Activation of group IVC phospholipase A(2) by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells

Abstract

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A(2) (PLA(2)) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA(2) in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA(2) inhibited both (3)H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA(2). Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA(2).

Fig. 1

³H-arachidonic acid (³H-AA) and/or ³H-oleic acid (³H-OA; Fig. 1e) release from human coronary artery endothelial cells induced by 3-, 4-, 5-, or 6-ring PAHs. Cells were pre-labeled with ³H-fatty acid and stimulated for 1 h with vehicle or various concentrations of PAHs as described in “Materials and methods”. Cumulative release of ³H-AA into the medium was measured by scintillation counting. a 3-ring PAHs (acenapthalene, anthracene, phenanthrene). b 4-ring PAHs (chrysene, pyrene, benzo[a]anthracene, fluoranthene). c 5-ring compounds (benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene). d 6-ring compounds (indeno[1,2,3-c,d] pyrene, benzo[g,h,i]perylene). e ³H-oleic acid and ³H-AA release from ECs treated with SRM 1597A. *Significantly different from vehicle-treated control, P < 0.05; n = 4

Fig. 2

³H-oleic acid (³H-OA) release from human coronary artery endothelial cells induced by 3-, 4-, 5-, or 6-ring PAHs. Cells were pre-labeled with ³H-OA and stimulated for 1 h with vehicle or various concentrations of PAHs as described in “Materials and methods”. Cumulative release of ³H-OA into the medium was measured by scintillation counting. a 3-ring PAHs. b 4-ring PAHs. c 5-ring PAHs. d 6-ring PAHs. *Significantly different from vehicle-treated control, P < 0.05; n = 4

Fig. 3

Apoptosis of human coronary artery endothelial cells induced by PAHs. Cells were treated with vehicle, 30 μM PAHs, 10 μM arachidonic acid (AA) or oleic acid (OA) or 0.8 mg/mL SRM-1597A for 2 h and apoptosis determined by histone fragmentation (a) or by detection of PARP cleavage as described in “Materials and methods”. *Significantly different from vehicle-treated control P < 0.05; n = 4. Two antibodies were used for detection of PARP cleavage: one that recognizes both the 116-kD and the 89-kD fragments of PARP (b) and one that recognizes only the 89-kD cleavage product (c)

Fig. 4

Characterization of groups IVA, B, and C PLA₂ isoform expression and activity in human coronary artery endothelial cells. a Group IVA, B, and C are expressed by endothelial cells. RT-PCR analysis of total RNA from human coronary artery endothelial cells using PLA₂ primers was performed as described in “Materials and methods”. A 10-μL aliquot of the PCR was analyzed on a 1.5% agarose gel containing ethidium bromide. Amplification of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was performed as a control using the same reaction conditions. DNA size markers are in bp. b Endothelial cells exhibit significant group IVC PLA₂ activity. Whole cell homogenates were incubated with ¹⁴C-AA-PC in the presence of calcium or EGTA. Cells were pretreated with the group IV and VI inhibitor methyl arachidonoyl fluorophosphonate (MAFP; 10 μM), the group VI inhibitor bromoenol lactone suicide substrate (BEL; 1 μM), or vehicle and release of ¹⁴C-labeled arachidonic acid measured as described in “Materials and methods”. *Significantly different from results obtained in the absence of inhibitor. P < 0.05; n = 3

Fig. 5

Silencing of group IVC PLA₂ inhibits ³H-OA release and histone fragmentation induced by PAHs or SRM1597A. a Western blot analysis of lysates from human coronary artery endothelial cells (HCAEC) transfected with siRNA against group IVC PLA₂ (lane 1) or control siRNA (lane 2). Silencing RNA against group IVC PLA₂ did not silence the other cytosolic PLA₂ enzymes. b ³H-OA release from HCAECs transfected with Superfect alone, untargeted RNA or siRNA group IVC PLA₂ and treated with PAHs listed in the legend to Fig. 3 or SRM 1597A. c Histone fragmentation in HCAECs transfected with Superfect alone, untargeted RNA or siRNA group IVC PLA₂ and treated with PAHs or SRM 1597A. *Significantly different from vehicle, #Significantly different from response obtained in the absence of siRNA, P < 0.05 n = 3

Fig. 6

Western blot analysis of PARP and caspase-3 cleavage in aorta from feral mice trapped from the Superfund site or control site. PARP cleavage was assessed from mice taken from the control site (a) and from the Superfund site (b). c demonstrates the cleavage of caspase-3 from mice inhabiting the superfund site vs. the control site. Lanes 1–3 are representative of aortic tissue from mice from the Superfund site, and lanes 4–6 are from control mice. These blots are representative of 10 mice from each site