Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.

BACKGROUND

Cell-based angiogenesis is a promising treatment for ischemic diseases; however, survival of implanted cells is impaired by the ischemic microenvironment. In this study, mesenchymal stem cells (MSCs) for cell transplantation were preconditioned with trimetazidine (TMZ). We hypothesized that TMZ enhances the survival rate of MSCs under hypoxic stimuli through up-regulation of HIF1-α.

RESULTS

Bone marrow-derived rat mesenchymal stem cells were preconditioned with 10 μM TMZ for 6 h. TMZ preconditioning of MSCs remarkably increased cell viability and the expression of HIF1-α and Bcl-2, when cells were under hypoxia/reoxygenation (H/R) stimuli. But the protective effects of TMZ were abolished after knocking down of HIF-1α. Three days after implantation of the cells into the peri-ischemic zone of rat myocardial ischemia-reperfusion (I/R) injury model, survival of the TMZ-preconditioned MSCs was high. Furthermore, capillary density and cardiac function were significantly better in the rats implanted with TMZ-preconditioned MSCs 28 days after cell injection.

CONCLUSIONS

TMZ preconditioning increased the survival rate of MSCs, through up-regulation of HIF1-α, thus contributing to neovascularization and improved cardiac function of rats subjected to myocardial I/R injury.

The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane (OMM) protein, serves as a mitochondrial gatekeeper, mediating the transport of nucleotides, Ca2+ and other metabolites across the OMM. VDAC1 also plays a central role in mitochondria-mediated apoptosis by facilitating the release of apoptotic proteins and by association with both pro- and anti-apoptotic proteins. Tumor cells, which are constantly exposed to hypoxic conditions, affect the cell via the transcription factor hypoxia-inducible factor (HIF) that induces transcriptional activity. In cultured cells and in lung cancer patients, hypoxia induces VDAC1 truncation at the C-terminus (VDAC1-ΔC). However, the molecular mechanisms involved in VDAC1-ΔC formation are unknown. Here, we show that hypoxia-induced VDAC1-ΔC formation is inhibited by the Ca2+ chelator BAPTA-AM, by calpain inhibitor-1, by inhibitor of the asparagine endopeptidase (AEP) and by si-RNA targeting HIF1-α or Ca2+-activated protease calpain-1 expression but not that of calpain-2. Finally, VDAC1-ΔC expressed in bacteria and reconstituted into a planar lipid bilayer exhibited decreased channel conductance relative to the full-length protein, yet retained voltage-dependent conductance. These findings suggest that hypoxia, acting via HIF-1α expression, leads to VDAC1 cleavage involving the activation of calpain 1 and AEP.

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

Keywords: Glucose metabolism; HIF-1α; LDH-A; NPC; PD-L1; Silibinin.

OBJECTIVE

To elucidate preventive effects of high molecular weight hyaluronan (HMWHA) on the joint capsule of immobilized knees in rats.

METHODS

Unilateral knee joints of rats were immobilized with an internal fixator. Either 50 μl of HMWHA (Im-HA group) or 50 μl of saline (control group) was administered intra-articularly once a week after surgery. Sagittal sections were prepared from the medial midcondylar region of the knee joints and assessed by histological, histomorphometric, and immunohistochemical methods. Gene expressions related to inflammation, fibrotic conditions, and hypoxia were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Tissue elasticity of the capsule from both groups was examined using a scanning acoustic microscope (SAM).

RESULTS

CD68 positive cells decreased in adhesion areas of the synovial membrane after 1 week in both groups. The length of the superficial layer in the synovial membrane of the Im-HA group was significantly longer than those in the control group over a period of 4 to 8 weeks with significantly small numbers of CD68 positive cells. The gene expressions of IL-6, IL-1β, TGF-β, CTGF, COL1a1, COL3a1, SPARC, and HIF1-α were significantly lower in the Im-HA group compared to those in the control group. The sound speed of the anterior and posterior synovial membrane increased significantly (a reduction in elasticity) in the control group compared to those in the Im-HA group during weeks 1 to 4.

CONCLUSIONS

This study demonstrated that HMWHA injections suppressed inflammatory, fibrotic, and hypoxic conditions observed in the immobilized joint capsule.

Decreased skeletal muscle capillarization is considered to significantly contribute to the development of pulmonary cachexia syndrome (PCS) and progressive muscle wasting in several chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). It is unclear to which extent the concurrent presence of systemic inflammation contributes to decreased skeletal muscle capillarization under these conditions. The present study was designed to examine in vitro the effects of the pro-inflammatory cytokine, tumor necrosis factor (TNF), on the regulation of hypoxia-angiogenesis signal transduction and capillarization in skeletal muscles. For this purpose, fully differentiated C2C12 skeletal muscle myocytes were stimulated with TNF and maintained under normoxic or hypoxic conditions. The expression levels of the putative elements of the hypoxia-angiogenesis signaling cascade were examined using qPCR, western blot analysis and immunofluorescence. Under normoxic conditinos, TNF stimulation increased the protein expression of anti-angiogenic von-Hippel Lindau (VHL), prolyl hydroxylase (PHD)2 and ubiquitin conjugating enzyme 2D1 (Ube2D1), as well as the total ubiquitin content in the skeletal muscle myocytes. By contrast, the expression levels of hypoxia-inducible factor 1‑α (HIF1-α) and those of its transcriptional targets, vascular endothelial growth factor (VEGF)A and glucose transporter 1 (Glut1), were markedly reduced. In addition, hypoxia increased the expression of the VHL transcript and further elevated the VHL protein expression levels in C2C12 myocytes following TNF stimulation. Consequently, an impaired angiogenic potential was observed in the TNF-stimulated myocytes during hypoxia. In conclusion, TNF increases VHL expression and disturbs hypoxia-angiogenesis signal transduction in skeletal muscle myocytes. The current findings provide a mechanism linking systemic inflammation and impaired angiogenesis in skeletal muscle. This is particularly relevant to further understanding the mechanisms mediating muscle wasting and cachexia in patients with chronic inflammatory diseases, such as COPD.

Menstrual endometrial breakdown induced by estradiol and progesterone withdrawal is regularly attributed to vasospasm of spiral arteries causing ischemia and hypoxia. We investigated whether hypoxia actually occurred in an in vivo model of menstruation. Three complementary approaches were used to look for signs of hypoxia in fragments of human functionalis xenografted to ovariectomized immunodeficient mice bearing pellets-releasing estradiol and progesterone, and then deprived of ovarian steroids. Hormone withdrawal 21 d after grafting induced menstrual breakdown and MMP expression within 4 d. Local partial oxygen pressure (pO2) was measured by electron paramagnetic resonance using implanted lithium phtalocyanine crystals. In mice with hormone maintenance until sacrifice, pO2 was low one week after grafting (14.8±3.4 mmHg) but increased twofold from the second week when tissue was largely revascularized. After 3 wk, pO2 was not modified by hormone withdrawal but was slightly increased on hormone reimpregnation 4 d after removal (34.7±6.1 mmHg) by comparison with hormone maintenance (27.1±8.6 mmHg). These results were confirmed using fluorescence quenching-based OxyLite measurements. In a further search for signs of hypoxia, we did not find significant HIF1-α immunostaining, nor pimonidazole adducts after hormone withdrawal. We conclude that hypoxia is not needed to trigger menstrual-like tissue breakdown or repair in human endometrial xenograft.

Functional imaging modalities like Diffusion-weighted imaging are increasingly used to predict tumor behavior like cellularity and vascularity in different tumors. Histogram analysis is an emergent imaging analysis, in which every voxel is used to obtain a histogram and therefore statistically information about tumors can be provided. The purpose of this study was to elucidate possible associations between ADC histogram parameters and several immunhistochemical features in rectal cancer. Overall, 11 patients with histologically proven rectal cancer were included into the study. There were 2 (18.18%) females and 9 males with a mean age of 67.1 years. KI 67-index, expression of p53, EGFR, VEGF, and Hif1-alpha were semiautomatically estimated. The tumors were divided into PD1-positive and PD1-negative lesions. ADC histogram analysis was performed as a whole lesion measurement using an in-house matlab application. Spearman's correlation analysis revealed a strong correlation between EGFR expression and ADCmax (p=0.72, P=0.02). None of the vascular parameters (VEGF, Hif1-alpha) correlated with ADC parameters. Kurtosis and skewness correlated inversely with p53 expression (p=-0.64, P=0.03 and p=-0.81, P=0.002, respectively). ADCmedian and ADCmode correlated with Ki67 (p=-0.62, P=0.04 and p=-0.65, P=0.03, respectively). PD1-positive tumors showed statistically significant lower ADCmax values in comparison to PD1-negative tumors, 1.93 ± 0.36 vs 2.32 ± 0.47×10-3mm2/s, p=0.04. Several associations were identified between histogram parameter derived from ADC maps and EGFR, KI 67 and p53 expression in rectal cancer. Furthermore, ADCmax was different between PD1 positive and PD1 negative tumors indicating an important role of ADC parameters for possible future treatment prediction.

Diving causes a transient reduction of vascular function, but the mechanisms behind this are largely unknown. The aim of this study was therefore to analyze genetic reactions that may be involved in acute changes of vascular function in divers. Rats were exposed to 709 kPa of hyperbaric air (149 kPa Po(2)) for 50 min followed by postdive monitoring of vascular bubble formation and full genome microarray analysis of the aorta from diving rats (n = 8) and unexposed controls (n = 9). Upregulation of 23 genes was observed 1 h after simulated diving. The differential gene expression was characteristic of cellular responses to oxidative stress, with functions of upregulated genes including activation and fine-tuning of stress-responsive transcription, cytokine/cytokine receptor signaling, molecular chaperoning, and coagulation. By qRT-PCR, we verified increased transcription of neuron-derived orphan receptor-1 (Nr4a3), plasminogen activator inhibitor 1 (Serpine1), cytokine TWEAK receptor FN14 (Tnfrsf12a), transcription factor class E basic helix-loop-helix protein 40 (Bhlhe40), and adrenomedullin (Adm). Hypoxia-inducible transcription factor HIF1 subunit HIF1-α was stabilized in the aorta 1 h after diving, and after 4 h there was a fivefold increase in total protein levels of the procoagulant plasminogen activator inhibitor 1 (PAI1) in blood plasma from diving rats. The study did not have sufficient power for individual assessment of effects of hyperoxia and decompression-induced bubbles on postdive gene expression. However, differential gene expression in rats without venous bubbles was similar to that of all the diving rats, indicating that elevated Po(2) instigated the observed genetic reactions.

Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.

Cellular responses to oxygen fluctuations are largely mediated by hypoxia-inducible factors (HIFs). Upon inhalation, the first organ inspired oxygen comes into contact with is the lungs, but the understanding of the pulmonary HIF oxygen-sensing pathway is still limited. In this review we will focus on the role of HIF1&alpha; and HIF2&alpha; isoforms in lung responses to oxygen insufficiency. In particular, we will discuss novel findings regarding their role in the biology of smooth muscle cells and endothelial cells in the context of hypoxia-induced pulmonary vasoconstriction. Moreover, we will also discuss recent studies into HIF-dependent responses in the airway epithelium, which have been even less studied than the HIF-dependent vascular responses in the lungs. In summary, we will review the biological functions executed by HIF1 or HIF2 in the pulmonary vessels and epithelium to control lung responses to oxygen fluctuations as well as their pathological consequences in the hypoxic lung.

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. However, it is unclear whether Ku80 regulates tumor growth in human melanoma. In this study, we screened a siRNA library targeting 6024 human genes and identified Ku80 as a potential therapeutic target in melanoma cells. Knockdown of Ku80 significantly suppressed melanoma cell proliferation and induced apoptosis, as well as enhanced the antitumor effect of melatonin in melanoma in vitro and in vivo. Overexpression of Ku80, however, promoted melanoma growth and increased the insensitivity of melanoma cells to melatonin. Mechanistically, we found that Ku80 bound to the PDK1 promoter and activated the transcription of PDK1. Moreover, we showed that the binding of Ku80 at the PDK-1 promoter was HIF1-α dependent, and melatonin degraded HIF1-α in melanoma cells. Furthermore, clinical data revealed that the expression of Ku80 and PDK-1 proteins were positively correlated and elevated in the tumor tissues of melanoma patients, and high expression of Ku80 predicted a poor prognosis in melanoma. Collectively, our study demonstrated that Ku80 promoted melanoma growth and regulated antitumor activity of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway, suggesting that Ku80 may be a potential molecular target for melanoma treatment.

Radiation-induced bystander effects (RIBE) may have potential implications for radiotherapy, yet the radiobiological impact and underlying mechanisms in hypoxic tumor cells remain to be determined. Using two human tumor cell lines, hepatoma HepG2 cells and glioblastoma T98G cells, the present study found that under both normoxic and hypoxic conditions, increased micronucleus formation and decreased cell survival were observed in non-irradiated bystander cells which had been co-cultured with X-irradiated cells or treated with conditioned-medium harvested from X-irradiated cells. Although the radiosensitivity of hypoxic tumor cells was lower than that of aerobic cells, the yield of micronucleus induced in bystander cells under hypoxia was similar to that measured under normoxia indicating that RIBE is a more significant factor in overall radiation damage of hypoxic cells. When hypoxic cells were treated with dimethyl sulfoxide (DMSO), a scavenger of reactive oxygen species (ROS), or aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), before and during irradiation, the bystander response was partly diminished. Furthermore, when only hypoxic bystander cells were pretreated with siRNA hypoxia-inducible factor-1α (HIF-1α), RIBE were decreased slightly but if irradiated cells were treated with siRNA HIF-1α, hypoxic RIBE decreased significantly. In addition, the expression of HIF-1α could be increased in association with other downstream effector molecules such as glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF), and carbonic anhydrase (CA9) in irradiated hypoxic cells. However, the expression of HIF-1α expression in bystander cells was decreased by a conditioned medium from isogenic irradiated cells. The current results showed that under hypoxic conditions, irradiated HepG2 and T98G cells showed reduced radiosensitivity by increasing the expression of HIF-1α and induced a syngeneic bystander effect by decreasing the expression of HIF-1α and regulating its downstream target genes in both the irradiated or bystander cells.

Keywords: HIF1 alpha; bystander; hepatoma cells; hypoxia; micronuclei; radiation.

Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

Background: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST.

Methods: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3.

Results: MPNSTs from Nf1-Arf^flox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition.

Conclusions: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention. Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets. Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP. Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention.

Keywords: let-7a; miR-143; microRNA; remote limb postconditioning; stroke.

Desferrioxamine (DFX), which is an iron chelator, mimics hypoxia by enhancing HIF1-alpha accumulation and upregulating inflammatory mediators. DFX is usually beneficial, with preventive effects related primarily to its ability to scavenge reactive oxygen species. However, toxic effects on skeletal and ocular organs have been reported. The cytokinesis block micronucleus test and alkaline single-cell gel (Comet) assay were used to evaluate the genotoxic effects of DFX on human blood lymphocytes. Cultured human lymphocytes treated with 130microM DFX for various periods of time showed significant differences in the incidence of micronucleated binucleate cells, as well as in the length and moment of the comet tail. Western blot analysis using antibodies to proteins involved in the p53-mediated response to DNA damage revealed that p53 was accumulated and DNA damage checkpoint kinases were activated in lymphocytes treated with DFX. On the other hand, the p53 downstream target proteins p21 and bax were not affected, which indicates that DFX does not promote the transactivational activity of p53. Apoptosis assays demonstrated DFX-induced apoptosis of lymphocytes via the caspase cascade. The observed increase in the sub-G1 fraction and enhanced caspase-3 activity indicate that DFX can promote apoptosis in human lymphocytes, and these results were confirmed by protein immunoblot analysis. As apoptotic cell death is preceded by the collapse of the mitochondrial membrane potential, we also measured the mitochondrial membrane potential (Deltapsi(m)) using DiOC6, which is a fluorescent membrane potential probe. The fluorescence intensity of DiOC6 in lymphocytes was significantly reduced in a time-dependent manner after DFX treatment. Taken together, these results indicate that DFX activates p53-mediated checkpoint signals and induces apoptosis via mitochondrial damage in human peripheral blood lymphocytes.

In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in biology not only for developmental organogenesis and carcinogenesis, but also for analyses on reconstitution and maintenance in a variety of biological environments surrounding the cells. In addition to providing architectural similarity to living organisms, 3D culture with a radial flow bioreactor (RFB) can also closely mimic the living hypoxic microenvironment under which specific organogenesis or carcinogenesis occurs. The findings of the present study under the RFB culture conditions show that cancer cells underwent a shift from aerobic to hypoxic energy metabolism, in addition to protein expression to maintain the 3D structure. In RFB-cultured cells, protein stability of hypoxia-inducible factor 1 (HIF1) α, a subunit of HIF1, was increased without upregulation of its mRNA. Under these conditions, PHD2, HIF-prolyl-4-hydroxy-lase 2 and a HIF1 downstream enzyme, were stabilized without affecting the mRNA levels via downregulation of FK506-binding protein 8. PHD2 accumulation, which occurred concomitant with HIF1 stabilization, may have compensated for the lack of oxygen under hypoxic conditions to regulate the HIF levels. 3D-culture-induced overexpression of carbonic anhydrase (another representative HIF downstream enzyme) was found to occur independently of cell density in RFB--cultured cells, suggesting that the RFB provided an adequately hypoxic microenvironment for the cultured cells. From these results, it was hypothesized that the key factors are regulatory molecules, which stabilize and degrade HIF molecules, thereby activating the HIF1 pathway under a hypoxic milieu.

OBJECTIVE

Obtaining high number of stem cells is of interest for cell based therapies. N-Acetyl-l-cysteine (NAC) acts as a source of sulfhydryl groups and an anti-oxidative agent. The aim of this study was to test different NAC concentration on proliferation and differentiation of deciduous teeth dental pulp stem cells (DTSCs) in vitro as well as to define the possible underlining mechanism of its effect.

METHODS

Number of viable, apoptotic and senescent DTSCs was determined after addition of NAC (0.1mM, 1.0mM, 2.0mM). Also, cell cycle analysis, HIF1-α expression, LDH isoenzymes, superoxide-dismutase (SOD) and catalase (CAT) activity, sulfhydryl groups content, the level of lipids' and proteins' oxidative damage and differentiation capacity of NAC treated DTSCs was determined.

RESULTS

DTSCs expressed HIF-1α in all conditions. The lowest NAC dose (0.1mM) increased the number of DTSCs by one fifth comparing to the control, most likely stimulating entry of cells into S phase of cell cycle and enhancing the activity of LDH5 isoenzyme. The highest NAC dose (2mM) inhibited DTSCs proliferation. Also, DTSCs had the lowest level of oxidative damage with 0.1mM NAC. All tested NAC concentrations enhanced DTSCs osteo-chondrogenesis.

CONCLUSIONS

The lowest NAC dose exerted significant positive effect on DTSCs proliferation as well as antioxidative protection creating beneficial environment for stem cells in vitro cultivation especially when their clinical use is important for stimulation of osteo-chondrogenesis.

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.

BACKGROUND

It is generally accepted that metabolic changes that take place in individuals exposed to high elevation are because of ambient hypoxia, which occurs as a consequence of a low total atmospheric pressure. The discovery of hypoxia inducible factor 1 (HIF1), a transcription factor, has been a breakthrough in the understanding of adaption to high altitudes.

OBJECTIVE

The purpose of the present review was to discuss specific epidemiological aspects of cardiovascular disease (CVD) risk factors and their mechanisms in vulnerable, understudied populations living at high altitudes.

RESULTS

Obesity prevalence has been inversely associated with elevation. HIF1 has been related to plasma leptin--a hormone secreted by adipose tissue that produces negative feedback on appetite--and inversely associated with obesity. Diverse factors, such as genetics, chronic hypoxia, diet and lifestyle behaviours, could have an influence on the high dyslipidaemia rates of high-altitude natives. Hypoxia could mediate the effects of altitude on human physiology, including lipid metabolism. Genetic studies suggest that dyslipidaemia could be related to the HIF1. Hypoxia inhibits oxidative phosphorylation and stimulates the oxygen signalling pathway through the HIF1. Low fasting glycaemia in individuals at high altitudes has been shown. An increased GLUT4 protein content in skeletal muscle in response to hypoxia has been reported and could be associated with lower glucose levels. Given the high prevalence of dyslipidaemia and the low prevalence of obesity and diabetes in these impoverished high-altitude communities, changes in lifestyle including decreased physical activity and the consumption of a more westernised diet would likely increase the prevalence of CVD related mortality.

CONCLUSIONS

Control over major CVD risk factors, when identified early, could be the key to reducing morbidity and mortality in patients with limited access to medical services such as Native populations.

Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

Aims: Experimental data suggest that visceral adipose tissue (VAT) dysfunction contributes to non-alcoholic fatty liver disease (NAFLD) development in obesity, however, data on humans are limited. Aims of this study were to investigate the relationship between NAFLD and VAT morphofunctional impairment and to determine whether the extent of VAT remodelling is associated with liver damage and metabolic alterations in obesity.

Methods: We analysed data from 40 obese individuals candidate to bariatric surgery in whom paired intraoperative liver and omental biopsies were performed for diagnosing NAFLD and VAT inflammation by immunohistochemistry and mRNA expression studies.

Results: Within our study population, NAFLD was significantly associated with greater VAT CD68⁺ macrophages infiltration (P = .04), fibrosis (P = .04) and impaired microvascular density (P = .03) as well as increased expression of markers of local hypoxia, apoptosis and inflammation (UNC5B, CASP7, HIF1-α, IL-8, MIP2, WISP-1, all P < .01). The degree of VAT inflammation correlated with the severity of hepatic injury (steatosis, inflammation, fibrosis; all P < .01) and impaired gluco-metabolic profile.

Conclusions: In obese patients, NAFLD is associated in a dose-dependent manner with signs of VAT remodelling, which reflect more severe clinical metabolic impairment. Our study depicts morphological alterations and novel mediators of VAT dysfunction, adding knowledge for future therapeutic approaches to NAFLD and its metabolic complications.

Keywords: NAFLD; adipose tissue; inflammation; obesity.