diagnostic and therapeutic strategies. Received 12 September 2001; received after revision 7 November 2001; accepted 7 November 2001     

PSF and CMF,autocrine factors that regulate gene expression during growth and early development ofDictyostelium

Throughout growth and development,Dictyostelium cells secrete autocrine factors that accumulate in proportion to cell density. At sufficient concentration, these factors cause changes in gene expression. VegetativeDictyostelium cells continuously secrete prestarvation factor (PSF). The bacteria upon which the cells feed inhibit their response to PSF, allowing the cells to monitor their own density in relation to that of their food supply. At high PSF/bacteria ratios, which occur during late exponential growth, PSF induces the expression of several genes whose products are needed for cell aggregation. When the food supply has been depleted, PSF production declines, and a second density-sensing pathway is activated. Starving cells secrete conditioned medium factor (CMF), a glycoprotein of Mr 80 kDa that is essential for the development of differentiated cell types. Antisense mutagenesis has shown that cells lacking CMF cannot aggregate, and preliminary data suggest that CMF regulates cAMP signal transduction. Calculations indicate that a mechanism of simultaneously secreting and recognizing a signal molecule, as used byDictyostelium to monitor cell density, could also be used to determine the total number of cells in a tissue.     

Renal vascular reactivity to ATP in hyper- and hypothyroid rats

The effects of adenosine triphosphate (ATP) on the renal vasculature of isolated kidneys from control, hyper- and hypothyroid rats were characterized. ATP responsiveness was evaluated in basal tone and in raised tone (phenylephrine 10^–6 M) preparations. These responses were compared with those obtained with barium chloride or sodium nitroprusside (SNP), used respectively as nonreceptor agonists for vasoconstriction or vasodilation. In preparations at basal tone, ATP produced dose-related vasoconstriction, which was increased in hyperthyroid kidneys, and was severely attenuated in kidneys from hypothyroid rats. In raised tone preparations from control rats ATP produced a dual response: vasoconstriction at low doses, which declined with increasing doses to give way to vasodilator responses; biphasic responses were found in some kidneys. Hyperthroid kidneys showed increased pressor responses and a vasodilator response similar to those seen in kidneys from control rats. However, in hypothyroid kidneys the vasodilator response was abolished. The responses to barium chloride and to SNP were significantly increased and decreased in hyper- and hypothyroid kidneys, respectively; vasoconstrictor responses to SNP were also found in hypothyroid kidneys. Hence the abnormal responses to ATP observed in both thyroid dysfunctions may be partially explained by unspecific alterations in the contractile machinery of the renal vasculature in these kidneys. However, ATP responsiveness (vasoconstriction at low tone and vasodilation at raised tone) was more severely affected in hypothyroid kidneys, suggesting that purinergic (P_2X and P_2Y) receptor activity may be decreased in these organs.     

The exercising heart at altitude

Maximal cardiac output is reduced in severe acute hypoxia but also in chronic hypoxia by mechanisms that remain poorly understood. In theory, the reduction of maximal cardiac output could result from: (1) a regulatory response from the central nervous system, (2) reduction of maximal pumping capacity of the heart due to insufficient coronary oxygen delivery prior to the achievement of the normoxic maximal cardiac output, or (3) reduced central command. In this review, we focus on the effects that acute and chronic hypoxia have on the pumping capacity of the heart, particularly on myocardial contractility and the molecular responses elicited by acute and chronic hypoxia in the cardiac myocytes. Special emphasis is put on the cardioprotective effects of chronic hypoxia. (Part of a multi-author review.)