Death receptor 3 mediates TNFSF15- and TNFα-induced endothelial cell apoptosis

Tumor necrosis factor superfamily 15 (TNFSF15) suppresses angiogenesis by specifically inducing apoptosis in proliferating endothelial cells. Death receptor 3 (DR3), a member of the TNF receptor superfamily (TNFRSF25), has been identified as a receptor for TNFSF15 to activate T cells. It is unclear, however, whether DR3 mediates TNFSF15 activity on endothelial cells. Here we show that siRNA-mediated knockdown of DR3 in an in vivo Matrigel angiogenesis assay, or in adult bovine aortic endothelial (ABAE) cell cultures, leads to resistance of endothelial cells to TNFSF15-induced apoptosis. Interestingly, DR3-depleted cells also exhibited markedly diminished responsiveness to TNFα cytotoxicity, even though DR3 is not a receptor for TNFα. Treatment of the cells with either TNFSF15 siRNA or a TNFSF15-neutralizing antibody, 4-3H, also results in a significant inhibition of TNFα-induced apoptosis. Mechanistically, DR3 siRNA treatment gives rise to an increase of ERK1/2 MAPK activity, and up-regulation of the anti-apoptotic proteins c-FLIP and Bcl-2, thus strengthening apoptosis-resisting potential in the cells. These findings indicate that DR3 mediates TNFSF15-induced endothelial cell apoptosis, and that up-regulation of TNFSF15 expression stimulated by TNFα is partly but significantly responsible for TNFα-induced apoptosis in endothelial cells.     

Suppression of endothelial cell activity by inhibition of TNFα

Introduction

TNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.

Methods

Human dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.

Results

Certolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).

Conclusion

Our data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.     

Reactive oxygen species in TNFα-induced signaling and cell death

TNFα is a pleotropic cytokine that initiates many downstream signaling pathways, including NF-κB activation, MAP kinase activation and the induction of both apoptosis and necrosis. TNFα has shown to lead to reactive oxygen species generation through activation of NADPH oxidase, through mitochondrial pathways, or other enzymes. As discussed, ROS play a role in potentiation or inhibition of many of these signaling pathways. We particularly discuss the role of sustained JNK activation potentiated by ROS, which generally is supportive of apoptosis and “necrotic cell death” through various mechanisms, while ROS could have inhibitory or stimulatory roles in NF-κB signaling.     

Nitrated fatty acids prevent TNFα-stimulated inflammatory and atherogenic responses in endothelial cells

Nitration products (nitroalkenes) of linoleic acid (LNO₂) and oleic acid (OA-NO₂) can act as endogenous PPARγ ligands with electrophilic properties to exert anti-inflammatory effects on atherosclerotic plaques in the vasculature. Here, we show that OA-NO₂ and LNO₂ prevent tumor necrosis factor α (TNFα)-stimulated inflammatory and atherogenic responses in human umbilical vein endothelial cells (HUVECs). Both OA-NO₂ and LNO₂ prevented TNFα-stimulated release of the cytokines, IL-6, IL-8, IL-12/p40, IFNγ, MCP-1, and IP-10, and inhibited NF-κB activation. OA-NO₂ and LNO₂ also blocked TNFα-induced expression of the adhesion molecules, ICAM-1, VCAM-1, and E-selectin, and suppressed monocyte adhesion to HUVECs. In each case, OA-NO₂ was more potent and efficacious than was LNO₂, possibly due to increased stability in aqueous media. Collectively, these results substantiate a new functional role for nitrated fatty acids, demonstrating that OA-NO₂ and LNO₂ exert an anti-inflammatory function against the inflammatory cascade initiated by the representative pro-inflammatory cytokine, TNFα.     

MCP-induced protein 1 suppresses TNFα-induced VCAM-1 expression in human endothelial cells

Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. We here report that treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor (TNF) α substantially increased the expression of MCP-induced protein 1 (MCPIP1). Overexpression of MCPIP1 protected ECs against TNFα-induced endothelial activation, as characterized by the attenuation in the expression of the adhesion molecule VCAM-1 and monocyte adherence to ECs. Conversely, small interfering RNA-mediated knock down of MCPIP1 increased the expression of VCAM-1 and monocytic adherence to ECs. These studies identified MCPIP1 as a feedback control of cytokines-induced endothelial inflammation.     

Shear stress regulates expression of death-associated protein kinase in suppressing TNFα-induced endothelial apoptosis

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)‐induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto‐inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm² for 6 h. Quantitative RT‐PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18‐h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho‐serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase‐3 and ‐7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up‐regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long‐term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine‐activated apoptosis. J. Cell. Physiol. 227: 2398–2411, 2012. © 2011 Wiley Periodicals, Inc.     

Claudin 1 mediates TNFα-induced gene expression and cell migration in human lung carcinoma cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.     

Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.     

Senescent endothelial cells are prone to TNF-α-induced cell death due to expression of FAS receptor

The senescent endothelial cells show various phenotypes which can increase the incidence of inflammatory cardiovascular diseases, but the fundamental basis for such phenotypic changes of senescing cells remains to be elucidated. This study was undertaken to find transmembrane receptors that might be highly expressed in senescent endothelial cells and play a key role in cell death signal transduction. Comparison of mRNA expression in young and senescent human umbilical vein endothelial cells, using a cDNA microarray method, provided a list of transmembrane receptors including the FAS receptor (tumor necrosis factor receptor superfamily member 6) whose expression levels were significantly increased by cellular senescence. Additional studies focused on FAS demonstrated that a high expression of FAS receptor in senescent endothelial cells is responsible for the susceptibility to apoptotic cell death, as the siRNA-mediated suppression of FAS expression in senescent cells prevented the cell death, and overexpression of exogenous FAS in young cells increased cell death. We also verified that FAS expression level was closely associated with the activation of caspase-3 and caspase-9 involved in apoptosis. The senescence-induced transmembrane receptors including the FAS receptor may provide novel therapeutic targets to prevent cardiovascular diseases.     

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury.This process is primarily regulate...     

A novel signaling pathway in TNFα-induced neutrophil apoptosis

Comment on: Geering B, et al. Blood 2011; 117:5953-62.     

Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells

Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.     

BimL displacing Bcl-xL promotes Bax translocation during TNFα-induced apoptosis

Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. Bim_L, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim_L activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of Bim_L in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x_L and Bax decreased after TNFα treatment, while that between Bcl-x_L and Bim_L increased. Bax did not co-immunoprecipitate with Bim_L before or after the TNFα treatment. In addition, the increased interaction between Bim_L and Bcl-x_L was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim_L redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.