The protective role of 5-hydroxymethyl-2-furfural (5-HMF) against acute hypobaric hypoxia

Our previous study showed that pretreatment with 5-hydroxymethyl-2-furfural (5-HMF) led to protection against hypoxic injury via a p-ERK-mediated pathway in vitro. Whether the protection of 5-HMF against hypoxia is effective in vivo is unknown. The present study is aimed to verify the role of 5-HMF in acute hypobaric hypoxia using Kunming mice as an in vivo model and further investigate the underlying mechanisms. Mice pretreated with or without 5-HMF for 1 h were exposed to acute hypobaric hypoxic condition for 6 h and then the survival time, the survival rate, the permeability of blood–brain barrier (BBB), the histological analysis in hippocampus and cortex, and the phosphorylation level of mitogen-activated protein kinases (ERK, JNK, and p38) were investigated. The results showed that 5-HMF significantly increased the survival time and the survival rate of mice. Accordingly, pretreatment with 5-HMF markedly attenuated acute hypobaric hypoxia-induced permeability of BBB (P < 0.01). In addition, the cellular damage extent of the hippocampus and the cortex induced by hypoxia for 6 h was also attenuated by pretreatment with 5-HMF, especially in the hippocampus CA1 region. Furthermore, the activation of ERK rather than JNK and p38 was involved in the protection of 5-HMF against acute hypobaric hypoxia. In summary, 5-HMF enhanced the survival capability of mice and decreased acute hypoxic damage to the brain, which may be associated with the effects on BBB and p-ERK.     

Oxygen tension regulates the maturation of the blood-brain barrier.

The oxygen tension during the development of vascular systems influences vascular vessel formation through regulating angiogenesis. We studied the effect of hypoxia/reoxygenation (H/R) to explain its role in concert with astrocytes involvement in the development of the blood-brain barrier (BBB). On the basis of the fact that the disappearance of hypoxic regions and the decreased expression of vascular endothelial growth factor (VEGF) were observed by immunohistochemistry in a development-dependent manner in rat cerebral cortex, we examined the effects of astrocytes on the BBB-like properties of ECV304 cells by exposing astrocytes to H/R. Conditioned medium of reoxygenated astrocytes inhibited [(3)H]thymidine incorporation and tube formation of ECV 304 cells. When astrocytes were exposed to reoxygenation, the expression of VEGF was reduced, whereas the expression of angiopoietin-1 and thrombospondin-1 was enhanced. Moreover, [(3)H]sucrose permeability assay revealed that astrocytes enhance the barrier function of ECV 304 cells in coculture model within 5 h of reoxygenation. Correspondingly, the occludin expression of ECV 304 cells was slightly increased by the conditioned medium of reoxygenated astrocytes. In conclusion, our study suggests that reoxygenation of astrocytes may act as a significant driving force for the maturation of the BBB during brain development through oxygen-regulated gene(s).     

Efficiency of homocysteine plus copper in inducing apoptosis is inversely proportional to gamma-glutamyl transpeptidase activity.

Hyperhomocysteinemia represents an independent risk factor for atherosclerosis, but the mechanisms leading to cellular dysfunctions remain unknown. Using ECV304 cells, we found that homocysteine (Hcy) plus copper (Cu2+) induced cytotoxic effects: loss of cell adhesion, increased permeability to PI, and the occurrence of morphologically apoptotic cells. This form of apoptosis, inhibited by Z-VAD-fmk, was associated with a loss of mitochondrial potential, a cytosolic release of cytochrome c, activation of caspase-3, degradation of poly(ADP-ribose)polymerase, and internucleosomal DNA fragmentation. However, the ability of Hcy plus Cu2+ to induce apoptosis decreased when the pretreatment culture time increased. As a positive correlation was found between the length of time of culture before treatment and the enhancement of gamma-glutamyl transpeptidase (gamma-GT) activity, we asked whether gamma-GT was involved in the control of Hcy plus Cu2+-induced apoptosis. Therefore, ECV304 cells were treated with either acivicin or dexamethasone, inhibiting and stimulating gamma-GT, respectively. In ECV304 cells and human umbilical venous endothelial cells, acivicin favored Hcy plus Cu2+-induced apoptosis whereas dexamethasone counteracted the apoptotic process. As acivicin and dexamethasone were also capable of modulating cell death in ECV304 cells treated with antitumoral drugs, our data emphasize that the involvement of gamma-GT in the control of apoptosis is not restricted to Hcy but also concerns other chemical compounds.     

Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

Puerarin induces mitochondria-dependent apoptosis in hypoxic human pulmonary arterial smooth muscle cells

Background

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs), and to determine the possible underlying mechanisms.

Methodology/Principal Findings

HPASMCs were cultured for 24 h in normoxia or hypoxia (5% O₂) conditions with and without puerarin. Cell number and viability were determined with a hemacytometer or a cell counting kit. Apoptosis was detected with a TUNEL test, rhodamine-123 (R-123) fluorescence, a colorimetric assay, western blots, immunohistochemical staining and RT-PCR. Hypoxia inhibited mitochondria-dependent apoptosis and promoted HPASMC growth. In contrast, after puerarin (50 µM or more) intervention, cell growth was inhibited and apoptosis was observed. Puerarin-induced apoptosis in hypoxic HPASMCs was accompanied by reduced mitochondrial membrane potential, cytochrome c release from the mitochondria, caspase-9 activation, and Bcl-2 down-regulation with concurrent Bax up-regulation.

Conclusions/Significance

Puerarin promoted apoptosis in hypoxic HPASMCs by acting on the mitochondria-dependent pathway. These results suggest a new mechanism of puerarin relevant to the management of clinical hypoxic pulmonary hypertension.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Effect of cigarette smoke and dexamethasone on Hsp72 system of alveolar epithelial cells

Smoking is the leading risk factor of chronic obstructive pulmonary disease (COPD) and lung cancer. Corticosteroids are abundantly used in these patients; however, the interaction of smoking and steroid treatment is not fully understood. Heat shock proteins (Hsps) play a central role in the maintenance of cell integrity, apoptosis and cellular steroid action. To better understand cigarette smoke-steroid interaction, we examined the effect of cigarette smoke extract (CSE) and/or dexamethasone (DEX) on changes of intracellular heat shock protein-72 (Hsp72) in lung cells. Alveolar epithelial cells (A549) were exposed to increasing doses (0; 0.1; 1; and 10 μM/μl) of DEX in the medium in the absence(C) and presence of CSE. Apoptosis, necrosis, Hsp72 messenger-ribonucleic acid (mRNA) and protein expression of cells were measured, and the role of Hsp72 on steroid effect examined. CSE reduced the number of viable cells by significantly increasing the number of apoptotic and necrotic cells. DEX dose-dependently decreased the ratio of apoptosis when CSE was administered, without change in necrosis. CSE − DEX co-treatment dose-dependently increased Hsp72 mRNA and protein expression, with the highest level measured in CSE + DEX (10) cells, while significantly lower levels were noted in all respective C groups. Pretreatment with Hsp72 silencing RNA confirmed that increased survival observed following DEX administration in CSE-treated cells was mainly mediated via the Hsp72 system. CSE significantly decreases cell survival by inducing apoptosis and necrosis. DEX significantly increases Hsp72 mRNA and protein expression only in the presence of CSE resulting in increased cellular protection and survival. DEX exerts its cell protective effects by decreasing apoptotic cell death via the Hsp72 system in CSE-treated alveolar epithelial cells.     

Planning and monitoring of patients for electrical cardioversion for atrial fibrillation

Objectives

This study evaluated the waiting list for elective electrical cardioversion (ECV) for persistent atrial fibrillation (AF), focusing on when and why procedures were postponed. We compared the effects of management of the waiting list conducted by physicians versus management by nurse practitioners (NPs) and we evaluated the safety of our anticoagulating policy by means of bleeding or thromboembolic complications during and after ECV.

Background

Not all patients selected for ECV receive their treatment at the first planned instance due to a variety of reasons. These reasons are still undocumented.

Methods

We evaluated 250 consecutive patients with persistent AF admitted to our clinic for elective ECV.

Results

Within 5 to 6 weeks, 186 of 242 patients (77%) received ECV. The main reason for postponing an ECV was an inadequate international normalised ratio (INR); other reasons included spontaneous sinus rhythm and switch to rate control. A total of 23 of the 147 patients (16%) managed by the research physician were postponed due to an inadequate INR at admission versus 4 out of 98 patients (4%) managed by NPs (p = 0.005)

Conclusion

An inadequate INR is the main reason for postponing an ECV. Management of ECV by NPs is safe and leads to less postponing on admission.     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.     

MAPK pathway mediates the protective effects of onychin on oxidative stress-induced apoptosis in ECV304 endothelial cells

Our recent studies have shown that onychin could protect rabbit aortic rings from lysophosphatidylcholine-induced injury by preserving endothelium-dependent relaxation and alleviating acute endothelial damage mediated by oxidative stress. However, the effect of onychin on apoptosis of endothelial cells induced by oxidative stress was not evaluated. In the present study, we investigated the effect of onychin on Hydrogen Peroxide (H2O2) induced apoptosis of ECV304 endothelial cells. Cultured human umbilical vein endothelial cell line (ECV304) was pretreated with vehicle (DMSO), genistein, or different concentrations of onychin (0.1, 0.3, 1, 3, and 10 micromol/L) for 30 minutes and then exposed to 1 mmol/L H2O2 for 24 hours. Cell apoptosis was determined by TUNEL and flow cytometric analysis. Meanwhile, Western-blot was used to measure the expression of phospho-ERK1/2, phospho-p38 and caspase-3. Our data showed that onychin treatment exhibited a protective effect on ECV304 endothelial cells from H2O2-induced apoptosis in a concentration-dependent manner. Moreover, onychin attenuated H2O2-induced phosphorylation of p38MAPK and increased H2O2-induced phosphorylation of ERK1/2. Furthermore, onychin decreased the activation of caspase-3. The opposing effects of onychin on phosphorylation levels of p38MAPK and ERK1/2, and its caspase-3 inhibition might play a role in the beneficial effect of onychin on endothelial injury.     

Trichostatin A,an Inhibitor of Histone Deacetylase,Inhibits the Viability and Invasiveness of Hypoxic Rheumatoid Arthritis Fibroblast‐Like Synoviocytes via PI3K/Akt Signaling

This study was undertaken to explore the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, on the viability, apoptosis, and invasiveness of hypoxic rheumatoid arthritis fibroblast‐like synoviocytes (RA FLSs). RA FLSs were exposed to hypoxia for 24 h in the presence or absence of 2 μM TSA and tested for cell viability, apoptosis, invasion, and gene expression. The involvement of the phosphatidylinositol‐3‐kinase (PI3K)/Akt pathway was checked. TSA significantly inhibited the viability and induced apoptosis of hypoxic RA FLSs, compared to vehicle control. TSA blocked hypoxia‐induced invasion of RA FLSs during Matrigel invasion assays and reduced the expression of matrix metalloproteinases (MMP‐2 and MMP‐9) and PI3K and phosphorylation of Akt. Overexpression of constitutively active Akt reversed TSA‐mediated suppression of invasiveness and downregulation of MMP‐2 and MMP‐9. Our results indicate the antisurvival and antiinvasive activities of TSA in hypoxic RA FLSs, which is associated with inactivation of PI3K/Akt signaling.     

Suppression of TNFalpha-mediated NFkappaB activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells.

Flavonoids are a group of naturally-occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor-kappaB (NFkappaB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFkappaB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFkappaB/inhibitor-kappaB (IkappaB) system in ECV304 cells activated with tumor necrosis factor-alpha (TNFalpha) were examined. We investigated the inhibitory action of six flavonoids on IkappaB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IkappaB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IkappaBalpha and IkappaBbeta in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFkappaB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFkappaB/IkappaB system.     

Phospholipase D protects ECV304 cells against TNFalpha-induced apoptosis

Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.     

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4 ± 4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ± 0.00; 0.28 ± 0.01, and 0.31 ± 0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ± 0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.     

Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide

Ligusticum chuanxiong and Angelica sinensis have been widely used in traditional Chinese medicine to treat some pathological settings such as atherosclerosis and hypertension. We determined the protective effect of the extract of Ligusticum chuanxiong and Angelica sinensis (ELCAS) on human umbilical vein endothelial cells (ECV304) damage induced by hydrogen peroxide. ECV304 cells were pre-treated with ELCAS and exposed to 5 mM hydrogen peroxide. The results show that ELCAS dose- and time-dependently protected ECV304 cells against hydrogen peroxide damage and suppressed the production of reactive oxygen species (ROS). The decrement of ROS may be associated with increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Western blot analysis revealed that ELCAS significantly increased the phosphorylation of ERK and promoted eNOS expression. These observations indicate that ELCAS protected ECV304 cells against hydrogen peroxide damage by enhancing the antioxidative ability, activating ERK and eNOS signaling pathway. Our data also provide new evidence of Ligusticum chuanxiong and Angelica sinensis in preventing both cardiovascular and cerebrovascular diseases.