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Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells
H C Thomas1, M W Lamé, S K Dunston
1Departments of Veterinary Pathology, Microbiology, and Immunology, University of California at Davis, California, USA.
Abstract:
In the monocrotaline (MCT) model of pulmonary hypertension, the pulmonary vascular endothelium is the likely early target of the reactive metabolite monocrotaline pyrrole (MCTP). Incubation of cultured bovine pulmonary arterial endothelial cells (BPAEC) with MCTP results in covalent binding to DNA, cell cycle arrest, and delayed but progressive cell death. The mode of cell death in MCTP-induced endothelial damage has not been addressed previously. Since DNA damage is frequently associated with apoptosis, the presence or absence of apoptosis in adherent BPAEC was determined by several techniques, including morphologic and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling. Two concentrations of MCTP (5 and 34.5 microgram/ml) along with a vehicle control were examined with each assay. Both concentrations of MCTP induced increasing numbers of cells to undergo apoptosis over time beginning as early as 6 h after exposure to MCTP in the high concentration group. Control and vehicle control cells exhibited small amounts of apoptosis (1-2%), which did not change over the duration of the experiment. Additionally, cell membrane integrity was assessed over time by either exposure to membrane-impermeant dyes or measuring LDH release. By either method, BPAEC had increased membrane permeability after about 48 h of either low or high concentration MCTP exposure. We conclude that both a low or high concentration of MCTP causes cell death in BPAEC by inducing apoptosis.
Insights
Monocrotaline pyrrole (MCTP) induces apoptosis, a form of programmed cell death, in bovine pulmonary arterial endothelial cells (BPAEC). This finding clarifies the mechanism of endothelial cell death in the monocrotaline model of pulmonary hypertension.
Area of Science:
- Cell Biology
- Toxicology
- Pulmonary Medicine
Background:
- The monocrotaline (MCT) model is used to study pulmonary hypertension.
- Monocrotaline pyrrole (MCTP) is a reactive metabolite implicated in MCT-induced pulmonary vascular endothelial damage.
- The precise mechanism of MCTP-induced endothelial cell death was previously uncharacterized.
Purpose of the Study:
- To determine the mode of cell death in bovine pulmonary arterial endothelial cells (BPAEC) exposed to MCTP.
- To investigate whether MCTP induces apoptosis in BPAEC.
Main Methods:
- Cultured BPAEC were exposed to two concentrations of MCTP (5 and 34.5 microgram/ml) or a vehicle control.
- Apoptosis was assessed using morphologic evaluation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNL).
- Cell membrane integrity was evaluated by dye exclusion and lactate dehydrogenase (LDH) release assays.
Main Results:
- Both low and high concentrations of MCTP significantly increased the number of apoptotic BPAEC over time.
- Apoptosis was detectable as early as 6 hours post-exposure in the high concentration group.
- Increased membrane permeability, indicative of cell damage, was observed around 48 hours after MCTP exposure.
Conclusions:
- Monocrotaline pyrrole (MCTP) induces cell death in BPAEC primarily through apoptosis.
- These findings elucidate a key mechanism in MCT-induced pulmonary endothelial cell injury and contribute to understanding pulmonary hypertension pathogenesis.