Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Cathepsins: key modulators of cell death and inflammatory responses

Apoptosis is a key mechanism in the build up and maintenance of both innate and adaptive immunity as well as in the regulation of cellular homeostasis in almost every organ and tissue. Central to the apoptotic process is a family of intracellular cysteine proteases with aspartate-specificity, called caspases. Nevertheless, there is growing evidence that other non-caspase proteases, in particular lysosomal cathepsins, can play an important role in the regulation of apoptosis. In this review, the players and the molecular mechanisms involved in the lysosomal apoptotic pathways will be discussed as well as the importance of these pathways in the immune system and the pathogenesis of diseases.

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Soluble TNF-alpha but not transmembrane TNF-alpha sensitizes T cells for enhanced activation-induced cell death

In addition to its proinflammatory effects, TNF-alpha exhibits immunosuppression. Here, we compared the capacities of transmembrane TNF-alpha (tmTNF) and soluble TNF-alpha (sTNF) in regulating expansion of activated T cells by apoptosis. Splenic CD4(+) T cells from wtTNF, TNF-alpha-deficient (TNF(-/-)) and TNF(-/-) mice expressing a non-cleavable mutant tmTNF showed comparable proliferation rates upon TCR-mediated stimulation. Activation-induced cell death (AICD), however, was significantly attenuated in tmTNF and TNF(-/-), compared with wtTNF CD4(+) T cells. Addition of sTNF during initial priming was sufficient to enhance susceptibility to AICD in tmTNF and TNF(-/-) CD4(+) T cells to levels seen in wtTNF CD4(+) T cells, whereas addition of sTNF only during restimulation failed to enhance AICD. sTNF-induced, enhanced susceptibility to AICD was dependent on both TNF receptors. The reduced susceptibility of tmTNF CD4(+) T cells for AICD was also evident in an in vivo model of adoptively transferred CD4(+) T-cell-mediated colonic inflammation. Hence, the presence of sTNF during T-cell priming may represent an important mechanism to sensitize activated T cells for apoptosis, thereby attenuating the extent and duration of T-cell reactivities and subsequent T-cell-mediated, excessive inflammation.

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

Sudden cardiac death in patients with silent myocardial ischemia after myocardial infarction (from the Swiss Interventional Study on Silent Ischemia Type II [SWISSI II])

The occurrence of sudden cardiac death (SCD) in patients with silent ischemia after myocardial infarction (MI) and the factors facilitating SCD are unknown. This study aimed to determine the factors facilitating SCD in patients with silent ischemia after MI. In the Swiss Interventional Study on Silent Ischemia Type II (SWISSI II), 201 patients with silent ischemia after MI were randomized to percutaneous coronary intervention (PCI) or medical management. The main end point of the present analysis was SCD. Multivariable regression models were used to detect potential associations between baseline or follow-up variables and SCD. During a mean follow-up of 10.3 +/- 2.6 years, 12 SCDs occurred, corresponding to an average annual event rate of 0.6%. On multivariate regression analysis, the decline in the left ventricular ejection fraction (LVEF) during follow-up was the only independent predictor of SCD (p = 0.011), other than age; however, the baseline LVEF was not. The decline in LVEF was greater in patients receiving medical management than in those who had received PCI (p <0.001), as well as in patients with residual myocardial ischemia or recurrent MI compared with patients without these findings (p = 0.038 and p <0.001, respectively). Compared with medical management, PCI reduced the rate of residual myocardial ischemia (p <0.001) and recurrent MI (p = 0.001) during follow-up. In conclusion, patients with silent ischemia after MI are at a substantial risk of SCD. The prevention of residual myocardial ischemia and recurrent MI using PCI resulted in better long-term LVEF and a reduced SCD incidence.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Life and death partners: apoptosis, autophagy and the cross-talk between them

It is not surprising that the demise of a cell is a complex well-controlled process. Apoptosis, the first genetically programmed death process identified, has been extensively studied and its contribution to the pathogenesis of disease well documented. Yet, apoptosis does not function alone to determine a cell's fate. More recently, autophagy, a process in which de novo-formed membrane-enclosed vesicles engulf and consume cellular components, has been shown to engage in a complex interplay with apoptosis. In some cellular settings, it can serve as a cell survival pathway, suppressing apoptosis, and in others, it can lead to death itself, either in collaboration with apoptosis or as a back-up mechanism when the former is defective. The molecular regulators of both pathways are inter-connected; numerous death stimuli are capable of activating either pathway, and both pathways share several genes that are critical for their respective execution. The cross-talk between apoptosis and autophagy is therefore quite complex, and sometimes contradictory, but surely critical to the overall fate of the cell. Furthermore, the cross-talk is a key factor in the outcome of death-related pathologies such as cancer, its development and treatment.

Different patterns of Siglec-9-mediated neutrophil death responses in septic shock

Sialic-acid-binding immunoglobulin-like lectin (Siglec) 9 mediates death signals in neutrophils. The objective of this study was to determine the heterogeneity of neutrophil death responses in septic shock patients and to analyze whether these ex vivo data are related to the severity and outcome of septic shock. In this prospective cohort study, blood samples of patients with septic shock (n = 26) in a medical-surgical intensive care unit (ICU) were taken within 24 h of starting the treatment of septic shock (phase A), after circulatory stabilization (phase B), and 10 days after admission or at ICU discharge if earlier (phase C). Neutrophil death was quantified in the presence and absence of an agonistic anti-Siglec-9 antibody after 24 h ex vivo. In phase A, two distinct patterns of Siglec-9-mediated neutrophil death were observed: resistance to neutrophil death (n = 14; Siglec-9 nonresponders) and increased neutrophil death (n = 12; Siglec-9 responders) after Siglec-9 ligation compared with neutrophils from normal donors. Experiments using a pharmacological pan-caspase-inhibitor provided evidence for caspase-independent neutrophil death in Siglec-9 responders upon Siglec-9 ligation. There were no differences between Siglec-9 responders and nonresponders in length of ICU or hospital stay of survivors or severity of organ dysfunction. Taken together, septic shock patients exhibit different ex vivo death responses of blood neutrophils after Siglec-9 ligation early in shock. Both the resistance and the increased susceptibility to Siglec-9-mediated neutrophil death tend to normalize within 72 h after shock. Further studies are required to understand the role of Siglec-9-mediated neutrophil death in septic shock.

Cell death in allergic diseases

Apoptosis, the most common form of cell death, is a key mechanism in the build up and maintenance of both innate and adaptive immunity. Central to the apoptotic process is a family of intracellular cysteine proteases with aspartate-specificity, called caspases. Caspases are counter-regulated by multiple anti-apoptotic molecules, and the expression of the latter in leukocytes is largely dependent on survival factors. Therefore, the physiologic rates of apoptosis change under pathologic conditions. For instance, in inflammation, the expression of survival factors is usually elevated, resulting in increased cell survival and consequently in the accumulation of the involved immune cells. In many allergic diseases, eosinophil apoptosis is delayed contributing to both blood and tissue eosinophilia. Besides eosinophils, apoptosis of other leukocytes is also frequently prevented or delayed during allergic inflammatory processes. In contrast to inflammatory cells, accelerated cell death is often observed in epithelial cells, a mechanism, which amplifies or at least maintains allergic inflammation. In conclusion, deregulated cell death is a common phenomenon of allergic diseases that likely plays an important role in their pathogenesis. Whether the apoptosis is too little or too much depends on the cell type. In this review, we discuss the regulation of the lifespan of the participating leukocytes in allergic inflammatory responses.