Apoptosis Of HUVECs Research Articles

Targeted inhibition of αvβ3 integrin with an RNA aptamer impairs endothelial cell growth and survival

αvβ3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. αvβ3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of αvβ3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-αvβ3 that binds recombinant αvβ3 integrin, for its ability to bind endogenous αvβ3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-αvβ3 binds αvβ3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-αvβ3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-αvβ3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-αvβ3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

Nitric Oxide Inhibition of Homocysteine-induced Human Endothelial Cell Apoptosis by Down-regulation of p53-dependent Noxa Expression through the Formation of S-Nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.

Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells

This study evaluated direct effects of peroxisome proliferatoractivated receptor gamma(PPARgamma) agonists, including thiazolidinediones (TZDs), on vascular cell apoptosis and related protein expression to test the hypothesis that these effects are dependent on i) the respective agent's structure and ii) endothelial cells' vascular origin. Exposure (48 h) of human umbilical vein endothelial cells (HUVECs, n=6) to up to 10 microM troglitazone (TRO), rosiglitazone, pioglitazone, and to up to 50 microM RWJ241947=MCC-555 (RWJ) inhibited (p<0.05) apoptosis by 8-25%, whereas 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2)) triggered (50 microM: + 400%, p<0.05) endothelial cell death versus control (=100%). Moreover, RWJ (50 microM) completely abrogated TNF-alpha(2000 U/ml) and stearic acid (200 microM) induced apoptosis in HUVECs . Similar results were obtained in human adult (saphenous) vein- and aortic endothelial cells, the latter showing no anti-apoptotic response to TRO. In HUVECs, TZDs' anti-apoptotic effects inversely correlated (r=-0.95, p<0.01) with increased (p<0.05) expression of the apoptosis-inhibitor bcl-2, whereas PGJ(2)-induced apoptosis was associated with upregulation of c-myc (+447%) and E2F-1 (+339%). Additionally, TZDs (by 25-39%) and PGJ(2) (-70%) reduced (p<0.05) expression of heat shock protein 60 (hsp60) showing no correlation with apoptosis (r=0.14, n.s.). Modulation of apoptosis by PPARgammaagonists differs in endothelial cells dependent on their vascular origin and the agonists' structure. Thiazolidinediones' ability to reduce both, endothelial apoptosis and hsp60 expression could well add to beneficial vascular effects attributed to these oral antidiabetic drugs.

Streptococcus pneumoniae R6x induced p38 MAPK JNK-mediated Caspase-dependent apoptosis in human endothelial cells

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence, kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.

Antioxidative stress–associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress

Adult and embryonic stem cells hold great promise for regenerative medicine. Expression profiling of stem cells revealed a characteristic imprint of genes, so-called "stemness" genes, providing resistance to stress. Circulating progenitor cells with an endothelial phenotype (EPCs) can be isolated from peripheral blood and contribute to neovascularization and endothelial regeneration. We investigated whether EPCs are equipped with an antioxidative defense to provide resistance against oxidative stress. EPCs exhibited a significantly lower basal reactive oxygen species (ROS) concentration as compared with mature umbilical vein endothelial cells (HUVECs). Incubation with H(2)O(2) (500 microM) or the redox cycler LY-83583 (10 microM) profoundly increased the ROS concentration to 3- and 4-fold and induced apoptosis in HUVECs. In contrast, H(2)O(2) and LY-83583 induced only a minor increase in intracellular ROS levels and apoptosis in EPCs. Consistently, the expression of the intracellular antioxidative enzymes catalase, glutathione peroxidase and manganese superoxide dismutase (MnSOD), was significantly higher in EPCs versus HUVECs and human microvascular endothelial cells. In accordance, combined inhibition of these antioxidative enzymes increased ROS levels in EPCs and impaired EPC survival and migration. Taken together, EPCs reveal a higher expression of antioxidative enzymes and, thus, are exquisitely equipped to be protected against oxidative stress consistent with their progenitor cell character.

Effects of the Indazolylpyrimidine GW786034 on Angiogenesis and MM Cell Growth: Therapeutic Implications.

Vascular endothelial growth factor (VEGF) and its receptors play an important role in the pathogenesis of multiple myeloma (MM). Specifically, VEGF, which is present in the MM bone marrow (BM) microenvironment, induces neovascularization; triggers tumor cell growth, survival, and migration; inhibits dendritic cell maturation; and induces osteoclastogenesis. Therefore, the VEGF receptor provides a potential novel therapeutic target in MM. Previous studies showed that GW654652, a pan VEGFR inhibitor, inhibits growth and migration of MM tumor cells (Podar et al. Blood . 2004; 103:3473–3479.). Here we describe the effects of GW786034, a derivative of GW654652, on MM cells to support its potential clinical evaluation in MM patients. GW786034 inhibits VEGF- triggered Flt-1 phosphorylation and activation of downstream signaling molecules, induces concentration-dependent MM cell and HUVEC apoptosis, and inhibits VEGF- triggered MM cell migration. Specifically, GW786034 induces cleavage of caspase-8 and PARP, but not caspase-9, and induces downregulation of the pro-apoptotic molecules survivin, cIAP1, 2, and Mcl-1. Furthermore, GW786034 decreases proliferation of MM cells induced by their adhesion to BMSCs, and inhibits VEGF-induced upregulation of ICAM-1 and VCAM-1, thereby abrogating their adhesion to HUVECs. Consistent with these results, GW786034 inhibited both tubuli formation and accumulation of tumor cells at vascular branching points in a Matrigel tube forming assay. Finally, GW786034 sensitizes both MM cells alone and tumor cells bound to BMSCs or HUVECs to melphalan and bortezomib. Taken together, these studies support clinical evaluations of GW786034 either alone or in combination with other agents in MM patients.

IFN-alpha sensitizes human umbilical vein endothelial cells to apoptosis induced by double-stranded RNA.

The ability of endothelial cells to mount an efficient antiviral response is important in restricting viral dissemination and eliminating viral infection from the endothelium and surrounding tissues. We demonstrate that dsRNA, a molecular signature of viral infection, induced apoptosis in HUVEC, and priming with IFN-alpha shortened the time between when dsRNA was encountered and when apoptosis was initiated. IFN-alpha priming induced higher levels of mRNA for dsRNA-activated protein kinase, 2'5'-oligoadenylate synthetase, and Toll-like receptor 3, transcripts that encode dsRNA-responsive proteins. dsRNA induced activation of dsRNA-activated protein kinase and nuclear translocation of transcription factors RelA and IFN regulatory factor-3 in IFN-alpha-primed HUVECs before the activation of intrinsic and extrinsic apoptotic pathways. These changes did not occur in the absence of dsRNA, and apoptosis resulting from incubation with dsRNA occurred much later when cells were not primed with IFN-alpha. The entire population of IFN-alpha-primed HUVECs underwent nuclear translocation of RelA and IFN regulatory factor-3 in response to dsRNA, whereas less than one-half of the population responded with apoptosis. When IFN-alpha-primed HUVECs were coincubated with dsRNA and proteasome inhibitors, all HUVECs were rendered susceptible to dsRNA-induced apoptosis. These studies provide evidence that many endothelial cells that are alerted to the risk of infection by IFN-alpha would undergo apoptosis sooner in response to dsRNA than non-IFN-alpha-primed cells, and this would enhance the likelihood of eliminating infected cells prior to the production of progeny virions.

A Role for Caspase-1 in Serum Withdrawal-Induced Apoptosis of Endothelial Cells

Mouse lung endothelial cells (MLEC) and HUVEC were used under serum withdrawal (SW) conditions as a model of endothelial cell (EC) apoptosis. Apoptosis was quantified by time-lapse video microscopy. Mouse lung ECs from caspase-1−/− mice had significantly reduced rates of SW-induced apoptosis compared with wild-type mice, specifically implicating caspase-1 in proapoptotic signaling in ECs. SW conditions induced HUVEC apoptosis with concomitant activation of caspase-1. Further studies demonstrated that the caspase-1 inhibitors z-VAD and z-YVAD significantly reduced the rate of SW-induced HUVEC apoptosis. HUVEC, when transfected with caspase-1, showed a highly significant increase in apoptosis. SW was associated with increases in reactive oxygen species production that were significantly inhibited by the antioxidant N-acetyl-l-cysteine, although rates of apoptosis and caspase-1 activation were unaffected. These results demonstrate the involvement of caspase-1 in SW-induced EC apoptosis, independently of reactive oxygen species production.

Structural requirements of cyclopentenone prostaglandins to induce endothelial cell apoptosis

Prostaglandins are a family of structurally related molecules formed by many cells in response to extrinsic stimuli. A member of this family, 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2), shows unique biological properties including anti-inflammatory, anti-viral, and anti-tumour activity, and has attracted much attention as a high affinity ligand for the peroxisome proliferator-activated receptor γ. Increasing evidence points to additional effects. We investigated several structurally related prostaglandins in comparison to 15d-PGJ 2 with respect to their apoptosis-inducing capacity in human umbilical endothelial cells (HUVEC). Cell viability was tested with a modified MTT assay and apoptosis was detected by Annexin V staining and cell cycle analysis by flow cytometry. Incubation of confluent HUVECs with 15d-PGJ 2 markedly reduced endothelial cell viability which was due to apoptosis. In contrast, none of the other PGs tested affected cell viability. Interestingly, the cyclopentenone ring alone dose-dependently reduced cell viability and significantly induced apoptosis in HUVECs with as low a concentration as 0.25 μM. In conclusion, we report that the cyclopentenone moiety of cyPGs is an essential component for the apoptosis-inducing properties of 15d-PGJ 2. For 15d-PGJ 2 the position of the cyclopentenone ring in conjunction with the side chains yields a molecule with unique biological properties.

The Effect of a Monoclonal Antibody Coupled to Ricin A Chain-Derived Peptides on Endothelial Cells in Vitro: Insights into Toxin-Mediated Vascular Damage

Immunotoxins (ITs) containing plant or bacterial toxins have a dose-limiting toxicity of vascular leak syndrome (VLS) in humans. The active A chain of ricin toxin (RTA), other toxins, ribosome-inactivating proteins, and the VLS-inducing cytokine IL-2 contain the conserved sequence motif (x)D(y) where x = L, I, G, or V and y = V, L, or S. RTA-derived LDV-containing peptides attached to a monoclonal antibody, RFB4, induce endothelial cell (EC) damage in vitro and vascular leak in two animal models in vivo. We have now investigated the mechanism(s) by which this occurs and have found that (1) the exposed D75 in the LDV sequence in RTA and the C-terminal flanking threonine play critical roles in the ability of RFB4-conjugated RTA peptide to bind to and damage ECs and (2) the LDV sequence in RTA induces early manifestations of apoptosis in HUVECs by activating caspase-3. These data suggest that RTA-mediated inhibition of protein synthesis (due to its active site) and apoptosis (due to LDV) may be mediated by different portions of the RTA molecule. These results suggest that ITs prepared with RTA mutants containing alterations in LDVT may kill tumor cells in vivo in the absence of EC-mediated VLS.

Autocrine FGF-2 is responsible for the cell density-dependent susceptibility to apoptosis of HUVEC : A role of a calpain inhibitor-sensitive mechanism.

To elucidate the factors affecting endothelial susceptibility to apoptosis, we studied the effects of cell density on endothelial cell apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2/basic FGF). On deprivation, more cells became apoptotic in a dense culture (5 x 10(2) cells/mm(2)) than in a sparse culture (1 x 10(2) cells/mm(2)) of human umbilical vein endothelial cells. FGF-2, hepatocyte growth factor, and vascular endothelial cell growth factor, but not insulin-like growth factor-I, decreased apoptosis in the dense culture to a level similar in the sparse culture. An anti-FGF-2 antibody significantly increased the apoptosis in the sparse culture, suggesting that the sparse culture was resistant to apoptosis because of the greater autocrine production of FGF-2. Western blot analysis and metabolic labeling revealed that the sparse culture has, in fact, more FGF-2 than the dense culture. The steady state level of mRNA for FGF-2 was not significantly different between the dense and sparse cultures. Among a panel of inhibitors for 2 major cytoplasmic proteolytic enzymes, calpain inhibitors increased FGF-2 in the dense culture, but proteasome inhibitors did not. Our findings demonstrate that cell density affects endothelial survival by regulating autocrine FGF-2 production through a calpain inhibitor-sensitive mechanism.

Generation of reactive oxygen intermediates, activation of NF-κB, and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide synthase?

Exposure to high glucose causes characteristic dysfunction and morphologic changes of the endothelium. To study the underlying mechanisms of glucotoxicity, human endothelial cells (HUVECs) were isolated from umbilical veins and cultivated under hyperglycemic conditions (10–30 mM) for up to 72 h. The generation of reactive oxygen intermediates (ROIs) was determined by histochemical staining of the cells by dichlorodihydrofluorescein. Activation of the transcription factor nuclear factor-kappa B (NF-κB) family was analyzed by the electromobility shift assay and by histochemical staining of the cells with rhodamine-labelled consensus sequences of activated NF-κB. Apoptotic cells were identified by morphologic analysis and DNA fragmentation. Incubation of HUVECs with high glucose led to rapid increase in the generation of ROIs. After an incubation of 2 to 6 h, NF-κB became activated, with the maximum at 4 h. Exposure of HUVECs to high glucose for up to 72 h caused a significant induction of apoptosis in HUVECs. The increased generation of ROIs, activation of apoptosis, and induction of apoptosis were also observed in cells incubated with 3-O-methyl-D-glucose, a glucose derivative that is taken up by the cells but not metabolized. Generation of ROIs, activation of NF-κB, and induction of apoptosis were not only prevented by antioxidants (thioctic acid, tocopherol, superoxide dysmutase-mimetic), but also by l-nitroarginine. These observations indicate that high glucose leads to an increase in generation of ROIs, an activation of NF-κB, and an induction of apoptosis by a glucose-specific and NO synthase-dependent mechanism. Our data suggest that peroxynitrite, which is rapidly formed from nitric oxide and superoxide anions, is the mediator of the cytotoxic effects of high glucose on endothelial cells. Because the induction of apoptosis by glucose was prevented by an antisense nucleotide to the p65NF-κB binding site, we assume that the ROI-mediated activation of NF-κB plays an important role for induction of apoptosis by glucose.

4-Hydroxynonenal induces dysfunction and apoptosis of cultured endothelial cells.

Lipolytic products of triglyceride-rich lipoproteins, i.e., free fatty acids, may cause activation and dysfunction of the vascular endothelium. Mechanisms of these effects may include lipid peroxidation. One of the major and biologically active products of peroxidation of n-6 fatty acids, such as linoleic acid or arachidonic acid, is the aldehyde 4-hydroxynonenal (HNE). To study the hypothesis that HNE may be a critical factor in endothelial cell dysfunction caused by free fatty acids, human umbilical endothelial cells (HUVEC) were treated with up to160 microM of linoleic or arachidonic acid. HNE formation was detected by immunocytochemistry in cells treated for 24 h with either fatty acid, but more markedly with arachidonic acid. To study the cellulareffects of HNE, HUVEC were treated with different concentrations of this aldehyde, and several markers of endothelial cell dysfunction were determined. Exposure to HNE for 6 and 9 h resulted in increased cellular oxidative stress. However, short time treatment with HNE did not cause activation of nuclear factor-kappaB (NF-kappaB). In addition, HUVEC exposure to HNE caused a dose-dependent decrease in production of both interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). On the other hand, HNE exerted prominent cytotoxic effects in cultured HUVEC, manifested by morphological changes, diminished cellular viability, and impaired endothelial barrier function. Furthermore, HNE treatment induced apoptosis of HUVEC. These data provide evidence that HNE does not contribute to NF-kappaB-related mechanisms of the inflammatory response in HUVEC, but rather to endothelial dysfunction, cytotoxicity, and apoptotic cell death.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.

Prevention of gastric damage by no-releasing aspirin (NCX-4016) is mediated by inhibition of ICE/-like proteases in gastric mucosa and endothelial cells

Background. Tumor necrosis factor-ct (TNFtx), a proinflammatory cytokine, is released in vivo and in vitro after exposure to non-steroidal anti-inflammatory drugs (NSAIDs). TNFct receptor binding leads to activation of multiple interleukin-1 converting enzyme (ICE)-like proteases (caspases) and endothelial cell death. Endothelial cell dysfunction is an early step in the process of NSAID-gastropathy. Nilric oxide(NO)-releasing NSAIDs, are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. NO-releasing agents (sodium nitroprusside [SNP] and L-Arginine) prevent TNF-induced ICE/caspase 1 activation in human endothelial ceils (HUVECs). Aims. To assess whether NO-NSAIDs modulate ICE-like proteases in vivo and in vitro and protect endothelial cells from TNFct-induced apoptosis. Methods. Macrophages, prepared from mouse spleen, were used to investigate TNFet release in vitro. Gastric damage was induced in rat by oral administration of 150 mg/kg aspirin or an equimolar dose (249 mg/kg) of NCX-4016 (NicOx, Milan, Italy). ICE-like activity was measured in cell Iysates and gastric homogenates using YVAD.AMC as substrate in a fluorimetric assay. HUVEC apoptosis was assessed by FACS-scan analysis. Results. In vitro studies demonstrated that aspirin, but not NCX-4016, released TNFct from mouse macrophages. Co-incubation with 1-100 laM SNP resulted in a concentrationdependent inhibition of aspirin-induced TNFct-release. In viw) aspirin administration caused a time-dependent increase in gastric mucosal damage and gastric mucosa ICE/caspase activity. Pretreating rats with 0.1 mM L-Arginine, SNP and Z.VAD.fmk, a pan-ICE/caspase inhibitor, prevented both gastric mucosal damage and ICE/caspase upregulation induced by aspirin. No gastric damage was detectable with any dose of NCX-4016 tested. Like aspirin NCX-4016 was largely metabolized to salicylate. In contrast to aspirin NCX-416 significantly increased plasma nitrite/nitrate concentrations and inhibited the aspirin-induced upregulation of ICE/caspase activity into the gastric mucosa. Although aspirin had no effect on HUVEC apoptosis induced by TNFcq NCX-4016 and SNP caused a concentrationdependent inhibition of apoptosis and prevented ICEqike pmteases activation as demonstrated by Westem blot analysis and fluorimetric assay. NCX-4016 inhibited IL-113 release from endotoxin-stimulated macrophages. Conclusions. 1) Activation of ICE/caspase pmteases is a key step in the process of NSAID-gastropathy; 2) NCX-4016 spares the gastric mucosa and protects endothelial cells from TNFetinduced apoptosis. The potential of NO-releasing NSAIDs to modulate ICE-like pmteases and IL-113 release might help to explain the anti-inflammatory activity of these compounds.

Mechanisms of action of antimalarials in inflammation: induction of apoptosis in human endothelial cells.

Antimalarials are beneficial therapeutic agents in systemic lupus and rheumatoid arthritis. These autoimmune diseases have abnormally low apoptosis of inflammatory cells. Both disorders have an abnormal angiogenesis. In the present report, antimalarials were demonstrated to selectively increase apoptosis of HUVECs in vitro. A 24-h exposure to 50 or 150 microM of the drugs was associated with a significant loss of substrate-adherent cells. Chloroquine exhibited an inhibitory effect on HUVEC proliferation over 7 days. Programmed cell death in HUVECs rendered nonadherent by chloroquine was confirmed by the induction of DNA fragmentation in floating cells. Northern blot analysis revealed a rapidly increased expression of the bcl-x(s) gene without any change in the expression of the bcl-2 gene, indicating that HUVECs under chloroquine were undergoing apoptosis. The onset of the apoptotic cascade in HUVECs appeared shortly after the addition of chloroquine. The effect of chloroquine on apoptosis was distinct from acute cell lysis and was restricted to HUVECs. Antimalarials also induced IL-1alpha production. In parallel, chloroquine alone did not increase the expression of IL-6. Anti-IL-1alpha Ab or IL-1Ra only marginally reversed chloroquine-induced depression of proliferation for the low drug concentration, but not the massive cell death effect at and above 50 microM. Taken together, these data may indicate that antimalarials repress angiogenesis. The autocrine mechanism involving IL-1alpha accounts only for a minor fraction of the full antiendothelial effect of chloroquine, which is mainly dependent on apoptosis.

Immune-modulating and anti-vascular activities of two xanthenone acetic acid analogues: A comparative study to DMXAA

In order to proliferate, solid tumours require the development and continuous expansion of an organised host-derived vascular network. The anti-vascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) emerged as derivative of the flavone-8-acetic acid (FAA) and xanthenone-4-acetic acid (XAA). Its anti-vascular activity is not based on direct cytotoxic effects, but is characterized by an immune-mediated component, through the activation of NF-kappaB pathway, and a direct anti-vascular action, involving the induction of endothelial cell apoptosis and changes in tumour vessel permeability. Despite promising pre-clinical results, DMXAA showed moderate anti-tumour activity in clinical trials. In this study, we compared to DMXAA the in vitro immune-modulating and the anti-vascular properties of two XAA analogues, AP/1649 and AP/1897. Their immune-stimulating activities were evaluated on a human monocyte cell line and their anti-vascular activities were studied by measuring the induction of HUVECs apoptosis and using DCE-MRI to determine tumour perfusion following drug treatment. Although the two molecules exerted an immune stimulation comparable to that produced by DMXAA, they showed reduced (AP/1649) or minimal (AP/1897) anti-vascular activity in vitro, and no anti-vascular effects in vivo. These results endorse the current theories concerning two independent actions exerted by DMXAA.