von Hippel-Lindau β-domain-luciferase fusion protein as a bioluminescent hydroxyproline sensor for a hypoxia-inducible factor prolyl hydroxylase assay

Hypoxia-inducible factor prolyl hydroxylases (HPHs) are responsible for hydroxylation of proline residues in hypoxia-inducible factor-α (HIF-α), resulting in von Hippel-Lindau (VHL)-mediated proteasome degradation of the hydroxylated proteins. Pharmacological inhibition of the enzyme leads to stabilization of HIF-α proteins and consequent activation of HIF, which provides therapeutic benefit for a variety of tissues undergoing ischemic stress. In an effort to develop a new assay for measuring HPH activity, we designed a fusion protein, VHL β-domain-luciferase. Recombinant fusion protein with a glutathione S-transferase (GST) tag was purified from Escherichia coli. GST-VHL β-domain-luciferase with C-terminal deletion (GVbL-CD) was obtained as a major product and found to have luciferase activity. In a GVbL-CD capture assay using HIF peptide-bound beads, at least a 13-fold increase in luciferase activity was elicited for HIF peptide with hydroxyproline compared with unhydroxylated HIF peptide. HPH inhibitory activities of known HPH inhibitors or HIF-1α inducers were assessed using this assay, whose results were in good agreement with those obtained from conventional methods. The competitive effect of 2-ketoglutarate on dimethyloxalylglycine-mediated HPH inhibition was assessed very well in the new assay. Taken together, the VHL β-domain protein with luciferase activity is of use for HPH activity assay.     

Caffeic acid phenethyl ester possesses potent cardioprotective effects in a rabbit model of acute myocardial ischemia-reperfusion injury

Although great achievements have been made in elucidating the molecular mechanisms contributing to acute myocardial ischemia/reperfusion (I/R) injury, an effective pharmacological therapy to protect cardiac tissues from serious damage associated with acute myocardial infarction, coronary arterial bypass grafting surgery, or acute coronary syndromes has not been developed. We examined the in vivo cardioprotective effects of caffeic acid phenethyl ester (CAPE), a natural product with potent anti-inflammatory, antitumor, and antioxidant activities. CAPE was systemically delivered to rabbits either 60 min before or 30 min after surgically inducing I/R injury. Infarct dimensions in the area at risk were reduced by >2-fold (P < 0.01) with CAPE treatment at either period. Accordingly, serum levels of normally cytosolic enzymes lactate dehydrogenase, creatine kinase (CK), MB isoenzyme of CK, and cardiac-specific troponin I were markedly reduced in both CAPE treatment groups (P < 0.05) compared with the vehicle-treated control group. CAPE-treated tissues displayed significantly less cell death (P < 0.05), which was in part due to inhibition of p38 mitogen-activated protein kinase activation and reduced DNA fragmentation often associated with caspase 3 activation (P < 0.05). In addition, CAPE directly blocked calcium-induced cytochrome c release from mitochondria. Finally, the levels of inflammatory proteins IL-1beta and TNF-alpha expressed in the area at risk were significantly reduced with CAPE treatment (P < 0.05). These data demonstrate that CAPE has potent cardioprotective effects against I/R injury, which are mediated, at least in part, by the inhibition of inflammatory and cell death responses. Importantly, protection is conferred when CAPE is systemically administered after the onset of ischemia, thus demonstrating potential efficacy in the clinical scenario.     

Dexmedetomidine alleviates cerebral ischemia-reperfusion injury in rats via inhibition of hypoxia-inducible factor-1α

Dexmedetomidine (Dex) was reported to reduce ischemia-reperfusion (I/R) injury in kidney and brain tissues. Thus, we aimed to study the role and mechanism of Dex in cerebral I/R injury by inhibiting hypoxia-inducible factor-1α (HIF-1α) and apoptosis. First, I/R injury models were established. Six groups were assigned after different treatments: sham, I/R, I/R+Dex, I/R+2-methoxyestradiol (2ME2) (HIF-1α inhibitor), I/R+CoCl ₂ (HIF-1α activator), and I/R+Dex+CoCl ₂ groups. Neurological function, cerebral infarction volume, survival, and apoptosis of brain cells were then analyzed. Besides, immunohistochemistry and Western blot analysis were used to detect the expression of HIF-1α, BCL-2[B-cell leukemia/lymphoma 2] adenovirus E1B interacting protein 3 (BNIP3), B-cell leukemia/lymphoma 2 (BCL2), BCL2[B-cell leukemia/lymphoma 2] associated X (Bax), and cleaved-caspase3 proteins in brain tissues. I/R rats showed cerebral infarction, increased neurological function score, number of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)-positive cells and HIF-1α–positive cells as well as decreased neurons. Inhibition of HIF-1α can reduce the apoptosis induced by I/R, and overexpression of HIF-1α can aggravate apoptosis in brain tissue of I/R rats. Furthermore, activation of HIF-1α expression blocks the inhibitory effect of Dex on neuronal apoptosis in I/R rats. Dex may inhibit the neuronal apoptosis of I/R rats by inhibiting the HIF-1α pathway and then improve the cerebral I/R injury in rats.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

Protective effects of caffeic acid phenethyl ester on skeletal muscle ischemia-reperfusion injury in rats

There is a great evidence that reactive oxygen species (ROS) play an important role in the pathophysiology of ischemia −reperfusion(I/R)injury in skeletal muscle.Caffeic acid phenethyl ester(CAPE)is a component of honeybeep ropolis.It has antioxidant, anti−inflammatory and free radical scavenger properties.The aim of this study is to determine the protective effects of CAPE against I/R injury in respect of protein oxidation, neutrophil in filtration, and the activities of xanthine oxidase(XO)and adenosine deaminase(AD)onan<invivomodel of skeletal muscle I/R injury.Rats were divided into three equal groups each consisting of sixrats:Sham operation, I/R, and I/R plus CAPE(I/R+CAPE)groups.CAPE was administered intraperitoneally 60 min before the beginning of the reperfusion.At the end of experimental procedure, blood and gastrocnemius muscle tissues were used for biochemical analyses.Tissue protein carbonyl(PC)levels and the activities of XO, myeloperoxidase(MPO) and AD in I/R group were significantly higher than that of control(p0.01, p0.05, p0.01, p0.005, respectively).Administration of CAPE significantly decreased tissue PC levels, MPO and XO activities in skeletal muscle compared to I/R group(p0.01, p0.05, p0.05, respectively).In addition, plasma creatine phosphokinase(CPK), XO and ADactivities were decreased in I/R+CAPE group compared to I/R group(p0.05, p0.05, p0.001). The results of this study revealed that free radical attacks may play an important role in the pathogenesis of skeletal muscle I/R injury. Also, the potent free radical scavenger compound, CAPE, may have protective potential in this process. Therefore, it can be speculated that CAPE or other antioxidant agents may be useful in the treatment of I/R injury as well as diffused traumatic injury of skeletal muscle.     

Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1β mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.     

缺血后处理降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的易感性及其机制

目的:探讨缺血后处理对高胆固醇血症基础上发生的心肌缺血/再灌注损伤的影响及其可能的机制。方法:建立食源性高胆固醇血症大鼠模型,运用TTC染色、酶活性检测等方法测定缺血/再灌注所致的心肌损伤,用实时定量RT-PCR方法检测心肌组织中低氧诱导因子-1α(HIF-1α)mRNA水平,用Western blot方法检测HIF-1α蛋白水平。结果:高胆固醇血症加重了缺血/再灌注造成的心肌损伤,而缺血后处理显著缩小了高胆固醇血症大鼠缺血/再灌注所致的心梗面积,降低了血清肌酸激酶(CK)的活性,减少了心肌细胞凋亡。同时,缺血后处理提高了高胆固醇血症大鼠缺血心肌组织中HIF-1α的蛋白水平。结论:缺血后处理可以降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的敏感性,其效应与心肌组织中HIF-1α的蛋白水平存在着相关性。     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

The effect of cathepsin K deficiency on airway development and TGF-β1 degradation

Background

Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.

Methods

The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.

Results

HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120_CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120_CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.

Conclusion

In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.     

The Suppressive Effect of Resveratrol on HIF-1α and VEGF Expression after Warm Ischemia and Reperfusion in Rat Liver

Background

Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in many human tumors and their metastases, and is closely associated with a more aggressive tumor phenotype. The aim of the present study was to investigate the effect of resveratrol (RES) on the expression of ischemic-induced HIF-1α and vascular endothelial growth factor (VEGF) in rat liver.

Methods

Twenty-four rats were randomized into Sham, ischemia/reperfusion (I/R), and RES preconditioning groups. I/R was induced by portal pedicle clamping for 60 minutes followed by reperfusion for 60 minutes. The rats in RES group underwent the same surgical procedure as I/R group, and received 20 mg/kg resveratrol intravenously 30 min prior to ischemia. Blood and liver tissue samples were collected and subjected to biochemical assays, RT-PCR, and Western blot assays.

Results

I/R resulted in a significant (P<0.05) increase in liver HIF-1α and VEGF at both mRNA and protein levels 60 minutes after reperfusion. The mRNA and protein expressions of HIF-1α and VEGF decreased significantly in RES group when compared to I/R group (P<0.05).

Conclusion

The inhibiting effect of RES on the expressions of HIF-1α and VEGF induced by I/R in rat liver suggested that HIF-1α/VEGF could be a promising drug target for RES in the development of an effective anticancer therapy for the prevention of hepatic tumor growth and metastasis.     

Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver, and liver injury. This study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild-type (WT), CYP2E1-knock-in (KI), and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair-fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolyl hydroxylase 2, which promotes HIF-1α degradation, were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol-fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were colocalized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells, which express CYP2E1, with ethanol plus arachidonic acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a greater extent than in HepG2 C34 cells, which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than in C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress, and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis, and liver injury.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Ischemia-reperfusion leads to depletion of glutathione content and augmentation of malondialdehyde production in the rat heart from overproduction of oxidants: Can caffeic acid phenethyl ester (CAPE) protect the heart?

During restoration of blood flow of the ischemic heart induced by coronary occlusion, free radicals cause lipid peroxidation with myocardial injury. Lipid peroxidation end-products, such as malondialdehyde (MDA), have been used to assess oxygen free radical-mediated injury of the ischemic-reperfused (I/R) myocardium in rats. This experimental study assessed the preventive effect of caffeic acid phenthyl ester (CAPE), antioxidant, on I/R-induced lipid peroxidation in the rat heart. We are also interested in the role of CAPE on glutathione (GSH) levels, an antioxidant whose levels are influenced by oxidative stress. I/R leads to the depletion of GSH which is the major intracellular nonprotein sulphydryl and plays an important role in the maintenance of cellular proteins and lipid in their functional state and acts primarily to protect these important structures against the threat of oxidation. In addition, we also examined morphologic changes in the heart by using light microscopy. The left coronary artery was occluded for 30 min and then reperfused for 120 min more before the experiment was terminated. CAPE (50 M kg^–1) was administered 10 min prior to ischemia and during occlusion by infusion. At the end of the reperfusion period, rats were sacrificed, and the heart was quickly removed for biochemical determination and histopathological analysis. I/R was accompanied by a significant increase in MDA production and decrease in GSH content in the rat heart. Administration of CAPE reduced MDA production and prevented depletion of GSH content. These beneficial changes in these biochemical parameters were also associated with parallel changes in histopathological appearance. These findings imply that I/R plays a causal role in heart injury due to overproduction of oxygen radicals or insufficient antioxidant and CAPE exert cardioprotective effects probably by the radical scavenging and antioxidant activities.     

Binge alcohol promotes hypoxic liver injury through a CYP2E1–HIF-1α-dependent apoptosis pathway in mice and humans

Binge drinking, a common pattern of alcohol ingestion, is known to potentiate liver injury caused by chronic alcohol abuse. This study was aimed at investigating the effects of acute binge alcohol on hypoxia-inducible factor-1α (HIF-1α)-mediated liver injury and the roles of alcohol-metabolizing enzymes in alcohol-induced hypoxia and hepatotoxicity. Mice and human specimens assigned to binge or nonbinge groups were analyzed for blood alcohol concentration (BAC), alcohol-metabolizing enzymes, HIF-1α-related protein nitration, and apoptosis. Binge alcohol promoted acute liver injury in mice with elevated levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and hypoxia, both of which were colocalized in the centrilobular areas. We observed positive correlations among elevated BAC, CYP2E1, and HIF-1α in mice and humans exposed to binge alcohol. The CYP2E1 protein levels (r = 0.629, p = 0.001) and activity (r = 0.641, p = 0.001) showed a significantly positive correlation with BAC in human livers. HIF-1α levels were also positively correlated with BAC (r = 0.745, p < 0.001) or CYP2E1 activity (r = 0.792, p < 0.001) in humans. Binge alcohol promoted protein nitration and apoptosis with significant correlations observed between inducible nitric oxide synthase and BAC, CYP2E1, or HIF-1α in human specimens. Binge-alcohol-induced HIF-1α activation and subsequent protein nitration or apoptosis seen in wild type were significantly alleviated in the corresponding Cyp2e1-null mice, whereas pretreatment with an HIF-1α inhibitor, PX-478, prevented HIF-1α elevation with a trend of decreased levels of 3-nitrotyrosine and apoptosis, supporting the roles of CYP2E1 and HIF-1α in binge-alcohol-mediated protein nitration and hepatotoxicity. Thus binge alcohol promotes acute liver injury in mice and humans at least partly through a CYP2E1–HIF-1α-dependent apoptosis pathway.     

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.     

Caffeic acid phenethyl ester changes the indices of oxidative stress in serum of rats with renal ischaemia–reperfusion injury

Oxygen‐derived free radicals have been implicated in the pathogenesis of renal injury after ischaemia–reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. To investigate whether treatment with either CAPE or alpha‐tocopherol modifies the levels of the endogenous indices of oxidant stress, we examined their effects on an in vivo model of renal ischaemia–reperfusion injury in rats. CAPE at 10 μmol kg^?1 or alpha‐tocopherol at 10 mg kg^?1 was administered intraperitoneally before reperfusion. Acute administration of both CAPE and alpha‐tocopherol altered the indices of oxidative stress differently in renal ischaemia–reperfusion injury. Copyright © 2001 John Wiley & Sons, Ltd.