Oxidized low-density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism

ARTICLE IN PRESS Journal of Biomechanics 42 (2009) 2394–2399

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Oxidized low-density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism Xiang Wang a,b,, Li Yang a, Yao Liu a, Wei Gao a, Weiyan Peng a, K.-L. Paul Sung a,b, Lanping Amy Sung b a Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Shapingba District 174], Chongqing 400044, PR China b Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412, USA

a r t i c l e in f o

a b s t r a c t

Article history: Accepted 23 May 2009

The oxidized low-density lipoprotein (Ox-LDL) plays an important role in atherosclerosis, yet it remains unclear if it damages circulating erythrocytes. In this study, erythrocyte deformability and its membrane proteins after Ox-LDL incubations are investigated by micropipette aspiration, thiol radical measurement, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results show that Ox-LDL incubation reduces the erythrocyte deformability, decreases free thiol radical contents in erythrocytes, and induces the cross-linking among membrane proteins. SDS-PAGE analysis reveals a high molecular weight (HMW) complex as well as new bands between spectrins and band 3 and reduced ratios between band 3 and other major membrane skeletal proteins. Analyses indicate that Ox-LDL makes erythrocytes harder to deform through a molecular mechanism by which the oxidation of free thiol radicals forms disulfide bonds among membrane skeletal proteins. & 2009 Elsevier Ltd. All rights reserved.

Keywords: Atherosclerosis Lipoprotein Erythrocyte Deformation Thiol radical Membrane proteins

1. Introduction It has been well established that oxidized low-density lipoprotein (Ox-LDL) plays an important role in atherosclerotic injury. The initial accumulation of Ox-LDL in the vascular endothelium may exceed the capacity for macrophages to remove them. The free radicals of excessive Ox-LDL would then injure cells with the formation of the necrotic extracellular lipid core—a key transitional step in lesion progression. Therefore, Ox-LDL may damage blood vessel endothelium by forming foam cells and developing the atherosclerotic focus (Schwartz et al., 1991; Ross, 1999; Azarsiz et al., 2003). The Ox-LDL also acts via reducing the endothelial NOS activity to suppress the anti-thrombogenic activity of the endothelial cells (ECs) (Chen et al., 2007), inducing EC apoptosis, and ultimately, inhibiting their proliferative activities and altering their migratory behaviors (Sugano et al., 2004; Chen et al., 2003; Vink et al., 2000). Thus, atherosclerosis pathogenesis has a close relationship with the existence of Ox-LDL and free radicals; specifically, the Ox-LDL free radicals that promote the formation of atherosclerotic plaques (Chow et al., 2002; Aikawa et al., 2002). Investigations also show that certain physical interactions exist between circulating erythrocytes and the vascular wall in the process of atherosclerosis. For example, erythrocyte aggregation is

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E-mail address: [email protected] (X. Wang). 0021-9290/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2009.05.026

increased in familial hypercholesterolemia after LDL oxidation (Ejima et al., 2000)10; Ox-LDL induces hemolysis; and oxygen radicals of Ox-LDL provoke the structural changes in erythrocytes (Azizova et al., 2002). Furthermore, Ox-LDL may affect blood viscosity in atherosclerosis patients via the oxidation of fibrinogen and thereby induce changes in blood rheology (Azizova et al., 2007). In the process of atherosclerosis, the higher shear stress in vivo decreases erythrocyte deformability and promotes the vascular stiffness and microcirculatory alteration (Lee et al., 2006; Yuan et al., 2001). In our previous research, we found that the oxidative damage originating from oxygen-free radicals influences erythrocyte mechanical properties (Wang et al., 1999). Deformability is necessary for erythrocytes to endure the stress in the entire circulatory system. From a biophysical point of view, erythrocyte deformation has a close relationship to the molecular structure of membrane proteins. To deform normally, its membrane skeleton must be able to undergo topological rearrangement, and some skeletal proteins may unfold (Mohandas and Evans, 1994). The membrane skeleton is a thin layer of spectrin/actin-based protein network anchored to the endo-face of the membrane. The basic repeating unit consists of a junctional complex (JC), where six spectrin converge and meet with a short actin filament, and is suspended by up to six suspension complexes (SCs) to the lipid bilayer. SC contains ankyrin, band 3, and protein 4.2. While band 3 is a multiple transmembranedomain anion exchanger (Zhang et al., 2003), protein 4.2 is tightly membrane-bound, and ankyrin has a b spectrin binding site. Together, this network is essential to the mechanical stability and

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2. Materials and methods 2.1. Preparation of Ox-LDL (Pulh et al., 1995) Preparation of LDL: LDL was isoelectrically precipitated at pH 5.12 from the plasma, while mixed with the same volume of heparin solution (heparin 100unit/ ml) and using 1% citric acid in saline to adjust the pH to 5.12. The LDL precipitate was separated from the supernatant by centrifugation at 9000g for 30 min and stored at 4 1C. Oxidative modification of LDL: the LDL can be oxidatively modified through lipid oxidation using catalytic copper ions (Cu2+ and Cu+). A large amount of hydroperoxy radicals (LOOd and LOd) can be formed through a chain reaction of lipid peroxidation. Seven mg of LDL were dissolved in 50 ml of copper chloride solution (CuCl2 80 mg/L in saline) and oxidized at 37 1C for 24 h in water bath. The reaction was ended by adding the same volume of EDTA solution (2 g/L in PBS, pH 7.4) and the Ox-LDL solution was dialyzed against PBS three times to remove excessive Cu2+ and stored at 4 1C. 2.2. Oxidation injury of erythrocytes through incubation with Ox-LDL solution Fresh human blood was obtained from the Blood Bank of Chongqing Red Cross and separated from the plasma by centrifugation at 3000 rpm (1500g) for 15 min. Concentrated erythrocytes were washed three times with PBS (1 mM EDTA, 20 mg/ dL PMSF, 2 mg/dL pepstatin, and 1.5 mg/ml BSA, pH 7.40), then suspended and adjusted to 10% HCT. The experimental groups include: Ox-LDL dosage group (Ox-LDL of 0, 1.3, 1.6, 1.8, and 2.0 mg/dL incubated for 72 h in 37 1C water bath), Ox-LDL injury time group (incubated with different durations in Ox-LDL of 1.8 mg/ dL at 37 1C in water bath for 24, 72, 96, 120, or 144 h) and control group (incubated without Ox-LDL at 37 1C in water bath for 24, 72, 96, 120, or 144 h). In order to keep blood sample for the same storage time, the blood samples of Ox-LDL dosage group and injury time group were taken from different donators.

the free thiol radical contents. In this experiment, erythrocyte membranes were solubilized by a solution containing 0.3 ml of 10% sodium dodecyl sulphate (SDS) and 4 ml of 10 mM PBS buffer. Then 0.1 ml of 10 mM DTNB in 10 mM PBS was added. Based on the absorbance at 415 nm after incubation at 37 1C in water bath for 15 min, the contents of SH-radical were determined using GSH as a standard. 2.6. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of erythrocyte membrane proteins The protein profiles of erythrocyte membranes before and after Ox-LDL incubations were analyzed by SDS-PAGE. The gradient gels (4–12%) and electrophoresis buffer were from Invitrogen (Carlsbad, CA, USA). After electrophoresis, the gels were stained with coomassie blue, scanned, and analyzed by using Biorad image analysis software used for SDS-PAGE (Bennett et al., 1980). 2.7. Statistical analysis The statistical analysis of experimental data was done by the Student’s t-test using Microsoft EXCEL software. Results are presented as mean7standard error mean (SEM), where n denotes the number of individual cells tested. Differences between sample means were tested using the Student’s t-test (unpaired, twotailed).

3. Results 3.1. The alterations of viscoelastic properties of erythrocytes by OxLDL The experimental results in Fig. 1 showed increased elastic moduli and viscous coefficient of erythrocytes after incubation with increasing Ox-LDL concentrations.

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Erythrocyte membranes were prepared with an established procedure based on the Dodge’s method (Dodge et al., 1963), using 40 volumes of hypotonic lysis buffer (10 mmol Tris-HCl, pH 7.4) at 0 1C. The lysed erythrocytes were centrifuged at 10,000g for 30 min at 0 1C and the ghost membranes were further washed twice with the same buffer. 2.5. Determination of SH-radicals in erythrocyte membranes SH-radicals of erythrocyte membrane proteins were quantified according to Anderson (Anderson, 1985) and Yamaguchi (Yamaguchi et al., 1994). Like the reduced glutathione hormone (GSH) molecules, the thiol radicals in erythrocyte membrane proteins react with 5, 59-dithiobis (2-nitrobenzoic acid) (DTNB) to form a product possessing an absorbance at 415 nm, which is then used to analyze

6 4.226*

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2.3. Measuring erythrocyte viscoelasticity by the micropipette aspiration system Erythrocyte viscoelasticity was measured using a micropipette aspiration system. The aspiration micropipette was carefully positioned to the surface of an individual erythrocyte. Then, through the pressure system, the erythrocyte was aspirated with one rank leap negative pressure and produced a small deformation. The deformation process of erythrocyte following the impacted times was recorded and analyzed with imaging system, timer, and TV video system. The radius of micropipette (Rp), the partial length aspirated, and the process of aspirated into micropipette of erythrocyte were determined and recorded by the image recording system. The erythrocyte membrane elastic modulus m and viscous coefficient Z were analyzed based on a ‘‘hemispherical cap model’’ (Chien et al.,1978; Evans and Hochmuth, 1976; Lima et al., 2006).

7.661*

8 μ (10-3 dyn/cm)

elastic deformability of the erythrocyte (Sung et al., 2000; Sung and Vera, 2003; Dahl et al., 2004; Discher et al., 1994). However, it remains unclear whether Ox-LDL may damage circulating erythrocytes, and investigation into this matter is necessary. If Ox-LDL does indeed damage circulating erythrocytes, its mechanism would require further examination at the cellular and molecular levels. Such studies would provide a better understanding of the physiological and clinical consequences of Ox-LDL-induced changes, not only in the endothelial walls, but also in the erythrocytes, which constitute 50% or less of human blood volume.

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Fig. 1. The changes of erythrocyte membrane mechanical properties after being treated with different Ox-LDL dosages at 37 1C for 72 h. (A) Elastic modulus m, (B) viscous coefficient Z. The cell number n ¼ 33. *Po0.001 (compared with normal group).

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The experimental results in Fig. 2 showed that the elastic moduli and viscous coefficient of the erythrocyte increased with increasing Ox-LDL incubation time in Ox-LDL of 1.8 mg/dL. In control groups, mechanical properties of erythrocytes had a relatively small change with incubation time in PBS compared within Ox-LDL incubation. We found that Ox-LDL injury has little influence in the erythrocyte diameter. The diameter or morphology was well maintained by the osmotic pressure of the suspension solution and incubation medium during the entire incubation period.

3.2. The thiol radical oxidation of erythrocyte membranes Ox-LDL produced in the lipid metabolism in vivo may significantly influence erythrocyte properties. Since many superoxides and oxygen-free radicals are produced in lipid metabolism, they are to be expected. They may induce the oxidation of membrane lipids and proteins, causing cross-linking among the different membrane proteins by thiol radical oxidation. That would, as a result, influence erythrocyte deformability as well as membrane fluidity and its function (Yamaguchi et al., 1994). Fig. 3 indicates that both the dosage and the time of Ox-LDL incubation have caused the decrease of free thiol radicals in erythrocyte membranes. The effect of concentrations is greater than that of the incubation time within the experimental set up.

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Fig. 2. The changes of erythrocyte membrane mechanical properties after being incubated with different durations in Ox-LDL of 1.8 mg/dL at 37 1C water bath for 24, 72, 96, 120, or 144 h. (A) Elastic modulus m, (B) viscous coefficient Z. Cell number n ¼ 39. *Po0.001 (compared with control group).

3.3. The cross-linking of erythrocyte membrane proteins by Ox-LDL The observed decrease of free thiol radicals may be the consequence of their cross-linking among erythrocyte membrane proteins. The oxidative damage may lead to a variation in the erythrocyte membrane protein profiles. SDS-PAGE analysis of the erythrocyte membrane proteins in Fig. 4 show that a new band of high molecular weight (HMW) appeared above a and b spectrin in both experimental groups (i.e., Ox-LDL dosage and injury time). Furthermore, the Ox-LDL dosage had a greater effect than the incubation duration on the formation of HMW and the alteration in band 3. With increasing Ox-LDL, HMW increased its intensity; meanwhile, band 3 declined along with some lower molecular weight proteins. The results indicate that the free superoxide radicals from Ox-LDL may attack the free thiol radicals of membrane proteins, such as band 3, inducing cross-linking among membrane proteins through formation of disulfide bonds of inter- and intra-molecular interactions (Fig. 5). The Ox-LDL injury time had a relatively little effect on the variation of crosslinking membrane proteins (Fig. 6). The experimental results suggest that oxidative reaction in erythrocyte membrane proteins induced by Ox-LDL may be an important molecular mechanism by which the viscoelasticity of the membrane and elastic deformation of the whole cell may be altered in the process of atherosclerosis.

4. Discussion Ox-LDL plays an important role in the process of atherosclerosis. While many researchers have focused their works on the oxidative damage of Ox-LDL to the vascular endothelium, few have investigated its impact on erythrocytes. In this study, we demonstrated that Ox-LDL has a significant effect on viscoelastic properties of erythrocytes, suggesting that under pathological conditions Ox-LDL may indeed injure erythrocytes, and reduce their deformability in circulation. We also found that Ox-LDL decreased free thiol radicals in erythrocyte membranes, which coincided with the increase of the cross-linking among membrane skeletal proteins. For the membrane to deform normally, the skeletal network with a ‘‘spoked’’ hexagonal topology must undergo equibiaxial and/or anisotropic extension (Sung and Vera, 2003), where spectrin and other skeletal proteins may also unfold. The inter- or intramolecular cross-linking of membrane proteins would limit topological rearrangement of the network and hinder erythrocyte deformation. Our SDS-PAGE analysis reveals that thiol radicals of the membrane are oxidized to form disulfide bonds causing protein cross-linking. In addition to band 3, a number of other bands, including those lower molecular weight bands (see Fig. 4 under ‘‘dosage’’) may also be sensitive to Ox-LDL. Membrane protein may be conjugated to form HMW complex. The appearance of a HMW band, with the decrease of band 3 and lower molecular weight bands suggests that free superoxide radicals from Ox-LDL attack the free thiol radicals of the membrane proteins, inducing the cross-linking among membrane proteins, through the formation of inter- and/or intra-molecular disulfide bonds. In short, new bands found in SDS-PAGE may be the result of new cross-links among membrane proteins. These changes may lead to erythrocyte stiffening after Ox-LDL exposure. Previous studies have shown that Ox-LDL induced hemolysis (Azizova et al., 2002), which suggests that oxygen radicals of Ox-LDL may provoke changes of erythrocyte structure. In this study, we indeed found that Ox-LDL changed erythrocyte structure by cross-linking membrane proteins. Under the

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Fig. 3. Changes of thiol radicals in RBC membranes after being injured with Ox-LDL. (A) Ox-LDL Dosage, (B) Ox-LDL injuring time. *Po0.001 (compared with normal group).

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Fig. 4. Erythrocyte membrane protein profiles analyzed by SDS-PAGE after incubating with various Ox-LDL dosages and times. A Control erythrocyte ghost membrane. B, C, D, and E are 1.3, 1.6, 1.8, and 2.0 mg/dL for 72 h, respectively. F, G, H, I and J are different Ox-LDL injuring time: 24,72, 96, 120 and 144 h at 1.8 mg/dL Ox-LDL.

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physiological condition, normal erythrocytes undergo reversible deformation owing to the flexible membrane skeleton network that allows cells to endure shear stress during circulation. Without the network, erythrocytes will lyse; over cross-linking of the network, erythrocytes will stiffen and break in circulation—also causing hemolysis. In addition to protein–protein cross-linking, oxidation of erythrocytes may also lead to the formation of lipid–protein conjugate(s); lipid–protein conjugates are also found in Ox-LDL. Thus, lipid–protein conjugation may represent a shared mechanism that induces phagocytosis by macrophages (Sambrano et al., 1994). Furthermore, oxidation of fibrinogen by Ox-LDL may also influence blood viscosity in atherosclerosis (Azizova et al., 2007). Our research concludes that Ox-LDL damages the erythrocyte membrane by cross-linking membrane proteins, thus reducing erythrocyte deformation. It is likely that reduction of erythrocyte deformability may shorten their life span, influence their function, and increase blood viscosity. All of these may lead to abnormal circulation and endothelium damage, and may activate platelet coagulation. Thus, while reduced erythrocyte deformation may not be the original cause, it may serve as an important factor in the progression of atherosclerosis. This investigation demonstrates that oxidative damage by OxLDL leads to changes in the erythrocyte membrane proteins and that the cross-linking of membrane proteins plays an important role in determining erythrocyte deformability. We conclude that the polymerization of membrane proteins may be the underlining molecular mechanism by which the deformability of erythrocytes is reduced when they are exposed to Ox-LDL.

Conflict of interest statement We would like to state that all author and co-authors of manuscript with the title ‘‘Oxidized Low-Density Lipoprotein (Ox-LDL) Impacts on Erythrocyte Viscoelasticity and Its Polymerizing Mechanism of Membrane Protein’’ do not have any conflict of interest. If you have any questions, do not be hesitated to contact with us.

Acknowledgement Dr. Xiang Wang was supported by National Natural Science Foundation of China (NSFC 10572159). This research is also partially supported by ‘‘111 Project’’ entitled ‘‘Biomechanics &

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