Apoptosis In Human Endothelial Cells Research Articles

Mechanism and consequences of invasion of endothelial cells by Staphylococcus aureus

It has become clear that Staphylococcus aureus is a facultative intracellular microorganism. Adherence and invasion are a prerequisite for endovascular infections caused by S. aureus, such as infective endocarditis. These phenomena may also be involved in the pathogenesis of invasive and metastatic infection upon hematogenous dissemination, such as osteomyelitis and abscess formation. The underlying molecular mechanism has been elucidated in detail, including its likely relevance in vivo. However, the mode of action of recently identified modulators of invasion, such as pls/Pls have not yet been clarified. The potential outcome for host cells and S. aureus following invasion are diverse. Surprisingly, induction of apoptosis in human endothelial cells is more complex than previously thought, since it appears to involve multiple virulence factors. In the light of increasing resistance to antimicrobial therapy, understanding the multifacetted pathogenesis of S. aureus infection in detail is needed for a better prevention and therapy.

Up‐regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose‐induced apoptosis in human endothelial cells

Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of endothelial nitric oxide synthase (eNOS). eNOS was recently demonstrated to be extensively regulated by Akt and heat shock protein 90 (HSP90). Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interactions between eNOS and HSP90 in high glucose-induced endothelial cell apoptosis. The protein interaction of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5 mM) or high-level (33 mM) glucose for different durations (2, 4, 6, and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were up-regulated by high glucose exposure for 2-4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we propose that, in HUVECs, during early phase of high glucose exposure, apoptosis can be prevented by enhancement of eNOS activity through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt. With longer exposure, dysregulation of eNOS activity would result in apoptosis. The present study provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus.

Angiotensin II-induced apoptosis in human endothelial cells is inhibited by adiponectin through restoration of the association between endothelial nitric oxide synthase and heat shock protein 90

Adiponectin can protect vessels from injury by promoting the activity of endothelial nitric oxide synthase (eNOS) with increased nitric oxide production. Recently, it was demonstrated that eNOS activity is highly regulated by heat shock protein 90 (HSP90). We tested the hypothesis that adiponectin can prevent endothelial cell injury produced by angiotensin II through promotion of the association between eNOS and HSP90. Cultured human umbilical vein endothelial cells (HUVECs) were treated with angiotensin II (2 μM) to induce apoptosis. In the presence of globular adiponectin, apoptosis was inhibited in a dose–response manner. Angiotensin II-induced apoptosis was also inhibited by treatment with an NO donor and by combined treatment with both angiotensin II type 1 and type 2 receptor blockers. Western blotting and immunoprecipitation of the lysates from the treated cells showed that globular adiponectin could restore the association between eNOS and HSP90 and enhance the phosphorylation of eNOS. In conclusion, angiotensin II-induced human endothelial cell apoptosis can be prevented by adiponectin through promotion and stabilization of the association between eNOS and HSP90.

Inhibition of the arachidonic acid metabolism blocks endothelial cell migration and induces apoptosis.

Previous studies have demonstrated that inhibitors of the arachidonic acid metabolism block migration and sensitise human glioma cells to treatment inducing apoptosis. This paradigm may provide a new concept for anti-invasive treatment strategies targeting invasive glioma cells. However, the effect of such treatment on other cellular elements in glial tumours such as endothelial cells is unknown. In this study we have analysed the expression of metabolites of the arachidonic acid pathway in endothelial cells in vitro and in vivo and we have assessed the influence of inhibitors of this pathway on motility, capillary like tube formation, and apoptosis in human endothelial cells. Human endothelial cells (HUVEC) in culture showed expression for thromboxane synthase and both isoforms of cyclo-oxygenase, COX-1 and COX-2. Immunostaining demonstrated low levels of COX-1 expression in capillaries and larger vessels of normal brain and moderately elevated levels of this enzyme in small vessels of brain tumours of various grades. Both thromboxane synthase and COX-2 expression was limited to endothelial cells found in anaplastic gliomas and glioblastomas. Thromboxane synthase inhibitors strongly decreased endothelial cell migration in HUVEC in vitro and capillary like tube formation was strongly inhibited by the compound at a similar dose range. The non-selective cyclo-oxygenase inhibitor ASA and the selective COX-2 inhibitor sulindac only had a minor effect on endothelial cell migration, however, the COX-2 inhibitor sulindac showed a synergistic effect with the thromboxane synthase inhibitor. Thromboxane synthase inhibitors induced apoptosis in endothelial cells as demonstrated by intracellular histone-complexed DNA fragmentation. These data suggest that inhibitors of thromboxane synthase influence migration and apoptosis in both human glioma cells and human endothelial cells. An anti-invasive treatment strategy using this class of compounds may therefore not only sensitise glioma cells to conventional treatments inducing apoptosis but may also be supported by an anti-angiogenic effect.

Crystalline silica induces apoptosis in human endothelial cells in vitro.

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.

Pneumatocoele formation during Pneumocystis carinii pneumonia in a man with HIV-1 infection

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O2-02 angiotensin II-induced apoptosis of human endothelial cells is inhibited by adiponectin through up-regulation of the association between endothelial nitric oxide synthase and heat shock protein 90

O2-02 angiotensin II-induced apoptosis of human endothelial cells is inhibited by adiponectin through up-regulation of the association between endothelial nitric oxide synthase and heat shock protein 90

Effect of vascular endothelial growth factor and epidermal growth factor on iatrogenic apoptosis in human endothelial cells

To study the effect of growth factors on iatrogenic apoptosis, we examined the influence of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on staurosporine-induced apoptosis in primary cultures of human umbilical vein endothelial cells (HUVEC). Apoptosis was evaluated by a cell viability test, the TUNEL-POD assay and the activation of the pro-apoptotic caspase-3. Staurosporine (10–100 nM) caused the activation of caspase-3. This effect was manifest after 2 hr of incubation and reached its maximum after 5 hr. Severe loss of viability followed within 18 hr. VEGF or EGF (10–100 ng/mL) added together with staurosporine decreased the activation of caspase-3. The loss of viability was 24 hr delayed. The action of growth factors was observed at 1% serum concentration but also at concentration optimal for HUVEC survival (10%, v/v). Furthermore, the inhibition of PI-3 kinase (PI-3K) by wortmannin or LY294002 as well as the inhibition of MEK by PD098059 or U0126 prevented the protective effect of VEGF and EGF. Western blotting analysis showed that after 3 hr of incubation with staurosporine the level of the anti-apoptotic protein Mcl-1 decreased and this effect was reverted by VEGF. It is concluded that VEGF and EGF antagonize the pro-apoptotic action of staurosporine by the combined signalling of PI-3K and ERKs pathways.

Redox-dependent apoptosis in human endothelial cells after adhesion of Plasmodium falciparum-infected erythrocytes.

During Plasmodium falciparum infection leading to cerebral malaria, mechanisms such as cytokine generation and cytoadherence of parasitized red blood cells (PRBC) to post-capillary venules are clearly involved. We demonstrated that PRBC adhesion to human lung endothelial cells (HLEC) upregulated TNF-alpha superfamily genes and genes related to apoptosis and inflammation. Apoptosis was confirmed by standard techniques (annexin-V binding, genomic DNA fragmentation, and caspases activation). This apoptotic process involved the cytoplasmic pathway from a death receptor (DR-6, Fas, TNF-R1) through caspase 8, and the mitochondrial pathway though Bad and caspase 9 activation. Oxidative stress has been implicated in apoptosis induction in various pathological models. Superoxide anion (O(2)*(-)) is a key molecule in the oxidative stress pathway which can form peroxynitrites (ONOO(-)) in association with nitric oxide (NO*). Even though the role of NO* in malaria physiopathology is still a matter of controversy, we demonstrated that PRBC-induced apoptosis in endothelial cells is mediated through an oxidative stress pathway. The inhibition of NO* synthesis protected the endothelial cells suggesting a deleterious role for NO*. In addition, the superoxide dismutase mimetic, MnTBAP, also protected the HLEC against PRBC-induced apoptosis, revealing the role of O(2)*(-) and ONOO(-).

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway.

The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (MPhi), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/MPhi when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/MPhi to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/MPhi refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.

Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells

We have recently demonstrated that the redox reactant pyruvate prevents hydrogen peroxide (H 2O 2)-induced endothelial apoptosis and that its anti-apoptotic feature is mediated partially through the mitochondrial compartment. However, little is known about molecular signal pathways that mediate the anti-apoptotic feature of pyruvate. A biochemical approach to elucidate such signal pathways was attempted in human umbilical vein endothelial cells (HUVECs). Effects of antioxidant pyruvate were compared with those of cytosolic reductant l-lactate, redox-neutral acetate, and malate-aspartate shuttle blocker aminooxyacetate. Various indices of endothelial apoptosis were correlated with cell viability. Submillimolar H 2O 2 caused >50% cell killing, as manifested by its oxidant insult. The massive cell death induced by H 2O 2 was inhibited by pyruvate but not by l-lactate or aminooxyacetate, suggesting a role of cytosolic NADH reducing equivalents, possibly via stimulated oxidant generation. The induction and nuclear translocation of p53 by H 2O 2 was blocked by pyruvate and appeared to be somewhat enhanced by l-lactate or aminooxyacetate in association with oxidant generation. Nuclear translocation of p53 accompanied the transactivation of bax and downregulation of bcl-2. The pyruvate-related redox manipulation inhibited the H 2O 2-induced p53 activation, restored the downregulated bcl-2 and the upregulated bax, and hence enhanced the bcl-2/bax expression ratio. In contrast, l-lactate, acetate, or aminooxyacetate had no such effect. These results indicate that pyruvate could modulate key regulatory signal pathways in cytosol and mitochondrial matrix, thereby inactivating endothelial death pathways. Furthermore, it is suggested that stabilizing the expression of bcl-2 and bax genes by metabolic antioxidants may be an effective strategy for endothelial protection against oxidative stress.

Migration inhibitory factor mediates angiogenesis via mitogen-activated protein kinase and phosphatidylinositol kinase.

In this study, we investigated the effects of migration inhibitory factor (rhMIF) on angiogenesis-related signaling cascades and apoptosis in human endothelial cells (ECs). We show that in vitro rhMIF induces migration and tube formation in Matrigel of human dermal microvascular endothelial cells (HMVECs), with potency comparable to that of basic fibroblast growth factor. In vivo, rhMIF induces angiogenesis in Matrigel plugs and in the corneal bioassay. Using panels of relatively specific kinase inhibitors, antisense oligonucleotides, and dominant-negative mutants, we show that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) are critical for MIF-dependent HMVEC migration, whereas Src and p38 kinases are nonessential. Moreover, we demonstrate that rhMIF induces time-dependent increases in phosphorylation levels of MEK1/2, Erk1/2, and Elk-1, as well as PI3K, and its effector kinase, Akt, in HMVECs. Studies with dominant-negative mutants and antisense oligonucleotides corroborate these effects in HMVECs. Furthermore, we demonstrate that rhMIF-induced angiogenesis in the rat cornea in vivo and in the ex vivo endothelial cell morphogenesis assay is also MAPK- and PI3K-dependent. Our findings support a role for MIF as an angiogenic factor and provide a rationale for the use of MIF as a therapeutic inducer of neovascularization in the development of collateral circulation in coronary artery disease.

Role of Raf in Vascular Protection from Distinct Apoptotic Stimuli

Raf kinases have been linked to endothelial cell survival. Here, we show that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) differentially activate Raf, resulting in protection from distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. bFGF activated Raf-1 via p21-activated protein kinase-1 (PAK-1) phosphorylation of serines 338 and 339, resulting in Raf-1 mitochondrial translocation and endothelial cell protection from the intrinsic pathway of apoptosis, independent of the mitogen-activated protein kinase kinase-1 (MEK1). In contrast, VEGF activated Raf-1 via Src kinase, leading to phosphorylation of tyrosines 340 and 341 and MEK1-dependent protection from extrinsic-mediated apoptosis. These findings implicate Raf-1 as a pivotal regulator of endothelial cell survival during angiogenesis.

Plasmodium falciparum–Infected Erythrocyte Adhesion Induces Caspase Activation and Apoptosis in Human Endothelial Cells

During Plasmodium falciparum infection leading to cerebral malaria, cytokine production and cytoadherence of parasitized erythrocytes (PRBCs) to postcapillary venules are involved. We demonstrate that PRBC adhesion induces apoptosis in human endothelial cells (HLECs). PRBC adhesion modulated HLEC gene expression in tumor necrosis factor-alpha superfamily genes (Fas, Fas L, and DR-6) and apoptosis-related genes (Bad, Bax, caspase-3,SARP 2, DFF45/ICAD, IFN-gamma receptor 2, Bcl-w, Bik, and iNOS). Apoptosis was confirmed by (1) morphological modifications by electron microscopy, (2) annexin V binding, (3) DNA degradation, by measuring intracytoplasmic nucleosomes, and (4) caspase activity. The apoptotic stimulus was physical contact between HLECs and PRBCs and not parasite-secreted molecules. In addition, it was found that cytoplasmic (caspase 8) and mitochondrial (caspase 9) pathways were involved in this process. These data not only describe the direct apoptotic effect of PRBC adhesion on endothelial cells but also provide new useful tools that allow an evaluation of potential pharmaceuticals.

1P-0257 Binding of thrombospondin 1 to CD36 induces apoptosis of human endothelial cells through activation of p38 mitogen activated protein kinase

1P-0257 Binding of thrombospondin 1 to CD36 induces apoptosis of human endothelial cells through activation of p38 mitogen activated protein kinase

Anandamide induces apoptosis in human endothelial cells: its regulation system and clinical implications

Anandamide (AEA), an endogenous cannabinoid, is generated by macrophages during shock conditions, and is thought to be a causative mediator of septic shock. Thus, we hypothesized that AEA plays a crucial role in endothelial cell (EC) injury. Here, we demonstrate that AEA induces apoptosis in a time-and dose-dependent manner in human umbilical vein endothelial cells (HUVECs). AEA triggered phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen activated protein kinase. AEA also showed a marked increase of interleukin Ibeta- converting enzyme (ICE)CED-3 family protease (caspase-3) activity. AEA-induced EC death was inhibited by a selective vanilloid receptor 1 (VR1) antagonist, capsazepine, and was enhanced by a VR1 agonist, capsaicin, indicating that AEA induces apoptosis in ECs via VR1. In conclusion, we propose that AEA may play a crucial role in EC injury under conditions of shock, and that the use of inhibitors of the AEA regulation system may have a therapeutic effect under these conditions.

A novel caspase dependent pathway is involved in apoptosis of human endothelial cells by Shiga toxins.

Shiga toxins have been shown to induce apoptosis on primary cultures, but not passaged ones, of human umbilical vein endothelial cells, independent of cytokine pre-treatment. Here, a peculiar pattern of caspase activation was observed; caspase-3 and -2, but not conventional upstream caspases, were activated at the initial phase of 6 hr, whereas a broad range inhibitor of caspases, VAD-fmk, but not mono-specific ones, suppressed DNA fragmentation and cell death. These results suggest additional analogous molecules, which have yet to be delineated, are involved. The requirement of retrograde uptake of toxins was also proved by the intervening effect of brefeldin A.

TIRAP mediates endotoxin-induced NF-κB activation and apoptosis in endothelial cells

Bacterial lipopolysaccharide (LPS) initiates multiple signaling events in vascular endothelial cells that can result in activation and/or cell death. LPS-induced activation of endothelial cells elicits a wide array of vascular endothelial responses, many of which are dependent on NF-κB activation. Several of the signaling molecules that mediate LPS-induced NF-κB activation, including Tlr-4, MyD88, and IRAK-1, have been similarly reported to mediate LPS pro-apoptotic signaling. Recently, a new signaling molecule, TIRAP, has been identified that mediates LPS-induced NF-κB signaling in monocytes and macrophages. Using a TIRAP dominant negative construct, we have identified a role for TIRAP in mediating LPS-induced NF-κB activation and apoptosis in human endothelial cells. These data identify TIRAP as a dual functioning signaling molecule and suggest the presence of a MyD88-independent LPS signaling pathway in human endothelial cells.

Synergistic induction of apoptosis in human endothelial cells by tumour necrosis factor-alpha and transforming growth factor-beta.

Serum deprivation stimulates endothelial apoptosis while albumin inhibits this and has been proposed as important in confining apoptotic remodelling to poorly perfused vessels. Tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta are also reported to induce endothelial apoptosis. To investigate the comparative roles of these stimuli, the effect of TNF-alpha and TGF-beta, alone or in combination, in the presence or absence of serum or albumin was studied. There was strong synergy between the cytokines in inducing human umbilical vein endothelial cell apoptosis, but only in the absence of serum. Synergy was destroyed by boiling cytokines and was not affected by polymyxin B. Dose response experiments revealed greater activity of TGF-beta(1) than TGF-beta(2). The synergy was protein synthesis dependent and apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and FACS analysis. Data suggests a role for synergistic activation of endothelial cell apoptosis by TNF-alpha and TGF-beta(1) but perhaps only in poorly perfused vessels deprived of serum factors.

Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.