Oxidized Low-Density Lipoprotein Induces Apoptosis of Human Endothelial Cells by Activation of CPP32-Like Proteases
A Mechanistic Clue to the ‘Response to Injury’ Hypothesis
Background Oxidized LDL (oxLDL) is believed to play a key role as a triggering molecule that causes injury to the endothelium as an early event in atherogenesis. However, the mechanisms by which oxLDL injures endothelial cells are entirely unknown. We speculate that oxLDL may activate a cellular suicide pathway that leads to apoptosis.
Methods and Results Human umbilical venous endothelial cells (HUVEC) were incubated with increasing doses of native or oxLDL for 18 hours. Apoptosis of HUVEC was measured with an ELISA specific for histone-associated DNA fragments and confirmed with DNA laddering. Native LDL had no effect, but incubation with oxLDL dose-dependently induced apoptosis of HUVEC. Induction of apoptosis by oxLDL was associated with increased CPP32-like protease activity, which is the major enzyme that initiates the proteolytical cascade leading to cell death. Specific inhibition of CPP32 activity completely abrogated oxLDL-induced apoptosis. The antioxidants N-acetylcysteine and the combination of vitamins C and E prevented oxLDL-induced apoptosis, abrogated the enhancement of CPP32-like protease activity, and inhibited the proteolytic cleavage of CPP32 into its active subunit p17.
Conclusions oxLDL activates the suicide pathway leading to apoptosis of endothelial cells by enhancing CPP32-like protease activity. The oxLDL-mediated activation of CPP32 appears to involve the elaboration of reactive oxygen species. Activation of the cell death effector CPP32 by oxLDL may provide a mechanistic clue to the “response-to-injury” hypothesis of atherogenesis.
Experiments in animals that have demonstrated increased endothelial cell turnover rates in lesion-prone regions in the arterial tree1 2 support the hypothesis that injury to the endothelium might precipitate the atherosclerotic process.3 oxLDL is believed to play a key role as a triggering molecule in the earliest phase of atherosclerosis.4 The results of in vitro studies have shown that oxidatively modified LDL causes injury to endothelial cells.5 However, the mechanisms by which oxLDL injures endothelial cells are entirely unknown.
Endothelial cell injury may lead to two distinct types of cell death: apoptosis or necrosis.6 Apoptosis refers to the morphological alterations exhibited by “actively” dying cells, including cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation,7 whereas necrosis is characterized by cellular swelling, rupture of plasma membrane, and cell lysis.6 The signal transduction leading to apoptosis is characterized by a complex array of biochemical pathways, whereby CPP32, a member of the interleukin-1β–converting enzyme–like protease family, appears to play a central role.8 9
It was the aim of the present study to investigate whether oxLDL induces apoptosis of endothelial cells via activation of CPP32-like proteases. Furthermore, we addressed any potentially protective effect of antioxidants on oxLDL-induced apoptosis as well as CPP32-like protease activity.
Preparation and Oxidation of LDL
Human LDL was isolated through sequential ultracentrifugation and oxidated as described previously.10 Antioxidant-free LDL (0.3 mg protein/mL) was incubated with CuSO4 (5 μmol/L) for 24 hours at 23°C. The degree of oxidation was assessed via two different methods: the increase of mobility on agarose gel (1.4 versus native LDL) and the formation of thiobarbituric acid–reactive substances (3.4±0.8 mmol/L).
HUVEC were purchased from Cell Systems/Clonetics and cultured in endothelial basal medium supplemented with hydrocortisone (1 μg/mL), bovine brain extract (12 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 ng/mL), epidermal growth factor (10 ng/mL), and 10% fetal calf serum (GIBCO) until the third passage. DNA fragmentation analysis was carried out as recently described11 12 with a cell death detection assay (Boehringer-Mannheim Biochemicals). For demonstration of the DNA ladder, 1×106 cells were removed from culture flask, washed with PBS, and incubated in lysis buffer (5 mmol/L Tris-HCl, pH 8, 20 mmol/L EDTA, and 0.5% Triton X-100) for 15 minutes at 4°C, and the DNA was isolated and radioactively labeled as previously described.11 12 LDH was measured as previously described.13
Measurement of CPP32-Like Protease Activity
HUVEC (5×105 cells) were lysed in buffer (1% Triton X-100, 0.32 mol/L sucrose, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 2 mmol/L dithiothreitol, and 10 mmol/L Tris-HCl, pH 8) for 15 min at 4°C, followed by centrifugation (20 000g, 10 minutes), and CPP32-like activity was measured in resulting supernatants as described previously.8 9
RNA was prepared according to the method of Liu et al,14 and RNA was blotted onto nylon membranes; the blots were hybridized with a radioactively labeled human CPP32 probe and human GAPDH probe and incubated for 24 hours. Then, the blots were washed (0.1% SDS/0.2% SSC) and exposed to x-ray films.
Proteins were prepared as described for detection of CPP32 activity, resolved on 15% SDS-polyacrylamide gels, and blotted onto nitrocellulose membranes.11 12 CPP32/p17 was detected through incubation with the primary antibody against human CPP32/p17 (Santa Cruz Biochemicals; 1:200 in BSA/0.5% Tween 20, for 1 hour) and horseradish peroxidase–conjugated anti-goat IgG antibody (1:2000 in BSA/0.5% Tween 20, for 1 hour), followed by enhanced chemiluminescence (Amersham International).
Statistical analysis was performed with ANOVA followed by a modified Bonferroni LSD test (SPSS Software).
Induction of Apoptosis by oxLDL
Incubation of HUVEC with oxLDL for 18 hours induced DNA fragmentation in a concentration-dependent manner with maximal effects at 10 μg/mL (Fig 1⇓, top). In contrast, native LDL did not induce apoptosis in the concentration range tested (Fig 1⇓, top). The induction of apoptosis by oxLDL was confirmed by demonstrating DNA fragmentation through agarose gel electrophoresis (data not shown). LDH release did not increase ≤10 μg/mL oxLDL (105±11% compared with control cells) excluding the induction of necrosis.
Involvement of CPP32-Like Proteases
Tumor necrosis factor-α as well as FAS-mediated apoptosis is mediated via activation of CPP32-like proteases.8 9 Therefore, we investigated the involvement of CPP32-like proteases in oxLDL-stimulated apoptosis. Incubation of HUVEC with oxLDL (10 μg/mL) significantly increased CPP32-like protease activity (133±12% of controls), whereas native LDL had no effect on CPP32-like protease activity (91±5%; P<.05 versus oxLDL). Furthermore, inhibition of CPP32-like activity by the specific tetrapeptide inhibitor Ac-DEVD-CHO (100 μmol/L) completely prevented the oxLDL-induced apoptosis (Fig 1⇑, bottom). Incubation of HUVEC with oxLDL (5 or 10 μg/mL) for 2 hours (data not shown) or 6 hours (Fig 2a⇓) did not alter mRNA levels of CPP32, indicating the lack of a transcriptional effect of oxLDL on CPP32. In contrast, oxLDL incubation stimulated the proteolytic cleavage of CPP32 into its active subunits, as demonstrated by the appearance of the CPP32 p17 subunit (Fig 2b⇓) and by a reduction in the full-length CPP32 of 44±9% (mean±SEM; n=4).
Inhibition of oxLDL-Induced Apoptosis by Antioxidants
To investigate a potential involvement of oxidative stress in apoptosis induction by oxLDL, we determined the effects of the antioxidants NAC and the combination of vitamins C and E, which has been shown to be more effective than the use of each vitamin alone.12 NAC (200 μmol/L) as well as vitamins C and E (10 μmol/L) prevented oxLDL-induced apoptosis (Fig 1⇑, bottom). Furthermore, the increase in CPP32-like protease activity triggered by oxLDL (133±12% of control) was drastically reduced by the antioxidants NAC (70±5.8% of control, P<.01) and vitamins C and E (96±8% of control, P<.05). The decreased CPP32-like protease activity correlated with a reduction in proteolytic cleavage of the CPP32 into the active p17 subunit (Fig 2b⇑).
The results of the present study demonstrate that oxLDL induces apoptosis of human endothelial cells via activation of a CPP32-like protease. Inhibition of proteolytical cleavage of CPP32 into its active subunits p12 and p17 completely abrogated oxLDL-induced apoptosis of HUVEC. These findings may provide a clue to the “response-to-injury” hypothesis of atherogenesis, which proposed an as-yet-undefined injury to the endothelium as the central mechanism for initiating atherosclerotic lesion development. Moreover, treatment of HUVEC with antioxidants prevented oxLDL-induced activation of CPP32 as well as apoptosis, suggesting that the generation of reactive oxygen species contributes to the induction of apoptosis by oxLDL.
Endothelial cells of lesion-prone regions are characterized by an increased cell turnover rate1 associated with an enhanced accumulation of LDL,2 suggesting a mechanistic link between endothelial cell injury and the susceptibility to atherosclerotic plaque development.3 oxLDL is a well-established triggering molecule in the atherosclerotic process.4 The present study demonstrates that even low concentrations of oxLDL activate a major signaling pathway, leading to endothelial cell apoptosis (ie, the proteolytical cleavage of CPP32-like proteases). CPP32, a member of the interleukin-1β–converting enzyme–like protease family, has been identified as one of the key enzymes responsible for apoptosis in human cells.9 On activation, proteolytical cleavage of CPP32 releases two subunits to compose the active, mature form of the enzyme, with the larger p17 subunit containing the catalytic site.9 We now demonstrate that oxLDL-induced apoptosis is associated with enhanced CPP32-like protease activity due to increased proteolytic cleavage. Moreover, a specific peptide aldehyde inhibitor of CPP32 completely inhibited oxLDL-induced apoptosis. Taken together, these findings provide strong evidence to link oxLDL-induced apoptosis of HUVEC to the activation of CPP32. Thus, oxLDL appears to injure endothelial cells via activation of the cell suicide pathway.
The findings of the present study may provide a clue to the molecular mechanisms underlying the “response-to-injury” theory of atherogenesis. Apoptosis, which refers to the morphological alterations of “actively” dying cells in contrast to necrosis, is believed to represent an important defense in the event of questionable or irreparable cell damage.7 This type of physiological cell death, due to the use of an intrinsic mechanism that exists for the specific purpose of committing suicide, enables removal of unwanted cells without incitation of an inflammatory response, which usually follows cell death by necrosis. Thus, apoptosis of endothelial cells may allow for an uncompromised regeneration of the endothelial cell layer despite injurious insults. This is exactly what has been observed in lesion-prone areas of the arterial tree, which are characterized by an increased cell turnover rate.1
oxLDL has been shown to increase the endothelial production of partially reduced oxygen species, including hydrogen peroxide, superoxide anions, and hydroxyl radicals.10 15 The generation of reactive oxygen species has been demonstrated to be involved in mediating apoptosis via activation of the cell death program in numerous cells,16 including endothelial cells.12 Antioxidants completely prevented the induction of apoptosis in response to stimulation with oxLDL. Thus, the oxidant stress imposed by oxLDL appears to activate the signaling pathways, leading to apoptosis of endothelial cells. However, the precise mechanisms by which oxLDL activates CPP32-like activity and cleavage remain to be determined.
In summary, the present investigation demonstrates that oxLDL activates the suicide pathway leading to apoptosis of endothelial cells. The oxLDL-induced enhancement of CPP32-like protease activity appears to involve the elaboration of reactive oxygen species. These findings may not only provide a mechanistic clue to the “response-to-injury” hypothesis leading to increased cell turnover rates but also considerably extend the role of oxLDL as a key triggering molecule for activation of endothelial cells.
Selected Abbreviations and Acronyms
|HUVEC||=||human umbilical venous endothelial cells|
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Di 600/2-1, Di 600/2-2, and Ga 431/2-1). We thank Christine Göbel for expert technical assistance.
- Received February 3, 1997.
- Revision received February 19, 1997.
- Accepted February 20, 1997.
- Copyright © 1997 by American Heart Association
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