Effects of MβCD on Lipoxygenase-Induced LDL Oxidation

Abstract

Beta-cyclodextrin (β-CD) has been applied as drug/food carriers or potential drugs for treating some diseases. Most recently, some evidence indicated that methyl-β-cyclodextrin (MβCD) and 2-hydroxypropyl-β-cyclodextrin (2-HPβCD), two major derivatives of β-CD, may inhibit atherogenesis, implying that cyclodextrins also can be potential drugs for treating atherosclerosis. It is well known that modification (e.g. oxidation) of low-density lipoprotein (LDL) is one of the most critical steps of atherogenesis. Lipoxygenase, an enzyme able to be expressed by atherosclerosis-related vascular cells, is generally regarded as a possible in vivo agent of LDL oxidation. In this study, the effects of MβCD on LDL oxidation induced by lipoxygenase were investigated by measuring the electrophoretic mobility, conjugated diene formation, malondialdehyde (MDA) production, and amino group blockage of LDL. We found that the lipids depleted from LDL by MβCD could be oxygenated more readily by lipoxygenase whereas the lipoxygenase-induced oxidation of the remaining lipid-depleted LDL decreased. The data imply that MβCD has an inhibitory effect on lipoxygenase-induced LDL oxidation and probably helps to inhibit atherogenesis.

Βeta-cyclodextrin (β-CD), a cyclic oligosaccharide consisting of seven a-(1,4)-linked glucopyranose subunits, has been widely applied in pharmaceuticals as a durg carrier.^1,2) Recently, methyl-β-cyclodextrin (MβCD) and 2-hydroxypropyl-β-cyclodextrin (2-HPβCD), two derivatives of β-CD with improved water solubility, have been directly used as potential drugs to treat the Niemann–Pick C1 disease^3,4) and cancers.^5,6)

More recently, Zimmer et al.⁷⁾ reported that 2-HPβCD can promote atherosclerosis regression via macrophage reprogramming. Our recent studies also imply that 2-HPβCD/MβCD probably helps to inhibit atherogenesis by impairing monocyte-endothelium adhesion⁸⁾ or lowering the oxidation of low-density lipoprotein (LDL).⁹⁾ These studies imply that cyclodextrin could be recruited as an effective drug to treat atherosclerosis.

Lipoxygenase is expressed in a variety of human cells including human vascular smooth muscle cells,¹⁰⁾ macrophages,¹¹⁾ and fibroblasts.¹²⁾ The enzyme has been found in human atherosclerotic lesions/plaques,^13,14) implying that lipoxygenase probably represents an agent of LDL oxidation in vivo and involves in atherogenesis.^15–18)

In our previous study about the effects of cyclodextrin on LDL oxidation,⁹⁾ copper, a widely used agent of LDL oxidation for in vitro experiments, was utilized to oxidize LDL. In this study, we sought to investigate the effects of cyclodextrin (MβCD as a representative) on LDL oxidation induced by lipoxygenase which is a possible in vivo agent of LDL oxidation.

Experimental

Reagents

Human plasma LDL (Cat. No. YB-001) was purchased from Yiyuan Biotechnologies (Guangzhou, China). MβCD (Cat. No. C4555) and lipoxygenase derived from soybean (SLO; Cat. No. L6632) were from Sigma-Aldrich (U.S.A.).

MβCD Treatment and LDL Oxidation

Before oxidation, ethylenediaminetetraacetic acid (EDTA) was removed from LDL solutions by dialysis. For MβCD treatment, 0.2 mg/mL LDL solutions were incubated with different concentrations of MβCD in phosphate buffered saline (PBS) at 37°C for 1 h. For LDL oxidation, native LDL and LDL treated with MβCD were directly mixed with lipoxygenase at a final concentration of 500 units/mL in PBS and incubated at 37°C.

Agarose Gel Electrophoresis

At different time points during LDL oxidation, the samples treated with or without MβCD and stained with Sudan Black B were electrophoresed on 0.5% agarose gels in 0.075 M sodium barbital buffer at 55 V for 1 h. Relative electrophoretic mobility (REM) was calculated as the ratio of the migration distance of oxidized LDL to that of native LDL based on Sudan Black staining. To determine the changes in the protein component of LDL during oxidation (the 8-h time points were tested) Coomassie brilliant blue R-250 was also utilized to stain the second gel loading the same samples as those stained with Sudan Black B and running in the sample electrophoresis chamber.

Measurements of Conjugated Dienes, Malondialdehyde (MDA), and Free Amino Groups

Conjugated diene formation was dynamically measured at 20-min intervals for 220 min at 37°C during LDL oxidation. Thiobarbituric acid reactive substances (TBARS) Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and 2,4,6-trinitrobenzene sulfonic acid (TNBSA) Assay (picrylsulfonic acid solution from Sigma) were utilized to measure MDA production and remaining free amino groups (the blockage of amino groups was calculated by comparing the remaining free amino groups of the oxidized product to those of the un-oxidized control) at 18 h after LDL oxidation, respectively. A UV-5100 spectrophotometer (Metash Instruments, Shanghai, China) was used to measure the absorbance of each sample at 234 (for conjugated dienes), 532 (for MDA), and 335 nm (for free amino groups), respectively.

Isolation and Measurement of Depleted Lipids from LDL by MβCD

An Amicon Ultra-0.5 mL, 10 kDa Centrifugal Filter Unit (Merck Millipore, U.S.A.) was used to remove the LDL remnants and obtain the depleted lipids according to the user’s manual. The depleted cholesterols were measured via a Beckman AU480 analyser (Beckman Coulter). The phospholipids was measured at 240 nm by using a UV-5100 spectrophotometer (Metash Instruments).

Statistical Analysis

Student t-test was used to determine the significance between different groups. A value of p<0.05 from at least three independent experiments was considered statistically significant.

Results and Discussion

To determine the effects of MβCD on lipoxygenase-induced LDL oxidation, the electrophoretic mobility (Fig. 1), conjugated diene formation (Fig. 2), and MDA production (Fig. 3) during LDL oxidation were measured firstly. Without MβCD treatment, the incubation of LDL with lipoxygenase derived from soybean (SLO) caused slight increases in electrophoretic mobility on agarose gel (the second lanes in Fig. 1A; the dark curve in Fig. 1B), conjugated diene formation (the dark curves in Fig. 2), and MDA production (the third bar in Fig. 3) compared with native LDL. The data is consistent with previous reports.^19,20)

Fig. 1. MβCD Enhances the Electrophoretic Mobility of SLO-Oxidized LDL in a Concentration-Dependent Manner

(A) Representative images of agarose gel electrophoresis at different time points (0, 4, 12, 24 h, respectively) during LDL oxidation induced by lipoxygenase derived from soybean (SLO). (B) Calculated relative electrophoretic mobility (REM) of the samples at different time points.

Fig. 2. MβCD Improves the SLO-Induced Formation of Conjugated Dienes

LDL samples were treated with or without MβCD before SLO-induced oxidation. The samples were measured at 20-min intervals for 220 min from the beginning of SLO-induced oxidation.

Fig. 3. MβCD Increases the SLO-Induced Malondialdehyde (MDA) Production in a Concentration-Dependent Manner

TBARS assay was performed at the 18-h time point of SLO-induced oxidation (* p<0.05).

Surprisingly, in a concentration-dependent manner MβCD caused significant increases in electrophoretic mobility (lanes 3–6 in Fig. 1A; the gray curves in Fig. 1B), conjugated diene formation (the gray curves in Fig. 2), and MDA production (bars 4–6 in Fig. 3) of SLO-oxidized LDL. Since conjugated diene formation and MDA production mainly reflect the oxidative status of LDL lipids the data imply that MβCD promoted SLO-induced oxidation of LDL lipids which is completely opposite to the effect of MβCD on copper-induced oxidation of LDL lipids.⁹⁾

Next, amino group blockage of LDL which reflects the oxidative status of LDL proteins were obtained. The data shows that MβCD decreased the percent of blocked amino groups of SLO-oxidized LDL in a concentration-dependent manner (Fig. 4A). It implies that MβCD inhibited SLO-induced oxidation of LDL proteins which coincides with the effect of MβCD on copper-induced oxidation of LDL proteins.⁹⁾

Fig. 4. MβCD Impairs the SLO-Induced Oxidation of LDL Protein

(A) MβCD decreased amino group blockage of LDL proteins during SLO-induced oxidation in a concentration-dependent manner. The assay was performed at the 18-h time point of SLO-induced oxidation (* p<0.05). (B) MβCD induced the separation between the lipid (left) and protein (right) components of LDL and decreased the electrophoretic mobility of LDL protein (right). The electrophoresis was performed at the 8-h time point of SLO-induced oxidation. Left panel: the gel stained with Sudan Black B; right panel: the gel loading the same samples and running in the same electrophoresis chamber but stained with Coomassie brilliant blue. (C) MβCD caused the concentration-dependent increases in the lipids drawn from LDL. The percentage of phospholipids (PL; left panel) or cholesterols (right panel) depleted from LDL by MβCD significantly increased in a concentration-dependent manner. * p<0.05; ** p<0.01; *** p<0.001.

To simultaneously display the lipid and protein components of LDL, agarose gel electrophoresis on two gels loading the same samples and running in the same electrophoresis chamber but stained with different dyes was performed. Figure 4B shows the electrophoretic results at the 8-h time point of SLO-induced oxidation. The following points are observed: (a) for all MβCD-treated LDLs (lanes 3–6 in Fig. 4B), the lipid bands separated from the protein bands; (b) MβCD promoted the electrophoretic mobility of LDL lipids in a concentration-dependent manner (left panel of Fig. 4B); (c) MβCD decreased the electrophoretic mobility of LDL proteins in a concentration-dependent manner (right panel of Fig. 4B).

To further confirm the effect of MβCD separating the lipid and protein components, we sought to isolate and measure the depleted lipids. The data shows that the percentage of phospholipids and cholesterols depleted from LDL significantly increased with the increase of MβCD concentration (Fig. 4C), confirming that MβCD is able to separate the lipid and protein components of LDL. The bucket-shaped (like a truncated cone) MβCD molecules have a hydrophobic central cavity and a hydrophilic outer surface.¹⁾ The hydrophobic cavity of MβCD is able to host lipids (or hydrophobic food/drug molecules when MβCD is recruited as a delivery system), therefore depleting/extracting lipids from LDL or the plasma membrane of cells.^21,22)

Lipoxygenase generally targets polyunsaturated fatty acid (PUFAs) (mainly linoleic acid) of LDL lipids. While PUFAs/lipids are tightly packed by apolipoproteins in intact LDL particles, they are not easily accessible to lipoxygenase, an enzyme with a relatively high molecular weight (108 kDa for SLO homodimer) and a relatively large size. However, once the lipid and protein components of LDL are partially or completely separated due to MβCD treatment (Figs. 4B, C) the exposed PUFAs/lipids will be more readily oxygenated by lipoxygenase (Figs. 1–3 and left panel of Fig. 4B) whereas the indirect oxidation (by radical intermediates derived from hydroperoxy lipids¹⁹⁾) of the remaining lipid-depleted but protein-containing LDL will become more difficult (Fig. 4A and right panel of Fig. 4B).

Therefore, MβCD induced the depletion/separation of lipids from apolipoprotein-containing LDL. Although the SLO-induced oxidation of the lipids depleted from LDL was promoted, the SLO-induced oxidation of the remaining protein-containing LDL was inhibited. Taken together, MβCD has an inhibitory effect on SLO-induced oxidation of LDL which is consistent with that on copper-induced LDL oxidation.⁹⁾

Acknowledgment

This study was supported by the Natural Science Foundation of Jiangxi Province of China (20151BAB205005).

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Loftsson T., Duchene D., Int. J. Pharm., 329, 1–11 (2007).
• 2) Lakkakula J. R., Macedo Krause R. W., Nanomedicine (Lond.), 9, 877–894 (2014).
• 3) Taylor A. M., Liu B., Mari Y., Liu B., Repa J. J., J. Lipid Res., 53, 2331–2342 (2012).
• 4) Lopez A. M., Terpack S. J., Posey K. S., Liu B., Ramirez C. M., Turley S. D., Clin. Exp. Pharmacol. Physiol., 41, 780–787 (2014).
• 5) Mohammad N., Malvi P., Meena A. S., Singh S. V., Chaube B., Vannuruswamy G., Kulkarni M. J., Bhat M. K., Mol. Cancer, 13, 204 (2014).
• 6) Gotoh K., Kariya R., Alam M. M., Matsuda K., Hattori S., Maeda Y., Motoyama K., Kojima A., Arima H., Okada S., Biochem. Biophys. Res. Commun., 455, 285–289 (2014).
• 7) Zimmer S., Grebe A., Bakke S. S., Bode N., Halvorsen B., Ulas T., Skjelland M., De Nardo D., Labzin L. I., Kerksiek A., Hempel C., Heneka M. T., Hawxhurst V., Fitzgerald M. L., Trebicka J., Bjorkhem I., Gustafsson J. A., Westerterp M., Tall A. R., Wright S. D., Espevik T., Schultze J. L., Nickenig G., Lutjohann D., Latz E., Sci. Transl. Med., 8, 333–350 (2016).
• 8) Ao M., Wu L., Zhou X., Chen Y., Biol. Pharm. Bull., 39, 1029–1034 (2016).
• 9) Ao M., Gan C., Shao W., Zhou X., Chen Y., Eur. J. Pharm. Sci., 91, 183–189 (2016).
• 10) Limor R., Kaplan M., Sharon O., Knoll E., Naidich M., Weisinger G., Keidar S., Stern N., J. Cell. Biochem., 108, 1203–1210 (2009).
• 11) Rydberg E. K., Krettek A., Ullstrom C., Ekstrom K., Svensson P. A., Carlsson L. M., Jonsson-Rylander A. C., Hansson G. I., McPheat W., Wiklund O., Ohlsson B. G., Hulten L. M., Arterioscler. Thromb. Vasc. Biol., 24, 2040–2045 (2004).
• 12) Ezaki M., Witztum J. L., Steinberg D., J. Lipid Res., 36, 1996–2004 (1995).
• 13) Ylä-Herttuala S., Rosenfeld M. E., Parthasarathy S., Sigal E., Särkioja T., Witztum J. L., Steinberg D., J. Clin. Invest., 87, 1146–1152 (1991).
• 14) Yla-Herttuala S., Rosenfeld M. E., Parthasarathy S., Glass C. K., Sigal E., Witztum J. L., Steinberg D., Proc. Natl. Acad. Sci. U.S.A., 87, 6959–6963 (1990).
• 15) Rankin S. M., Parthasarathy S., Steinberg D., J. Lipid Res., 32, 449–456 (1991).
• 16) Kühn H., Belkner J., Zaiss S., Fährenklemper T., Wohlfeil S., J. Exp. Med., 179, 1903–1911 (1994).
• 17) Huo Y., Zhao L., Hyman M. C., Shashkin P., Harry B. L., Burcin T., Forlow S. B., Stark M. A., Smith D. F., Clarke S., Srinivasan S., Hedrick C. C., Pratico D., Witztum J. L., Nadler J. L., Funk C. D., Ley K., Circulation, 110, 2024–2031 (2004).
• 18) Wittwer J., Hersberger M., Prostaglandins Leukot. Essent. Fatty Acids, 77, 67–77 (2007).
• 19) Kühn H., Belkner J., Suzuki H., Yamamoto S., J. Lipid Res., 35, 1749–1759 (1994).
• 20) Pirillo A., Reduzzi A., Ferri N., Kuhn H., Corsini A., Catapano A. L., Atherosclerosis, 214, 331–337 (2011).
• 21) Wu L., Huang J., Yu X., Zhou X., Gan C., Li M., Chen Y., J. Membr. Biol., 247, 189–200 (2014).
• 22) Zhao S., Liao H., Ao M., Wu L., Zhang X., Chen Y., FEBS Open Bio, 4, 190–199 (2014).