Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Hypoxia induces BMP‐2 expression via ILK,Akt, mTOR,and HIF‐1 pathways in osteoblasts

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP‐2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP‐2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG‐63, hFOB and bone marrow stromal cells M2‐10B4. Integrin‐linked kinase (ILK) inhibitor (KP‐392), Akt inhibitor (1L‐6‐hydroxymethyl‐chiro‐inositol‐2‐[(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF‐1 protein. The binding of HIF‐1α to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia‐induced HIF‐1α activation and subsequent BMP‐2 up‐regulation. Taken together, our results provide evidence that hypoxia enhances BMP‐2 expression in osteoblasts by an HIF‐1α‐dependent mechanism involving the activation of ILK/Akt and mTOR pathways. J. Cell. Physiol. 223:810–818, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O₂) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl₂ or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal ?893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (?282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.     

Midkine prevented hypoxic injury of mouse embryonic stem cells through activation of Akt and HIF-1α via low-density lipoprotein receptor-related protein-1

Stem cell functions are dramatically altered by oxygen in tissue culture, which means the antioxidant/oxidant balance is critical for protection as well as toxicity. This study examined the effect of the heparin-binding growth factor midkine (MK) on hypoxia-induced apoptosis and related signal pathways in mouse embryonic stem cells (mESCs). Hypoxia (60 h) increased lactate dehydrogenase release and apoptosis, and reduced cell viability and proliferation. These effects were reversed by MK (100 ng/ml). MK also reversed hypoxia-induced increases of intracellular reactive oxygen species, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Blockage of JNK and p38 MAPK using small interference (si)RNAs produced a decrease in apoptosis. A loss of mitochondrial membrane potential, increases of cytochrome c release from mitochondria to cytosol, and cleaved caspase-3 expression, as well as decreases in cIAP-2 and Bcl-2 were also reversed by MK. Hypoxia alone and hypoxia with MK increased low-density lipoprotein receptor-related protein-1 (LRP-1) mRNA and protein expression. Hypoxia with MK rapidly increased serine/threonine protein kinase (Akt) phosphorylation which reversed by LRP-1 Ab (0.1 μg/ml) and prolonged heme oxygenase-1 (HO-1) expression. In addition, hypoxia with MK increased the expression of hypoxia-inducible factor-1α (HIF-1α). Moreover, inhibition of Akt, HO-1, and HIF-1α signaling pathways abolished the MK-induced blockage of apoptosis. In conclusion, MK partially prevented hypoxic injury of mESCs through activation of Akt, HO-1, and HIF-1α via LRP-1.     

Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling

Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra‐venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF‐1α and SDF‐1. Using small interference RNA, we knocked down the expression of HIF‐1α in MSCs to study the role of HIF‐1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF‐1α not only abolished the migration of MSCs, but also reduced the expression of SDF‐1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF‐1α mediated SDF‐1 expression. These findings provide new insight into the role of HIF‐1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC‐based therapeutics for wound healing.     

Involvement of β1‐integrin via PIP complex and FAK/paxillin in dexamethasone‐induced human mesenchymal stem cells migration

Although glucocorticoids strongly affect numerous biological processes including cell growth, development, and homeostasis, their effects on migration of human mesenchymal stem cells (hMSCs) are unclear. Therefore, we investigated the role of dexamethasone (DEX) and its related signaling pathways on migration of hMSCs. We found that DEX, at 10^?8 to 10^?6 M, significantly increased migration after a 24 h incubation, and DEX (10^?6 M) increased migration at >12 h. Moreover, DEX (10^?6 M) increased the level of glucocorticoid receptor (GR)‐α mRNA and protein expression, but not GR‐β mRNA. The increases in DEX‐induced migration were inhibited by the GR antagonist mifepristone (10^?7 M). In addition, DEX increased integrin‐linked kinase (ILK) and α‐parvin expression but did not change PINCH‐1/2 expression in lysate. DEX also increased formations of complex with ILK and α‐parvin, and ILK and PINCH‐1/2 as shown by immunoprecipitation, which were all inhibited by mifepristone. DEX‐induced migration was blocked by ILK and α‐parvin small interfering(si)RNAs. In addition, DEX increased focal adhesion kinase (FAK) and paxillin expression, which were attenuated by ILK and α‐parvin siRNAs. DEX‐induced cell migration was inhibited by FAK/paxillin siRNAs. DEX also increased β1‐integrin expression, which was blocked by FAK/paxillin siRNAs. In addition, DEX‐induced cell migration was inhibited by β1‐integrin siRNA. Downregulation of ILK, α‐parvin, FAK/paxillin and β1‐integrin expression by siRNAs decreased DEX‐induced filamentous(F)‐actin organization and migration of hMSCs. In conclusion, DEX partially stimulates hMSC migration by the expression of β1‐integrin through formation of a PINCH‐1/2/ILK/α‐parvin complex (PIP complex), and FAK and paxillin expression. J. Cell. Physiol. 226: 683–692, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

A potential mechanism for short time exposure to hypoxia-induced DNA synthesis in primary cultured chicken hepatocytes: Correlation between Ca(2+)/PKC/MAPKs and PI3K/Akt/mTOR

Less information is available concerning the molecular mechanisms of cell survival after hypoxia in hepatocytes. Therefore, this study examined the effect of hypoxia on DNA synthesis and its related signal cascades in primary cultured chicken hepatocytes. Hypoxia increased [3H] thymidine incorporation, which was increased significantly after 0-24 h of hypoxic exposure. Indeed, the percentage of cell population in the S phase was increased in hypoxia condition. However, the release of LDH indicating cellular injury was not changed under hypoxic conditions. Hypoxia increased Ca2+ uptake and PKC translocation from the cytosol to the membrane fraction. Among the PKC isoforms, hypoxia stimulated the translocation of PKC alpha and epsilon. Hypoxia also phosphorylated the p38 and p44/42 mitogen-activated protein kinases (MAPKs), which were blocked by the inhibition of PKC. On the other hand, hypoxia increased Akt and mTOR phosphorylation, which was blocked in the absence of intra/extracellular Ca2+. The inhibition of PKC/MAPKs or PI3K/Akt pathway blocked the hypoxia-induced [3H] thymidine incorporation. However, hypoxia-induced Ca2+ uptake and PKC translocation was not influenced by LY 294002 or Akt inhibitor and hypoxia-induced MAPKs phosphorylation was not changed by rapamycin. In addition, LY 294002 or Akt inhibitor has no effect on the phosphorylation of MAPKs. It suggests that there is no direct interaction between the two pathways, which cooperatively mediated cell cycle progression to hypoxia in chicken hepatocytes. Hypoxia also increased the level of the cell cycle regulatory proteins [cyclin D(1), cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4] and p-RB protein but decreased the p21 and p27 expression levels, which were blocked by inhibitors of upstream signal molecules. In conclusion, short time exposure to hypoxia increases DNA synthesis in primary cultured chicken hepatocytes through cooperation of Ca2+/PKC, p38 MAPK, p44/42 MAPKs, and PI3K/Akt pathways.     

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.     

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β₁, β₂ and α₅ on EPCs. Adhesion to FN is reduced by blocking β₁ and α₅ (49% and 2% of control, P < 0.05) whereas α₄‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α₅ for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α₅ is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α₅‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.     

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.     

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.     

Long non‐coding RNA GAPLINC promotes angiogenesis by regulating miR‐211 under hypoxia in human umbilical vein endothelial cells

In this study, we investigated the role of a long non‐coding RNA GAPLINC in angiogenesis using human umbilical vein endothelial cells (HUVEC). We found that hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) increased the expression of GAPLINC in HUVEC cells. Moreover, GAPLINC overexpression down‐regulated miR‐211 and up‐regulated Bcl2 protein expression. Further rescue experiments confirmed that hypoxia directly increased GAPLINC expression. GAPLINC overexpression also increased cell migration and vessel formation which promoted angiogenesis, and these changes were attributed to the increased expression of vascular endothelial growth factor receptors (VEGFR) and delta‐like canonical notch ligand 4 (DLL4) receptors. Finally, we demonstrated that GAPLINC promotes vessel formation and migration by regulating MAPK and NF‐kB signalling pathways. Taken together, these findings comprehensively demonstrate that overexpression of GAPLINC increases HUVEC cells angiogenesis under hypoxia condition suggesting that GAPLINC can be a potential target for critical limb ischaemia (CLI) treatment.