Effects of over‐expression of HIF‐1alpha in bone marrow‐derived mesenchymal stem cells on traumatic brain injury

Mesenchymal stem cells (MSCs) have been shown to play therapeutic effect in traumatic brain injury (TBI). To augment the therapeutic effect, MSCs could be engineered to over‐express genes that are beneficial for treatment. In the present study, we over‐expressed hypoxia inducible factor (HIF)‐1alpha in bone marrow derived MSCs (BM‐MSCs) and sought to investigate whether HIF‐1alpha could enhance the therapeutic effect of MSCs in a mouse model of TBI. Balb/c mice were subjected to controlled cortical impact injury and MSCs were transplanted intravenously at 6 h after injury. The lesion volume and brain water content were measured and the neurological function was assessed by modified neurologic severity score tests. Double‐labeled immunofluorescence for BrdU and NeuU was performed to determine angiogenesis and neurogenesis. The expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) was measured by quantitative RT‐PCR and western blotting. After TBI, mice received BM‐MSCs over‐expressing HIF‐1alpha showed significantly more functional recovery, reduced brain damage, increased angiogenesis and neurogenesis and increased expression of VEGF and EPO, compared with control mice or mice treated with non‐transduced BM‐MSCs. Over‐expression of HIF‐1alpha enhanced BM‐MSCs induced improvement of neurological recovery after TBI, by stimulating angiogenesis and neurogenesis.     

HMGB1‐induced angiogenesis in perforated disc cells of human temporomandibular joint

High mobility group 1 protein (HMGB1), a highly conserved nuclear DNA‐binding protein and inflammatory mediator, has been recently found to be involved in angiogenesis. Our previous study has demonstrated the elevation of HMGB1 in the tissue of perforated disc of temporomandibular joint (TMJ). Here, we investigated a novel mediator of HMGB1 in regulating hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) to mediate angiogenesis in perforated disc cells of TMJ. HMGB1 increased the expression of HIF‐1α and VEGF in a dose‐ and time‐dependent manner in these cells. Moreover, immunofluorescence assay exhibits that the HIF‐1α were activated by HMGB1. In addition, HMGB1 activated extracellular signal‐related kinase 1/2 (Erk1/2), Jun N‐terminal kinase (JNK), but not P38 in these cells. Furthermore, both U0126 (ErK inhibitor) and SP600125 (JNK inhibitor) significantly suppressed the enhanced production of HIF‐1α and VEGF induced by HMGB1. Tube formation of human umbilical vein endothelial cells (HUVECs) was significantly increased by exposure to conditioned medium derived from HMGB1‐stimulated perforated disc cells, while attenuated with pre‐treatment of inhibitors for VEGF, HIF‐1α, Erk and JNK, individually. Therefore, abundance of HMGB1 mediates activation of HIF‐1α in disc cells via Erk and JNK pathway and then, initiates VEGF secretion, thereby leading to disc angiogenesis and accelerating degenerative change of the perforated disc.     

miR‐148a inhibits early relapsed colorectal cancers and the secretion of VEGF by indirectly targeting HIF‐1α under non‐hypoxia/hypoxia conditions

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR‐148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR‐148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl₂ to determine the underlying mechanism of miR‐148a. The effects of miR‐148a on the phosphoryl‐ERK (pERK)/hypoxia‐inducible factor‐1α (HIF‐1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR‐148a on angiogenesis was demonstrated through a tube formation assay. Sixty‐three CRC tissues (28 early relapse and 35 non‐early relapse) were analysed to assess the relationship between miR‐148a and HIF‐1α/VEGF. The protein expression of pERK/HIF‐1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR‐148a (all P < 0.05). The protein expression of VEGF/HIF‐1α was strongly inversely associated with the expression of miR‐148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR‐148a significantly obliterated angiogenesis. miR‐148a suppresses VEGF through down‐regulation of the pERK/HIF‐1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR‐148a down‐regulation increased the early relapse rate of CRC. This demonstrates that miR‐148a is a potential diagnostic and therapeutic target.     

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.     

MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

The macrolide compound MFTZ‐1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti‐tumour activities. In this study, we further examined the effects of MFTZ‐1 on hypoxia‐inducible factor‐1α (HIF‐1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ‐1 reduced HIF‐1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ‐1 did not affect the degradation of HIF‐1α protein or the level of HIF‐1α mRNA. By contrast, MFTZ‐1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol‐3‐kinase (PI3K)‐Akt and p42/p44 mitogen‐activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ‐1 abrogated the HIF‐1α‐driven increase in VEGF mRNA and protein secretion. MFTZ‐1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ‐1 can reduce constitutive, HIF‐1α‐independent VEGF secretion and concurrently antagonize inducible, HIF‐1α‐dependent VEGF secretion. Moreover, MFTZ‐1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low‐concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ‐1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti‐angiogenesis of MFTZ‐1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2‐defective HL60/MX2 cells, we showed that MFTZ‐1 affected HIF‐1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion possibly via PI3K‐Akt and MAPK pathways, eliciting anti‐angiogenesis independently of its Top2 inhibition.     

Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway

Cardiac vascular microenvironment is crucial for cardiac remodelling during the process of heart failure. Sphingosine 1‐phosphate (S1P) tightly regulates vascular homeostasis via its receptor, S1pr1. We therefore hypothesize that endothelial S1pr1 might be involved in pathological cardiac remodelling. In this study, heart failure was induced by transverse aortic constriction (TAC) operation. S1pr1 expression is significantly increased in microvascular endothelial cells (ECs) of post‐TAC hearts. Endothelial‐specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis in myocardium. In vitro experiments demonstrated that S1P/S1pr1 praxis activated AKT/eNOS signalling pathway, leading to more production of nitric oxide (NO), which is an essential cardiac protective factor. Inhibition of AKT/eNOS pathway reversed the inhibitory effect of EC‐S1pr1‐overexpression on angiotensin II (AngII)‐induced cardiomyocyte (CM) hypertrophy, as well as on TGF‐β‐mediated cardiac fibroblast proliferation and transformation towards myofibroblasts. Finally, pharmacological activation of S1pr1 ameliorated TAC‐induced cardiac hypertrophy and fibrosis, leading to an improvement in cardiac function. Together, our results suggest that EC‐S1pr1 might prevent the development of pressure overload‐induced heart failure via AKT/eNOS pathway, and thus pharmacological activation of S1pr1 or EC‐targeting S1pr1‐AKT‐eNOS pathway could provide a future novel therapy to improve cardiac function during heart failure development.     

Extracellular high‐mobility group box 1 mediates pressure overload‐induced cardiac hypertrophy and heart failure

Inflammation plays a key role in pressure overload‐induced cardiac hypertrophy and heart failure, but the mechanisms have not been fully elucidated. High‐mobility group box 1 (HMGB1), which is increased in myocardium under pressure overload, may be involved in pressure overload‐induced cardiac injury. The objectives of this study are to determine the role of HMGB1 in cardiac hypertrophy and cardiac dysfunction under pressure overload. Pressure overload was imposed on the heart of male wild‐type mice by transverse aortic constriction (TAC), while recombinant HMGB1, HMGB1 box A (a competitive antagonist of HMGB1) or PBS was injected into the LV wall. Moreover, cardiac myocytes were cultured and given sustained mechanical stress. Transthoracic echocardiography was performed after the operation and sections for histological analyses were generated from paraffin‐embedded hearts. Relevant proteins and genes were detected. Cardiac HMGB1 expression was increased after TAC, which was accompanied by its translocation from nucleus to both cytoplasm and intercellular space. Exogenous HMGB1 aggravated TAC‐induced cardiac hypertrophy and cardiac dysfunction, as demonstrated by echocardiographic analyses, histological analyses and foetal cardiac genes detection. Nevertheless, the aforementioned pathological change induced by TAC could partially be reversed by HMGB1 inhibition. Consistent with the in vivo observations, mechanical stress evoked the release and synthesis of HMGB1 in cultured cardiac myocytes. This study indicates that the activated and up‐regulated HMGB1 in myocardium, which might partially be derived from cardiac myocytes under pressure overload, may be of crucial importance in pressure overload‐induced cardiac hypertrophy and cardiac dysfunction.     

Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.     

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.     

Beta‐lapachone inhibits pathological retinal neovascularization in oxygen‐induced retinopathy via regulation of HIF‐1α

Retinal neovascularization in retinopathy of prematurity (ROP) is the most common cause of blindness for children. Despite evidence that hypoxia inducible factor (HIF)‐1α ‐VEGF axis is associated with the pathogenesis of ROP, the inhibitors of HIF‐1α have not been established as a therapeutic target in the control of ROP pathophysiology. We investigated the hypothesis that degradation of HIF‐1α as a master regulator of angiogenesis in hypoxic condition, using β‐lapachone, would confer protection against hypoxia‐induced retinopathy without affecting physiological vascular development in mice with oxygen‐induced retinopathy (OIR), an animal model of ROP. The effects of β‐lapachone were examined after intraocular injection in mice with OIR. Intraocular administration of β‐lapachone resulted in significant reduction in hypoxia‐induced retinal neovascularization without retinal toxicity or perturbation of developmental retinal angiogenesis. Our results demonstrate that HIF‐1α–mediated VEGF expression in OIR is associated with pathological neovascularization, not physiological angiogenesis. Thus, strategies blocking HIF‐1α in the developing eye in the pathological hypoxia could serve as a novel therapeutic target for ROP.     

Qiliqiangxin protects against anoxic injury in cardiac microvascular endothelial cells via NRG‐1/ErbB‐PI3K/Akt/mTOR pathway

Cardiac microvascular endothelial cells (CMECs) are important angiogenic components and are injured rapidly after cardiac ischaemia and anoxia. Cardioprotective effects of Qiliqiangxin (QL), a traditional Chinese medicine, have been displayed recently. This study aims to investigate whether QL could protect CMECs against anoxic injury and to explore related signalling mechanisms. CMECs were successfully cultured from Sprague‐Dawley rats and exposed to anoxia for 12 hrs in the absence and presence of QL. Cell migration assay and capillary‐like tube formation assay on Matrigel were performed, and cell apoptosis was determined by TUNEL assay and caspase‐3 activity. Neuregulin‐1 (NRG‐1) siRNA and LY294002 were administrated to block NRG‐1/ErbB and PI3K/Akt signalling, respectively. As a result, anoxia inhibited cell migration, capillary‐like tube formation and angiogenesis, and increased cell apoptosis. QL significantly reversed these anoxia‐induced injuries and up‐regulated expressions of NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mammalian target of rapamycin (mTOR), hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in CMECs, while NRG‐1 knockdown abolished the protective effects of QL with suppressed NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. Similarly, LY294002 interrupted the beneficial effects of QL with down‐regulated phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. However, it had no impact on NRG‐1/ErbB signalling. Our data indicated that QL could attenuate anoxia‐induced injuries in CMECs via NRG‐1/ErbB signalling which was most probably dependent on PI3K/Akt/mTOR pathway.     

Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.     

The valosin‐containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload

Hypertension‐induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin‐containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4‐month male spontaneously hypertensive rats (SHRs) vs. age‐matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild‐type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin‐containing protein VCP expression was also observed to be time‐ and dose‐dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac‐specific overexpression of VCP showed a significant repression in TAC‐induced LVH vs. litter‐matched WT controls upon 2‐week TAC. TAC‐induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.