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.

Survival in patients with high-risk prostate cancer is predicted by miR-221, which regulates proliferation, apoptosis, and invasion of prostate cancer cells by inhibiting IRF2 and SOCS3

A lack of reliably informative biomarkers to distinguish indolent and lethal prostate cancer is one reason this disease is overtreated. miR-221 has been suggested as a biomarker in high-risk prostate cancer, but there is insufficient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancer-related death, extending and validating earlier findings. By mechanistic investigations we showed that miR-221 regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediated activation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, two oncogenes that negatively regulate this signaling pathway. miR-221 expression sensitized prostate cancer cells for IFN-γ-mediated growth inhibition. Our findings suggest that miR-221 offers a novel prognostic biomarker and therapeutic target in high-risk prostate cancer.

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.

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.

Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein-Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways.

In the human urothelium and suburothelium, intradetrusor botulinum neurotoxin type A does not induce apoptosis: preliminary results

Intradetrusor injections of botulinum neurotoxin type A (BoNTA) are emerging as the preferred second-line treatment for neurogenic and idiopathic overactive bladder (OAB). In animal experiments, intradetrusor BoNTA injections have been shown to cause apoptosis in the bladder urothelium and suburothelium but not the detrusor.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Inhibition of the kynurenine-NAD+ pathway leads to energy failure and exacerbates apoptosis in pneumococcal meningitis

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1? and -? were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo

The role of N-myc downstream regulated gene-1 (NDRG1) in cancer has recently gained interest, as potential regulator of cell death and tumor suppressor. Although its normal function in the pancreas is largely unknown, loss of NDRG1 expression is associated with a more aggressive tumor phenotype and poor outcome in pancreatic cancer patients.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Bacteremia causes hippocampal apoptosis in experimental pneumococcal meningitis

Bacteremia and systemic complications both play important roles in brain pathophysiological alterations and the outcome of pneumococcal meningitis. Their individual contributions to the development of brain damage, however, still remain to be defined.