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.     

Studies on the glycoprotein modification in erythrocyte membrane during experimental cerebral malaria

Plasmodium berghei ANKA (Pb ANKA) is a lethal strain of malaria that causes experimental cerebral malaria (ECM) in rodent models. Pathology of the disease is associated with the sequestration of the infected rbc (irbc) in the micro vessels of brain. In the present study, we analyzed the nature of the glycoprotein modification occurring in irbc membrane during erythrocytic stages of Pb ANKA infection. Titration of naturally occurring glycoproteins with concanavalin A (Con A) and wheat germ agglutinin (WGA) lectins revealed an enhanced lectin binding ability for the irbc membrane preparations. Partial characterization of the Con A specific determinants (alpha-d-methyl mannoside specificity) by lectin affinity chromatography followed by 2D electrophoresis and WGA specific determinants (sialic acid specificity) by Western analysis revealed the association of novel lectin specific determinants in irbc membrane. To correlate the biochemical changes with the morphological changes, SEM of irbc, and TEM of sequestered irbc were performed. These ultra structural studies revealed variable and irregular surface protrusions and deep surface indentations on irbc. These observations implicate that altered glycoprotein profiles may lead to cytoarchitectural changes in irbc membrane and such changes may be essential to establish contact with the host endothelial cells. These observations may be central to the microvascular sequestration and pathology of ECM.     

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells

 Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase. Received: 20 November 2000 / Accepted: 8 March 2001     

AlphaB-crystallin inhibits glucose-induced apoptosis in vascular endothelial cells

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM). Transfection of pcDNA3-rat alphaB-crystallin into these cells inhibited high glucose-induced apoptosis by approximately 36%, such an effect was not observed when cells were transfected with an empty vector. AlphaB-crystallin transfection inhibited by about 35% of high glucose induced activation of caspase-3. High concentration of glucose enhanced formation of reactive oxygen species (ROS) in these cells but this was significantly (p < 0.001) curtailed by transfection of alphaB-crystallin. Results of our study indicate that alphaB-crystallin effectively inhibits both ROS formation and apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.     

Plasmodium falciparum isolates from patients with uncomplicated malaria promote endothelial inflammation

The ability of Plasmodium falciparum infected erythrocytes (Pf-IEs) to activate endothelial cells has been described; however, the interaction of the endothelium with Pf-IEs field isolates from patients has been less characterized. Previous reports have shown that isolates alter the endothelial permeability and apoptosis. In this study, the adhesion of 19 uncomplicated malaria isolates to Human Dermal Microvascular Endothelial Cells (HDMEC), and their effect on the expression of ICAM-1 and proinflammatory molecules (sICAM-1, IL-6, IL-8, and MCP-1) was evaluated.P. falciparum isolates adhered to resting and TNFα-activated HDEMC cells at different levels. All isolates increased the ICAM-1 expression on the membrane (mICAM-1) of HDMEC and increased the release of its soluble form (sICAM-1), as well the production of IL-6, IL-8 and MCP-1 by HDMEC with no signs of cell apoptosis. No correlation between parasite adhesion and production of cytokines was observed.In conclusion, isolates from uncomplicated malaria can induce a proinflammatory response in endothelial cells that may play a role during the initial inflammatory response to parasite infection; however, a continuous activation of the endothelium can contribute to pathogenesis.     

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。     

Conventional and experimental treatment of cerebral malaria

The most severe complication of Plasmodium falciparum infection is cerebral malaria (CM). Cerebral malaria implies the presence of neurological features, especially impaired consciousness. The treatment of CM is limited to: (i) a few conventional anti-malarial drugs (quinine or artemisinins), (ii) adjunctive treatments (initial stabilisation, blood exchange transfusion, osmotic diuretics and correction of hypoglycaemia, acidosis and hypovolaemia) and (iii) immunomodulation. There are clear procedures concerning treatment of CM, which include the use of the anti-plasmodial drugs. Adjunctive treatments are permissible but there is no single official guideline and immune intervention is a possibility currently being examined in rodent models only. The suggested immunomodulation approach is based on the strong likelihood that CM is the result of an immunopathological process. P. falciparum initiates the multifactorial chain of events leading to lethal CM and, after a certain stage, it is impossible to stop the progression even by using anti-malarial drugs. We present evidence that CM is a result of a dysregulated immune response. Therefore, it might be prevented by early modulation of discrete factors that participate in this process. In experimental systems, some immunomodulators delay or prevent CM without affecting the parasitaemia. Therefore, in the future the ultimate treatment of CM may be a combination of an anti-malarial and an immunomodulator. However, the overall effect of an immunomodulator would need to be carefully examined in view of concomitant infections, especially in malaria endemic areas.     

Plasmodium berghei ANKA: erythropoietin activates neural stem cells in an experimental cerebral malaria model

Cerebral malaria (CM) causes substantial mortality and neurological sequelae in survivors, and no neuroprotective regimens are currently available for this condition. Erythropoietin (EPO) reduces neuropathology and improves survival in murine CM. Using the Plasmodium berghei model of CM, we investigated if EPO’s neuroprotective effects include activation of endogenous neural stem cells (NSC). By using immunohistochemical markers of different NSC maturation stages, we show that EPO increased the number of nestin⁺ cells in the dentate gyrus and in the sub-ventricular zone of the lateral ventricles, relative to control-treatment. 75% of the EPO-treated CM mice displayed migration as nestin⁺ NSC. The NSC showed differentiation towards a neural cell lineage as shown by PSA-NCAM binding and NSC maturation and lineage commitment was significantly affected by exogenous EPO and by CM in the sub ventricular zone. These results indicate a rapid, EPO-dependent activation of NSC during CM pathology.     

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 methemoglobin promotes cyto-adherence of uninfected RBC to endothelial cells: Insight into cerebral malaria pathology

The endothelial cell barrier is tightly regulated, and disruption or the leaky behavior of the barrier leads to pathology. Disturbance of blood-brain barrier is observed during viral infection, cerebral malaria, and acute hemorrhagic encephalitis. Red blood cells (RBCs) bind to the endothelial cells (ECs) and their affinity towards ECs enhances in the presence of Plasmodium falciparum infection. ECs stimulated with methemoglobin (MetHb; 20 µM) for 1 hour exhibit high levels of cyto-adherence receptors CD36 and ICAM-1 on their cell surface compared with unstimulated cells. These ECs have acquired affinity towards uninfected RBCs in flow at arterial shear stress. SEM analysis indicates that EC–RBC cyto-adherence involved multiple attachment points. Initially, ECs bind single layer of RBCs and the number of RBCs increases over time to give high-order cyto-adherence with more than 30 RBCs adhered to each endothelial cell. The cyto-adherence complexes are stable to high shear stress and can withstand shear stress up to 450 dyne/cm ². MetHb-treated ECs exhibited high reactive oxygen species level, and preincubation of ECs with antioxidant (NAC or mannitol) abolished the formation of EC–RBC cyto-adherence complexes. In addition, gallic acid (present in red wine) and green tea extract has inhibited the formation of EC–RBC cyto-adherence complex. A better understanding of gallic acid and tea polyphenol targeting pathological cyto-adherence may allow us to develop a better adjuvant therapy for cerebral malaria and other noninfectious diseases.     

Pathogenic T cells in cerebral malaria

Malaria remains a major global health problem and cerebral malaria (CM) is one of the most serious complications of this disease. Recent years have seen important advances in our understanding of the pathogenesis of cerebral malaria. Parasite sequestration, a hallmark of this syndrome, is thought to be solely responsible for the pathological process. However, this phenomenon cannot explain all aspects of the pathogenesis of CM. The use of an animal model, Plasmodium berghei ANKA in mice, has allowed the identification of specific pathological components of CM. Although multiple pathways may lead to CM, an important role for CD8+ T cells has been clarified. Other cells, including platelets, and mediators such as cytokines also have an important role. In this review we have focused on the role of T cells, and discuss what remains to be studied to understand the pathways by which these cells mediate CM.     

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。     

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells

 Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin’s cells. CD8⁺ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca²⁺-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells. Received: 5 December 1996 / Accepted: 16 January 1997     

Proinflammatory factors present in sera from patients with acute dengue infection induce activation and apoptosis of human microvascular endothelial cells: possible role of TNF-alpha in endothelial cell damage in dengue

There is evidence that severe dengue disease is associated with alterations of the microvascular endothelium. We examined the hypothesis that activation and damage of microvascular endothelial cells (EC) could be induced by inflammatory mediators present in dengue patient's sera. We cultured human microvascular EC (HMEC-1) in vitro with sera from patients with acute dengue infection. Sera from patients with acute dengue induced an increase in ICAM-1 expression on HMEC-1. This effect was greater with samples from the acute febrile phase than with samples from the convalescent phase of the disease. Acute dengue sera had elevated levels of TNF-alpha and the endothelial activating effect of acute dengue sera was inhibited up to 80% by pre-treatment with monoclonal antibodies against TNF-alpha. Furthermore, acute dengue sera induced apoptosis in HMEC-1. These findings support the pathophysiologic significance of microvascular EC and serum inflammatory mediators in dengue.     

Nerve growth factor promotes formation of lumen-like structures in vitro through inducing apoptosis in human umbilical vein endothelial cells

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.     

Coincident parasite and CD8 T cell sequestration is required for development of experimental cerebral malaria

Cerebral malaria (CM) is a fatal complication of Plasmodium falciparum infection. Using a well defined murine model, we observed the effect on disease outcome of temporarily reducing parasite burden by anti-malarial drug treatment. The anti-malarial treatment regime chosen decreased parasitaemia but did not cure the mice, allowing recrudescence of parasites. These mice were protected against CM, despite their parasitaemia having increased, following treatment cessation, to levels surpassing that associated with CM in mice not treated with the drug. The protection was associated with reduced levels of cytokines, chemokines, CD8⁺ T cells and parasites in the brain. The results suggest that the development of the immunopathological response that causes CM depends on a continuous stimulus provided by parasitised red blood cells, either circulating or sequestered in small vessels.