Bleomycin induces the extrinsic apoptotic pathway in pulmonary endothelial cells

Abstract

Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe2+) and O2. The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-xL, Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4–8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.

Endothelial Cells Recruit Macrophages and Contribute to a Fibrotic Milieu in Bleomycin Lung Injury

“…Similar to SSc, endothelial cells are believed to be a major target of BLM-induced injury when administered via intravenous or subcutaneous routes. Bleomycin can induce apoptosis in cultured endothelial cells (5), but whether endothelial cells undergo apoptosis after BLM treatment in vivo has not been clearly shown, except for one report where subendothelial blebbing was described (6). Although endothelial cells are widely accepted as a primary target of the drug, the extent to which endothelial cells contribute to BLM-induced pulmonary inflammation and fibrosis, and the extent of vasculopathy in this model, are debated.…”

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…In most eukaryotic cells, ⌬ m is the major component of the mitochondrial electrochemical transmembrane potential and, as such, is involved in pulmonary endothelial mitochondrial ATP generation, regulation of calcium homeostasis, apoptosis, nitric oxide signaling, and other functions (19,58,60,61). Dissipation of ⌬ m is considered a hallmark of mitochondrial dysfunction in diverse cell types, including pulmonary endothelial cells exposed to oxidative stresses and bleomycin (24,33,43,54,69). On the other hand, pulmonary endothelial ⌬ p is implicated in regulating channel-mediated calcium entry as a key signaling response to mechanical stimuli, vasoactive substances, oxidative stress, ischemia, and hypoxia (14,17,31,49,59,67,70).…”

“…dysfunction in diverse cell types, including pulmonary endothelial cells exposed to oxidative stresses and bleomycin (24,33,43,54,69). On the other hand, pulmonary endothelial ⌬ p is implicated in regulating channel-mediated calcium entry as a key signaling response to mechanical stimuli, vasoactive substances, oxidative stress, ischemia, and hypoxia (14,17,31,49,59,67,70).…”

Pulmonary Hypertension Is a Probable NO/ONOO−Cycle Disease: A Review

“…e most directly affected of these is the PARP mechanism because it is greatly stimulated by the single-and double-strand breaks in DNA that are produced by bleomycin. In addition, apoptosis, which is sometimes involved in NO/ONOO − cycle diseases, is also triggered by bleomycin [183]. It can be seen from this, that most of the NO/ONOO − cycle is triggered by bleomycin, such that this alone strongly suggests a NO/ONOO − cycle mechanism for PAH.…”

“…e drug bleomycin has been shown to initiate some cases of PAH [162,181]. It is known to increase several mechanisms involved in the NO/ONOO − cycle including stimulating poly (ADP-ribose) polymerase (PARP), oxidative stress, superoxide generation, in�ammatory cytokines, oxidative stress, partial uncoupling of the NOSs (which is presumably caused by BH4 depletion), mitochondrial dysfunction, and NF-B elevation [181][182][183][184][185]. Superoxide is speci�cally implicated in having a causal role in bleomycininitiated PAH because overexpression of superoxide dismutase in a mouse model lessens subsequent pulmonary hypertension, �brosis, and vascular remodeling following bleomycin treatment [185].…”