Overexpression Of Hypoxia-inducible Factor-1α Research Articles

ED-71 ameliorates bone regeneration in type 2 diabetes by reducing ferroptosis in osteoblasts via the HIF1α pathway

Eldecalcitol (ED-71), a novel active form of vitamin D, shows potential in treating osteoporosis. However, its underlying mechanisms of action remain to be determined. This study aimed to investigate the effect of ED-71 on bone regeneration and to illustrate its mode of action. The in-vitro model was developed using rat primary osteoblasts cultured under high-glucose conditions, and these cells were treated with ED-71. Additionally, an in vivo model of cranial bone defects was established in type 2 diabetic rats, and ED-71 was administered by gavage. The results demonstrated that ED-71 prevented osteoblast cell death, enhanced rat primary osteoblasts' osteogenic capacity, and attenuated the overexpression of hypoxia-inducible factor 1α (HIF1α) induced by high glucose levels. Furthermore, ED-71 increased glutathione peroxidase 4 (GPX4) levels and inhibited ferroptosis in response to hyperglycemic stimulation. Notably, interference with the HIF1α activator and ferroptosis activator Erastin significantly reduced the therapeutic effects of edetate osteolysis. These findings were further tested in vivo experiments. These results suggest that ED-71 activates the HIF1α pathway in vivo and in vitro, effectively relieving the ferroptosis induced by high glucose. Significantly, ED-71 may improve osteogenic disorders caused by diabetes.

Coronary and carotid artery dysfunction and KV7 overexpression in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS.

Overexpression of hypoxia-inducible factor-1α in hidradenitis suppurativa: the link between deviated immunity and metabolism

Hypoxia-inducible factor-1α (HIF-1α) is the master transcription factor of glycolysis, Th17 cell differentiation and suppression of regulatory T cells. In the skin and serum of patients with psoriasis vulgaris, increased expression of HIF-1α has been reported, whereas HIF-1α expression in the skin and serum of patients with hidradenitis suppurativa (HS) has not yet been studied. The objective of the study is to demonstrate is there a role for HIF-1α in the pathogenesis of hidradenitis suppurativa, and its relation to HS severity. Twenty patients suffering from hidradenitis suppurativa were included in the study. Punch biopsies were taken from lesional skin for the determination of HIF-1α expression by immunohistochemical staining, and HIF-1α gene expression by quantitative reverse transcription real time PCR. Quantification of HIF-1α protein concentration was done by enzyme-linked immunosorbent assay. Twenty socio-demographically cross-matched healthy volunteers served as controls. We found increased serum levels of HIF-1α. Literature-derived evidence indicates that the major clinical triggering factors of HS, obesity, and smoking are associated with hypoxia and enhanced HIF-1α expression. Pro-inflammatory cytokines such as tumor necrosis factor-a via upregulation of nuclear factor kappaB enhance HIF-1α expression. HIF-1α plays an important role for keratinocyte proliferation, especially for keratinocytes of the anagen hair follicle, which requires abundant glycolysis providing sufficient precursors molecules for biosynthetic pathways. Metformin via inhibition of mTORC1 as well as adalimumab attenuate HIF-1α expression, the key mediator between Th17-driven deviated immunity and keratinocyte hyperproliferation. In accordance with psoriasis, our study identifies HS as an HIF-1α-driven inflammatory skin disease and offers a new rationale for the prevention and treatment of HS by targeting HIF-1a overexpression.

Overexpression of Hypoxia-Inducible Factor-1α and Its Relation with Aggressiveness and Grade of Oral Squamous Cell Carcinoma.

Hypoxia-inducible factor-1α (HIF-1α) has been shown to be involved in cancer metastasis in several cancer types. There is however conflicting evidence of HIF-1α expression with oral cancer prognosis. Therefore, this study set out to investigate HIF-1α overexpression and its relationship with the aggressiveness and grade of oral squamous cell carcinoma (OSCC) and to explore the diagnostic potential of HIF-1α overexpression in OSCC in a cohort of Pakistani patients. Immunostaining of HIF-1α was performed on 54 OSCC and 14 normal oral mucosa (NOM) tissue samples and various cut-offs were used to evaluate its immunohistochemical expression. HIF-1α expression in OSCC samples was significantly higher than in controls, with minimal immunoreactivity in NOM. HIF-1α overexpression was significantly associated with increased tumor size (p = 0.046). However, no association was found between HIF-1α overexpression and increasing Broder's histological grade or TNM stage. The cut-off >10% cells with moderate to marked intensity carried a sensitivity of 70% and a specificity of 100% to distinguish between tumor and control. ROC curve analysis of HIF-1α weighted histoscores showedHIF-1α overexpression as a highly sensitive and specific diagnostic test (p < 0.001, AUC = 0.833). HIF-1α overexpression is a tumor-specific finding associated with increased tumor size and carries a potential diagnostic role.

Overexpression of hypoxia-inducible factor 1α and excessive vascularization in the peri-implantation endometrium of infertile women with chronic endometritis.

Chronic endometritis (CE) contributes to impaired endometrial receptivity and is closely associated with poor in vitro fertilization (IVF) outcomes. However, the mechanisms underlying CE are unclear. Here, we investigated the role of the hypoxic microenvironment and endometrial vascularization in the peri-implantation endometrium of infertile women with CE. This retrospective study involved 15 fertile women and 77 infertile patients diagnosed with CE based on CD138+ ≥1/10 high-power fields (HPFs). The CE patients were divided into Group 1 (CD138+ 1-4/10 HPFs, 53 cases) and Group 2 (CD138+ ≥5/10 HPFs, 24 cases). The expression levels of hypoxia-inducible factor 1α (HIF1α), vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in peri-implantation endometrium were assessed by qRT-PCR and western blot analyses. Spatial levels of HIF1α, VEGFA, and VEGFR2 in various endometrial compartments was determined using immunohistochemistry and H-score analysis. Microvascular density (MVD) was determined using CD34 staining and scored using Image J. Finally, we used qRT-PCR to assess changes in the expression of HIF1α, VEGFA, and VEGFR2 in CE patients after treatment with first-line antibiotics. Relative to Group 1 and control group, during the implantation window, protein and mRNA levels of HIF1α, VEGFA, and VEGFR2 were markedly high in Group 2 (P<0.05). H-score analysis showed that HIF1α, VEGFA, and VEGFR2 in the luminal, glandular epithelium, and stromal compartments were markedly elevated in Group 2, comparing to control group and Group 1 (P<0.05). Moreover, markedly elevated MVD levels were observed in Group 2. Notably, the above indexes did not differ significantly in the control group versus Group 1. Treatment with antibiotics significantly suppressed the endometrial HIF1α and VEGFA levels in CE-cured patients. Here, we for the first time report the upregulation of HIF1α, VEGFA, and VEGFR2, as well as excessive endometrial vascularization in the peri-implantation endometrium of CE patients. Our findings offer new insights into reduced endometrial receptivity in CE-associated infertility.

Occludin Promotes Adhesion of CD8+ T Cells and Melanocytes in Vitiligo via the HIF-1α Signaling Pathway.

Loss of melanocytes induced by activated CD8+ T cells is the pathological hallmark of vitiligo. Melanocyte-specific CD8+ T cells are recruited to the skin via chemokines, thereby releasing perforin, granzyme, and other cytotoxic substances that destroy the melanocytes. However, the mechanism of CD8+ T cells to adhere to melanocytes is unknown. Previous transcriptome sequencing results published by our group showed that the occluding (OCLN) gene was significantly upregulated in CD8+ T cells from skin lesions of vitiligo. Occludin is a crucial component of the tight junction between cells; in cells without tight junction, occludin mediates the adhesion of two cells in the form of a self-ligand. This study demonstrated that OCLN gene expression was elevated in the CD8+ T cells of vitiligo patients, and occludin mediates the adherence of CD8+ T cells to melanocytes. Besides, pathological changes in vitiligo skin lesions reveal that CD8+ T cells continuously persist in the skin lesions, which is related to the persistence of the disease. In this regard, we found that fibroblasts from vitiligo patients significantly express occludin, which may participate in the continuous retention of CD8+ T cells in the skin lesions. The pathogenesis of vitiligo is closely related to oxidative stress, and our data suggest that overexpression of hypoxia-inducible factor-1α (HIF-1α) increases the expression of occludin. Besides, ChIP-qPCR of CD8+ T cells revealed that HIF-1α directly binds to the OCLN promoter. Thus, occludin upregulation promotes the adhesion of CD8+ T cells and melanocytes via the HIF-1α signaling pathway. Our study results suggested a critical role for OCLN in the occurrence, progression, and maintenance of vitiligo. Therefore, inhibiting the expression of OCLN gene may be a potential targeted treatment strategy.

Hypoxic Hepatocellular Carcinoma Cells Acquire Arsenic Trioxide Resistance by Upregulating HIF-1α Expression.

Although arsenic trioxide (ATO) is used in the treatment of advanced hepatocellular carcinoma (HCC) in clinical trials, it is not satisfactory in terms of improving HCC patients' overall survival. Intratumoral hypoxia and overexpression of hypoxia-inducible factor-1α (HIF-1α) may result in ATO resistance and tumor progression. We investigated the mechanisms involving HIF-1α expression and acquired ATO chemoresistance in HCC cells and mice. The therapeutic effects of ATO in normoxic and hypoxic HCC cells were assessed using cell viability and apoptosis assays in vitro and a xenograft model in vivo. mRNA and protein expression of HIF-1α, P-glycoprotein, and VEGF were measured by qRT-PCR and western blotting. HIF-1α inhibition was performed to investigate the mechanism of ATO resistance. VEGF secretion was tested using ELISA and tube formation assays. Compared to normoxic cells, hypoxic HCC cells were more resistant to ATO, with higher IC50 values and less apoptosis, and upregulated HIF-1α protein expression, accompanied with the enhancement of P-glycoprotein and VEGF synthesis after ATO treatment. VEGF secretion was elevated in the supernatant of ATO-treated HCC cells, and this change can potentiate angiogenesis in vitro. HIF-1α inhibition attenuated ATO resistance and angiogenesis and promoted the anticancer effects of ATO both in vitro and in vivo by downregulating therapy-induced P-glycoprotein and VEGF overexpression. Hypoxic HCC cells acquire ATO resistance by upregulating HIF-1α levels; thus, combining ATO with a HIF-1α-targeting agent may lead to enhanced antitumor effects in HCC.

Hypoxia-inducible factor 1-alpha enhances the secretome to rejuvenate adult cardiosphere-derived cells

ObjectiveAfter cardiac injury, endogenous repair mechanisms are ineffective. However, cell-based therapies provide a promising clinical intervention based on their ability to restore and remodel injured myocardium due to their paracrine factors. Recent clinical trials have demonstrated that adult cardiosphere-derived cell therapy is safe for the treatment of ischemic heart failure, although with limited regenerative potential. The limited efficiency of cardiosphere-derived cells after myocardial infarction is due to the inferior quality of their secretome. This study sought to augment the therapeutic potential of cardiosphere-derived cells by modulating hypoxia-inducible factor-1α, a regulator of paracrine factors. MethodsCardiosphere-derived cells were isolated and expanded from the right atrial appendage biopsies of patients undergoing cardiac surgery. To study the effect of hypoxia-inducible factor-1α on the secretome, cardiosphere-derived cells were transduced with hypoxia-inducible factor-1α–overexpressing lentivirus, and various cardioprotective factors within the secretome were quantified using enzyme-linked immunosorbent assays. Comparative analysis of the regenerative potential of cardiosphere-derived cells was performed in a rat myocardial infarction model. ResultsMechanistically, overexpression of hypoxia-inducible factor-1α in adult cardiosphere-derived cells led to the enrichment of the secretome with vascular endothelial growth factor A, angiopoietin 1, stromal cell–derived factor 1α, and basic fibroblast growth factor. Intramyocardial administration of cardiosphere-derived cells transduced with hypoxia-inducible factor-1α after myocardial infarction significantly improved left ventricular ejection fraction, fractional shortening, left ventricular end-systolic volume, and cardiac output. Functional improvement of the rat heart correlated with improved adaptive remodeling of the infarcted myocardium by enhanced angiogenesis and decreased myocardial fibrosis. We also showed that hypoxia-inducible factor-1α expression in cardiosphere-derived cells was adversely affected by aging. ConclusionsHypoxia-inducible factor-1α improves the functional potency of cardiosphere-derived cells to preserve myocardial function after myocardial infarction by enriching the cardiosphere-derived cells' secretome with cardioprotective factors. This strategy may be useful for improving the efficacy of allogeneic cell-based therapies in future clinical trials.

Ultrasmall Barium Titanate Nanoparticles for Highly Efficient Hypoxic Tumor Therapy via Ultrasound Triggered Piezocatalysis and Water Splitting.

Hypoxia in a solid tumor microenvironment (TME) can lead to the overexpression of hypoxia-inducible factor-1α (HIF-1α), which correlates to tumor metastasis. Reactive oxygen species (ROS) induced tumor cell apoptosis is becoming a promising method in tumor treatment. Currently, the ROS generating systems, e.g., photodynamic treatment and sonodynamic treatment, highly depend on oxygen (O2) in the tumor microenvironment (TME). However, the level of O2 in TME is too low to produce enough ROS. Herein, we developed an ultrasmall DSPE-PEG2000 coated barium titanate nanoparticle (P-BTO) for tumor treatment based on ultrasound triggered piezocatalysis and water splitting. Interestingly, irradiated by ultrasound, the surface of ultasmall P-BTO nanoparticles produced imbalance charges, which induced a cascade of redox reaction processes to simultaneously generate ROS and O2, the latter one was hardly generated in large-sized barium titanate nanoparticles. The as-synthesized P-BTO reached the highest accumulation in the tumor site at 4 h after intravenous injection. The results showed that the produced O2 significantly alleviated the hypoxia of TME to down-regulate the expression of HIF-1α, and the produced ROS can efficiently kill tumor cells. Moreover, the tumor metastasis was also inhibited, providing a different way to treat triple-negative breast cancer, which was easily metastatic and lacked effective treatments in the clinic.

Effect of hypoxia inducible factor 1α overexpression on differentiation of stem cells derived from human exfoliated deciduous teeth into vascular endothelial cells

To investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells. SHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis. After identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly ( P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher ( P<0.05), the relative expression of p-GSK3β protein was significantly lower ( P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased ( P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group ( P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower ( P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups ( P<0.05). Overexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.

Prognostic significance of hypoxia-inducible factor-1α expression in advanced pharyngeal cancer without human papillomavirus infection.

This study aimed to clarify the association between both hypoxia-inducible factor-1α and glucose transporter type-1 expression and survival outcome in advanced pharyngeal cancer without human papillomavirus infection. Twenty-five oropharyngeal and 55 hypopharyngeal cancer patients without human papillomavirus infection were enrolled. All patients had stage III-IV lesions and underwent concurrent chemoradiotherapy or surgery. Hypoxia-inducible factor-1α and glucose transporter type-1 expression were investigated in primary lesions by immunohistochemistry. There were 41 and 39 cases with low and high hypoxia-inducible factor-1α expression, and 28 and 52 cases with low and high glucose transporter type-1 expression, respectively. There was no significant correlation between hypoxia-inducible factor-1α and glucose transporter type-1 expression. In univariate analysis, nodal metastasis, clinical stage and high hypoxia-inducible factor-1α expression, but not glucose transporter type-1 expression, predicted significantly worse prognosis. In multivariate analysis, hypoxia-inducible factor-1α overexpression was significantly correlated with poor overall survival, disease-specific survival and recurrence-free survival. High hypoxia-inducible factor-1α expression was an independent risk factor for poor prognosis for advanced human papillomavirus-unrelated pharyngeal cancer.

Deferoxamine Ameliorates Compressed Spinal Cord Injury by Promoting Neovascularization in Rats.

The therapeutic effect of deferoxamine (DFO) for spinal cord injury (SCI) has been demonstrated in previous studies; however, the exact mechanism of action is still unclear. Here, we hypothesized that DFO ameliorates spinal cord compression by promoting neovascularization. Using an SCI model of moderate compression, rats were intraperitoneally injected with 30mg/kg or 100mg/kg DFO for 1-2weeks, and significant neovascularization was found in the injured spinal cord, showing overexpression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), and an increase in the number of new blood vessels. In addition, SCI in rats was significantly ameliorated after treatment with DFO, with less motor dysfunction, increased spared neural tissue, and improved electrophysiological conduction. By contrast, the ameliorative effect of DFO on SCI was suppressed when DFO-induced neovascularization was blocked by lenvatinib, a vascular endothelial growth factor receptor inhibitor, further suggesting that the primary pharmacological effect of DFO in SCI is the promotion of neovascularization. Therefore, we concluded that DFO effectively alleviated SCI by promoting neovascularization in the injured spinal cord. Considering that DFO is an FDA-approved free radical scavenger and iron chelator, it may represent a promising alternative strategy for SCI therapy in the future.

Overexpression of hypoxia-inducible factor 1α is associated with neutrophilic inflammation in chronic rhinosinusitis with nasal polyps

ObjectiveThis study aimed to assess the possible role of hypoxia-inducible factor 1α (HIF-1α) in promoting neutrophilic inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) patients. MethodsWe examined HIF-1α expression in sinonasal tissues from CRSwNP patients and healthy controls by using immunohistochemistry, qRT-PCR, and western blot. Next, the stimulatory effects of several cytokines (IFN-γ, IL-17A, IL-6, etc.) and reagents (dexamethasone (DEX), clarithromycin (CAM) and curcumin (CUM)) on HIF-1α expression in cultured normal nasal epithelial cells (NECs) were also evaluated. Moreover, the effects of CAM and glucocorticoid on nasal symptoms and signs of uncontrolled neutrophilic CRSwNP patients were evaluated. ResultsThe mRNA and protein expression of HIF-1α were significantly increased in polyp tissues compared with healthy controls (P < 0.05), and the HIF-1α level in polyp tissues was positively associated with IL-17A production and tissue neutrophilia (P < 0.05). Moreover, in cultured NECs, HIF-1α expression was upregulated in the presence of IL-17A and IL-6 (P < 0.05). Both CAM and CUM showed an additive effect with DEX in inhibiting HIF-1α expression (P < 0.05). Moreover, combined glucocorticoid and CAM significantly improved nasal symptoms and signs compared with glucocorticoid alone in uncontrolled neutrophilic CRSwNP patients (P < 0.05). ConclusionOur findings indicate that HIF-1α is associated with neutrophilic inflammation and glucocorticoid resistance in CRSwNP patients.

Effects of inhibition of miR-155-5p in neural stem cell subarachnoid transplant on rats with cerebral infarction

Cerebral infarction is a leading cause of death, which calls for effective prevention and treatment. Transplant of neural stem cells (NSCs) is a potential therapeutic treatment to cerebral infarction although its efficacy still needs to be improved. Overexpression of Hypoxia-inducible factor 1α (HIF-1α) has been shown to enhance the protective effects of stem cell transplant on cerebral infarction. The expression of HIF-1α is predicted to be regulated by miR-155-5p. Therefore, we regulated the expression of miR-155-5p in NSCs and evaluated the effects of miR-155-5p-regulated NSC transplant on cerebral infarction. We inhibited miR-155-5p expression in NSCs by overexpressing miR-155-5p inhibitor. HIF-1α expression, cell viability and the expression of apoptosis markers were examined. We established the middle cerebral artery occlusion (MCAO) rat model, and the infarct volume, neurobehavioral outcomes, inflammation and oxidative stress were evaluated after NSC transplant. miR-155-5p directly targeted HIF-1α and negatively regulated its expression. Inhibition of miR-155-5p enhanced cell viability and prevented cell apoptosis. Transplant of miR-155-5p-inhibited NSCs significantly decreased infarct volume, improved neurobehavioral outcomes of MCAO rats. Transplant of miR-155-5p-inhibited NSCs significantly inhibited inflammation and oxidative stress. Inhibition of miR-155-5p in NSCs resulted in enhanced protection against cerebral infarction after NSC transplant.

Effects of Inhibition of miR-155-5p in Neural Stem Cell Subarachnoid Transplant on Rats with Cerebral Infarction.

Cerebral infarction is a leading cause of death, which calls for effective prevention and treatment. Transplant of neural stem cells (NSCs) is a potential therapeutic treatment to cerebral infarction although its efficacy still needs to be improved. Overexpression of hypoxia-inducible factor 1α (HIF-1α) has been shown to enhance the protective effects of stem cell transplant on cerebral infarction. The expression of HIF-1α is predicted to be regulated by miR-155-5p. Therefore, we regulated the expression of miR-155-5p in NSCs and evaluated the effects of miR-155-5p-regulated NSC transplant on cerebral infarction. We inhibited miR-155-5p expression in NSCs by overexpressing miR-155-5p inhibitor. HIF-1α expression, cell viability, and the expression of apoptosis markers were examined. We established the middle cerebral artery occlusion (MCAO) rat model, and the infarct volume, neurobehavioral outcomes, inflammation, and oxidative stress were evaluated after NSC transplant. miR-155-5p directly targeted HIF-1α and negatively regulated its expression. Inhibition of miR-155-5p enhanced cell viability and prevented cell apoptosis. Transplant of miR-155-5p-inhibited NSCs significantly decreased infarct volume and improved neurobehavioral outcomes of MCAO rats. Transplant of miR-155-5p-inhibited NSCs significantly inhibited inflammation and oxidative stress. Inhibition of miR-155-5p in NSCs resulted in enhanced protection against cerebral infarction after NSC transplant.

Effect of lentiviral vector-mediated overexpression of hypoxia-inducible factor 1 alpha delivered by pluronic F-127 hydrogel on brachial plexus avulsion in rats

Brachial plexus avulsion often results in massive motor neuron death and severe functional deficits of target muscles. However, no satisfactory treatment is currently available. Hypoxia-inducible factor 1α is a critical molecule targeting several genes associated with ischemia-hypoxia damage and angiogenesis. In this study, a rat model of brachial plexus avulsion-reimplantation was established, in which C5-7 ventral nerve roots were avulsed and only the C6 root reimplanted. Different implants were immediately injected using a microsyringe into the avulsion-reimplantation site of the C6 root post-brachial plexus avulsion. Rats were randomly divided into five groups: phosphate-buffered saline, negative control of lentivirus, hypoxia-inducible factor 1α (hypoxia-inducible factor 1α overexpression lentivirus), gel (pluronic F-127 hydrogel), and gel + hypoxia-inducible factor 1α (pluronic F-127 hydrogel + hypoxia-inducible factor 1α overexpression lentivirus). The Terzis grooming test was performed to assess recovery of motor function. Scores were higher in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups (in particular the gel + hypoxia-inducible factor 1α group) compared with the phosphate-buffered saline group. Electrophysiology, fluorogold retrograde tracing, and immunofluorescent staining were further performed to investigate neural pathway reconstruction and changes of neurons, motor endplates, and angiogenesis. Compared with the phosphate-buffered saline group, action potential latency of musculocutaneous nerves was markedly shortened in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Meanwhile, the number of fluorogold-positive cells and ChAT-positive neurons, neovascular area (labeled by CD31 around avulsed sites in ipsilateral spinal cord segments), and the number of motor endplates in biceps brachii (identified by α-bungarotoxin) were all visibly increased, as well as the morphology of motor endplate in biceps brachil was clear in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Taken together, delivery of hypoxia-inducible factor 1α overexpression lentiviral vectors mediated by pluronic F-127 effectively promotes spinal root regeneration and functional recovery post-brachial plexus avulsion. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, China.

The Role of CD39/CD73/Ado/A2AR Axis and HIF-1α in Chronic Lymphocytic Leukemia

Introduction: CD39/CD73/ADO/A2A system plays important roles in tumor growth and therapy. Extracellular adenosine (ADO) receptor A2A (A2AR) is the dominant receptor expressed on chronic lymphocytic leukemia (CLL) cells. ADO has been shown to protect CLL cells from spontaneous and drug-induced apoptosis through A2AR activation. Overexpression of hypoxia inducible factor-1α (HIF-1α), an important mediator controlling the expression of a wide variety of apoptotic genes, has been observed in bone marrow leukemic cells from CLL patients. However, the reciprocal action of A2AR and HIF-1α in CLL remains elusive. This study was aimed to explore the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α in CLL.Materials and Methods: Peripheral blood (PB) and bone marrow (BM) samples were collected from 30 healthy volunteers (control) and 20 patients who were diagnosed with CLL for the first time at the Hematology Department in our hospital. CD39/CD73/ADO/A2AR axis-related protein and HIF-1α protein expressions in CLL PB and BM tissues were examined by Western-blot. CD39, CD73, A2AR and HIF-1α expression on CLL cells were also determined by FACS. CLL cell line (Lym-2) was purchased from ATCC and incubated in DMEM medium supplemented with 10% FBS. The protein expressions of CD39, CD73 and A2AR on Lym-2 cells were confirmed by Western-blot. To study the impacts of A2AR activation and inactivation on CLL cells, CLL cells were treated with A2AR agonist CGS21680 or antagonist SCH58261, followed by determination of cell proliferation and HIF-1α protein expression.Results: The protein expressions of CD39, CD73, A2AR and HIF-1α were higher in CLL group than those in control group (BM: CD39 1.471 vs 0.926, CD73 1.097 vs 0.489, A2AR 1.139 vs 0.342 and HIF-1α 0.940 vs 0.362, P<0.05 Figure 1A; PB : CD39 1.809 vs 1.331, CD73 1.039 vs 0.653, A2AR 1.738 vs 1.119 and HIF-1α 1.336 vs 1.010, P<0.05 Figure 1B). Moreover, CD73 and HIF-1α protein expression was found to have relationship with disease stage. Patients who belonged to stage IV (Rai) and stage B/C (Binet) exhibited higher levels of CD73 and HIF-1α than patients who belonged to stage I to III (Rai) and stage A (Binet) . Data from FACS analysis showed that the proportion of CD39+CLL cells was higher than CD73+CLL cells and HIF-1α+CLL cells (P<0.05, Figure 2A). When compared to control group, the proportions of CD39+cells and CD73+ cells were significant higher in CLL group (Figure 2B). Western-blot results showed that Lym-2 cells expressed CD39, CD73 and A2AR simultaneously (Figure 3A). The results from CLL cells and A2AR agonist/antagonist incubation showed that when CGS21680 concentration was of ≥ 10uM, cell proliferation was promoted obviously (Figure 3B). On the contrary, SCH58261 could inhibit CLL cells proliferation when its concentration was of ≥5uM at a concentration dependent manner (Figure 3C). Moreover, HIF-1α protein expression was also affected by A2AR agonist at a concentration dependent manner(0uM 0.851, 10uM 1.116, 20uM 1.420, 50uM 1.41, Figure 3D).Conclusion: Our study showed that ADO produced by CD39 and CD73 could affect CLL cells proliferation and HIF-1α expression through A2AR-mediated mechanisms. The exploration of the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α could help us to find a novel approach to CLL therapy.Disclosures: No relevant conflicts of interest to declare. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metformin suppresses hypoxia‑induced migration via the HIF‑1α/VEGF pathway in gallbladder cancer in�vitro and in�vivo

Hypoxia plays a crucial role in cancer development and progression. Overexpression of hypoxia-inducible factor-1α (HIF‑1α) has been demonstrated in a hypoxic microenvironment in various tumor types. Metformin has been identified as an antitumor drug in various tumor types. However, its role in cellular migration in a hypoxic microenvironment, and the associated regulatory mechanism, have yet to be fully elucidated. The present study aimed to investigate the clinical significance of HIF‑1α, and its biological role, in gallbladder cancer (GBC). Furthermore, the role of metformin in cellular migration, and its underlying mechanism in GBC, were also identified. Real‑time quantitative polymerase chain reaction analysis and immunohistochemistry experiments revealed that HIF‑1α was significantly upregulated in GBC tissues. HIF‑1α overexpression was closely associated with lymph node metastasis and tumor‑lymph node‑metastasis (TNM) stages. HIF‑1α was able to promote cell migration in a hypoxic microenvironment by overexpressing vascular endothelial growth factor (VEGF) in GBC‑SD cells, an effect which was partly reversed by small‑interfering RNA HIF‑1α (siHIF‑1α) and 2‑methoxyestradiol. Further experiments demonstrated that metformin inhibited hypoxia‑induced migration via HIF‑1α/VEGF invitro. In addition, metformin suppressed GBC growth and downregulated the expression of HIF‑1α and VEGF in a GBC‑SD cell xenograft model. Taken together, these results suggest that HIF‑1α may contribute to tumor migration via the overexpression of VEGF in GBC, while metformin is able to inhibit tumor migration by targeting the HIF‑1α/VEGF pathway.

RETRACTED: Effect of overexpression of hypoxia-inducible factor-1α induced by hyperoxia invivo in LNCaP tumors on tumor growth rate [APJTM 8/10 (2015) 813-820

RETRACTED: Effect of overexpression of hypoxia-inducible factor-1α induced by hyperoxia invivo in LNCaP tumors on tumor growth rate [APJTM 8/10 (2015) 813-820

Aberrant expression of miR-153 is associated with overexpression of hypoxia-inducible factor-1α in refractory epilepsy.

Evidence suggest that overexpression of hypoxia-inducible factor-1α (HIF-1α) is linked to multidrug resistance of epilepsy. Here we explored whether aberrant expression of HIF-1α is regulated by miRNAs. Genome-wide microRNA expression profiling was performed on temporal cortex resected from mesial temporal lobe epilepsy (mTLE) patients and age-matched controls. miRNAs that are putative regulator of HIF-1α were predicted via target scan and confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). Mimics or miRNA morpholino inhibitors were transfected in astrocytes and luciferase reporter assay was applied to detect HIF-11α expression. Microarray profiling identified down-regulated miR-153 as a putative regulator of HIF-1α in temporal cortex resected from surgical mTLE patients. RT-qPCR confirmed down-regulation of miR-153 in plasma of mTLE patients in an independent validation cohort. Knockdown of miR-153 significantly enhanced expression of HIF-1α while forced expression of miR-153 dramatically inhibited HIF-1α expression in pharmacoresistant astrocyte model. Luciferase assay established that miR-153 might inhibit HIF-1α expression via directly targeting two binding sites in the 3′UTR region of HIF-1α transcript. These data suggest that down-regulation of miR-153 may contribute to enhanced expression of HIF-1α in mTLE and serve as a novel biomarker and treatment target for epilepsy.