Selective iNOS-inhibition does not influence apoptosis in ruptured canine cranial cruciate ligaments

Abnormal patterns of cell death, including increased apoptosis, can influence homeostasis of ligaments and could be involved in the pathogenesis of cranial cruciate ligament (CCL) rupture. Increased nitric oxide (NO) production has been implicated as a stimulus to increased apoptosis in articular cartilage. This study investigated apoptotic cell death in ruptured canine CCL (CCL group, n = 15), in ruptured CCL of dogs treated with oral L-N6-(1-iminoethyl)-lysine (L-NIL), a selective NO-synthetase(NOS)-inhibitor, (L-NIL group, n = 15) and compared the results with normal canine CCL (control group, n = 10). Orally administered L-NIL at a dosage of 25mg/m2 of body surface area was effective in inhibiting NO production in the articular cartilage of dogs in the L-NIL group, but it did not significantly influence the increased quantity of apoptotic cells found in ruptured CCL specimens. The results of this study suggest that apoptosis of ligamentocytes in the canine CCL is not primarily influenced by increased NO production within the stifle joint.

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.

Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation

BACKGROUND Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. METHODS Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry. RESULTS In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days. CONCLUSIONS We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

Suppression of OCT4B enhances sensitivity of lung adenocarcinoma A549 cells to cisplatin via increased apoptosis

BACKGROUND Resistance to chemotherapy in lung adenocarcinoma remains a major obstacle. We examined the potential role of Octamer-binding transcription factor-4B (OCT4B) in enhancing sensitivity of lung adenocarcinoma cells to cisplatin. MATERIALS AND METHODS RNAi interference was used to examine the role of OCT4B in cisplatin-treated A549 cells. Cells were transfected with OCT4B siRNA prior to a 48-h cisplatin treatment. Propidium iodide (PI) and caspase-3 staining were used to determine cell viability and apoptosis. Cell-cycle analysis was performed to evaluate alterations in phase distribution. RESULTS OCT4B suppression in cells increased the number of non-viable, PI(+), and apoptotic, caspase-3(+) cells in the presence and absence of cisplatin treatment. Importantly, cisplatin treatment of OCT4B-suppressed cells resulted in a marked transition of cells from G0/G1 to G2/M phase. CONCLUSION Silencing of OCT4B confers sensitivity to cisplatin treatment in A549 cells via cell-cycle regulation, increased proliferation and enhancement of cisplatin-induced apoptosis. OCT4B clearly protects A549 cells from apoptosis.

Co-activation of JAK/STAT and p38 MAPK pathways by type I interferon enhances apoptosis in human basophils

It has previously been published that interferon-α (type I IFN) improves clinical symptoms of asthma patients. Since human basophils are major inflammatory cells in maintaining chronic allergic asthma we investigate whether type I IFN affect human blood basophils. Furthermore, previous studies have shown that spontaneous apoptosis of human basophils is slow due to constitutive expression of anti-apoptotic BCL-2 family members. In addition, IL-3 exceptionally promotes survival of basophils by enhancing constitutive expression of BCL-2 family members and by inducing de-novo expression of Pim-1 kinase. Thus, we also assessed whether type I IFN might overcome IL-3-induced survival of human basophils. Our data show that type I IFN enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or type II IFN treated cells. Furthermore, we demonstrate that both type I IFN and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. Analyses of signaling pathways reveal that type I IFN promote prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of type I IFN is abolished. On the other hand, type I IFN do not reduce IL-3-induced de novo expression of Pim-1 and BCL-2. This is in line with our observation that IL-3-induced survival is dominant over type I IFN-enhanced apoptosis. In addition, phosphorylation of p38 MAPK in type I IFN treated cells is comparable to non-treated cells. Particularly however, inhibition of this p-p38 activity abrogates apoptosis as well. We conclude that type I IFN-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38 MAPK pathways. Our study identifies a so far unknown effect of type I IFN and may explain the improved clinical symptoms of asthma patients treated with type I IFN.

Apoptosis in Bovine viral diarrhea virus (BVDV)-induced mucosal disease lesions: a histological, immunohistological, and virological investigation

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal "mucosal disease" (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

Vitamin B6 reduces hippocampal apoptosis in experimental pneumococcal meningitis

BACKGROUND Bacterial meningitis caused by Streptococcus pneumoniae leads to death in up to 30% of patients and leaves up to half of the survivors with neurological sequelae. The inflammatory host reaction initiates the induction of the kynurenine pathway and contributes to hippocampal apoptosis, a form of brain damage that is associated with learning and memory deficits in experimental paradigms. Vitamin B6 is an enzymatic cofactor in the kynurenine pathway and may thus limit the accumulation of neurotoxic metabolites and preserve the cellular energy status. The aim of this study in a pneumococcal meningitis model was to investigate the effect of vitamin B6 on hippocampal apoptosis by histomorphology, by transcriptomics and by measurement of cellular nicotine amide adenine dinucleotide content. METHODS AND RESULTS Eleven day old Wistar rats were infected with 1x10(6) cfu/ml of S. pneumoniae and randomized for treatment with vitamin B6 or saline as controls. Vitamin B6 led to a significant (p > 0.02) reduction of hippocampal apoptosis. According to functional annotation based clustering, vitamin B6 led to down-regulation of genes involved in processes of inflammatory response, while genes encoding for processes related to circadian rhythm, neuronal signaling and apoptotic cell death were mostly up-regulated. CONCLUSIONS Our results provide evidence that attenuation of apoptosis by vitamin B6 is multi-factorial including down-modulation of inflammation, up-regulation of the neuroprotective brain-derived neurotrophic factor and prevention of the exhaustion of cellular energy stores. The neuroprotective effect identifies vitamin B6 as a potential target for the development of strategies to attenuate brain injury in bacterial meningitis.

Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited

Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membrane-based organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented nuclei), by immunochemistry for active caspase-3 and deltaC-APP, and by proteolytic caspase-3 activity. Co-incubation of the cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.

Dexamethasone aggravates hippocampal apoptosis and learning deficiency in pneumococcal meningitis in infant rats.

In an infant rat model of pneumococcal meningitis the effect of dexamethasone on neuronal injury in the hippocampus and on learning disability after recovery from the disease was examined. Treatment with dexamethasone or vehicle was started 18 h after infection, concomitant with antibiotics. Neuronal apoptosis in the hippocampal dentate gyrus 34 h after infection was significantly aggravated by dexamethasone treatment compared with vehicle controls (p = 0.02). Three weeks after acute pneumococcal meningitis, learning capacity of animals was assessed in the Morris water maze. The results showed a significantly impaired learning performance of infected animals treated with dexamethasone compared with vehicle controls (p = 0.01). Dexamethasone had no effect on hippocampal injury or learning in uninfected controls. Thus, dexamethasone as adjuvant therapy increased hippocampal cell injury and reduced learning capacity in this model of pneumococcal meningitis in infant rats.

Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

Inhibition of meningitis-associated neutrophil apoptosis by TNF-α depends on functional PI3-kinase in monocytes

In bacterial meningitis, neutrophils cope with bacterial infection but also lead to tissue damage. The balance of beneficial and harmful effects may depend on the lifespan of the neutrophils in the CNS. Here, we show that CSF of patients with meningococcal meningitis contains a neutrophil apoptosis-inhibiting capacity that correlates with TNF-α content. In vitro experiments show that Neisseria meningitidis as well as LPS derived from these bacteria regulated neutrophil apoptosis mainly by stimulating TNF-α production in monocytes. Whereas LPS-induced PI3K-dependent survival signals in monocytes are critical for neutrophil survival, PI3K signaling in granulocytes did not contribute to the increased lifespan of neutrophils. We conclude that LPS-driven PI3K signaling in monocytes regulates neutrophil apoptosis and thereby, may be crucial in the initiation of secondary brain damage in bacterial meningitis.