Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells

BackgroundApoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5′-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC.Methods and resultsTo simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po₂ < 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/?0.4; P < 0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/?0.5; P < 0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αβ-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP.ConclusionThe data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.     

IL-4 induces apoptosis of endothelial cells through the caspase-3-dependent pathway

The present study was designed to investigate the hypothesis that interleukin-4 (IL-4) can induce apoptosis of human endothelial cells and to study regulatory pathways of this process. Indeed, DNA ladder assay and flow cytometry study showed that IL-4 can induce apoptosis of endothelial cells in a time- and dose-dependent manner. In addition, IL-4 markedly increased activity of caspase-3, and inhibition of this enzyme suppressed IL-4-induced apoptosis in a dose-dependent manner. These results provide the first evidence that IL-4 can induce apoptosis of human endothelial cells. In addition, the data indicate that the caspase-3-dependent pathway is critically involved in this process.     

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.     

Integrin ανβ3 modulates lipopolysaccharide-induced hyperpermeability in cardiac microvascular endothelial cells

ABSTRACT

Previous studies suggest an association of cardiac microvascular endothelial cells (CMECs) hyperpermeability with sepsis-related cardiac injury. Our results showed that CMECs permeability was dependent upon concentration and time of lipopolysaccharides (LPS) stimulation. Integrin ανβ3 expression decreased after LPS stimulation. Pretreatment with anti-integrin ανβ3 antibody enhanced LPS-induced hyperpermeability. Upregulation of integrin ανβ3 decreased LPS-induced hyperpermeability. F-actin remodeling was enhanced after LPS stimulation and was inhibited by up-regulation of integrin ανβ3. Inhibition of Src or Rac1 reduced CMECs permeability after LPS stimulation, but there were no differences in the phosphorylation of Src and Rac1 when over-expressing or blocking integrin β3. After pretreatment with Src or Rac1 inhibitor, no significant difference was found in the expression of integrin ανβ3 in LPS-induced CMECs. These finding suggested that integrin ανβ3 overexpression decreased LPS-stimulated CMECS permeability by inhibition of cytoskeletal remodeling, but the mechanism might not be mediated via Src/Rac1 signaling.     

Caveolar structure and protein sorting are maintained in NIH 3T3 cells independent of glycosphingolipid depletion

Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.     

Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes

There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.     

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes. Takai and Matikainen contributed equally to this work.     

脂多糖调控对大鼠心脏微血管内皮细胞的转录组分析

目的探索脂多糖(LPS)对大鼠心脏微血管内皮细胞(rCMECs)转录组的调节作用。方法对照组(正常培养rCMECs),LPS组(100 ng/mL LPS处理6 h的rCMECs),每组进行3个生物学重复转录组测序。得到差异基因后,使用实时定量PCR对部分差异基因mRNA的表达进行验证。分别对上调和下调基因进行GO和KEGG富集,并对差异基因进行共表达网络分析。采用独立t检验进行统计学分析。结果LPS处理后,265个基因表达上调,118个基因的表达下调。前10个最显著上调基因为:Mt2a、Cyp7b1、Sod2、Icam1、Ccl2、AC128848.1、Mt1、Cebpd、Serpinb2和Tnfrsf11b。前10个最显著下调基因为:Cavin2、Ankrd1、Edn1、Prss35、Lmod1、Dhrs3、Ttc22、Sema6a、Map2k3和Sema7a。定量PCR的结果表明Mt2a、Sod、Ccl2、Cxcl1、Icam1和Vcaml基因的表达得到了上调(P均<0.01);而Cavin2、Ankrd1、Edn1和Prss35基因表达下调(P均<0.05)。GO和KEGG富集的结果表明,上调基因与内皮细胞对炎性免疫细胞的趋化作用和黏附作用密切相关;而下调基因则是与钙离子信号和G蛋白相关通路以及内皮通透性增加有关。此外,差异基因进行共表达网络分析发现Sod2处于核心位置,提示其可能与LPS诱导的rCMECs的各种变化密切相关。结论LPS调控了rCMECs中大量与炎性免疫细胞进入心肌组织相关基因的表达。     

Diabetes alters intracellular calcium transients in cardiac endothelial cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.     

Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells

Green tea polyphenol-(-)epigallocatechin-3-gallate (EGCG)-is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In this study we describe a novel observation that EGCG displayed strong inhibitory effects on the proliferation and viability of HTB-94 human chondrosarcoma cells in a dose-dependent manner and induced apoptosis. Investigation of the mechanism of EGCG-induced apoptosis revealed that treatment with EGCG resulted in DNA fragmentation, induction of caspase-3/CPP32 activity, and cleavage of the death substrate poly(ADP-ribose)polymerase (PARP). Pretreatment of cells with a synthetic pan-caspase inhibitor (Z-VAD-FMK) and a caspase-3-specific inhibitor (DEVD-CHO) prevented EGCG-induced PARP cleavage. The induction of apoptosis by EGCG via activation of caspase-3/CPP32-like proteases may provide a mechanistic explanation for its antitumor effects.     

Macrophage-mediated clearance of cells undergoing caspase-3-independent death

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide--namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments - are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages - both by CD14-dependent and CD14-independent mechanisms--do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.     

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca(2+) activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca(2+) activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

Hamster Bcl-2 protein is cleaved in vitro and in cells by caspase-9 and caspase-3

Full-length cDNA of hamster bcl-2 (771 nt) was cloned by RT-PCR and inserted into pGEX-4T-1 to produce the recombinant hamster Bcl-2 protein. The purified recombinant Bcl-2 protein (26.4 kDa) was used as a substrate for the active human caspase-3 and caspase-9 in vitro. It is shown here that Bcl-2 is efficiently cleaved by caspase-3 to a 23 kDa fragment. Although not possessing a putative caspase-9 cleavage site in its sequence, hamster Bcl-2 was also cleaved by caspase-9 into exactly the same 23 kDa cleavage product, indicating that cleavage occurred at the same site. Caspase-3- and caspase-9-mediated cleavage of Bcl-2 was efficiently blocked by caspase-3 (zDEVD) and caspase-9 (zLEHD) inhibitor, respectively. We also show that caspase-9/-3-mediated cleavage of Bcl-2 occurs in vivo during apoptosis in CHO-HSV-TK cells after exposure to the antiviral drug ganciclovir.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.     

Metabolic compartmentation and substrate channelling in muscle cells

The published experimental data and existing concepts of cellular regulation of respiration are analyzed. Conventional, simplified considerations of regulatory mechanism by cytoplasmic ADP according to Michaelis-Menten kinetics or by derived parameters such as phosphate potential etc. do not explain relationships between oxygen consumption, workload and metabolic state of the cell. On the other hand, there are abundant data in literature showing microheterogeneity of cytoplasmic space in muscle cells, in particular with respect to ATP (and ADP) due to the structural organization of cell interior, existence of multienzyme complexes and structured water phase. Also very recent experimental data show that the intracellular diffusion of ADP is retarded in cardiomyocytes because of very low permeability of the mitochondrial outer membrane for adenine nucleotidesin vivo. Most probably, permeability of the outer mitochondrial membrane porin channels is controlled in the cellsin vivo by some intracellular factors which may be connected to cytoskeleton and lost during mitochondrial isolation. All these numerous data show convincingly that cellular metabolism cannot be understood if cell interior is considered as homogenous solution, and it is necessary to use the theories of organized metabolic systems and substrate-product channelling in multienzyme systems to understand metabolic regulation of respiration. One of these systems is the creatine kinase system, which channels high energy phosphates from mitochondria to sites of energy utilization. It is proposed that in muscle cells feed-back signal between contraction and mitochondrial respiration may be conducted by metabolic wave (propagation of oscillations of local concentration of ADP and creatine) through cytoplasmic equilibrium creatine and adenylate kinases and is amplified by coupled creatine kinase reaction in mitochondria. Mitochondrial creatine kinase has experimentally been shown to be a powerful amplifier of regulatory action of weak ADP fluxes due to its coupling to adenine nucleotide translocase. This phenomenon is also carefully analyzed.It is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry. P. Mitchell The Ninth Sir Hans Krebs Lecture, Dresden, July 2, 1978.     

Metabolic control and compartmentation in single living cells

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.     

Nitrate transport and compartmentation in cereal root cells

Measurement of cytosolic nitrate is one of the factors requiredfor the resolution of factors controlling nitrate uptake andassimilation in plants and for identifying likely nitrate transportmechanisms at both the plasma membrane and tonoplast. This paperreviews methods and reported measurements of cytosolic nitratein higher plants and concludes that nitrate-selective microelectrodesare the best approach. These microelectrodes have been usedto measure intracellular nitrate activities in barley and maizeroot cells. Triplebarrelled electrodes, incorporating a pH-sensingbarrel have been used to identify the compartmental locationof the nitrate-selective tip giving unequivocal estimates ofvacuolar and cytosolic nitrate activities. The microelectrodemeasurements are used to discuss the possible mechanisms ofnitrate transport at both the tonoplast and plasma membrane.The energetics of possible proton-coupled transport systemsare described and the feasibility of the mechanism is discussed. Key words: Cytosol, compartmentation, Hordeum vulgare L, nitrate, roots, Zea mays L