Augmentation of erythropoietin enhancer-mediated hypoxia-inducible gene expression by co-transfection of a plasmid encoding hypoxia-inducible factor 1α for ischemic tissue targeting gene therapy

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a potential treatment for ischemic diseases. However, VEGF expression should be tightly regulated to avoid side effects such as tumor growth. Previously, our group developed the erythropoietin (Epo) enhancer–SV40 promoter system for hypoxia-specific gene expression. In the present study, the activity of the Epo enhancer–SV40 promoter system was further enhanced without significant decrease in its specificity by co-transfection of the hypoxia-inducible factor 1α (HIF1α) gene. pSV-HIF1α was constructed by the insertion of the HIF1α cDNA into pSI. At a 1:1 ratio, co-transfection of pSV-HIF1α and pEpo-SV-Luc increased the promoter activity of the Epo enhancer–SV40 promoter system, showing at least three times higher gene expression under hypoxia as compared with the pEpo-SV-Luc single-plasmid transfection. Furthermore, co-transfection showed significant hypoxia specificity. Also, co-transfection of pEpo-SV-VEGF with pSV-HIF1α showed the enhanced VEGF expression without loss of hypoxia specificity, as compared with pEpo-SV-VEGF single-plasmid transfection. Furthermore, pSV-HIF1α induced the endogenous hypoxia-responsive genes such as angiopoietin-1, which would be beneficial for therapeutic angiogenesis. Therefore, with hypoxia specificity and higher gene expression, co-transfection of pSV-HIF1α and pEpo-SV-VEGF may be useful for ischemia targeting gene therapy.     

Investigational hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHI) for the treatment of anemia associated with chronic kidney disease

Introduction: In the last decade, concerns have been raised around the use of erythropoiesis-stimulating agents (ESAs) and intravenous iron in chronic kidney disease (CKD) patients, especially when given at high doses. Moreover, treatment with ESA is expensive.

Areas covered: We searched PubMed for original articles, reviews, and editorials having as a topic anemia, CKD, hypoxia inducible factor, hepcidin, iron, and hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHI). HIF-PHI are a new class of small molecules activating HIF-alfa isoforms (the main mediators of the effects of hypoxia on the body). This causes the secretion of endogenous erythropoietin and increased iron availability. Differing from ESA, HIF-PHI are administered orally. Preliminary data from phase-II clinical studies have shown their efficacy and safety in the short term.

Expert Opinion: HIF-PHI are a new promising class of drugs. The results of large, phase-III clinical studies are awaited to prove their efficacy and safety on cardiovascular events and cancer development in the long term. Their capability of penetrating the ESA market in the future will be influenced also by their selling price. The oral administration of HIF-PHI will be weighed to the ‘intra-lines’ infusion of ESA in hemodialysis or to the infrequent subcutaneous injections of long-acting ESA.     

Erythropoietin production in healthy volunteers subjected to controlled hypobaric hypoxia: further evidence against a role for adenosine

Aims Objective of this study was to investigate whether adenosine modulates renal erythropoietin production.
Methods In the present study erythropoietin production was stimulated by hypobaric hypoxia by subjecting healthy volunteers to a simulated altitude of 4000  m in a low pressure chamber for 5.5  h. During exposure to hypoxia the subjects received i.v. in a randomized, single-blind, cross-over fashion the non-specific adenosine antagonist theophylline, the adenosine reuptake inhibitor dipyridamole and placebo.
Results Contrary to the working hypothesis, theophylline did not decrease and dipyridamole did not further boost erythropoietin concentrations.
Conclusions The results are in agreement with our earlier study using haemorrhage as a controlled physiological stimulus of erythropoietin production, and would question a major role for adenosine as a mediator of renal erythropoietin production.     

Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O(2) level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1alpha involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1alpha (HIF-1alpha). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1alpha in microglia.     

Radiosensitising agents for the radiotherapy of cancer: advances in traditional and hypoxia targeted radiosensitisers

Background: Radiotherapy is utilised for the treatment of ～ 50% of patients with solid tumours, but its efficacy is limited by normal tissue toxicity and by the intrinsic or acquired radioresistance of many tumours. The combination of radiotherapy with chemotherapeutic agents that preferentially sensitise tumour cells to its cytotoxic effects has thus long been considered as a strategy to enhance cancer therapy. However, current chemoradiotherapy protocols remain highly unsatisfactory. Therefore, continuing efforts are being conducted to identify improved radiosensitising agents. Objective: To survey the patent literature and associated peer-reviewed publications of the past 4 years pertaining to the development of novel radiosensitising agents, with a focus on anticancer drugs traditionally used as radiosensitisers and on agents targeting radioresistant hypoxic tumour cells. Methods: Patents were searched with a set of relevant keywords using several search engines (ep.espacenet.com/, www.freepatentsonline.com/, patft.uspto.gov/). A Medline search on the same topics was performed in parallel. Results/conclusion: A total of 37 patents/applications were retrieved. Of these, 14 concern the use of conventional anticancer cytotoxic drugs for tumour radiosensitisation. The other patents mostly disclose novel hypoxic radiosensitisers, bioreductive drugs and inhibitors of hypoxia-inducible factor-1. Whether these advances will translate into clinically valuable radiosensitisers is, however, unclear.     

In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A

Ochratoxin A (OTA) is a mycotoxin often found in cereals and agricultural products. There is unequivocal evidence of renal carcinogenicity of OTA in male rats, although the mechanism of action is unknown. At present, available data support an epigenetic mechanism (DNA non-reactive) resulting from oxidative stress and cytotoxicity, because a direct OTA interaction with DNA has not been demonstrated. Genotoxic mechanism (DNA-reactive vs. DNA non-reactive) may have implications on human risk assessment. Therefore, the aim of the present work was to identify biological pathways modulated by OTA in vitro in a human renal cell line (HK-2) to contribute to the elucidation of the mechanism of OTA toxicity. For that purpose, cells were exposed to 50 microM OTA during 6 and 24 h, and gene expression profiles were analyzed using Affymetrix Human Genome U133 A 2.0 Gene Chips. Under the same experimental conditions, genotoxicity was evaluated by the modified comet assay using FPG and Endo III to detect oxidative DNA damage, and intracellular ROS level by the H(2)DCF assay. After 6 h, with slight cytotoxicity (83% survival), genes involved in mitochondrial electron transport chain were up-regulated; and after 24 h, with a more pronounced cytotoxicity (51% survival), genes implicated in oxidative stress response were also up-regulated. Increase in intracellular ROS level and oxidative DNA damage was evident at both exposure times being more pronounced with high cytotoxicity. On the contrary, up-regulation of genes implicated in DNA damage response, as cell cycle control or apoptosis, was not detected at any exposure time. In conclusion, these results support a DNA non-reactive mechanism of OTA genotoxicity.     

Neuroprotective effects of PMC, a potent alpha-tocopherol derivative, in brain ischemia-reperfusion: reduced neutrophil activation and anti-oxidant actions

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of alpha-tocopherol for anti-oxidation. It is more hydrophilic than other alpha-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4-12 microM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 microM) also significantly inhibited neutrophil migration stimulated by leukotriene B(4) (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 microM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1alpha activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.     

Erythropoietin production in healthy volunteers subjected to controlled haemorrhage: evidence against a major role for adenosine

1 This study was carried out to assess the role of adenosine in the regulation of human erythropoietin (EPO) production. To this end we investigated in healthy volunteers whether the nonspecific adenosine antagonist theophylline increases and the adenosine uptake inhibitor dipyridamole decreases EPO production in response to an haemorrhage of 750  ml.
2 Healthy male nonsmokers received i.v. in a parallel, randomized, single-blind trial theophylline (loading dose 5  mg  kg⁻¹ over 20  min, followed by 0.5  mg  kg⁻¹ min⁻¹), dipyridamole (0.21  mg  kg⁻¹ h⁻¹) or placebo (0.9% NaCl) for 6  h following the phlebotomy. EPO concentrations were followed up to 72  h after phlebotomy.
3 Following blood loss EPO concentrations increased during all treatments. The AUC_EPO (0,72  h) were not statistically significantly different (theophylline: 398±30, dipyridamole: 301±15, placebo: 332±57 [mu  ml⁻¹ h]). Creatinine clearance and urinary cAMP excretion were unaltered by any treatment. Urinary excretion of adenosine was significantly increased during infusion of dipyridamole. Plasma renin activitiy was significantly increased during theophylline infusion.
4 In our model of controlled, physiological stimulation of EPO production by haemorrhage, adenosine appears unlikely to play a major role as a mediator of renal EPO production.     

Microarray analysis of hepatic gene expression in pyrazole-mediated hepatotoxicity: identification of potential stimuli of Cyp2a5 induction

Cytochrome P450 2a5 (Cyp2a5) expression is induced during liver damage caused by hepatotoxins such as pyrazole, however, the mechanism underlying this overexpression is unclear. In order to identify pathophysiological and cellular responses to pyrazole that might alter Cyp2a5 expression, we examined the effect of pyrazole on mouse hepatic gene expression in C57BL/6 mice using Affymetrix 430 2.0 microarrays. Over 3000 differentially expressed genes were identified 24-h after pyrazole treatment that were associated with a variety of cellular pathways. Upregulated genes were primarily involved in the splicing and processing of RNA and the unfolded protein response pathway, while downregulated genes were associated with amino acid and lipid metabolism, and generation of precursor metabolites for energy production. We also examined the effects of pyrazole on cellular pathways linked to metabolic and histopathological changes observed with pyrazole toxicity. Increased mRNA levels were observed for genes involved in bilirubin production, whereas the major genes of the urea cycle were strongly decreased. Changes in genes involved in carbohydrate metabolism were also observed which could explain pyrazole-induced glycogen depletion and decreased serum glucose. In addition, over 100 genes involved in the cellular stress response were upregulated by pyrazole treatment, including genes involved in the unfolded protein response and redox status. Based on these results and previous evidence concerning the regulation of Cyp2a5, we have identified several pathophysiological changes including altered energy homeostasis, hyperbilirubinemia, ER stress, and altered redox status that are associated with CYP2A5 overexpression and may represent potential stimuli for the induction of Cyp2a5.     

Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development

The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo.     

Concentration-dependent bifunctional effect of TGF-beta 1 on immunoglobulin production: a role for Smad3 in IgA production in vitro

Injury to the liver results in rapid induction of transforming growth factor-beta1 (TGF-beta(1)) consistent with a role for TGF-beta(1) in repairing damaged tissue. In addition to its ubiquitous role in injury repair, TGF-beta(1) is also well established as a critical regulator of immune homeostasis; however, its mechanisms of action remain enigmatic. We have previously demonstrated that the hepatotoxic chlorinated hydrocarbon, carbon tetrachloride, suppresses helper T-lymphocyte function in a TGF-beta(1)-dependent manner. Here, we report that, in opposition to its immunosuppressive effects at picomolar concentrations, femtomolar concentrations of TGF-beta(1) augment T cell-dependent anti-sRBC IgM antibody forming cell (AFC) and T cell-independent DNP-Ficoll-induced AFC responses. These data support a concentration-dependent bifunctional effect by TGF-beta(1) on humoral immune responses in vitro. We further investigated a putative mechanistic role for Smad3, an intracellular mediator of TGF-beta(1) signaling, in propagating the inhibitory effects of TGF-beta(1) on humoral immune responses. Relative to wild type littermates, splenocytes from mice homologous for a null mutation in the gene encoding the TGF-beta receptor-activated Smad3 (Smad3(Exon8-/-)) were less sensitive to inhibition by TGF-beta(1) following anti-sRBC- and LPS-sensitization in vitro. In agreement, inhibition of IgM protein production by TGF-beta(1) was also dampened in LPS-sensitized Smad3(Exon8-/-) splenic B cells. Moreover, stimulation of IgA by TGF-beta(1) was abrogated in LPS-sensitized Smad3(Exon8-/-) splenocytes suggesting an additional role for Smad3 in regulating IgA production in vitro. Our results suggest that the effects of TGF-beta(1) on humoral immune responses fundamentally differ in a concentration-dependent manner and are mediated, in part, through Smad3 signaling.