Objective: To perform cluster analysis of MRI signs of cerebral microangiopathy (small vessel disease, SVD) and to clarify the relationship between the isolated groups and circulating markers of inflammation and angiogenesis.

Material and methods: The identification of groups of MRI signs (MRI types) using cluster hierarchical agglomerative analysis and iterative algorithm of k-means and assessment of their relationship with serum concentrations of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor 1-α (HIF1-α) determined by ELISA were performed in 96 patients with SVD (STRIVE, 2013) (65 women, average age 60.91±6.57 years).

Results: Cluster analysis of MRI signs identified two MRI types of SVD with Fazekas grade 3 of white matter hyperintensity (WMH). MRI type 1 (n=18; 6 women, mean age 59.1±6.8 years) and MRI type 2 (n=22, 15 f., mean age 63.5±6.2 years) did not differ by age, sex, severity of hypertension, presence of other risk factors. MRI type 1 had a statistically significantly more pronounced WMH in the periventricular regions, multiple lacunes and microbleeds, atrophy, severe cognitive impairment and gait disorders compared with MRI type 2. Its formation was associated with a decrease in VEGF-A level. MRI type 2 had the significantly more pronounced juxtacortical WMH, white matter lacunes, in the absence of microbleeds and atrophy, and less severe clinical manifestations compared with MRI type 1. Its formation was associated with an increase in TNF-α level.

Conclusion: Clustering of diagnostic MRI signs into MRI types of SVD with significant differences in the severity of clinical manifestations suggests the pathogenetic heterogeneity of age-related SVD. The relationship of MRI types with circulating markers of different mechanisms of vascular wall and brain damage indicates the dominant role of depletion of angiogenesis in the formation of MRI type 1 and increased inflammation in the formation of MRI type 2. Further studies are needed to clarify the criteria and diagnostic value of differentiation of MRI types of SVD, and also their mechanisms with the definition of pathogenetically justified prevention and treatment of various forms of SVD.

Цель исследования: Провести кластерный анализ МРТ-признаков церебральной микроангиопатии (ЦМА) и уточнить связь выделенных групп с циркулирующими маркерами воспаления и ангиогенеза.

Материал и методы: У 96 больных с ЦМА (STRIVE, 2013) (65 женщин, средний возраст — 60,91±6,57 года) с помощью кластерного иерархического агломеративного анализа и итерационного алгоритма k-средних оценивалось выделение групп МРТ-признаков (МРТ-типы) и их связь с сывороточными концентрациями фактора некроза опухоли-альфа (TNF-α), трансформирующего фактора роста (TGF-β1), сосудистого эндотелиального фактора роста (VEGF-А), гипоксией индуцируемого фактора (HIF1-α), определенных методом иммуноферментного анализа ELISA.

Результаты: Кластерный анализ МРТ-признаков выделил два МРТ-типа ЦМА стадии Fazekas 3. МРТ-тип 1 (n=18; 6 женщин, средний возраст — 59,1±6,8 года) и МРТ-тип 2 (n=22, 15 женщин, средний возраст — 63,5±6,2 года) не различались между собой по возрасту, полу, тяжести артериальной гипертензии, наличию других факторов риска. МРТ-тип 1 по сравнению с МРТ-типом 2 имел статистически значимо более выраженную гиперинтенсивность белого вещества (ГИБВ) в перивентрикулярных отделах, множественные лакуны и микрокровоизлияния, атрофию, тяжелые когнитивные расстройства и нарушения ходьбы. Его формирование было связано со снижением VEGF-A. МРТ-тип 2 по сравнению с МРТ-типом 1 имел статистически значимо более выраженные юкстакортикальную ГИБВ, лакуны в белом веществе, при отсутствии микрокровоизлияний и атрофии, меньшей тяжести клинических проявлений. Его формирование было связано с повышением TNF-α.

Заключение: Кластеризация диагностических МРТ-признаков в МРТ-типы ЦМА со значимыми различиями в тяжести клинических проявлений позволяет предполагать патогенетическую гетерогенность возраст-зависимой ЦМА. Связь МРТ-типов с циркулирующими маркерами разных механизмов повреждения сосудов и мозга указывает на доминирующее значение истощения ангиогенеза в формировании МРТ-типа 1 и воспаления в формировании МРТ-типа 2. Целесообразны дальнейшие исследования по уточнению критериев и диагностической ценности дифференцирования МРТ-типов ЦМА, а также их механизмов с определением патогенетически обоснованной профилактики и лечения разных форм ЦМА.

Keywords: cerebral microangiopathy; neuroimaging; small vessel disease; tumor necrosis factor-alpha; vascular endothelial growth factor; white matter hyperintensity.

Objective: To investigate the effects of overexpression of long noncoding RNA (lncRNA) MEG3 on the proliferation and invasion of glioblastoma U251 cells by suppressing the expression of hypoxia inducible factor 1α(HIF1α).

Methods: The expression of lncRNA MEG3 and HIF1α mRNA were examined in human fetal glial cells (HFGCs) and U251 cells using realtime quantitative PCR (qRT-PCR), and the expression of HIF1α protein was detected with Western blotting.U251 cells in normal culture or transfected with pcDNA3.1 vector (NC group) or pcDNA3.1-MEG3 vector via lipofectamine2000 were exposed to hypoxia for 12h, and the expressions of HIF1α mRNA and protein were detected with qRT-PCR and Western blotting, respectively.MTT assay and Transwell assay were employed to examine the influence of MEG3 overexpression on the proliferation and invasion of U251 cells.

Results: The expression of MEG3 was significantly lower and HIF1α mRNA and protein expressions were significantly higher in U251 cells than in HFGCs (P < 0.05).In U251 cells, overexpression of MEG3 significantly decreased the mRNA and protein expressions of HIF1α(P < 0.05).Hypoxic exposure for 12h also resulted in significantly lowered expression of HIF1α protein in U251 cells (P < 0.05).Overexpression of MEG3 obviously suppressed the proliferation and invasiveness of U251 cells (P < 0.05).

Conclusions: MEG3 overexpression inhibits the proliferation and invasion of U251 cells through suppressing the expression of HIF1α mRNA and protein, suggesting that MEG3 may serve as a potential therapeutic target for glioblastomas.

Keywords: glioblastoma; hypoxia inducible factor 1 alpha; invasion; long noncoding RNA MEG3; proliferation.

The introduction of novel frontline agents in multiple myeloma (MM), like immunomodulatory drugs and proteasome inhibitors, has improved the overall survival of patients. Yet, MM is still not curable, and drug resistance (DR) remains the main challenge. To improve the understanding of DR in MM, we established a resistant cell line (MOLP8/R). The exploration of DR mechanisms yielded an overexpression of HIF1&alpha;, due to impaired proteasome activity of MOLP8/R. We show that MOLP8/R, like other tumor cells, overexpressing HIF1&alpha;, have an increased resistance to the immune system. By exploring the main target genes regulated by HIF1&alpha;, we could not show an overexpression of these targets in MOLP8/R. We, however, show that MOLP8/R cells display a very high overexpression of LCP1 gene (l-Plastin) controlled by HIF1&alpha;, and that this overexpression also exists in MM patient samples. The l-Plastin activity is controlled by its phosphorylation in Ser5. We further show that the inhibition of l-Plastin phosphorylation restores the sensitivity of MOLP8/R to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Our results reveal a new target gene of DR, controlled by HIF1&alpha;.

Hypoxia pathway and aberrant miRNA expression profile play crucial roles in the development of various cancers. Recent studies have emphasized that there are many collaborations in this cases because both of them are involved in cancer cell processes, including differentiation, metastasis, and cell proliferation and signaling pathways. Further studies have elucidated that miRNAs affect the hypoxia route, and more interestingly, the hypoxia pathway also affects miRNAs expression profile. The literature review summarizes the fundamental roles of hypoxia-related miRNAs in different cancers. The mutual interactions between miRNAs and hypoxia are a new layer of complexity in cancer hypoxia, which might be helpful in controlling cancer progression. It is also possible that the hypoxia pathway is initiated by miRNAs. In contrast, the hypoxia pathway regulates the expression of a large group of miRNAs. In this review for the first time, we discussed the significant interactions between hypoxia and miRNAs in order to determine new perspectives for new therapeutic aims in the field of cancer.

Keywords: HIF1 alpha; Hypoxia; cancer; metastasis; miRNAs; signaling pathways.

Oxidative microenvironment in fibrotic liver alleviates the efficacious outcome of mesenchymal stem cells (MSCs)-based cell therapy. Recent evidence suggests that pharmacological pretreatment is a rational approach to harness the MSCs with higher therapeutic potential. Here, we investigated whether Vitamin E pretreatment can boost the antifibrotic effects of Wharton's jelly-derived MSCs (WJMSCs). We used rat liver-derived hepatocytes injured by CCl₄ treatment in co-culture system with Vitamin E pretreated-WJMSCs (Vit E-WJMSCs) to evaluate the hepatoprotective effect of Vit E-WJMSCs. After 24 h of co-culturing, we found that Vit E-WJMSCs rescued injured hepatocytes as hepatocyte injury-associated medium (AST, ALT, and ALP) and mRNA (Cyp2e1, <em>Hif1</em>-<em>α</em>, and Il-1β) markers reduced to normal levels. Subsequently, CCl₄-induced liver fibrosis rat models were employed to examine the antifibrotic potential of Vit E-WJMSCs. After 1 month of cell transplantation, it was revealed that Vit E-WJMSCs transplantation ceased fibrotic progression, as evident by improved hepatic architecture and functions, more significantly in comparison to naïve WJMSCs. In addition, Vit E-WJMSCs transplantation decreased the expressions of fibrosis-associated gene (Tgf-β1, <em>α</em>-Sma, and Col1<em>α</em>1) markers in the liver parenchyma. Intriguingly, the results of tracing experiments discovered that more WJMSCs engrafted in the Vit E-WJMSCs treated rat livers compared to naïve WJMSCs treated livers. These findings implicate that pretreatment of WJMSCs with Vitamin E improves their tolerance to hostile niche of fibrotic liver; thereby further enhancing their efficacy for hepatic fibrosis.

Keywords: Fibrosis; Hepatocytes; Pretreatment; Vitamin E; Wharton’s jelly-derived mesenchymal stem cells.

Iron is regarded as one of the most important nutriments, and many diseases are related to iron deficiency or its overload. Approximately 70% of iron in the body is located in heme, functioning as hemoglobin, myoglobin, and cytochrome P450. Iron itself also has many catalytic functions through the iron-sulfa cluster. It is believed that iron and/or heme plays significant roles in regulation of genes, however, little about the mechanism has been elucidated. Recently, not only iron but also heme has been identified as important regulators of gene activation via oxygen sensing. For example, iron controls the oxygen response of HIF-1 activity by two mechanisms; in cytosol, the half life of HIF-1 alpha is determined by hydroxylation of Pro, and transcriptional activity of HIF1 alpha in nuclei is disturbed by hydroxylation of Asn. Hemoproteins in prokaryotes such as FixL, Dos, and HemAT were found to be oxygen sensors, however, little has been reported in eukaryotes. Our finding on Bach1 seems to be the first report of heme and oxygen-mediated regulation of genes in vertebrates. Understanding of these newly identified mechanisms in iron- and heme-controlled genes is essential in the field of nutritional science. We therefore summarize here the recent findings indicating mechanisms of iron as transcriptional regulators.

The incidence of ischemic heart disease (IHD) in patients with OSAS is estimated at around 20%. This greatly affect a common risk factors for both diseases: male gender, obesity, age and diabetes and hypertension. Attention is drawn to the possibility of genetic determinants of IHD. The aim of study was to answer the question whether the presence of polymorphisms of selected genes possibly related to IHD may be useful to isolate the group of patients with OSAS, especially vulnerable as a complication of IHD. Materials and methods. The study included 600 people with OSAS, which was isolated in patients with IHD (127 people). The remaining 473 individuals were observed as a control group. The polymorphism of three genes were evaluated to find possible influence on the occurrence of IHD or myocardial infarction as follows: SREBF1 (sterol regulatory element binding transcription factor 1), REBF2 (sterol regulatory element binding transcription factor 2) and HIF1 (hypoxia inducible factor 1, alpha subunit). Results. Analysis of relationship between polymorphisms of selected genes and the diagnosis of IHD in the whole group of patients with OSAS showed a relationship only for the gene SREBF1 finding the lowest frequency of its occurrence in AA homozygotes (at 13.6%) and twice with GG homozygotes (26.1%). Conclusions. Rating polymorphisms studied genes did not reveal their relationship to the occurrence of IHD in patients with OSAS, both in the whole group as well as separate subgroups.

OBJECTIVE

To investigate the changes in the expression of hypoxia induction factor-1alpha (HIF1-alpha) in myocardial tissues in severely scalded rats during early postburn stage.

METHODS

Male Wistar rats inflicted with 40% TBSA III degree scald were employed as the model. The myocardial tissue samples were harvested from the left and right ventricles at different postburn time points, and samples were also obtained from normal rats as control. The mRNA and protein expressions of HIF-1alpha in rat myocardial tissue were determined by RT-PCR and Western blot analysis respectively.

RESULTS

There was a difference of HIF-1alpha expression between left and right ventricles of the normal rats at both transcriptional and translational levels, and the mRNA and protein expressions of HIF-1alpha in the myocardial tissue of scalded rats were increased dramatically at early postburn stage.

CONCLUSIONS

The tolerance to ischemia and hypoxia of the rat left ventricle was higher than that of the right ventricle under normal condition. An increase in HIF1alpha expression in rat myocardial tissue could be induced in severely scalded rats.

<AbstractText>To assess the association of classic vascular risk factors, indicators of cerebral arteries wall damage and stress induction, and their role in early vascular and brain damage in middle age subjects without vascular events.</AbstractText><AbstractText>87 patients were evaluated (49 women, 38 men, mean age 51.2±6.5). The following vascular risk factors were assessed: hypertension, diabetes, total cholesterol and low density lipoproteins levels, obesity and smoking. Patients underwent ultrasound of neck arteries, brain MRI and laboratory testing of blood parameters, probably associated with vascular wall damage: CRP, TNF-<em>α</em>, sICAM-1, sVCAM, <em>HIF1</em>-<em>α</em>, NO, VAP-1, VEGF-A, VEGF-C, sVEGF-R1, sVEGF-R2, TGF-β1, general antioxidant status.</AbstractText><AbstractText>Mediating role of stress parameters in risk factors formation, initiation and maintenance of mechanisms of vascular damage was demonstrated. Hypercortisolemia suggested the association with age, atheromatosis, local inflammatory reactions via the TGF-β1-HIF-1-VEGF family, systemic inflammation response via CRP, and elevated epinephrine levels were associated with TNF-<em>α</em>-mediated systemic inflammation. The association of TNF-<em>α</em> and MRI signs of cerebral small vessel disease (SVD) in non-hypertensive patients may indicate that TNF-<em>α</em>-mediated inflammation and increased permeability of vessel wall is an independent cause and potential biomarker of early small vessel damage. Influence of hypertension on age-dependent SVD is probably maintained by local vascular wall damage mechanisms via the TGF-β1-HIF-1-VEGF family. However, hypertension heterogeneity and association of early cerebral vessels damage with various protective reactions require further clarification of the conditions for using these parameters as possible biomarkers of early SVD.</AbstractText>

Suspected deep tissue injury (DTI) is a new category of pressure ulcer (PU), and defined as an ulcer that developed from a deep tissue (subcutaneous tissue) region and deteriorates towards the superficial skin. DTI is a serious clinical problem because it cannot be detected at an early stage and rapidly deteriorates to a deep PU. Consequently, there is a requirement for the identification of novel biomarkers to detect damage to the deep tissue including deep muscle tissue. For this purpose, it is essential to understand the molecular and cellular mechanisms of DTI formation and deterioration. This article reviews the recent progress in studies on the hypoxia-related mechanisms of DTI, and introduces our attempts to establish novel biomarkers for detecting deep muscle damage. Hypoxia-inducible factor 1 alpha subunit (HIF1-alpha) is a widely used marker for hypoxic conditions. We detected increased expression and localization of HIF1-alpha in the deep muscle tissue of PU model rats, indicating that HIF1alpha is a key molecule in DTI and a valuable biomarker for hypoxia in DTI in the research field. From the biochemical aspect, we focused on creatine phosphokinase (CPK). CPK is an intracellular enzyme related to energy metabolism, and its level in serum has been extensively used as a diagnostic marker for muscle injury. We attempted to estimate muscle injury from the CPK levels in exudates, which can be collected non-invasively and reflect the microenvironmental conditions. Our results using PU model rats suggested that exudate CPK could be a more sensitive biomarker than serum CPK for deep muscle tissue injury, and could be applicable for clinical diagnosis of DTI.

Perinatal hypoxia-ischaemia is one of the leading factors that negatively influence the development of the central nervous system. Our aim was to investigate the effects of sex on the outcomes of acute neonatal hypoxia (ANH) in rat pups. Male and female Wistar rats were exposed to a hypoxic condition (8% oxygen for 120 min) at postnatal day 2 (P2). Immediately after ANH an increase in HIF1-α gene expression was observed in the rat brains, independently of sex. Brain-derived neurotrophic factor (BDNF) and glutathione peroxidase-4 gene expression was increased in female animals only. Hypoxic pups of both sexes showed a decreased reduced/oxidised glutathione (GSH/GSSG) ratio in the blood and only males had an increased GSH content in the whole brain immediately after hypoxia. Furthermore, an increased BDNF content in the brain was found in both male and female rat pups at 0 h and in serum 4 h after hypoxia, but at 4 h after hypoxia only males had an increased BDNF level in the brain. Only hypoxic males displayed retarded performance in the righting reflex, but in a negative geotaxis test hypoxic pups of both sexes had an increased turnaround time. Moreover, hypoxic female but not male pups demonstrated less weight gain than control littermates for the entire observation period (until P18). These results demonstrate that ANH at P2 leads to both molecular and physiological impairments in a sex-specific manner and the described model could be used to represent mild hypoxic brain damage in very preterm infants.

Perinatal hypoxia-ischemia is one of the most common causes of perinatal brain injury and subsequent neurological disorders in children. The aim of this work was to evaluate the potential antioxidant and neuroprotective effects of N-arachidonoyl-dopamine (NADA) in the model of acute neonatal hypoxia (ANH) in rat pups. Male and female Wistar rats were exposed to a hypoxic condition (8% oxygen for 120 min) at postnatal day 2 (P2). Transcription factor HIF1-α and glutathione peroxidases GPx2 and GPx4 gene expression was increased in rat brains in the hypoxic group compared to control 1.5 h but not 4 days after ANH. There were no post-hypoxic changes in reduced (GSH) and oxidised (GSSG) glutathione levels in the brain of rat pups 1.5 h and 4 d after hypoxia. Hypoxic rats displayed retarded performance in the righting reflex and the negative geotaxis tests. ANH resulted in increased ambulation in Open field test and impaired retention in the Barnes maze task under stressful conditions as compared with the control group. Treatment with NADA significantly attenuated the delayed development of sensorimotor reflexes and stress-evoked disruption of memory retention in hypoxic rats but had no effect on the hypoxia-induced hyperactivity. In rats exposed to hypoxia, treatment with NADA decreased GPx2 gene expression and increased GSH/GSSG ratio in whole brains 1.5 h after ANH. These results suggest that the long-lasting beneficial effects of NADA on hypoxia-induced neurobehavioural deficits are mediated, at least in part, by its antioxidant properties.

In this study, we aimed to explore the mechanism of glutathione peroxidase 3 (GPX3) in the growth of malignant melanoma (MM) cells by hypoxia-inducible factor-1α (HIF1-α) and HIF2-α regulating the metabolism through reactive oxygen species (ROS). The messenger RNA and protein expression of GPX3, HIF1-α, HIF2-α in tissues, and cell lines were measured by reverse transcription-quantitative PCR and Western blot analysis. A375 cells were transfected with GPX3 overexpression plasmid, small interfering RNA (siRNA) targeting GPX3, or siRNA targeting HIF1-α/HIF2-α to upregulate or downregulate the expression of GPX3 or HIF1-α/HIF2-α. The effects of H₂ O₂ and N-acetylcysteine (NAC) on the levels of HIF1-α and HIF2-α after overexpression of GPX3 were studied. The cell viability was detected by Cell Counting Kit-8. The levels of ROS, glucose uptake and lactic acid production, oxidative phosphorylation, and glycolysis of cells were measured for assessment of cellular metabolism. The expression of GPX3 decreased, while ROS, HIF1-α, and HIF2-α increased in MM tissues and cells. Overexpression of GPX3 inhibited the viability of MM cells and the growth of melanoma xenografts. The overexpression of GPX3 reduced the glucose uptake, extracellular lactic acid content, and extracellular acidification rate and increased the oxygen consumption rate level. Overexpression of GPX3 could reduce the levels of HIF1-α and HIF2-α, which could regulate metabolic levels. GPX3 reduced ROS level in MM to inhibit HIF1-α and HIF2-α. The addition of H₂ O₂ increased while NAC reduced the protein levels of HIF1-α and HIF2-α in the cells overexpressing GPX3. Our study demonstrates that GPX3 inhibits the growth of MM cells through its inhibitory effect on cell metabolic disorder by inhibiting HIF1-α via regulating ROS.

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been suggested as a potential therapy for extensive bilobar liver tumors, although in some circumstances this technique may induce tumor progression, a fact still not well studied. Our aim was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular CC531 cell line inoculation. ALPPS induced: tumor progression on deportalized lobe and metastases; expression of hepatic vasculogenic factors (HIF1-α and VEGF); and a dramatic increase of Kupffer cells (KCs) and tumor-associated macrophages (TAMs). Interestingly, KCs expressed COX-2 (M1 polarization), while TAMs expressed mainly arginase-1 (M2 polarization). ALPPS also induced a decrease of tumor-infiltrating lymphocytes and an increase of intrahepatic T lymphocytes. Thus, ALPPS technique seems to induce a hypoxic environment, which enhances hepatic HIF1-α and VEGF expression and may promote KCs and TAMs polarization. Consequently, the regenerative stimulus seems to be driven by a pro-inflammatory and hypoxic environment, in which M1 intrahepatic macrophages expressing COX-2 and T-Lymphocytes play a key role, facts which may be related with the tumor progression observed.

Are hypoxia-inducible factors (HIF) responsible for the potentiation of inhibin alpha subunit (INHA) gene expression in primary cultures of human term cytotrophoblasts under low-oxygen tension?

Both HIF1A and endothelial PAS domain protein 1 (EPAS1) are involved in the potentiation of INHA gene upregulation in cytotrophoblasts cultured under hypoxia.

During the in vitro differentiation of cytotrophoblasts into syncytiotrophoblasts under 21% O2, INHA expression increases. This expression is further increased when cells are cultured under low-oxygen tension (e.g. 2.5% O2). Moreover, in pregnancy-related diseases, such as pre-eclampsia or intrauterine growth restriction (IUGR), in which hypoxia is suspected to be responsible for the abnormal placental development, maternal serum concentration of inhibin A is elevated.

Cytotrophoblasts were isolated and purified from human term placentas (n = 6). Cells were cultured under 21% O2, and allowed to differentiate for 48 h. A first group of cells was treated for 16 h under 21% O2 with dimethyloxalylglycine (DMOG) or deferoxamine (DFX), molecules that mimic hypoxia by inhibiting HIF1 proteasomal degradation. Involvement of HIF1A and EPAS1 (also known as HIF2A), two HIF isoforms expressed in trophoblasts, was shown by treating another group of cells cultured under 2.5% O2 with specific inhibitors of HIF1A and EPAS1 for 16 h. INHA mRNA expression was assessed by real-time PCR and secreted inhibin A was quantified by ELISA. The role of HIF1A and EPAS1 in INHA transcriptional regulation was further confirmed by cotransfecting primary cytotrophoblasts with a luciferase reporter plasmid containing a 3.9 kb INHA promoter and plasmids allowing overexpression of HIF1A and EPAS1.

Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n≥ 4). The methods used were hormone measurements in the cell supernatants by enzyme-linked immunosorbent assay, real-time quantitative PCR, western blotting, immunofluorescence microscopy and transient transfection.

HIF1 protein stabilization with DMOG and DFX increased 21% O2-induced INHA mRNA and protein upregulation (P < 0.05 versus control), while hypoxia-induced INHA upregulation was repressed by HIF1A and EPAS1 inhibitors (P < 0.05 versus control). In transfection experiments of primary term cytotrophoblasts, cloned INHA promoter transcriptional activity was increased by 2.5% O2 compared to 21% O2 (P < 0.05). Overexpression of both HIF1A and EPAS1 under 21% O2 increased cloned INHA transcriptional activity (P < 0.001 versus control).

Not applicable.

HIF1A and EPAS1 may regulate INHA expression by binding to an hypoxia-responsive element within the promoter, but we were unable to identify such an element. Inhibition of HIF1A and EPAS1 did not completely suppress upregulation of INHA expression suggesting that other transcription factors, not identified or studied here, are involved.

Our data suggest that the effect of HIF1 proteins on INHA gene promoter activity may be indirect. By demonstrating the role of HIF1A and especially EPAS1 in INHA gene upregulation under hypoxia, the results suggest that HIF1 proteins may become new therapeutic targets in the treatment of pregnancy-related diseases such as pre-eclampsia or IUGR.

This work was fully supported by 'Fetus for Life' charity. C. Depoix was supported by a fellowship 'Fonds de Recherche Clinique' from 'Fondation Saint-Luc', Belgium. The authors declare that there is no conflict of interest regarding the publication of this paper.

Background: Peritoneal dialysis (PD) is a treatment for end stage renal disease patients, but it can also cause peritoneal fibrosis. Nestin is known as a neural stem cell marker and it has many functions. The hypoxia induced factor (HIF) signaling pathway can be activated under hypoxia conditions, leading to the overexpression of some angiogenesis related genes. The aim of our study is to demonstrate Nestin's role in the development of peritoneal fibrosis (PF), and to provide a new target (Nestin) to treat PF.

Methods: PD mice models were constructed by an intraperitoneal administration of PDS at 10 ml/100g/d for 4 weeks. Nestin-positive cells were isolated from peritonea of Nestin-GFP mice by flow cytometry. The relationship of Nestin and HIF1-α-VEGFA pathway was detected by Nestin knockdown, Co-immunoprecipitation and immunofluorescence. Also, proteasomal activity was demonstrated by CHX and MG132 application, followed by Western blotting and Co-immunoprecipitation.

Results: In our experiments, we found that Nestin expression resulted in PF. Also, HIF1-α/VEGFA pathway was activated in PF. Nestin knockdown reduced the level of HIF1-α. Nestin directly bound to HIF1-α and protected HIF1-α from proteasomal degradation. Overexpression of HIF1-α reverts the fibrosis levels in Nestin-knockdown cells. In brief, Nestin inhibited the degradation of HIF1-α by mitigating its ubiquitination level, leading to the activation of HIF1-α signaling pathway, and eventually promoted PF.

Conclusion: We found a novel mechanism of PF that Nestin promotes by protecting HIF1-α from proteasomal degradation. Taken together, our key findings highlight a novel mechanism by which the silencing of Nestin hinders HIF1- α -induced PF.

Keywords: HIF1-α pathway; angiogenesis; nestin; peritoneal dialysis; peritoneal fibrosis.

Lung injury is a serious condition encountered following hepatic ischemia/reperfusion (IR). This study aimed to explore whether a dipeptidyl peptidase-4 inhibitor agent vildagliptin (V) could alleviate the lung injury caused by hepatic IR in a rat model and if so elucidate its molecular protective mechanism. Three groups of rats were used. Sham group: received normal saline and exposed to a sham operation, IR group: received normal saline and subjected to the operation of hepatic I (45 min)/ R (180 min), V+IR group: received for 10 days intraperitoneal injection of V (10 mg/kg/day). After reperfusion, liver and lung were collected for biochemical and histological evaluation. Hepatic IR exhibited significant elevation in serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) enzyme levels, serum and lung malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) in addition to lung nitric oxide (NO) levels, hypoxia-inducible factor 1-alpha (HIF-1α) mRNA and protein levels, hepatocyte growth factor (HGF) mRNA expression, and inducible nitric oxide synthase (iNOS) mRNA and protein expressions in lung tissue along with a marked reduction in the serum and lung content of catalase in comparison to the sham group. Moreover, liver and lung injury in the IR group was detected by histopathological examination. Vildagliptin ameliorated markedly the biochemical changes as well as liver and lung architecture in comparison to the IR group. Vildagliptin mitigated the induced lung injury by hepatic IR via suppression of oxidative stress markers, pro-inflammatory cytokine TNF-α as well as the HIF1-α/iNOS/HGF expressions in lung tissue.

Keywords: HGF; HIF1-α; Hepatic ischemia/reperfusion; Lung injury; Vildagliptin; iNOS.

In the central nervous system injury induces cellular reprogramming and progenitor proliferation, but the molecular mechanisms that limit regeneration and prevent tumorigenesis are not completely understood. We previously described a zebrafish optic pathway tumor model in which transgenic Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors. Key pathways driving injury-induced glial reprogramming and regeneration contributed to tumor formation. In this study, we examine a time course of proliferation and present new analyses of the Tg(flk1:RFP)is18/+ dysplastic retina and tumor transcriptomes. Retinal dysplasia was first detected in 3-month-old adults, but was not limited to a specific stem cell or progenitor niche. Pathway analyses suggested a decrease in cellular respiration and increased expression of components of Hif1-α, VEGF, mTOR, NFκβ, and multiple interleukin pathways are associated with early retinal dysplasia. Hif-α targets VEGFA (vegfab) and Leptin (lepb) were both highly upregulated in dysplastic retina; however, each showed distinct expression patterns in neurons and glia, respectively. Phospho-S6 immunolabeling indicated that mTOR signaling is activated in multiple cell populations in wild-type retina and in the dysplastic retina and advanced tumor. Our results suggest that multiple pathways may contribute to the continuous proliferation of retinal progenitors and tumor growth in this optic pathway tumor model. Further investigation of these signaling pathways may yield insight into potential mechanisms to control the proliferative response during regeneration in the nervous system.

Introduction: Malignant neoplasm of the endometrium is the most common malignant neoplasm of the female reproductive system. Toll Like Receptors (TLR) play a significant role in innate and late-immunity against infections or damaged tissues. TLRs are also involved in the development of tumors in their natural microenvironment. TLRs play an important role in angiogenesis, necessary for survival and growth of the tumor. Hypoxia playing a critical role in angiogenesis, carcinogenesis, tumor progression and distant metastasis is primarily mediated through hypoxia inducible factors (HIFs). Vascular endothelial growth factor family proteins (VEGF) are also strongly involved in tumor angiogenesis and their action is strongly associated with TLR receptors.

Objectives: The aim of the study was to correlate the expression of selected TLRs and VEGFR's as well as HIF1α with clinicopathological data of endometrial cancer patients.

Material and methods: 123 neoplastic endometrial samples were included in the study. 51 samples of healthy endometrium served as control. The expression of TLR1, TLR2, TLR3, TLR4, VEGFR1 and VEGFR2, VEGF-A and HIF1α was examined after RNA isolation at the mRNA level by Real Time-PCR.

Results: We have noted a significant correlation between the expression of selected TLR and VEGFR's and clinical stage as well as pathological grading of endometrial cancer.

Conclusions: Received correlations confirm a significant contribution of some TLR expression and the receptor for VEGF in the pathogenesis of epithelial endometrial cancer.

Keywords: HIF1-α; TLR family; VEGF; VEGFR1; VEGR2; endometrial cancer.

A germline mutation in the Von-Hippel Lindau (VHL) gene predisposes carriers to development of abundantly vascularised tumours in the retina, cerebellum, spine, kidney, adrenal gland and pancreas. Most VHL patients die from the consequences of cerebellar haemangioblastoma or renal cell carcinoma. The VHL gene is a tumour suppressor gene and is involved in angiogenesis by regulation of the activity of hypoxia-inducible factor 1-alpha (HIF1-alpha). Clinical diagnosis of VHL can be confirmed by molecular genetic analysis of the VHL gene, which is informative in virtually all VHL families. A patient with (suspicion for) VHL is an indication for genetic counselling and periodical examination.

Hypoxia-inducible factor 1 (HIF-1) is responsible in regulating oxygen homeostasis in tissues. HypoxiaInducible Factor α (HIF1-α) is a central effector of the hypoxic response. HIF-1α protein overexpression has been shown to have prognostic relevance in breast cancer. HIF-1α polymorphism is associated with increased breast susceptibility reported by several case controls studies but results remained controversial. Therefore, we studied the relationship between the HIF1α gene polymorphism with the breast cancer risk in Saudi Arabia. <P> Methods: This study was consisted of 114 histologically confirmed Breast cancer patients and 117 sex -matched healthy women. HIF-1α genotyping was done by Amplification refractory mutation system PCR method. The HIF-1α gene genotypes were correlated with different clinicopathological characteristics of breast cancer patients. <P> Results: A significant difference was observed in genotype distribution of HIF-1α gene variation C1772T between breast cancer cases and sex matched healthy controls (p=0.001). Our findings showed that the HIF- 1α variant was associated with an increased risk of Breast cancer for HIF-1α CC vs CT genotype OR = 0. 38, 95% CI = (0. 22 -0. 65), P = 0.005) in codominant inheritance model. The significant association was reported for HIF1A for genotypes CC vs (CT+ TT) OR = 0. 39, 95% CI = (0. 231 -0. 67), P = 0.007) in dominant inheritance model tested. In case of recessive inheritance model, a significant association of HIF-1 alpha gene variants was reported for CC VS -(CC+ CT) vs TT) OR = 3.10, 95% CI = (0. 12- 77.03), P = 0.56). During the allelic comparison, A allele significantly increased the risk of Breast cancer with odd ratio (OR = 0. 66, 95% CI = 0. 53 -1. 21, P = 0.04) and risk ratio RR= 0. 51 (0. 32 -0. 80) P= 0.004). A significant association of HIF1α polymorphism was reported with stage as well as distant metastasis of the disease. <P> Conclusion: A significant association of HIF- 1α-CT heterozygosity and T allele significantly increased the susceptibility and is associated with the metastasis of Breast cancer. Further studies with larger data set and well-designed models are required to validate our findings.

Keywords: Amplification refractory mutation system PCR; Hypoxia-inducible factor 1 (HIF-1); Saudi Arabia; breast cancer; gene variation; susceptibility.

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it.

Background: Chronic and/or intermittent exposure to hypobaric hypoxia reportedly exerts cardioprotective effects against ischemia-reperfusion injury. However, few studies have focused on the cardioprotective effects of acute and/or short-term hypobaric and hypoxic exposures. This study investigated the effects of acute hypobaric hypoxia on myocardial ischemia-reperfusion injury.

Materials and methods: Rats were assigned to groups receiving normobaric normoxia (NN group), hypobaric hypoxia (HH group), or normobaric hypoxia (NH group). HH group rats were exposed to 60.8 kPa and 12.6% fraction of inspired oxygen in a hypobaric chamber for 6 h. NH group rats were exposed to hypoxic conditions under normal pressure. After each exposure, 30 min of myocardial ischemia was followed by 60 min of reperfusion. Cardiac function and infarct size were determined after reperfusion. Expression of hypoxia-inducible factor 1 alpha (HIF1α) was also measured.

Results: Cardiac function was better preserved in the HH and NH groups than in the NN group (p < 0.01 each). Median infarct size/area at risk was significantly lower in the HH group (50%, interquartile range [IQR] 48-54%; p < 0.01 vs. NN group) and NH group (45%, IQR 36-50%; p < 0.01 vs. NN group) than in the NN group (72%, IQR 69-75%). HIF1α expression was significantly higher in the HH group (p < 0.05 vs. NN group) and NH group (p < 0.01 vs. NN group) than in the NN group.

Conclusions: Exposure to acute and/or short-term hypobaric and hypoxic conditions might exert cardioprotective effects against myocardial ischemia-reperfusion injury via HIF1α modulation.