- Email: [email protected]
Virtually same oxidizability of LDL but higher Lp(a) levels in arterial compared to venous plasma
Chemistry and Physics of Lipids 184 (2014) 38–41
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Virtually same oxidizability of LDL but higher Lp(a) levels in arterial compared to venous plasma Gerd Hoerl a , Gerhard Ledinski a , Gerd Kager a , Michael Thalhammer b , Martin Koestenberger c , Guenther Juergens a , Thomas Gary d , Gerhard Cvirn a, * a
Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, Graz A-8010, Austria Department of Surgery, Medical University of Graz, Graz, Austria Department of Pediatrics, Medical University of Graz, Graz, Austria d Department of Angiology, Medical University of Graz, Graz, Austria b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2014 Received in revised form 2 September 2014 Accepted 15 September 2014 Available online 18 September 2014
Plaque formation is confined to the arterial trunk. We assumed that due to the higher aeration of arterial compared to venous blood, higher levels of the atherogenic agent oxidized LDL might be present in arteries, contributing to plaque formation. We aimed to compare (i) the basal oxidative status of LDL in arterial and venous blood and (ii) the susceptibility of arterial and venous LDL to oxidation. The basal oxidative status of LDL was determined by measuring lipid hydroperoxide (LPO) concentrations, plasma levels of auto-antibodies against oxidized LDL, and by measuring oxidation-specific epitopes on LDL particles. The oxidizability of arterial vs. venous LDL (catalyzed by copper) was estimated by monitoring the time-course of conjugated dienes formation. Interestingly, we found the same basal oxidative status of LDL in arterial and venous plasma. LPO concentrations and levels of auto-antibodies against oxidized LDL were similar in arterial and venous plasma and amounts of oxidation-specific epitopes were similar on the respective LDL particles. Moreover, we found similar susceptibilities of arterial and venous LDL to (copper-mediated) oxidation. Lag-times until the onset of conjugated diene formation were slightly shorter in arterial compared to venous LDL in the presence of 5 mM, but not in the presence of 1 mM CuCl2. Additionally, we found significantly higher levels of the atherogenic lipoprotein(a) in arterial plasma. We conclude that not higher oxidizability of arterial LDL but higher arterial lipoprotein(a) levels might help to explain why sclerosis is confined to the arterial trunk. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Conjugated diene formation Lipid peroxidation Lipoprotein(a) Oxidation-specific immune epitopes Plaque formation
1. Introduction Oxidation of low-density lipoprotein (LDL) plays an important role in the development of atherosclerosis. LDL in its native form is not atherogenic. However, circulating native LDL and LDL in the subendothelial space in arteries have been shown to be subject to oxidation by mechanisms involving free radicals and/or lipoxygenases (Esterbauer et al., 1992). Foremost, LDL lipids undergo peroxidation. Subsequently, certain aldehydes generated during lipid peroxidation modify the apolipoprotein B100, the protein part of LDL. The resulting oxidized form of LDL (oxLDL) is readily internalized
Abbreviations: BHT, butylated hydroxytoluene; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay; EC, endothelial cells; EDTA, ethylenediaminetetraacetic acid; HDL, high-density lipoprotein; IgG, immune globulin G; LDL, low-density lipoprotein; LPO, lipid hydroperoxides; oxLDL, oxidized low-density lipoprotein; PBS, phosphate buffered saline; WB, whole blood. * Corresponding author. Tel.: +43 316 380 4174; fax: +43 316 380 9610. E-mail address: [email protected] (G. Cvirn). http://dx.doi.org/10.1016/j.chemphyslip.2014.09.004 0009-3084/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
by macrophages through a so-called “scavenger receptor” pathway (Stocker and Keaney, 2004). The macrophages change to foam cells, initiating processes resulting in atherosclerosis. In accordance, numerous studies have shown that high plasma levels of oxLDL are associated with atherosclerosis (Savioiu et al., 2009), high cardiovascular risk (Park et al., 2011), vulnerability to rupture of atherosclerotic lesion (Nishi et al., 2002), and with acute myocardial infarction (Nordin Fredrikson et al., 2003). Since the oxygen content in arteries (approximately 20 mL/dL) is significantly higher than in veins (approximately 15 mL/dL), we hypothesized that oxidation of LDL, a prerequisite for atherogenesis, more easily occurs in arterial than in venous blood. This assumed mechanism might help to explain the much higher incidence of sclerosis in the arterial tree. It was therefore the aim of our pilot study to comparatively evaluate the basal oxidative status of LDL in arterial and venous blood and to compare the susceptibility of arterial and venous LDL for oxidation. The basal oxidative status of LDL was determined by measuring lipid hydroperoxide (LPO) concentrations, plasma
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
levels of auto-antibodies against oxidized LDL, and by measuring oxidation-specific epitopes on LDL particles. The oxidizability of arterial vs. venous LDL (catalyzed by copper) was estimated by monitoring the time-course of the formation of conjugated dienes. It has to be stated that copper-mediated oxidation is an in vitro method which might not reflect all aspects of LDL oxidative modification in vivo.
39
2.6. Determination of plasma levels of auto-antibodies against oxidized LDLs Auto-antibodies against native and oxidatively-modified LDL were determined by time-resolved fluoroimmunoassay. Details are stated in the Appendix A. 2.7. Determination of oxidation-specific immune epitopes on LDL particles
2. Materials and methods 2.1. Patients Arterial and venous blood was simultaneously drawn from 10 patients routinely treated for atherosclerosis in the peripheral arteries or in the cerebral vascular arteries. Patients’ characteristics are listed in Table 1. The fibrinogen levels in our patients were mean 406 mg/dL, indicating that our patients were in a chronic inflammatory state. This pilot study was approved by the appropriate institutional review board. Due to ethical reasons, no group consisting of non-atherosclerotic subjects was included in this pilot study. Particularly drawing blood from arteries in healthy controls is not endorsed by the internal review board. 2.2. Collection of whole blood (WB) and preparation of plasma Blood (4.9 mL) from the antecubital vein and the radial artery was collected into S-Monovette EDTA-tubes additionally containing 10 mL Pefabloc (12.6 mg/L) and 50 mL Trasylol (10,000 IU/ 100 mL). The WB was centrifuged at room temperature for 15 min at 1200 g to obtain platelet poor plasma. 2.3. Determination of lipid and lipoprotein standard values A standard lipid and lipoprotein panel was collected in our routine clinical laboratory including total lipids, total cholesterol, VLDL, LDL, HDL, LDL- and HDL-cholesterol, triglycerides, phospholipids, lipid phosphor, lipoprotein(a), and vitamin E (Mora, 2009).
The monoclonal antibody MAB/OB 04 (200 mL/well diluted 1:200 in PBS) against oxidatively modified LDL and a polyclonal rabbit anti-human apoB100 (200 mL/well, 1:10000 in PBS) were used as detection antibodies (Jürgens et al., 1990). The data are expressed as the ratio oxidatively-modified LDL counts/native LDL counts. Details are stated in the Appendix A. 2.8. Determination of conjugated diene formation LDL were dialyzed excessively against a PBS without EDTA and diluted to a final concentration of 0.2 mg LDL/mL. CuCl2 (1 or 5 mM final concentration) was added to LDL and the formation of conjugated dienes was continuously monitored (in triplicate) at 234 nm in a temperature-controlled (37 C) spectrophotometer (Hitachi U-2000) using 1 cm quartz cuvette. It has been shown that the time course of diene formation fully reflects the lipid hydroperoxides time profile, and thus, the progression of the oxidation process (Esterbauer et al., 1989). 2.9. Statistics Calculations were performed by SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean SD. Paired t-test or non-parametric paired Wilcoxon test, depending on normal or skewed distribution of data, were used to compare arterial and venous data. A P-value less than 0.05 was considered as statistically significant.
2.4. Preparation of LDL
3. Results
LDL was obtained from arterial or venous plasma by potassium bromide density gradient sequential ultracentrifugation (Jürgens et al., 1986). Details are stated in the Appendix A.
3.1. Lipid and lipoprotein standard values in arterial vs. venous plasma
2.5. Determination of lipid hydroperoxides (LPO) The amount of LPO present in LDL of arterial or venous origin was determined with a spectrophotometric assay for lipid hydroperoxides in serum lipoproteins (El-Saadani et al., 1989). Details are stated in the Appendix A.
With the exception of lipoprotein(a), no differences were found in the lipid and lipoprotein patterns between arterial and venous plasma, shown in Table 2. HDL and HDL-cholesterol showed a trend toward higher levels in arterial compared to venous plasma (P = 0.053 and 0.052, respectively). Plasma levels of lipoprotein(a), however, were significantly higher in arterial compared to venous plasma (P = 0.029). 3.2. Basal oxidative status of LDL in arterial vs. venous plasma
Table 1 Patients’ characteristics. A total of 10 atherosclerotic patients were included in the study. All patients (n = 10) Age (years), mean SD Male, % BMI (kg/m2), mean SD Current smokers, % Statin therapy, % Fibrinogen (mg/dL), mean SD Glucose (mg/dL), mean SD Treatment Arteria carotis interna, % Peripheral arteries, %
70.8 7.1 80 25.9 3.8 60 40 406 101 124.8 22.1 70 30
We found the same basal oxidative status of LDL in arterial and venous plasma in terms of LPO levels, plasma levels of autoantibodies against oxidatively modified LDL, and in the amount of oxidation-specific epitopes on the respective LDL-particles, shown in Table 2. 3.3. Susceptibility to oxidation of arterial LDL vs. venous LDL We found slightly shorter lag-times until the onset of conjugated diene formation (following the addition of 5 mM CuCl2) in arterial LDL compared to venous LDL, listed in Table 2. In the presence of 1 mM CuCl2, we found the same susceptibility of arterial and venous LDL to oxidation (P = 0.097), shown in Table 2.
40
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
Table 2 Lipid and lipoprotein patterns in arterial vs. venous plasma. Arterial values
Venous values
P-values (arterial vs. venous)
Standard values Total lipids (mg/dL) Total cholesterol (mg/dL) VLDL (mg/dL) LDL (mg/dL) HDL (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Total cholesterol/HDL-cholesterol LDL-cholesterol/HDL-cholesterol Triacylglycerides (mg/dL) Phospholipids (mg/dL) Lipid phosphor (mg/dL) Lipoprotein(a) (mg/dL) Vitamin E (nmol/mg LDL)
582.2 153.0 159.4 193.3 188.8 87.8 30.3 5.4 3.1 156.5 190.0 7.6 21.6 2.6
(453.4–714.5) (137.0–170.2) (131.1–192.0) (175.2–222.6) (126.9–227.9) (79.7–101.3) (20.3–36.5) (4.3–5.8) (2.7–3.5) (130.5–176.0) (161.8–204.5) (6.5–8.2) (9.5–25.6) (2.2–3.0)
549.6 146.4 162.0 191.5 161.0 87.1 25.8 5.2 3.3 153.5 191.5 7.7 17.2 2.9
(457.4–658.4) (118.2–153.8) (125.8–201.8) (149.7–226.2) (119.3–200.6) (68.1–103.0) (19.1–32.1) (4.6–5.8) (2.8–4.09 (126.5–183.3) (150.5–198.5) (6.0–7.9) (7.3–25.7) (2.2–3.0)
0.147 0.091 0.201 0.312 0.053 0.311 0.052 0.195 0.125 0.155 0.112 0.112 0.029 0.553
Specific values LPO (nmol/mg LDL) oxLDL-epitopes (ratio counts MAB OB 04/Anti Apo B) Autoantibodies against oxLDL (ratio counts ox LDL/reference LDL) Lag time of diene formation (min) (1 mM CuCl2) Lag time of diene formation (min) (5 mM CuCl2)
9.2 0.12 1.28 156.0 74.3
(6.9–9.5) (0.09–0.13) (1.24–1.41) (146.9–158.2) (69.2–76.3)
9.3 0.10 1.30 159.2 75.6
(7.7–10.2) (0.09–0.14) (1.23–1.50) (150.1–166.1) (73.3–79.8)
0.177 0.885 0.592 0.097 0.010
4. Discussion Plaque formation preferentially occurs in arteries and rarely in veins. Several mechanisms apparently prone arteries to plaque formation: (i) higher blood pressure and blood flow in arteries vs. veins exert higher shear stress on arterial than on venous endothelial cells (ECs). This high shear stress could easily lead to vessel injury, the initial step of atherogenesis; (ii) it has been shown that arterial EC, but not venous ECs, produce adhesion molecules (a prerequisite for atherogenesis) after being stressed by moderately and highly oxLDL (Amberger et al., 1997); (iii) it has been shown that angiotensin II induces formation of functional fractalkine (CX(3)CL1) in arterial but not venous endothelia (Rius et al., 2013). This fractalkine aggravates platelet adhesion, a mandatory mechanism in the initiation of atherosclerotic lesion formation (Flier and Schäfer, 2012); (iv) arterial blood derived LDL has been shown to increase platelet aggregation and macrophage cholesterol content in comparison to lipoprotein derived from venous blood (Keidar et al., 1989). Herein, we investigated a further possible mechanism that might help to explain the facilitated formation of plaques in arteries. We hypothesized that, due to higher aeration of the arterial blood, elevated levels of atherogenic oxLDL might be present in arteries. However, we found the same basal levels of oxLDL in arterial and venous plasma and, additionally, a similar susceptibility of arterial vs. venous LDL for (copper-catalyzed) oxidation. Solely shorter lag times until diene formation in the presence of 5 mM CuCl2 indicated a slightly higher oxidizabilty of arterial compared to venous LDL. However, the shortening was marginal and not present when 1 mM of CuCl2 were used as trigger of oxidation. Due to ethical reasons, no control group (non-atherosclerotic subjects) was investigated in the present pilot study. However, the findings of previous studies support the assumption that the LDL’s basal oxidative status in our atherosclerotic patients is comparable with that of healthy subjects (Dieber-Rothenauer et al.,1991). Same vitamin E and LPO levels were found in these healthy subjects (Gieseg and Esterbauer, 1994). Interestingly, we found significantly higher levels of lipoprotein (a) in arterial vs. venous plasma. Lipoprotein(a) consists of an LDL particle capable of promoting atherosclerosis and a plasminogenlike apolipoprotein(a) particle capable of increasing the risk of thrombosis (Deb and Caplice, 2004). Lipoprotein(a) has been shown to contribute to the development of atherosclerosis and/or
thrombosis in numerous in vitro, animal and epidemiological studies. A stepwise increase in risk of myocardial infarction has been observed (Kamstrup et al., 2008). We suggest that the elevated lipoprotein(a) levels in arteries compared to veins might be a further mechanism sensitizing arteries for the formation of atherosclerotic plaques. However, in all but one patient, the lipoprotein(a) levels were below the 30 mg/dL threshold where risk for cardiovascular diseases increases strongly (Kamstrup et al., 2009). Moreover, we found a strong trend towards higher HDL levels in arteries (P = 0.053). The biological significance of this trend is unclear. One possibility is that this increase in HDL is the result of HDL not being able to readily leave the lumen of the arterial vessel and to penetrate into the subintimal space and therefore to perform its anti-oxidative and anti-inflammatory functions. This might be a further mechanism explaining why sclerosis is limited to the arterial trunk. Studies dealing with the differences between arterial and venous lipoproteins are scarce. Keidar et al. have shown an enhanced capability of arterial LDL to support platelet aggregation when compared to venous LDL (Keidar et al., 1989). In contrast to our pilot study, Aviram et al. have shown that apolipoprotein(a) levels were reduced by 15% in arterial in comparison to venous blood (Aviram et al., 1987). On the other hand, Kronenberg et al. have shown that lipoprotein(a) concentrations in arteries are approximately 9% higher than in veins (Kronenberg et al., 1997). This is in agreement with our pilot study, albeit we found approximately 20% of increase. 5. Conclusions In conclusion, our pilot study may suggest a further explanation for the presence of atherosclerotic lesions in arteries but not in veins: higher lipoprotein(a) levels in arterial vs. venous plasma. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.2014.09.004. References Amberger, A., Maczek, C., Jürgens, G., Michaelis, D., Schett, G., Trieb, K., Eberl, T., Jindal, S., Xu, Q., Wick, G., 1997. Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins. Cell Stress Caperones 2, 94–103. Aviram, M., Viener, V., Brook, J.G., 1987. Reduced plasma high-density lipoprotein and increased platelet activity in arterial versus venous blood. Postgrad. Med. J. 63, 91–94. Dieber-Rothenauer, M., Puhl, H., Waeg, G., Striegl, G., Esterbauer, H., 1991. Effect of oral supplementation with D-a-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J. Lipid Res. 32, 1325–1332. Deb, A., Caplice, N.M., 2004. Lipoprotein(a): new insights into mechanism of atherogenesis and thrombosis. Clin. Cardiol. 27, 258–264. El-Saadani, M., Esterbauer, H., el-Sayed, M., Goher, M., Nassar, A.Y., Jürgens, G., 1989. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J. Lipid Res. 30, 627–630. Esterbauer, H., Striegl, G., Puhl, H., Rotheneder, M., 1989. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic. Res. Commun. 6, 67–75. Esterbauer, H., Gebicki, J., Puhl, H., Jürgens, G., 1992. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13, 341– 390. Flier, U., Schäfer, A., 2012. Fractaline – a local inflammatory marker aggravating platelet aggregation at the vulnerable plaque. Thromb. Haemost. 108, 457–463. Gieseg, S.P., Esterbauer, H., 1994. Low density lipoprotein is saturable by pro-oxidant copper. FEBS Lett. 343, 188–194. Jürgens, G., Lang, I., Esterbauer, H., 1986. Modifications of human low density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim. Biophys. Acta 875, 103–114.
41
Jürgens, G., Ashy, A., Esterbauer, H., 1990. Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein(a) and very-low-density lipoprotein. Biochem. J. 265, 605–608. Kamstrup, P.R., Benn, M., Tybjaerg-Hansen, A., Nordestgaard, B.G., 2008. Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population. Circulation 117, 176–184. Kamstrup, P.R., Tybjaerg-Hansen, A., Steffensen, R., Nordestgaard, B.G., 2009. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. J. Am. Med. Assoc. 301, 2331–2339. Keidar, S., Aviram, M., Grenadier, E., Markiewicz, W., Brook, J.G., 1989. Arterial blood derived low density lipoprotein increases platelet aggregation and macrophage cholesterol content in comparison to lipoprotein derived from venous blood. Artery 16, 62–71. Kronenberg, F., Trenkwalder, E., Lingenhel, A., Friedrich, G., Lhotta, K., Schober, M., Moes, N., Konig, P., Utermann, G., Dieplinger, H., 1997. Renovascular arteriovenous differences in Lp(a) plasma concentrations suggest removal of Lp(a) from the renal circulation. J. Lipid Res. 38, 1755–1763. Mora, S., 2009. Are advanced lipoprotein testing and subfractionation clinically useful? Circulation 119, 2396–2404. Nishi, K., Itabe, H., Uno, M., Kitazato, K.T., Horiguchi, H., Shinno, K., Nagahiro, S., 2002. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler. Thromb. Vasc. Biol. 22, 1649–1654. Nordin Fredrikson, G., Hedblad, B., Berglund, G., Nilsson, J., 2003. Plasma oxidized LDL: a predictors for acute myocardial infarction? J. Intern. Med. 253, 425–429. Park, S.H., Kim, J.Y., Lee, J.H., Park, H.Y., 2011. Elevated oxidized low-density lipoprotein concentrations in postmenopausal women with the metabolic syndrome. Clin. Chim. Acta 412, 435–440. Rius, C., Piqueras, L., Gonzalez-Navarro, H., Albertos, F., Company, C., Lopez-Gines, C., Ludwig, A., Blanes, J.I., Morcillo, E.J., Sanz, M.J., 2013. Arterial and venous endothelia display differential functional fraktaline (CX3CL1) expression by angiotensin-II. Arterioscler. Thromb. Vasc. Biol. 33, 96–104. Savioiu, G., Dragan, S., Cristescu, C., Serban, C., Noveanus, L., Ionescu, D., Nicola, T., Duicu, O., Raducan, A., Voicu, M., 2009. Measurement of plasma ox-LDL and carotid IMT may represent useful markers for atherosclerosis and may represent potential targets for therapeutic interventions. Rev. Med. Chir. Soc. Med. Nat. Iasi 113, 397–401. Stocker, R., Keaney, J.F., 2004. Role of oxidative modifications in atherosclerosis. Physiol. Rev. 84, 1382–1448.
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Virtually same oxidizability of LDL but higher Lp(a) levels in arterial compared to venous plasma Gerd Hoerl a , Gerhard Ledinski a , Gerd Kager a , Michael Thalhammer b , Martin Koestenberger c , Guenther Juergens a , Thomas Gary d , Gerhard Cvirn a, * a
Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, Graz A-8010, Austria Department of Surgery, Medical University of Graz, Graz, Austria Department of Pediatrics, Medical University of Graz, Graz, Austria d Department of Angiology, Medical University of Graz, Graz, Austria b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2014 Received in revised form 2 September 2014 Accepted 15 September 2014 Available online 18 September 2014
Plaque formation is confined to the arterial trunk. We assumed that due to the higher aeration of arterial compared to venous blood, higher levels of the atherogenic agent oxidized LDL might be present in arteries, contributing to plaque formation. We aimed to compare (i) the basal oxidative status of LDL in arterial and venous blood and (ii) the susceptibility of arterial and venous LDL to oxidation. The basal oxidative status of LDL was determined by measuring lipid hydroperoxide (LPO) concentrations, plasma levels of auto-antibodies against oxidized LDL, and by measuring oxidation-specific epitopes on LDL particles. The oxidizability of arterial vs. venous LDL (catalyzed by copper) was estimated by monitoring the time-course of conjugated dienes formation. Interestingly, we found the same basal oxidative status of LDL in arterial and venous plasma. LPO concentrations and levels of auto-antibodies against oxidized LDL were similar in arterial and venous plasma and amounts of oxidation-specific epitopes were similar on the respective LDL particles. Moreover, we found similar susceptibilities of arterial and venous LDL to (copper-mediated) oxidation. Lag-times until the onset of conjugated diene formation were slightly shorter in arterial compared to venous LDL in the presence of 5 mM, but not in the presence of 1 mM CuCl2. Additionally, we found significantly higher levels of the atherogenic lipoprotein(a) in arterial plasma. We conclude that not higher oxidizability of arterial LDL but higher arterial lipoprotein(a) levels might help to explain why sclerosis is confined to the arterial trunk. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Conjugated diene formation Lipid peroxidation Lipoprotein(a) Oxidation-specific immune epitopes Plaque formation
1. Introduction Oxidation of low-density lipoprotein (LDL) plays an important role in the development of atherosclerosis. LDL in its native form is not atherogenic. However, circulating native LDL and LDL in the subendothelial space in arteries have been shown to be subject to oxidation by mechanisms involving free radicals and/or lipoxygenases (Esterbauer et al., 1992). Foremost, LDL lipids undergo peroxidation. Subsequently, certain aldehydes generated during lipid peroxidation modify the apolipoprotein B100, the protein part of LDL. The resulting oxidized form of LDL (oxLDL) is readily internalized
Abbreviations: BHT, butylated hydroxytoluene; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay; EC, endothelial cells; EDTA, ethylenediaminetetraacetic acid; HDL, high-density lipoprotein; IgG, immune globulin G; LDL, low-density lipoprotein; LPO, lipid hydroperoxides; oxLDL, oxidized low-density lipoprotein; PBS, phosphate buffered saline; WB, whole blood. * Corresponding author. Tel.: +43 316 380 4174; fax: +43 316 380 9610. E-mail address: [email protected] (G. Cvirn). http://dx.doi.org/10.1016/j.chemphyslip.2014.09.004 0009-3084/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
by macrophages through a so-called “scavenger receptor” pathway (Stocker and Keaney, 2004). The macrophages change to foam cells, initiating processes resulting in atherosclerosis. In accordance, numerous studies have shown that high plasma levels of oxLDL are associated with atherosclerosis (Savioiu et al., 2009), high cardiovascular risk (Park et al., 2011), vulnerability to rupture of atherosclerotic lesion (Nishi et al., 2002), and with acute myocardial infarction (Nordin Fredrikson et al., 2003). Since the oxygen content in arteries (approximately 20 mL/dL) is significantly higher than in veins (approximately 15 mL/dL), we hypothesized that oxidation of LDL, a prerequisite for atherogenesis, more easily occurs in arterial than in venous blood. This assumed mechanism might help to explain the much higher incidence of sclerosis in the arterial tree. It was therefore the aim of our pilot study to comparatively evaluate the basal oxidative status of LDL in arterial and venous blood and to compare the susceptibility of arterial and venous LDL for oxidation. The basal oxidative status of LDL was determined by measuring lipid hydroperoxide (LPO) concentrations, plasma
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
levels of auto-antibodies against oxidized LDL, and by measuring oxidation-specific epitopes on LDL particles. The oxidizability of arterial vs. venous LDL (catalyzed by copper) was estimated by monitoring the time-course of the formation of conjugated dienes. It has to be stated that copper-mediated oxidation is an in vitro method which might not reflect all aspects of LDL oxidative modification in vivo.
39
2.6. Determination of plasma levels of auto-antibodies against oxidized LDLs Auto-antibodies against native and oxidatively-modified LDL were determined by time-resolved fluoroimmunoassay. Details are stated in the Appendix A. 2.7. Determination of oxidation-specific immune epitopes on LDL particles
2. Materials and methods 2.1. Patients Arterial and venous blood was simultaneously drawn from 10 patients routinely treated for atherosclerosis in the peripheral arteries or in the cerebral vascular arteries. Patients’ characteristics are listed in Table 1. The fibrinogen levels in our patients were mean 406 mg/dL, indicating that our patients were in a chronic inflammatory state. This pilot study was approved by the appropriate institutional review board. Due to ethical reasons, no group consisting of non-atherosclerotic subjects was included in this pilot study. Particularly drawing blood from arteries in healthy controls is not endorsed by the internal review board. 2.2. Collection of whole blood (WB) and preparation of plasma Blood (4.9 mL) from the antecubital vein and the radial artery was collected into S-Monovette EDTA-tubes additionally containing 10 mL Pefabloc (12.6 mg/L) and 50 mL Trasylol (10,000 IU/ 100 mL). The WB was centrifuged at room temperature for 15 min at 1200 g to obtain platelet poor plasma. 2.3. Determination of lipid and lipoprotein standard values A standard lipid and lipoprotein panel was collected in our routine clinical laboratory including total lipids, total cholesterol, VLDL, LDL, HDL, LDL- and HDL-cholesterol, triglycerides, phospholipids, lipid phosphor, lipoprotein(a), and vitamin E (Mora, 2009).
The monoclonal antibody MAB/OB 04 (200 mL/well diluted 1:200 in PBS) against oxidatively modified LDL and a polyclonal rabbit anti-human apoB100 (200 mL/well, 1:10000 in PBS) were used as detection antibodies (Jürgens et al., 1990). The data are expressed as the ratio oxidatively-modified LDL counts/native LDL counts. Details are stated in the Appendix A. 2.8. Determination of conjugated diene formation LDL were dialyzed excessively against a PBS without EDTA and diluted to a final concentration of 0.2 mg LDL/mL. CuCl2 (1 or 5 mM final concentration) was added to LDL and the formation of conjugated dienes was continuously monitored (in triplicate) at 234 nm in a temperature-controlled (37 C) spectrophotometer (Hitachi U-2000) using 1 cm quartz cuvette. It has been shown that the time course of diene formation fully reflects the lipid hydroperoxides time profile, and thus, the progression of the oxidation process (Esterbauer et al., 1989). 2.9. Statistics Calculations were performed by SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean SD. Paired t-test or non-parametric paired Wilcoxon test, depending on normal or skewed distribution of data, were used to compare arterial and venous data. A P-value less than 0.05 was considered as statistically significant.
2.4. Preparation of LDL
3. Results
LDL was obtained from arterial or venous plasma by potassium bromide density gradient sequential ultracentrifugation (Jürgens et al., 1986). Details are stated in the Appendix A.
3.1. Lipid and lipoprotein standard values in arterial vs. venous plasma
2.5. Determination of lipid hydroperoxides (LPO) The amount of LPO present in LDL of arterial or venous origin was determined with a spectrophotometric assay for lipid hydroperoxides in serum lipoproteins (El-Saadani et al., 1989). Details are stated in the Appendix A.
With the exception of lipoprotein(a), no differences were found in the lipid and lipoprotein patterns between arterial and venous plasma, shown in Table 2. HDL and HDL-cholesterol showed a trend toward higher levels in arterial compared to venous plasma (P = 0.053 and 0.052, respectively). Plasma levels of lipoprotein(a), however, were significantly higher in arterial compared to venous plasma (P = 0.029). 3.2. Basal oxidative status of LDL in arterial vs. venous plasma
Table 1 Patients’ characteristics. A total of 10 atherosclerotic patients were included in the study. All patients (n = 10) Age (years), mean SD Male, % BMI (kg/m2), mean SD Current smokers, % Statin therapy, % Fibrinogen (mg/dL), mean SD Glucose (mg/dL), mean SD Treatment Arteria carotis interna, % Peripheral arteries, %
70.8 7.1 80 25.9 3.8 60 40 406 101 124.8 22.1 70 30
We found the same basal oxidative status of LDL in arterial and venous plasma in terms of LPO levels, plasma levels of autoantibodies against oxidatively modified LDL, and in the amount of oxidation-specific epitopes on the respective LDL-particles, shown in Table 2. 3.3. Susceptibility to oxidation of arterial LDL vs. venous LDL We found slightly shorter lag-times until the onset of conjugated diene formation (following the addition of 5 mM CuCl2) in arterial LDL compared to venous LDL, listed in Table 2. In the presence of 1 mM CuCl2, we found the same susceptibility of arterial and venous LDL to oxidation (P = 0.097), shown in Table 2.
40
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
Table 2 Lipid and lipoprotein patterns in arterial vs. venous plasma. Arterial values
Venous values
P-values (arterial vs. venous)
Standard values Total lipids (mg/dL) Total cholesterol (mg/dL) VLDL (mg/dL) LDL (mg/dL) HDL (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Total cholesterol/HDL-cholesterol LDL-cholesterol/HDL-cholesterol Triacylglycerides (mg/dL) Phospholipids (mg/dL) Lipid phosphor (mg/dL) Lipoprotein(a) (mg/dL) Vitamin E (nmol/mg LDL)
582.2 153.0 159.4 193.3 188.8 87.8 30.3 5.4 3.1 156.5 190.0 7.6 21.6 2.6
(453.4–714.5) (137.0–170.2) (131.1–192.0) (175.2–222.6) (126.9–227.9) (79.7–101.3) (20.3–36.5) (4.3–5.8) (2.7–3.5) (130.5–176.0) (161.8–204.5) (6.5–8.2) (9.5–25.6) (2.2–3.0)
549.6 146.4 162.0 191.5 161.0 87.1 25.8 5.2 3.3 153.5 191.5 7.7 17.2 2.9
(457.4–658.4) (118.2–153.8) (125.8–201.8) (149.7–226.2) (119.3–200.6) (68.1–103.0) (19.1–32.1) (4.6–5.8) (2.8–4.09 (126.5–183.3) (150.5–198.5) (6.0–7.9) (7.3–25.7) (2.2–3.0)
0.147 0.091 0.201 0.312 0.053 0.311 0.052 0.195 0.125 0.155 0.112 0.112 0.029 0.553
Specific values LPO (nmol/mg LDL) oxLDL-epitopes (ratio counts MAB OB 04/Anti Apo B) Autoantibodies against oxLDL (ratio counts ox LDL/reference LDL) Lag time of diene formation (min) (1 mM CuCl2) Lag time of diene formation (min) (5 mM CuCl2)
9.2 0.12 1.28 156.0 74.3
(6.9–9.5) (0.09–0.13) (1.24–1.41) (146.9–158.2) (69.2–76.3)
9.3 0.10 1.30 159.2 75.6
(7.7–10.2) (0.09–0.14) (1.23–1.50) (150.1–166.1) (73.3–79.8)
0.177 0.885 0.592 0.097 0.010
4. Discussion Plaque formation preferentially occurs in arteries and rarely in veins. Several mechanisms apparently prone arteries to plaque formation: (i) higher blood pressure and blood flow in arteries vs. veins exert higher shear stress on arterial than on venous endothelial cells (ECs). This high shear stress could easily lead to vessel injury, the initial step of atherogenesis; (ii) it has been shown that arterial EC, but not venous ECs, produce adhesion molecules (a prerequisite for atherogenesis) after being stressed by moderately and highly oxLDL (Amberger et al., 1997); (iii) it has been shown that angiotensin II induces formation of functional fractalkine (CX(3)CL1) in arterial but not venous endothelia (Rius et al., 2013). This fractalkine aggravates platelet adhesion, a mandatory mechanism in the initiation of atherosclerotic lesion formation (Flier and Schäfer, 2012); (iv) arterial blood derived LDL has been shown to increase platelet aggregation and macrophage cholesterol content in comparison to lipoprotein derived from venous blood (Keidar et al., 1989). Herein, we investigated a further possible mechanism that might help to explain the facilitated formation of plaques in arteries. We hypothesized that, due to higher aeration of the arterial blood, elevated levels of atherogenic oxLDL might be present in arteries. However, we found the same basal levels of oxLDL in arterial and venous plasma and, additionally, a similar susceptibility of arterial vs. venous LDL for (copper-catalyzed) oxidation. Solely shorter lag times until diene formation in the presence of 5 mM CuCl2 indicated a slightly higher oxidizabilty of arterial compared to venous LDL. However, the shortening was marginal and not present when 1 mM of CuCl2 were used as trigger of oxidation. Due to ethical reasons, no control group (non-atherosclerotic subjects) was investigated in the present pilot study. However, the findings of previous studies support the assumption that the LDL’s basal oxidative status in our atherosclerotic patients is comparable with that of healthy subjects (Dieber-Rothenauer et al.,1991). Same vitamin E and LPO levels were found in these healthy subjects (Gieseg and Esterbauer, 1994). Interestingly, we found significantly higher levels of lipoprotein (a) in arterial vs. venous plasma. Lipoprotein(a) consists of an LDL particle capable of promoting atherosclerosis and a plasminogenlike apolipoprotein(a) particle capable of increasing the risk of thrombosis (Deb and Caplice, 2004). Lipoprotein(a) has been shown to contribute to the development of atherosclerosis and/or
thrombosis in numerous in vitro, animal and epidemiological studies. A stepwise increase in risk of myocardial infarction has been observed (Kamstrup et al., 2008). We suggest that the elevated lipoprotein(a) levels in arteries compared to veins might be a further mechanism sensitizing arteries for the formation of atherosclerotic plaques. However, in all but one patient, the lipoprotein(a) levels were below the 30 mg/dL threshold where risk for cardiovascular diseases increases strongly (Kamstrup et al., 2009). Moreover, we found a strong trend towards higher HDL levels in arteries (P = 0.053). The biological significance of this trend is unclear. One possibility is that this increase in HDL is the result of HDL not being able to readily leave the lumen of the arterial vessel and to penetrate into the subintimal space and therefore to perform its anti-oxidative and anti-inflammatory functions. This might be a further mechanism explaining why sclerosis is limited to the arterial trunk. Studies dealing with the differences between arterial and venous lipoproteins are scarce. Keidar et al. have shown an enhanced capability of arterial LDL to support platelet aggregation when compared to venous LDL (Keidar et al., 1989). In contrast to our pilot study, Aviram et al. have shown that apolipoprotein(a) levels were reduced by 15% in arterial in comparison to venous blood (Aviram et al., 1987). On the other hand, Kronenberg et al. have shown that lipoprotein(a) concentrations in arteries are approximately 9% higher than in veins (Kronenberg et al., 1997). This is in agreement with our pilot study, albeit we found approximately 20% of increase. 5. Conclusions In conclusion, our pilot study may suggest a further explanation for the presence of atherosclerotic lesions in arteries but not in veins: higher lipoprotein(a) levels in arterial vs. venous plasma. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
G. Hoerl et al. / Chemistry and Physics of Lipids 184 (2014) 38–41
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.2014.09.004. References Amberger, A., Maczek, C., Jürgens, G., Michaelis, D., Schett, G., Trieb, K., Eberl, T., Jindal, S., Xu, Q., Wick, G., 1997. Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins. Cell Stress Caperones 2, 94–103. Aviram, M., Viener, V., Brook, J.G., 1987. Reduced plasma high-density lipoprotein and increased platelet activity in arterial versus venous blood. Postgrad. Med. J. 63, 91–94. Dieber-Rothenauer, M., Puhl, H., Waeg, G., Striegl, G., Esterbauer, H., 1991. Effect of oral supplementation with D-a-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J. Lipid Res. 32, 1325–1332. Deb, A., Caplice, N.M., 2004. Lipoprotein(a): new insights into mechanism of atherogenesis and thrombosis. Clin. Cardiol. 27, 258–264. El-Saadani, M., Esterbauer, H., el-Sayed, M., Goher, M., Nassar, A.Y., Jürgens, G., 1989. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J. Lipid Res. 30, 627–630. Esterbauer, H., Striegl, G., Puhl, H., Rotheneder, M., 1989. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic. Res. Commun. 6, 67–75. Esterbauer, H., Gebicki, J., Puhl, H., Jürgens, G., 1992. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13, 341– 390. Flier, U., Schäfer, A., 2012. Fractaline – a local inflammatory marker aggravating platelet aggregation at the vulnerable plaque. Thromb. Haemost. 108, 457–463. Gieseg, S.P., Esterbauer, H., 1994. Low density lipoprotein is saturable by pro-oxidant copper. FEBS Lett. 343, 188–194. Jürgens, G., Lang, I., Esterbauer, H., 1986. Modifications of human low density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim. Biophys. Acta 875, 103–114.
41
Jürgens, G., Ashy, A., Esterbauer, H., 1990. Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein(a) and very-low-density lipoprotein. Biochem. J. 265, 605–608. Kamstrup, P.R., Benn, M., Tybjaerg-Hansen, A., Nordestgaard, B.G., 2008. Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population. Circulation 117, 176–184. Kamstrup, P.R., Tybjaerg-Hansen, A., Steffensen, R., Nordestgaard, B.G., 2009. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. J. Am. Med. Assoc. 301, 2331–2339. Keidar, S., Aviram, M., Grenadier, E., Markiewicz, W., Brook, J.G., 1989. Arterial blood derived low density lipoprotein increases platelet aggregation and macrophage cholesterol content in comparison to lipoprotein derived from venous blood. Artery 16, 62–71. Kronenberg, F., Trenkwalder, E., Lingenhel, A., Friedrich, G., Lhotta, K., Schober, M., Moes, N., Konig, P., Utermann, G., Dieplinger, H., 1997. Renovascular arteriovenous differences in Lp(a) plasma concentrations suggest removal of Lp(a) from the renal circulation. J. Lipid Res. 38, 1755–1763. Mora, S., 2009. Are advanced lipoprotein testing and subfractionation clinically useful? Circulation 119, 2396–2404. Nishi, K., Itabe, H., Uno, M., Kitazato, K.T., Horiguchi, H., Shinno, K., Nagahiro, S., 2002. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler. Thromb. Vasc. Biol. 22, 1649–1654. Nordin Fredrikson, G., Hedblad, B., Berglund, G., Nilsson, J., 2003. Plasma oxidized LDL: a predictors for acute myocardial infarction? J. Intern. Med. 253, 425–429. Park, S.H., Kim, J.Y., Lee, J.H., Park, H.Y., 2011. Elevated oxidized low-density lipoprotein concentrations in postmenopausal women with the metabolic syndrome. Clin. Chim. Acta 412, 435–440. Rius, C., Piqueras, L., Gonzalez-Navarro, H., Albertos, F., Company, C., Lopez-Gines, C., Ludwig, A., Blanes, J.I., Morcillo, E.J., Sanz, M.J., 2013. Arterial and venous endothelia display differential functional fraktaline (CX3CL1) expression by angiotensin-II. Arterioscler. Thromb. Vasc. Biol. 33, 96–104. Savioiu, G., Dragan, S., Cristescu, C., Serban, C., Noveanus, L., Ionescu, D., Nicola, T., Duicu, O., Raducan, A., Voicu, M., 2009. Measurement of plasma ox-LDL and carotid IMT may represent useful markers for atherosclerosis and may represent potential targets for therapeutic interventions. Rev. Med. Chir. Soc. Med. Nat. Iasi 113, 397–401. Stocker, R., Keaney, J.F., 2004. Role of oxidative modifications in atherosclerosis. Physiol. Rev. 84, 1382–1448.