Rationale:: Atherosclerosis is a widespread and devastating disease, but the origins of cells within atherosclerotic plaques are not well defined. Objective:: To investigate the specific contribution of vascular smooth muscle cells (SMCs) to atherosclerotic plaque formation by genetic inducible fate mapping in mice. Methods and Results:: Vascular SMCs were genetically pulse-labeled using the tamoxifen-dependent Cre recombinase, CreER ^T2 , expressed from the endogenous SM22α locus combined with Cre-activatable reporter genes that were integrated into the ROSA26 locus. Mature SMCs in the arterial media were labeled by tamoxifen treatment of young apolipoprotein E–deficient mice before the development of atherosclerosis and then their fate was monitored in older atherosclerotic animals. We found that medial SMCs can undergo clonal expansion and convert to macrophage-like cells that have lost classic SMC marker expression and make up a major component of advanced atherosclerotic lesions. Conclusions:: This study provides strong in vivo evidence for smooth muscle-to-macrophage transdifferentiation and supports an important role of SMC plasticity in atherogenesis. Targeting this type of SMC phenotypic conversion might be a novel strategy for the treatment of atherosclerosis, as well as other diseases with a smooth muscle component.

Patients with high-grade serous ovarian cancer (HGSC) frequently receive platinum-based chemotherapeutics, such as cisplatin. Cisplatin binds to DNA and induces DNA-damage culminating in mitochondria-mediated apoptosis. Interestingly, mitochondrial DNA is critically affected by cisplatin but its relevance in cell death induction is scarcely investigated. We find that cisplatin sensitive HGSC cell lines contain higher mitochondrial content and higher levels of mitochondrial ROS (mtROS) than cells resistant to cisplatin induced cell death. In clonal sub-lines from OVCAR-3 mitochondrial content and basal oxygen consumption rate correlate with sensitivity to cisplatin induced apoptosis. Mitochondria are in two ways pivotal for cisplatin sensitivity because not only knock-down of BAX and BAK but also the ROS scavenger glutathione diminish cisplatin induced apoptosis. Mitochondrial ROS correlates with mitochondrial content and reduction of mitochondrial biogenesis by knock-down of transcription factors PGC1α or TFAM attenuates both mtROS induction and cisplatin induced apoptosis. Increasing mitochondrial ROS by inhibition or knock-down of the ROS-protective uncoupling protein UCP2 enhances cisplatin induced apoptosis. Similarly, enhancing ROS by high-dose ascorbic acid or H₂O₂ augments cisplatin induced apoptosis. In summary, mitochondrial content and the resulting mitochondrial capacity to produce ROS critically determine HGSC cell sensitivity to cisplatin induced apoptosis. In line with this observation, data from the human protein atlas (www.proteinatlas.org) indicates that high expression of mitochondrial marker proteins (TFAM and TIMM23) is a favorable prognostic factor in ovarian cancer patients. Thus, we propose mitochondrial content as a biomarker for the response to platinum-based therapies. Functionally, this might be exploited by increasing mitochondrial content or mitochondrial ROS production to enhance sensitivity to cisplatin based anti-cancer therapies.

The activation of caspase-8, a crucial upstream mediator of death receptor signaling, was investigated in epirubicin- and Taxol-induced apoptosis of B-lymphoma cells. This study was performed because the CD95/Fas receptor-ligand interaction, recruitment of the Fas-associated death domain (FADD) adaptor protein, and subsequent activation of procaspase-8 have been implicated in the execution of drug-induced apoptosis in other cell types. Indeed, active caspase-8 was readily detected after treatment of mature and immature B-lymphoid cells with epirubicin or Taxol. However, neither constitutive nor drug-induced expression of the CD95/Fas ligand was detectable in B-lymphoma cells. Furthermore, overexpression of a dominant-negative FADD mutant (FADDdn) did not block caspase-8 processing and subsequent DNA fragmentation, indicating that drug-induced caspase-8 activation was mediated by a CD95/Fas-independent mechanism. Instead, caspase-8 cleavage was slightly preceded by activation of caspase-3, suggesting that drug-induced caspase-8 activation in B-lymphoma cells is a downstream event mediated by other caspases. This assumption was confirmed in 2 experimental systems—zDEVD-fmk, a cell-permeable inhibitor of caspase-3–like activity, blocked drug-induced caspase-8 cleavage, and depletion of caspase-3 from cell extracts impaired caspase-8 cleavage after in vitro activation with dATP and cytochrome c. Thus, these data indicate that drug-induced caspase-8 activation in B-lymphoma cells is independent of death receptor signaling and is mediated by postmitochondrial caspase-3 activation.

Local anaesthetics are known to induce apoptosis in clinically relevant concentrations. Hitherto, it is unknown what determines the apoptotic potency of local anaesthetics. Therefore, we compared apoptosis induction by local anaesthetics related to their physicochemical properties in human neuronal cells. Neuroblastoma cells (SHEP) were incubated with eight local anaesthetics, two of the ester and six of the amide types. At least, five concentrations of each local anaesthetic were evaluated. After incubation for 24 h, rates of cells in early apoptotic stages and overall cell death were evaluated by annexin V and 7-amino-actinomycin D double staining by flow cytometry. The concentrations that led to half-maximal neurotoxic effects (LD₅₀) were calculated and compared for all local anaesthetics. All local anaesthetics were neurotoxic in a concentration-dependent manner. All drugs induced similar rates of early apoptotic cell formation at low concentrations, whereas at high concentrations, late apoptotic or necrotic cell death predominated. Comparison of LD₅₀ values of the different local anaesthetics resulted in the following order of apoptotic potency from high to low toxicity: tetracaine>bupivacaine>prilocaine=mepivacaine=ropivacaine>lidocaine>procaine=articaine. The toxicity correlated with octanol/buffer coefficients and also with experimental potency of the local anaesthetic, but was unrelated to the structure (ester or amide type). All commonly used local anaesthetics induce neuronal apoptosis in clinically used concentrations. The neurotoxicity correlates with lipid solubility and thus with the conduction blocking potency of the local anaesthetic, but is independent of the chemical class (ester/amide).

The stilbene phytochemicals resveratrol and piceatannol have been reported to possess substantial antitumorigenic and antileukemic activities, respectively. Although recent experimental data revealed the proapoptotic potency of resveratrol, the molecular mechanisms underlying the antileukemic activity have not yet been studied in detail. In the present study, we show that resveratrol, as well as the hydroxylated analog piceatannol, are potent inducers of apoptotic cell death in BJAB Burkitt-like lymphoma cells with an ED₅₀ concentration of 25 μM. Further experiments revealed that treatment of BJAB cells with both substances led to a concentration-dependent activation of caspase-3 and mitochondrial permeability transition. Using BJAB cells overexpressing a dominant-negative mutant of the Fas-associated death domain (FADD) adaptor protein to block death receptor-mediated apoptosis, we demonstrate that resveratrol- and piceatannol-induced cell death in these cells is independent of the CD95/Fas signaling pathway. To explore the antileukemic properties of both compounds in more detail, we extended our study to primary, leukemic lymphoblasts. Interestingly, piceatannol but not resveratrol is a very efficient inducer of apoptosis in this ex vivo assay with leukemic lymphoblasts of 21 patients suffering from childhood lymphoblastic leukemia (ALL).