Group B Streptococcus induces macrophage apoptosis by calpain activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.     

Group B Streptococcus induces apoptosis in macrophages

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS beta-hemolytic activity, suggesting that GBS beta-hemolysin could be involved in apoptosis. beta-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.     

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.     

TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes approximately 30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-kappaB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.     

Fas induces apoptosis in human coronary artery endothelial cells in vitro

Background  

Published work suggests that some types of endothelial cells undergo apoptosis in response to ligation of the receptor Fas (CD95, APO1) but other types are resistant. Because heterogeneity among endothelial cells from different tissues, has been demonstrated, the purpose of this study was to determine, if Fas ligation and/or activation by human Fas ligand induces apoptosis and caspase activities, in cultured human coronary artery endothelial cells, and the differences between TNF-a and FAS induced apoptosis in these cells.     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains

A novel hemorrhagic metalloprotease, halysase, isolated from the snake venom of Gloydius halys induces apoptosis in endothelial cells. The purified metalloprotease is a monomeric glycoprotein with an isoelectric point of 4.8. Analysis of the cDNA sequence encoding halysase revealed that the enzyme consists of multifunctional domains including a proprotein domain, a protease domain, a disintegrin-like domain and a cysteine-rich domain. The metalloprotease has a DECD sequence in the disintegrin-like domain instead of the typical RGD sequence. Halysase strongly inhibits proliferation of human umbilical vein endothelial cells in a dose-dependent manner as well as adhesion of the cells to extracellular matrix proteins. The enzyme specifically hydrolyzes not only extracellular matrix proteins such as fibronectin, vitronectin, and type IV collagen, but also integrins alpha1beta1 and alpha5beta1. The apoptosis of endothelial cells induced by halysase is closely associated with activation of caspase-3 and decreased level of Bcl-X(L)/Bax. Apohalysase, which lacks metalloprotease activity, is also able to induce the apoptosis. Several lines of experimental evidence suggest that the protease domain and the disintegrin-like domain of halysase cooperatively contribute to the induction of endothelial cell apoptosis.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling

Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues.     

Streptococcus pneumoniae induces exocytosis of Weibel-Palade bodies in pulmonary endothelial cells

Invasive pneumococcal infections due to Streptococcus pneumoniae lead to inflammatory infiltration of leucocytes into lung alveolus, meninges and to septic dissemination within the vascular system. The lung microvasculature is covered by pulmonary endothelial cells containing Weibel‐Palade bodies (WPB) releasing procoagulant von Willebrand factor (vWF) and other proteins in response to inflammatory stimuli. The influence of pathogenic pneumococci on secretion of WPB proteins is unknown. Here, we report that adherence of S. pneumoniae to primary human pulmonary microvascular endothelial cells (HPMEC) stimulates the WPB exocytosis and the secretion of vWF and interleukin 8 (IL‐8). Moreover, infection analyses performed with pneumococcal mutants deficient in the expression of cytotoxic pneumolysin demonstrated that, in addition to direct bacterial adherence, sublytic concentrations of pneumolysin stimulated vWF secretion. The release of vWF was induced after infection with pneumococci from both the apical and the basal cell surfaces, which implies a stimulation of WPB exocytosis in both directions: from inside the vasculature and also following invasive pneumococcal transmigration from pulmonary tissue into the bloodstream. In conclusion, this study demonstrates that the most relevant pulmonary pathogen S. pneumoniae induces release of proinflammatory and procoagulative components directly contributing to pathophysiological processes leading to fatal tissue injury during course of infection.     

Beta blockade induces apoptosis in cultured capillary endothelial cells

Continuous beta blockade stimulates deposition of collagen in the pulmonary alveolar interstitium of adult rats. It also causes changes to the capillary endothelial cell compartment reminiscent of programmed cell death. To test whether beta blockade results in endothelial cell apoptosis, cultures of capillary endothelial cells were treated with both a wide-spectrum beta blocker and a beta-2-specific antagonist. Apoptosis was measured in these cultures using both terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and annexin-V assays. Both forms of beta blockade stimulated programmed cell death in these cultures. To test whether the apoptotic effect of beta blockade was related to interstitial collagen deposition, capillary endothelial cells were cocultured with beta-blocked pulmonary fibroblast monolayers. Cocultured endothelial cells were substantially protected from apoptosis after beta blockade; coculture over plain tissue culture plastic or over exogenous collagen films had no effect on programmed cell death in endothelial cells. These results suggest that both pulmonary endothelial and interstitial cells are vulnerable to injury from beta blockade but that paracrine interactions between these cells may protect the peripheral lung from substantive damage.     

Manganese induces apoptosis of human B cells: caspase-dependent cell death blocked by bcl-2.

Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis. Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.     

Docosahexaenoic acid induces ciap1 mRNA and protects human endothelial cells from stress-induced apoptosis

Induction of apoptosis represents a potential reaction of endothelial cells (ECs) after injury of the vascular endothelium. Beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in vascular diseases are widely recognized although the responsible mechanisms are not fully understood. Because it is not known whether PUFAs modulate EC apoptosis, we investigated the effects of n-3 and n-6 PUFAs on 4-hydroxynonenal (HNE)-induced EC apoptosis by annexin V staining and caspase-3 activation assays. Pretreatment with the n-3 fatty acid docosahexaenoic acid (DHA) reduced HNE-induced EC apoptosis. DHA-treated cells did not show the pronounced drop in intracellular GSH after HNE exposure seen in vehicle- or n-6 arachidonic acid-treated cells. This is most likely due to increased GSH levels in DHA-treated cells. Furthermore, DHA pretreatment increased ciap1 mRNA levels and transfection of cIAP1 small interfering RNA abolished the protective effect of DHA in HNE-induced apoptosis in HUVECs. Thus pretreatment of HUVECs with DHA reduces HNE-induced oxidative stress and apoptosis, and the protective effects of DHA seem to be dependent on cIAP1. The results provide a possible new mechanism for the atheroprotective effects of n-3 fatty acids in vascular disease.     

Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-alpha, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.     

Ap4A induces apoptosis in human cultured cells.

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'-P1,P3-triphosphate to diadenosine 5',5'-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.     

PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells

3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.     

Methylglyoxal and high glucose co-treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.