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.     

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.     

HIF-1α Activation by Intermittent Hypoxia Requires NADPH Oxidase Stimulation by Xanthine Oxidase

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O₂ saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O₂ for 30 sec) and normoxia (21% O₂ for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47^phox and p67^phox to the plasma membrane and their interaction with gp91^phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.     

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.     

NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro

Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.     

Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

Reactive oxygen species (ROS) and pro-inflammatory cytokines are crucial in ventricular remodelling, such as inflammation-associated myocarditis. We previously reported that tumour necrosis factor-α (TNF-α)-induced ROS in human aortic smooth muscle cells is mediated by NADPH oxidase subunit Nox4. In this study, we investigated whether TNF-α-induced ventricular remodelling was mediated by Nox2 and/or Nox4. An intravenous injection of murine TNF-α was administered to a group of mice and saline injection was administered to controls. Echocardiography was performed on days 1, 7 and 28 post-injection. Ventricular tissue was used to determine gene and protein expression of Nox2, Nox4, ANP, interleukin (IL)-1β, IL-2, IL-6, TNF-α and to measure ROS. Nox2 and Nox4 siRNA were used to determine whether or not Nox2 and Nox4 mediated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in adult human cardiomyocytes. Echocardiography showed a significant increase in left ventricular end-diastolic and left ventricular end-systolic diameters, and a significant decrease in the ejection fraction and fractional shortening in mice 7 and 28 days after TNF-α injection. These two groups of mice showed a significant increase in ventricular ROS, ANP, IL-1β, IL-2, IL-6 and TNF-α proteins. Nox2 and Nox4 mRNA and protein levels were also sequentially increased. ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems. Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes. Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

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.     

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.     

Transforming growth factor‐β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.     

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.     

Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species

Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.     

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.     

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.     

High‐fat diet induces cardiac remodelling and dysfunction: assessment of the role played by SIRT3 loss

Mitochondrial dysfunction plays an important role in obesity‐induced cardiac impairment. SIRT3 is a mitochondrial protein associated with increased human life span and metabolism. This study investigated the functional role of SIRT3 in obesity‐induced cardiac dysfunction. Wild‐type (WT) and SIRT3 knockout (KO) mice were fed a normal diet (ND) or high‐fat diet (HFD) for 16 weeks. Body weight, fasting glucose levels, reactive oxygen species (ROS) levels, myocardial capillary density, cardiac function and expression of hypoxia‐inducible factor (HIF)‐1α/‐2α were assessed. HFD resulted in a significant reduction in SIRT3 expression in the heart. Both HFD and SIRT3 KO mice showed increased ROS formation, impaired HIF signalling and reduced capillary density in the heart. HFD induced cardiac hypertrophy and impaired cardiac function. SIRT3 KO mice fed HFD showed greater ROS production and a further reduction in cardiac function compared to SIRT3 KO mice on ND. Thus, the adverse effects of HFD on cardiac function were not attributable to SIRT3 loss alone. However, HFD did not further reduce capillary density in SIRT3 KO hearts, implicating SIRT3 loss in HFD‐induced capillary rarefaction. Our study demonstrates the importance of SIRT3 in preserving heart function and capillary density in the setting of obesity. Thus, SIRT3 may be a potential therapeutic target for obesity‐induced heart failure.     

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.