3.(F).1. Effect o f copper ion concentration.
Addition o f Cu^^ to the experimental medium promoted LDL oxidation
in cell free controls and in the presence o f RMC [figure 3.4] in a dose
Cell free RMC 70 - 60 - 30 - 20 - 10 - 1 33 0.133 Cu^* Concentration (nM) Figure 3.4 O xidation of LDL: effect o f concentration. Experimental medium containing 200 pg/ml o f LDL, was incubated with or without RMC in the presence o f CuCl: at the concentration given in the bar graph for 24 h under the conditions described in section 3.(C).1. The TBARS were measured in the supernatants and expressed as nmol of TBARS/m g LDL. Data are the means ± SD o f duplicate experiments.
The TBARS generated in the presence o f cells were consistently higher than in cell free controls. The experimental medium R PM I-1640 is formulated without the addition o f copper and iron salts and has been reported to contain undetectable concentrations o f transition metal ions as measured by atomic absorption (Esterbauer et al. 1990). There is an absolute requirement for the presence o f trace amounts o f transition metal ions in incubation media for cells to oxidise LDL (Jurgens et al. 1987). The copper concentration used [5 nmol per 200 fig LDL, (in an incubation volume o f 1 ml)] in this study was similar to that used for smooth muscle cell mediated LDL oxidation experiments (Heinecke et al. 1986). It has been reported that the transition metal ion concentration needed to promote LDL oxidation in complex media such as R PM I-1640 (composition given in table 3.2) needs to be somewhat higher than that required for oxidation o f LDL in PBS (1.3 nmol Cu per 200 pg LDL)
because the available copper concentration may be somewhat diminished due to binding to media components such as histidine residues (Esterbauer et al
1990).
3.(F).2. Time course of LDL oxidation.
LDL oxidation was followed over time, by measuring the TBARS generated in RMC and cell free experimental medium at the time points indicated in figures 3.5 and 3.6.
60 RM C Cell free 50 40 30 20 10 0 0 3 6 9 12 15 18 21 24 27 Tim e (hours)
Figure 3.5 Time course of LDL oxidation in the presence of CuCh (5 gM).
RMCs or cell free dishes were incubated with experimental medium containing 200 M-g/ml of LDL and 5p.M CuCb at 37 °c. Medium was removed at the time points indicated on the graph and the extent LDL oxidation estimated by measuring the concentration o f TBARS generated. The results represent the means ± SD o f duplicate experiments.
Oxidation o f LDL proceeded at a relatively slow rate for 3-6 hours (the lag period) followed by a rapid rise in the levels o f TBARS generated (the propagation phase). This phase was followed by a plateau phase in which the concentration o f TBARS measured was relatively stable. It is important to note that copper induced LDL oxidation followed continuously by measuring the generation o f diene-conjugates (absorption at 234 nm), lipid hydroperoxides and TBARS proceeds at a faster rate in PBS than in RPM I and by 24 hours is in the decomposition phase. It is also difficult to get a accurate determination o f the lag-phase o f LDL oxidation by measuring TBARS. Continuous monitoring o f LDL oxidation over time is problematic in experiments involving cells as the most commonly used method involves monitoring the generation o f diene-conjugates by measuring the absorption o f an LDL sample at 234 nm in a spectrophotom eter continuously. Therefore it is
C ell free RMC 0 5 Time (hours) Figure 3.6 T im e course of LDL oxidation in the p resen ce o f F eC h (5 gM ). RMCs or cell free dishes were incubated with experimental medium containing 2 0 0 gg/m l of
LDL and 5gM FeCL at 37 °c. Medium was removed at the time points indicated on the graph and the extent LDL oxidation estimated by measuring the concentration o f TBARS generated. Each time point represents means ± SD o f duplicate experiments
difficult to conclude whether the length o f the lag period before LDL oxidation
enters the auto-catalytic phase is altered by the incubation o f LDL with RMC.
The most frequently used assay to measure LDL oxidation in this study was the
TEA assay and the level o f TBARS generated was relatively stable at 24 hours.
Therefore most o f the experiments were performed using an incubation period
o f 24 hours.
3.(F).3. LDL oxidation is promoted by RMC.
Several independent experiments performed showed that RMC
promoted LDL oxidation to a greater extent than cell free controls.
Freshly isolated LDL had <0.1 nmol TBARS/mgLDL and had similar
electrophoretic mobilities to serum LDL (data not shown). However, in
experiments where Cu^^ was not added to the LDL containing experimental
medium, there was detectable TBARS [figure 3.7] lipid hydroperoxides [figure
3.8] and an increase in electrophoretic mobility [figure 3.9] in media analysed
after a 24 hour incubation with cell free dishes or RMCs. The results obtained
for cell incubations, although higher than cell free controls did not reach
60 50 - d 40 20 - 10 - Cell free RMC Cell free + Cu^* RMC + Cu2+
X
RMCs or cell free dishes were incubated with experimental medium containing 2 0 0 pg/m l of
LDL at 37 °c for 24 h with or without CuCb (5pM ) and the medium was assayed for TBARS as described in section 3.(C).1. Results presented are the mean ± SD o f 5 independent experiments. *p=0.202 (cell free vs RMC) **p=0.024 (cell free+CX^ vs RMC+CX^).
Thiobarbituric acid reactive substances generated in experimental media containing LDL (200 pg/ml) and (SpM) after an incubation period o f 24 hours with cultures o f RMCs were 43 % higher than in cell free controls and this results was statistically significant at a value o f p=0.024.
Figure.3.7 Thiobarbituric acid assay results.
700 Cell free RMC Cell free+Cu' RMC+Cu^* 600 - 500 - cm 400 - £ 300 - 200 - 100 - Figure 3.8 Lipid
hydroperoxide assay results. RMCs or cell free dishes were incubated with experimental medium containing 2 0 0 pg/ml
o f LDL at 37 °c for 24 h with or without CuCb (5pM ) and the medium was assayed for LPO as described in section 3.(C).2. Results presented are the mean ±SD o f 4 independent experiments. *p=0.261 (cell free vs RMC) **p-Q 03 (cell free+Cu^^ vs RM C+Cu^l.
A similar result was obtained when total lipid hydroperoxides were measured in LDL containing media incubated with RMCs in the presence o f Cu^^, yielding values 48 % higher than cell free controls and this result was statistically significant at a p value o f 0.03.
The electrophoretic mobility o f LDL incubated with RM Cs in the presence o f Cu^^ (relative to native LDL) was also 28 % greater than LDL incubated in the presence o f Cu^^ in the absence o f cells (p=0.049)
7 -
f:
i : :
1 H 0 CZZl Cell free RMC Cell free + Cu^* RMC + Cu^+Î
Figure 3.9 Electrophoretic mobility results.
RMCs or cell free dishes were incubated with experimental medium containing 2 0 0 fj.g/ml of
LDL at 37 ^c for 24 h with or without CuCb (5|o.M) and an aliquot o f the medium was subjected to agarose gel electrophoresis as described in section 3.(C).3. The mobility o f each sample was compared with the mobility o f a native LDL sample. The results presented are the means ± SD o f 3 independent experiments. *p=0.15, **p=0.049.
3.(F).4. LDL oxidation in the presence of RM C: Effect of EDTA and
BHT.
The divalent cation chelator EDTA (100 pM ) substantially decreased cell induced and cell free LDL oxidation (figure 3.10). This decrease in oxidation was statistically significant. However, TBARS were still detectable in
cell free and RMC (1.36 ± 0.014 and 7.44 ± 1.75 nmol TBARS/mg LDL respectively), media, incubated in the presence o f EDTA. This suggests that either copper-EDTA complexes may still be able to promote LDL oxidation, that the LDL used in the experiments contained low levels o f pre-formed lipid peroxides or that experimental solutions may have contained low levels o f iron ions. It has been reported that copper-EDTA complexes and Iron-EDTA complexes promote reactions that result in LDL oxidation in Hams F-10 medium. H owever concentrations greater than 10 pM EDTA were found to inhibit oxidation o f LDL (Lamb and Leake, 1992a).
Cell free control RMC control Cell free + EDTA RMC + EDTA 50 - 20 - 10 - Figure 3.10 LDL oxidation by RMC: effect of EDTA RMCs or cell free dishes were incubated with experimental medium containing 200 |ig/ml of LDL and CuCL (5}iM) at 37 °c in the
presence or absence of EDTA (100 [iM) for 24 h. The supernatants were assayed for TBARS as described above. The results in the figure are the means ± SD of 4 experiments. *p=0.004 (cell free control vs cell free + EDTA). **p=0.014 (RMC control vs RMC+EDTA).
The chain breaking antioxidant BHT (20 pM ) caused a substantial and statistically significant reduction in both cell free and RMC induced LDL oxidation (figure 3.11). There were low concentrations o f TBARS detectable in cell free and RMC media incubated in the presence o f BHT (0.22 ± 0.083
and 0.436 ± .141 nmol TBARS/mg LDL respectively). This was probably due to the presence o f pre-formed endogenous lipid peroxides in LDL or peroxides introduced during LDL isolation procedures.
50
40 -
30 -
10 H
Cell free control RMC control Cell free + BHT RMC + BHT
Figure 3.11 LDL oxidation by RMC: effect of BHT RMCs or cell free dishes were incubated with experimental medium containing 200 gg/ml of LDL and CuCb (5pM) at 37 °c in the presence or absence of BHT (20 pM) for 24 h. The supernatants were assayed for TBARS as described above. The results in the figure are the means ± SD of 4 experiments.
*p=0.004 (cell free control vs cell free + BHT). **p=0.014 (RMC control vs RMC+ BHT).
3.(F).5. Effect of the NADPH oxidase inhibitor DPI on LDL oxidation.
Diphenyl iodonium chloride, an inhibitor o f the NADPH oxidase enzyme system did not significantly affect LDL oxidation in a cell free system or in the presence o f RMC (figure 3 .12). Incubation o f cells in the presence o f DPI did not have any obvious effect on cell morphology on examination o f the cultures using a phase-contrast microscope, following the 24 hour incubation period. Cell protein measured after incubation was not statistically significantly different from the protein concentration o f cells incubated in experimental alone (data not shown). The inclusion o f 0.1 % ethanol, which was used as solvent for DPI and ET Y A, in the experimental medium did not appreciably affect cell- free or RMC induced LDL oxidation.
SO 7 0 6 0 n bO $4 0 .c . 3 0 2 0 10
1= ] Cell free control RMC control Cell free + DPI Bsma RMC + DPI
Figure 3.12 Effect of DPI on LDL oxidation
RMCs or cell free dishes were incubated with experimental medium containing 200 [j.g/ml of LDL and CuClz (5pM) at 37 ®c in the presence or absence of DPI (100 |iM) for 24 h. The supernatants were assayed for TBARS as described above. The results in the figure are the means ± SD of 4 experiments. *p=0.872 (cell free control vs cell free + DPI),**p=0.335 (RMC control vs RMC+ DPI).
3.(F).6. Effect of the lipoxygenase inhibitor ETYA on LDL oxidation.
5,8,11,14-eicosatetraynoic acid at a concentration o f 150 \xM did not cause a statistically significant decrease in cell free LDL oxidation as measured by the TEA assay (figure 3.13). LDL oxidation in the presence o f RMC was decreased by 74% when ETYA (150 pM ) was included in the incubation medium and this reduction was statistically significance (p=0.008). However, there was a significant reduction in cell protein o f wells where ETYA was included in the incubation medium and this decrease was dependent on the dose o f ETYA added (figure 3.14). The reduction in cell protein was probably due to the toxic effects o f ETYA.. Therefore it is difficult to conclude from the results obtained from this series o f experiments whether inhibiting RMC lipoxygenase activity has any effect on oxidation o f LDL by these cells.
80 70 - 60 - 50 - E 40 - 30 - 20 - 10 - 0
Cell free control RMC control Cell free + ETYA RMC + ETYA
Figure 3.13 Effect of ETYA on LDL oxidation
RMCs or cell free dishes were incubated with experimental medium containing 200 pg/ml of LDL and CuCb (5|iM) at 37 °c in the presence or absence of ETYA (150 pM) for 24 h. The supernatants were assayed for TBARS as described above. The results in the figure are the means ± SD of 4 experiments. *p=0.300 (cell free control vs cell free + ETYA),**p=0.008 (RMC control vs RMC+ ETYA). 140 -I 120 - ? 100 60 - 40 - 20 - 0.15 150 ETYA C o n e. (^M)
Figure 3.14 Effect of ETYA on rat mesangial cell protein
RMCs were incubated with experimental medium containing 200 pg/ml of LDL and CuClz (5pM) at 37 "^C in the presence or absence of ETYA at the concentrations indicated on the graph for 24 h. The medium was removed, the cells solubilised and the protein measured by the Lowry assay. The data presented are the median and range of 2 experiments. *p = NS. **p < 0.01 (control vs ETYA) using the Mann-Whitney test
3.(F).7. Effect of SOD on oxidation of LDL
Superoxide dismutase at a concentration o f 100 pg/ml inhibited RMC induced LDL oxidation by 81% (figure 3.15) and this reduction was statistically significant (p=0.002). There was no evidence o f cytotoxicity on examining cells under phase-contrast microscopy after incubation with SOD. The cell protein also remained unchanged when compared with cell proteins o f wells where SOD was excluded. The oxidation o f LDL in the absence o f cells was also inhibited by up to 75 %. This result suggests that the inhibition of LDL oxidation by SOD may be due to a general antioxidant effect as suggested by Jessup et al (Jessup 1993). However, it cannot be fully discounted that SOD does not inhibit cell induced LDL oxidation by dismissing superoxide anions generated by cells. 80 70 - 60 - ^ 50 -
Î
E 40 - « S 3 0 - 20 - 10 - 0I I Cell free control