Study On The Mechanism Of Minimally Modified Low-density Lipoprotein-induced Apoptosis In Vascular Endothelial Cells

Apoptosis, as one of the main pathological features of atherosclerosis (AS), plays a key role in the formation and development of atherosclerotic plaques. Endothelial cells (ECs) apoptosis is the initial step in the process of AS. Therefore, understanding the mechanism by which ECs undergo apoptosis will contribute to the prevention and therapies of AS. Increasing evidences suggested that oxidative modification of low-density lipoprotein (LDL) might be responsible for apoptosis in the vessel wall. Nevertheless, the biological relevance of apoptotic signaling pathways induced by oxidized LDL for the evolution of atherosclerotic lesion remains unknown, because the majority of previous studies used extensively oxidized LDL (ox-LDL), which was generated by incubation with copper and not exist in early atherosclerotic lesions. Contrary to ox-LDL, minimally modified oxidized LDL (mm-LDL) prepared by iron, has many characteristics of modified LDL generated on the artery wall during the initial stages of AS. It has been demonstrated to play important roles in many aspects of the pathological process of AS. It is therefore believed that cells in early atherosclerotic lesions are not mainly exposed to ox-LDL but to mm-LDL. So we chose mm-LDL for our present study. To investigate whether mm-LDL could induce ECs apoptosis and elucidate the potential signal transduction pathway involved in this process, the present study was performed in several parts as follows:1. To investigate whether mm-LDL could induce apoptosis in human umbilical vein endothelial cells (HUVECs).HUVECs were obtained from umbilical cord veins by trypsin digest. Human serum LDL was isolated by two-step ultracentrifugation, mm-LDL was prepared by incubation with iron. HUVECs were treated with different concentration of LDL or mm-LDL for different time. Cell viability was measured by MTT assay. Phase-contrast microscopy, fluorescence microscopy and transmission electron microscopy (TEM) were used to observe mm-LDL-induced morphological changes. Apoptosis ratio was detected by flow cytometry (FCM).The results showed that after HUVECs were exposed to various concentration of mm-LDL (lOOug/ml ~ 300 ug/ml) for 12h, 24h and 48h respectively, cell viability was inhibited significantly in a concentration- and time- dependent manner. The maximum inhibition (59.3%) was observed at 48h of treatment with 300 ng/ml mm-LDL. In contrast, up to 300 jxg/ml n-LDL exerted no influence on cell viability. Nuclei in normal cells exhibited diffused Hoechst33258 staining of the chromatin. In contrast, a large number of cells treated with mm-LDL underwent a reduction in size and became round in shape. Nuclei were severely shrunken, fragmented and compacted with condensed chromatin. TEM analysis showed that untreated HUVECs revealed dispersed chromatin and rich in mitochondria. mm-LDL-treated cells illustrated that prominent chromatin condensation, pyknosis and fragmentation. Cytoplasmic alteration included vacuole-like cytoplasm and dilation of mitochondria. The FCM assay also suggested the induction of apoptosis by revealing a sub-Gl peak. Apoptotic ratio of cells treated with mm-LDL (100 ug/ml, 200 Hg/ml and 300 ug/ml) for 48h were 7.2+1.6 %, 20.7 + 3.1 % and 42.3 + 4.6 % respectively. These results suggested that mm-LDL-induced cell death was due to apoptosis.2. To further investigate the role of calcium and phospholipase A2 (PLA2) in the cellular signaling transduction mechanism by which mm-LDL induced apoptosis of HUVECs.Fluo-3/AM staining was used to detect the change of [Ca2+]i in HUVEC. PLA2 activity was determined by measuring the release of [3H]-arachidonic acid (3H-AA) from prelabeled cells. RT-PCR analysis was used to measure the mRNA expression of cytosolic PLA2 (cPLA2). Phosphorylation of cPLA2 was analyzed using Western Blots. Immunofluorescence was used to detect the subcellular translocation of cPLA2.The results showed that after mm-LDL (100 |xg/ml) was added, the intracellular calcium concentration increased rapidly. The fluorescence intensity reached the peak value within 55s, and then gradually declined to the baseline level. Preincubation with extracellular Ca2+ chelator EGTA decreased resting level of [Ca2+]i and significantly reduce the [Ca2+]i elevations induced by mm-LDL by 35.5%. mm-LDL stimulated 3H-AArelease in a dose- and time-dependent manner. This result could be attributed to the increase in PLA2 activity. A loss (24.3%) of AA release in response to mm-LDL in the presence of 5 mmol/L EGTA, an observation highly implied that the activation of PLA2 by mm-LDL was calcium-dependent. In the presence of SPLA2 special inhibitor p-BPB (20 umol/L), the releases of AA did not reduce. On the contrary, pretreated with cPLA2 inhibitor AACOCF3 (15 umol/L) lessen the releases of AA by 35%, which indicated that mm-LDL induced activation of CPLA2. Western Blots analysis showed that most of the CPLA2 from untreated HUVECs was seen to be in the more rapidly migrating unphosphorylated form. Treatment with mm-LDL (300 ug/ml) increased the proportion of phosphorylated CPLA2. This effect was detected from 0.5h and reached its maximum at 2h. The proportion of phosphorylated CPLA2 decreased from 4h but is still higher than that of the control cells. Also, the time course of CPLA2 phosphorylation correlated with that of CPLA2 activation. These data led us to conclude that CPLA2 phosphorylation was involved in mm-LDL induced activation of CPLA2. Cells stained with anti-cPLA2 antibody were observed under fluorescence microscopy. In the resting cells, the enzyme appeared diffusely distributed in the cytoplasm. After 4h treatment with mm-LDL, fluorescence in nuclear increase intensively, which indicate the translocation of CPLA2 to nuclear. Pretreated with EGTA (5mmol/L) could partly prevent CPLA2 from translocation to nuclear. In addition, RT-PCR showed that the expression of CPLA2 mRNA did not change after mm-LDL treatment, which indicated that mm-LDL-induced regulation of CPLA2 did not happen at the transcription level. Compared with that of mm-LDL (300 ug/ml) treated HUVECs, inhibition of calcium influx by EGTA or inhibition of CPLA2 activity by AACOCF3 reduced the apoptotic ratio by 41.8% and 33.6%, respectively. Addition of exogenous AA (50 umol/L), the predominant product of CPLA2 activation to AACOCF3-treated HUVECs restored mm-LDL-induced apoptosis. Taken together, these results demonstrated that calcium and CPLA2 involved in mm-LDL-induced apoptosis in HUVECs.3. To further investigate the role of AA, the product of CPLA2 activation, in mm-LDL-induced apoptosis in HUVECs, we observed the effect of exogenous AA on theapoptosis of HUVECs, also detect the changes of cellular status of oxidative stress and the protein expression of three apoptosis-related genes (bcl-2, bax and p53) during this process.HUVECs were incubated with exogenous AA (25 umol/L ~ 200 umol/L) for different time. Cell viability was analyzed by MTT assay. Cell damage degree was accessed by the release of lactate dehydrogenase (LDH). Gimersa staining was used to show morphological changes in apoptotic cells. Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to detect DNA fragmentation. Agarose gel electrophoresis analyze DNA ladder. Apoptosis ratio was detected by FCM. Cellular status of oxidative stress was assessed by measure the intracellular level of malondialdehyde (MDA) and the reduced glutathione (GSH). Effect of AA on the protein expression of apoptosis-related genes was analyzed using Western Blots.The results showed that low concentration of AA (25 umol/L) exerted no influence on cell viability and the release of LDH. Higher concentrations of AA (50 umol/L ~ 200 umol/L) reduced cell viability and increased the release of LDH in a time- and dose-dependent manner. Compared with the control cells, TUNEL-positive cells increased significantly after AA treatment. HUVECs treated with AA (150 umol/L) for 24h showed typical apoptotic morphological features after Gimersa staining. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length was observed after AA treatment, and "DNA ladder" became clearer with the addition of AA increasing. FCM analysis showed that the apoptotic ratio in HUVECs treated with AA (50 umol/L, 100 umol/L and 150 umol/L) for 20h were 20.7 + 3.6%, 38.6 ±4.3% and 52.5 + 8.6% respectively. These data together indicated that higher concentration of AA could induce apoptosis in HUVECs. Our experiment also found that the MDA content increase significantly in a dose-dependent manner upon AA treatment and the opposite tendency was found for the reduced-GSH. Furthermore, pretreatment with antioxidant a-tocopherol (50 umol/L) attenuated AA-induced increase in cellular MDA content and decrease in cellular reduced-GSH content inhibited the formation of "DNA ladder" and reduced the apoptotic ratio. These finds indicated that oxidative stress was involved in AA-inducedapoptosis in HUVECs. Western Blots analysis showed that incubation of cells with AA dramatically reduced the level of Bcl-2 protein in a dose-dependent manner, while no change of Bax expression was detected, which therefore resulted in the relative decrease of Bcl-2 / Bax ratio. Preincubation with a-tocopherol prevented AA-induced downregulation of Bcl-2 and increased the Bcl-2 / Bax ratio, which may contributed to its anti-apoptosis effect. We also did not see significant difference of p53 protein between the control and AA-treated cells. Our data indicated that AA induced apoptosis in HUVECs via downregulation of Bcl-2.In conclusion, our study demonstrated firstly that mm-LDL could induce apoptosis in HUVECs in vitro. CPLA2 that activated by increase of intracellular calcium plays an important role in this process and AA might be an important lipid mediator in this pathway. Furthermore, we also proposed that the use of specific CPLA2 inhibitor might be a favorable therapeutic approach in the treatment of AS.