Intimal thickening after arterial balloon injury is increased by intermittent repetitive hypoxia, but intermittent repetitive hyperoxia is not protective

Atherosclerosis 185 (2006) 254–263

Intimal thickening after arterial balloon injury is increased by intermittent repetitive hypoxia, but intermittent repetitive hyperoxia is not protective Antony K. Lau a , Xavier Chaufour b , Craig McLachlan b , Steven B. Leichtweis a , David S. Celermajer a,c , Colin Sullivan b , Roland Stocker a,d,e,∗ a

The Heart Research Institute, University of Sydney, Sydney, Australia Department of Vascular Surgery, University of Sydney, Sydney, Australia c Department of Medicine, University of Sydney, Sydney, Australia Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, UNSW Sydney NSW 2052, Australia e Department of Hematology, Prince of Wales Hospital, Sydney NSW 2052, Australia b

d

Received 22 January 2005; received in revised form 26 May 2005; accepted 21 June 2005 Available online 2 August 2005

Abstract Hypoxia increases and hyperoxia decreases experimental atherosclerosis, but it is unclear if repetitive hypoxic and hyperoxic insults affect intimal thickening after arterial injury. Rabbits on 2% cholesterol diet for 6 weeks underwent balloon injury to the abdominal aorta (AA) after week 3, and were then exposed to normoxia (n = 6), or 12 h daily of intermittent repetitive hypoxia (n = 6) or hyperoxia (n = 6). After week 6, damaged AA and undamaged thoracic aorta (TA) were assessed for intimal thickening and lipid content. Compared with normoxia, hypoxia and hyperoxia did not alter the rise in serum cholesterol related to cholesterol feeding. However, compared to normoxia, hypoxia markedly increased the intima-to-media ratio in AA (1.18 ± 0.09 versus 1.96 ± 0.14, P < 0.01) and TA (0.15 ± 0.02 versus 0.41 ± 0.01, P < 0.01) whereas hyperoxia had no effect on AA disease and increased intimal thickening in TA (0.26 ± 0.03, P < 0.01). Hyperoxia promoted positive arterial remodeling in both TA and AA, resulting in larger luminal size. The cholesterol content in AA was increased by hypoxia and decreased by hyperoxia, but decreased by both treatments in TA. Lipophilic antioxidants and the proportion of arterial lipids that was oxidized were not altered by hypoxia or hyperoxia. These results suggest that intermittent repetitive hyperoxia is not protective and intermittent repetitive hypoxia promotes arterial disease in normal and injured arteries independent of lipid peroxidation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Hyperoxia; Hypoxia; Lipid peroxidation; Restenosis

1. Introduction The ‘response-to-injury’ hypothesis of atherosclerosis [1] states that a key initiating event of the disease is endothelial damage, caused by cardiovascular disease risk factors, many of which are associated with increased oxidative stress. Oxidative modification of lipoproteins is commonly considered central to the development of atherosclerosis [2], ∗

Corresponding author. Tel.: +61 2 9385 1309; fax: +61 2 9385 1389. E-mail address: [email protected] (R. Stocker).

0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.06.040

although there is emerging evidence suggesting that lipoprotein lipid oxidation may not directly cause but rather represent a response to inflammatory processes in atherogenesis [3]. There is also older literature not immediately compatible with the oxidative modification hypothesis of atherosclerosis. Thus, exposure of hyperlipidemic rabbits to hyperoxia in the form of either continuous 28% oxygen [4], 100% oxygen for 2 h daily [5], or hyperbaric oxygen (100% oxygen at 2.5 atm for 2 h daily) [6] decreased aortic atherosclerosis and a reduction in serum hyperlipidemia was observed in the first two studies. Similarly, the ‘anoxemia theory’ of atheroscle-

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

rosis, proposed nearly 60 years ago, suggested arterial wall hypoxia to initiate atherosclerosis [7]. In cholesterol-fed rabbits, exposure to sustained hypoxia for 8 weeks promoted serum hyperlipidemia and aortic atherosclerosis [8], whereas in rabbits fed a normal diet, intermittent hypoxia for 3 weeks promoted arteriosclerotic changes in the aorta [9]. One study [10] compared 8 weeks of intermittent (5 h daily) hyperoxia (40% oxygen) or hypoxia (5–10% oxygen) versus room air controls in a small number of WHHL rabbits, and found no differences in plasma cholesterol and no correlation between plasma lipids and lesion formation. Hyperoxia decreased aortic lesions compared with hypoxia, but these two groups did not differ significantly from controls. None of the studies performed have identified potential mechanism(s) by which sustained hypoxia and hyperoxia affect atherosclerosis. In humans, neither disease associated with sustained hypoxia (e.g., chronic anemia, non-smoking-related lung disease and cyanotic congenital heart disease) nor hyperoxia from oxygen therapy, have been shown to affect atherogenesis. Instead, obstructive sleep apnea (OSA), a common condition that involves heavy snoring causing airway obstruction and intermittent profound hypoxia, is strongly associated with increased coronary disease and stroke [11–13]. However, the frequent coexistence of major cardiovascular risk factors in patients with OSA including obesity, hyperlipidemia, hypertension, smoking and insulin resistance [14], has made it difficult to confirm whether repetitive hypoxia poses an independent risk. With the major growth in vascular interventions, it would also be important to assess if hypoxia and hyperoxia may affect arterial healing after these procedures. The aim of our study was to assess the effects of intermittent repetitive hypoxia and hyperoxia on the extent of vascular disease in vivo, using an animal model (hypercholesterolemic rabbits) of atherosclerosis (undamaged thoracic aorta) and intimal hyperplasia (balloon-damaged abdominal aorta), and to define the biochemical composition of lesions in relation to the extent of lipoprotein lipid peroxidation.

2. Methods 2.1. Study protocol The local Animal Welfare Committee approved the present study. Male New Zealand White rabbits (∼3 kg) were fed chow supplemented with 2% (w/w) cholesterol (USP grade, ICN) for 6 weeks. Each rabbit consumed on average 150 g chow per day. There were three study groups of six rabbits each, exposed to normoxia (room air controls), repetitive hypoxia, or repetitive hyperoxia. For the first 3 weeks, all animals were housed in room air. After 3 weeks, balloon injury of the rabbit abdominal aorta was performed. Under general anaesthesia, a 3F Fogarty embolectomy catheter (Baxter, Sydney, Australia) was inserted into the right femoral artery, advanced 25 cm proximally to the level of diaphragm, the balloon inflated with 0.2 mL saline and then withdrawn, a

255

step repeated twice and resulting in complete denudation of the ballooned vessel area [15]. Animals in the hypoxia and hyperoxia groups were then exposed to intermittent repetitive hypoxia and hyperoxia from balloon injury (week 3) to euthanasia (week 6), with animals spending 12 h in room air cages followed by 12 h in climate-controlled chambers. The day–night cycle (diurnal rhythm) was reproduced by turning the lights on for room air cages and off for the climate-controlled chambers, to simulate conditions in patients with OSA. 2.2. Automated environmental chambers Two fully automated, air-sealed environmental chambers were constructed to perform these studies, one for repetitive hypoxia (Fig. 1A) and the other for repetitive hyperoxia (Fig. 2A). Each consisted of a wooden box divided into three equal compartments to house three animals by sliding partitions constructed of grill-type steel mesh that allow free distribution of air or gas mixtures. The chambers were housed in an air-conditioned room at 23 ± 1 ◦ C and the interior temperature with three animals housed varied from 24 to 26 ◦ C. Each chamber was equipped with an internal fan that was continuously turned on for circulation of air. The concentrations of oxygen and carbon dioxide inside the chambers were measured continuously by an oxymeter (Normocap Oxy, Datex Engstrom, Helsinki, Finland). The system was controlled via a PC computer using custom-made software written using Microsoft Visual Basic programming language that communicated with the electronic solenoid valves of the chambers through a custom-made relay termination pane via an analogue/digital card (Analog Devices, Norwood, MA, USA). The hypoxia chamber has been described previously in detail [16]. The 60-min cycle of this system (Fig. 1B) consisted of breathing room air for 12 min, 45 min of hypoxia and room air was restored in the last 3 min. At the start of each cycle, the RA-valve and B-valve were open to room air with the blower-suction motor (B) operating, filling the chamber with room air. At 12 min into the cycle, these valves were closed, the motor turned off and the N-valve was opened allowing influx of nitrogen (15 L/min) into the chamber. At 23 min, the N-valve was closed and hypoxia was maintained until at 57 min when the B-valve and RA-valve were reopened and motor re-activated to restore the chamber with room air. The hyperoxia chamber was connected to large oxygen cylinder via the O-valve that periodically opened to allow influx of oxygen to generate hyperoxic conditions. The cyclical nature of these conditions was achieved, as after the O-valve was closed, oxygen concentrations within the chamber would gradually fall back to baseline. Unlike the hypoxia chamber that was open to room air at the end of each cycle, the hyperoxia chamber was completely isolated from room air. At the start of the 90-min cycle of this system (Fig. 2B), the O-valve was opened allowing influx of oxygen (15 L/min),

256

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

Fig. 1. Schematic diagram of the hypoxia chamber. Chamber design (A) with RA-V, valve connected to room air; B, blower-suction motor; B-V, valve connected to blower-suction motor; N-V, valve connected to nitrogen cylinder; F, flow meter of nitrogen cylinder. Valves are opened (open bars) and closed (black bars) at specified times during each cycle (B) to generate the O2 (open circles) and CO2 (closed circles) concentrations as shown in (C).

generating hyperoxia. The O-valve closed at 12 min and oxygen concentration would peak and gradually return towards room air. About half of the cycle was spent in hyperoxic conditions. Soda lime (Dragersorb 800, Dragerwerk Aktiengesellschaft, Germany) was incorporated in the inlet side of the blower system to prevent accumulation of carbon dioxide. At 45 min during the cycle, the blower is activated for 9 min to draw internal air through the soda lime to decrease the rise in carbon dioxide concentration. Silica gel in the roof

of the chamber absorbed prevented rising humidity. To validate the degree of hypoxia and hyperoxia produced by these chambers, arterial blood was collected from animals at peak hypoxia or hyperoxia via an indwelling 22-G cannula in the central ear artery during the last 12-h cycle that the animals spent in the chambers. The blood was immediately placed on ice, and arterial blood gas and electrolytes determined by using selective electrodes in a blood-gas electrolyte analyzer (NovaStat, Waltham, MA).

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

257

Fig. 2. Schematic diagram of the hyperoxia chamber. Chamber design (A) with O-V, valve connected to oxygen cylinder; F, flow meter of oxygen cylinder; B, blower-suction motor; L, soda lime container; S, silica gel container. Valves are opened (open bars) and closed (black bars) at specified times during each cycle (B) to generate the O2 (open circles) and CO2 (closed circles) concentrations as shown in (C).

2.3. Histology Animals were euthanized after 6 weeks. Segments of mid TA (from the level of 6th to 10th intercostals arteries) and lower AA (from second lumbar to iliac arteries) were fixed in situ by 10% formalin perfused under physiological pres-

sure. After harvesting, sections were stored in 70% (v/v) ethanol and then paraffin-embedded [15]. Cross-sections (5 ␮m) were taken at non-branched levels and stained with Verhoeff Hematoxylin. Digital images were obtained and planimetry performed blinded (Adobe Photoshop V5.0) by tracing the lumen, internal and external elastic laminae, to

258

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

determine the areas of the lumen, intima and media. The sum of these areas indicated the arterial size to assess adventitial remodeling. The intima-to-media ratio (IMR) was calculated [15]. Data of each specimen were averaged from nine random cross-sections.

Table 1 Arterial blood gas analyses

2.4. Biochemistry

Arterial blood was collected at the time of maximal hypoxia or hyperoxia during the last cycle of the 12-h exposure and analyzed for the partial pressure of oxygen (pO2 ) and carbon dioxide (pCO2 ) in mmHg, and bicarbonate concentration (HCO3 − ) in mmol/L, confirming markedly systemic hypoxia or hyperoxia in animals from the respective chambers, with those exposed to hypoxia developing a respiratory alkalosis with metabolic compensation. ** P < 0.01 vs. normoxia controls. †† P < 0.01 hypoxia vs. hyperoxia.

Venous blood was collected at baseline (week 0), before (week 3) and after (week 6) exposure to hypoxia and hyperoxia. Serum was assayed for non-esterified cholesterol (NEC), high-density lipoprotein (HDL)-cholesterol and triglycerides using an automated direct enzymatic method (Boehringer Mannheim, Germany). Segments of TA and AA (proximal to the segments for histology) were harvested for biochemical analyses. They were perfused with ice-cold PBS containing 5 ␮M BHT, homogenized and analyzed by HPLC as described in detail previously [17]. Lipids were detected by reverse phase-HPLC with UV210 nm , cholesterylester hydroperoxides (CE-OOH) by HPLC with post-column chemiluminescence, and ␣tocopherol (␣-TOH) and ubiquinone-10 (CoQ10 ) by HPLC with electrochemical detection [17]. 2.5. Statistical analysis Data are expressed as mean ± S.E.M. Groups were compared using ANOVA, and a P-value of <0.05 denoted statistical significance.

3. Results 3.1. Hypoxia chamber At the start of each 60-min cycle (Fig. 1C), the chamber was at room air with the concentrations of oxygen at 21% and carbon dioxide at 0%. At 12 min, nitrogen was pumped into chamber and oxygen concentrations progressively dropped to 10.7% at 28 min into the cycle. Thereafter, oxygen concentrations rose gradually to 13% at the end of the cycle. Carbon dioxide concentrations peaked at 0.6%, which was non-hazardous. At 57 min into the cycle, room air was pumped into the chamber and the oxygen and carbon dioxide concentrations returned quickly to room air levels by the end of the cycle. Thus, hypoxic conditions of varying degrees were experienced in the chamber for 48 min of the 60-min cycle, and for two-thirds of that time the oxygen concentration was below 13%.

pO2 Normoxia Hypoxia Hyperoxia

pCO2

99 ± 1 32 ± 1 44 ± 6** 25 ± 1** 296 ± 6**,†† 29 ± 1††

pH

HCO3 −

7.45 ± 0.05 7.50 ± 0.06** 7.42 ± 0.07††

23 ± 1 19 ± 1** 23 ± 1††

whereas carbon dioxide levels rose to its peak of 0.4%. Carbon dioxide concentrations fell from 0.4% residual from the previous cycle to 0% with influx of oxygen during the next cycle. Thus, hyperoxic conditions with oxygen concentrations above 30% were experienced for about half of the cycle, and above 50% for about one-quarter of the cycle. 3.3. Arterial blood The arterial blood gas analyses confirmed that animals were exposed to markedly hypoxic and hyperoxic conditions (Table 1). The arterial partial pressure of oxygen (pO2 ) of animals in the hypoxic chamber reached a low of 44 ± 1 mmHg, whilst those in the hyperoxic chamber reached a high of 296 ± 6 mmHg at the respective peak of their cycle, compared with rabbits at room air with 99 ± 1 mmHg. Rabbits in the hypoxia chamber had a significantly lower partial pressure of carbon dioxide (pCO2 ), higher pH, and lower bicarbonate concentration than those at room air, consistent with the hyperventilatory response to systemic hypoxemia. Rabbits in the hyperoxia chamber had unaltered pCO2 and bicarbonate levels. 3.4. Serum lipids With 2% cholesterol dietary supplementation, serum NEC rose significantly in all three groups of rabbits from a baseline (week 0) of ∼1 to −17 mmol/L, paralleled by the rise in HDL-cholesterol (Table 2). By comparison, serum triglycerides remained largely unchanged. In general, there were no significant differences between the three groups before and after exposure to hypoxia and hyperoxia, except for slightly lower HDL-cholesterol and triglycerides in the hyperoxia group at week 6. 3.5. Aortic histology

3.2. Hyperoxia chamber At the start of each 90-min cycle (Fig. 2C), oxygen influx resulted in concentrations rising rapidly from 21% to 81% at 12 min into the cycle. Oxygen concentrations subsequently returned to room air levels by the end of the 90-min cycle,

In the undamaged TA of normoxic rabbits, very small areas of discrete intimal thickening appearing like fatty streaks were interspersed around the luminal surface, with thin, normal-looking intima lining most of the luminal surface in between these lesions (Fig. 3A). In contrast, inti-

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

259

Table 2 Serum lipids Week

Normoxia Hypoxia Hyperoxia

Cholesterol (mmol/L)

HDL-cholesterol (mmol/L)

Triglycerides (mmol/L)

0

3

6

0

3

6

0

3

6

0.8 ± 0.1 1.0 ± 0.3 0.8 ± 0.1

13 ± 1.3 12 ± 2.0 13 ± 1.1

17 ± 2.5 18 ± 2.3 16 ± 0.9

0.4 ± 0.1 0.6 ± 0.1 0.4 ± 0.1

9.5 ± 1.4 8.7 ± 2.1 6.9 ± 1.4

14 ± 2 15 ± 1 10 ± 1*

0.8 ± 0.1 0.8 ± 0.1 1.0 ± 0.1

0.8 ± 0.1 0.6 ± 0.1 0.5 ± 0.1

0.7 ± 0.1 1.0 ± 0.3 0.5 ± 0.1

Serum lipids were measured at baseline (week 0), at the time of aortic balloon injury (week 3) and at euthanasia (week 6), showing a significant rise in serum cholesterol and HDL-cholesterol with cholesterol feeding with triglycerides remaining unchanged, and neither hypoxia nor hyperoxia altered these parameters compared with controls. Results represent mean ± S.E.M. of six separate serum samples per group and treatment time point. * P < 0.05 vs. normoxia controls.

Fig. 3. Effects of repetitive hypoxia or hyperoxia on intimal thickening in normal thoracic (TA) and balloon-injured abdominal aorta (AA). Representative transverse aortic sections showing that both hypoxia and hyperoxia increased intimal thickening in TA, whilst only hypoxia but not hyperoxia increased disease in AA. The IMR of animals exposed to normoxia (open bars), hypoxia (grey bars) and hyperoxia (black bars) are shown. ** P < 0.01 vs. normoxia controls; †† P < 0.01 hypoxia vs. hyperoxia.

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

260

Table 3 Histological analyses of normal thoracic (TA) and balloon-injured abdominal aorta (AA) Arterial area (mm2 )

Medial area (mm2 )

Intimal area (mm2 )

Luminal area (mm2 )

IMR

TA Normoxia Hypoxia Hyperoxia

24 ± 1 23 ± 1 27 ± 2*,†

2.9 ± 0.1 2.3 ± 0.1** 2.8 ± 0.1††

0.3 ± 0.0 0.7 ± 0.0** 0.6 ± 0.1**,††

20 ± 1 20 ± 1 24 ± 2*,†

0.15 ± 0.02 0.41 ± 0.01** 0.26 ± 0.03**,††

AA Normoxia Hypoxia Hyperoxia

11 ± 1 11 ± 1 14 ± 1*,†

1.0 ± 0.1 0.9 ± 0.0 1.0 ± 0.1

1.4 ± 0.1 2.0 ± 0.1** 1.6 ± 0.2†

8±1 8±1 H ± 1*,†

1.18 ± 0.09 1.96 ± 0.14** 1.29 ± 0.10††

Aortic histology was assessed in the undamaged thoracic (TA) and damaged abdominal aorta (AA) at the end of the intervention, as described in Section 2. Results represent mean ± S.E.M. of nine separate aortic segments per site and for each treatment. * P < 0.05 vs. normoxia controls. ** P < 0.01 vs. normoxia controls. † P < 0.05 hypoxia vs. hyperoxia. †† P < 0.01 hypoxia vs. hyperoxia.

3.6. Aortic biochemistry

In AA, intimal thickening (r = −0.496, P = 0.022) and IMR (r = 0.642, P = 0.002) correlated with increased aortic NEC, but not with other lipid parameters except CEOOH/CE that, again, correlated inversely with intimal thickening (r = −0.459, P = 0.036) with a similar trend with IMR (r = −0.385, P = 0.085). The concentrations of α-TOH (r = 0.467, P = 0.033) and CoQ10 in TA (r = 0.496, P = 0.022) and CoQ10 in AA (r = 0.643, P = 0.002) correlated moderately with IMR, demonstrating that antioxidants were not deficient as vascular disease increased. Thus, increased disease was associated with an increased content of lipophilic antioxidants and a decreased extent of lipid peroxidation. Positive arterial remodeling in both TA and AA correlated with increased medial (TA: r = 0.505, P = 0.020; AA: r = 0.555, P = 0.009) and luminal (TA: r = 0.997, P < 0.001; AA: r = 0.985, P = < 0.001) areas, but did not correlate with any lipid parameters.

3.6.1. Thoracic versus abdominal aorta Non-oxidized lipids (NEC and CE) and oxidized lipids (CE-OOH) were significantly increased in the more diseased AA than TA (P < 0.01), although parent lipid-standardized CE-OOH (CE-OOH/CE) were not different, i.e., the proportion of lipid that was oxidized was the same in undamaged TA and damaged AA (Table 4). Once CE was expressed per parent NEC, the data was not different between TA and AA, or between treatment groups, reflecting influx of CE into the arterial wall as lipoproteins. The amount of lipophilic antioxidants ␣-TOH and CoQ10 , when lipid-standardized, were significantly decreased in the AA compared with TA (P < 0.01), indicating less available antioxidants per oxidizable lipid present. Overall, correlation between histological disease and lipids was poor. In TA, intimal thickening and IMR did not correlate with serum cholesterol or aortic lipids, including CE-OOH. The only exception was CE-OOH/CE that, interestingly, had an inverse correlation with intimal thickening (r = 0.473, P = 0.030) and IMR (r = −0.47, P = 0.032).

3.6.2. Thoracic aorta In normal TA, rabbits exposed to hypoxia and hyperoxia had less NEC but a trend towards higher ratios of CE to NEC compared with normoxia. This suggests a possible trend towards increased lipoprotein influx into the arterial wall of hypoxic rabbits consistent with the greater intimal thickening. Neither the absolute amount of oxidized lipid (CE-OOH) nor the proportion of lipid present that were oxidized (CEOOH/CE) were different between treatment groups indicating that repetitive hypoxia and hyperoxia did not affect the process of lipid peroxidation. In fact, the CE-OOH/CE of hypoxic rabbits was lower than normal, although this did not reach significance (P = 0.24). The concentration of ␣-TOH and CoQ10 were higher in TA of hypoxic rabbits than controls, although not reaching statistical significance (P = 0.056 and 0.072, respectively), and when lipid-standardized the levels became similar between groups. This suggests that lipophilic antioxidants entered the arterial wall with lipid, consistent with the fact that the antioxidants are carried in lipoproteins.

mal thickening was increased in rabbits exposed to hypoxia or hyperoxia, and hypoxia was associated with decreased medial area (Table 3). Thus, both hypoxia (0.41 ± 0.01) and hyperoxia (0.26 ± 0.03) significantly increased IMR compared with normoxic controls (0.15 ± 0.02) (Fig. 3C). In the damaged AA, substantial intimal thickening developed in a diffuse circumferential pattern (Fig. 3B) consistent with the global injury produced by balloon denudation. Rabbits with hypoxia treatment (1.96 ± 0.14) had significantly increased intimal area and IMR than the other groups, whilst hyperoxia treatment (1.29 ± 0.10) did not differ from normoxia (1.18 ± 0.09) (Fig. 3C). Hyperoxia, but not hypoxia, promoted positive arterial remodeling, indicated by increased total arterial area, resulting in larger luminal size (Table 3).

Aortic lipids, antioxidants and oxidized lipids were measured in the undamaged thoracic (TA) and damaged abdominal aorta (AA) at the end of the intervention, as described in Section 2. Results represent mean ± S.E.M. of six separate aortic segments per site and for each treatment. * P< 0.05 vs. normoxia controls. † P < 0.05 hypoxia vs. hyperoxia.

0.13 ± 0.02 0.17 ± 0.04 0.22 ± 0.02* 26 ± 2 42 ± 2* 35 ± 2* 70 ± 7 176 ± 41* 51 ± 3*,† AA Normoxia Hypoxia Hyperoxia

22 ± 4 33 ± 7 17 ± 1

31 ± 4 23 ± 6 33 ± 2

3667 ± 1415 3912 ± 1247 2546 ± 549

16 ± 3 11 ± 1 15 ± 3

210 ± 59 244 ± 74 159 ± 35

0.9 ± 0.1 0.9 ± 0.2 0.9 ± 0.2

0.66 ± 0.08 0.66 ± 0.04 0.55 ± 0.05 45 ± 11 73 ± 9 50 ± 10 1.7 ± 0.2 1.9 ± 0.4 1.4 ± 0.3 49 ± 8 34 ± 4 34 ± 4 TA Normoxia Hypoxia Hyperoxia

8±2 11 ± 1 10 ± 3

18 ± 6 34 ± 4 28 ± 5

773 ± 259 753 ± 68 933 ± 97

13 ± 4 7±1 11 ± 2

115 ± 28 191 ± 22 103 ± 12†

CoQ10 (pmol/mgp) CE-OOH (pmol/mgp) CE/NEC × 100 CE (nmol/mgp) NEC (nmol/mgp)

Table 4 Biochemical analyses of normal thoracic (TA) and balloon-injured abdominal aorta (AA)

CE-OOH/CE × 100

␣-TOH (pmol/mgp)

␣-TOH/CE

CoQ10 /CE × 100

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

261

3.6.3. Abdominal aorta The damaged AA contained increased lipids (NEC and CE) and oxidized lipids (CE-OOH). Aortic NEC concentration was further increased by hypoxia treatment but decreased by hyperoxia. Similar trends were seen with CE concentration, although not reaching statistical significance. Total CE-OOH was unchanged with hypoxia and decreased with hyperoxia, although once lipid-standardized, the differences were no longer apparent. Total and lipid-standardized ␣-TOH concentrations were not different between treatment groups. Total CoQ10 was increased in both the hypoxic and hyperoxic groups, but only the latter remained significant when lipid-standardized.

4. Discussion Here, we show that intermittent repetitive hypoxia significantly increased intimal disease in both normal and ballooninjured arteries, whilst intermittent repetitive hyperoxia promoted positive arterial remodeling but did not protect against but instead increased atherosclerosis in undamaged arteries. These effects occurred independent of the content of lipids, and in particular, the extent of lipid peroxidation. Thus, our data provide strong evidence that the disease promoting effect of hypoxia is not related to endothelial damage allowing influx of lipids nor oxidative stress resulting in increased lipoprotein lipid peroxidation. Sustained hypoxia (10% O2 ) has been reported to increase plasma cholesterol and atherosclerosis in cholesterol-fed rabbits [8,18]. In contrast but consistent with other studies using cyclical hypoxia and hyperoxia [6,10], we found that neither intermittent repetitive hypoxia nor hyperoxia affected the levels of serum lipids. Hence, the hypercholesterolemic effect of hypoxia appears to occur in sustained but not intermittent exposure. In normal TA with intact endothelium, diet-induced hypercholesterolemia was sufficient to cause mild atherosclerosis in 6 weeks. In damaged AA with endothelial denudation, intimal thickening and lipid accumulation in the arterial wall was much greater. Thus, as shown in our previous work [15] integrity of the endothelium is critical to the processes of intimal hyperplasia and lipid accumulation. Consistent with published data [6,10], repetitive hypoxia significantly increased intimal hyperplasia in both normal and balloon-injured arteries, but the mechanism(s) of these pro-atherogenic effects is unclear. Given the neutral effect on serum lipids, it is likely to impact directly on disease-initiating processes within the arterial wall. Hypoxia causes several potentially detrimental effects on endothelial and vascular smooth muscle cells. The activity of xanthine oxidase on the endothelial cell surface can be increased by hypoxia [19]. This could increase the generation of superoxide anion, neutralize nitric oxide and increase oxidative stress in the arterial wall. In addition, hypoxia increases secretion of the vasoconstrictor endothelin from

262

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263

the endothelium [20], consistent with the finding that in response to hypoxia, human pulmonary arteries constrict and endothelium-dependent relaxation of porcine pulmonary aortas is reversed to a contractile response [21]. Hypoxia could also contribute to atherogenesis by increasing adhesion of monocytes to endothelial cells [22], and by inducing the pro-proliferative, synthetic phenotype of smooth muscle cells [23]. Hypoxia depletes cellular energy stores and reduced glutathione that constitute a major antioxidant defense, and this can result in increased sensitivity of vascular cells to conditions of oxidative stress [24]. In addition, hypoxia may increase the production of reactive oxygen species by vascular cells, and both mitochondria [25] and NAD(P)H oxidase [26] have been implicated as a source of oxidants. Furthermore, hypoxia increases the activity of 15-lipoxygenase-2 in human macrophages, resulting in increased oxidation of LDL [27]. Therefore, hypoxia could conceivably exacerbate vascular disease by increasing local oxidative stress and LDL oxidation. We assessed lipid peroxidation in the arterial wall as a potential marker reflecting such putative increased oxidative stress. We found similar proportions of CE were oxidized to CE-OOH, the major type of oxidized lipid in lipoproteins undergoing oxidation, between the different treatment groups. Also, the lipophilic antioxidants, ␣-TOH and CoQ10 , were not deficient and in fact increased in line with the amount of lipids present. This is consistent with the fact that these antioxidants reside in circulating lipoproteins, and thus their entry into the arterial wall would parallel that of lipoproteinderived lipids [28]. We conclude that changes in tissue oxygen levels have no direct effect on aortic lipoprotein lipid peroxidation, and thus the disease promoting effects are not explained by this mechanism. Hence, these findings do not support the ‘oxidative modification theory’ of atherosclerosis, according to which oxidative modification of lipoprotein lipids within the artery wall is an early disease-promoting event [2]. Intermittent repetitive hyperoxia was not protective in our study, instead significantly increasing atherosclerosis in undamaged arteries and without effect on balloon-injured arteries. Previously, hyperoxia has been reported to be antiatherosclerotic in some studies [6], but it can also increase intimal thickening in rat pulmonary arteries [29]. A possible explanation for this discrepancy may lie in the shorter duration of hyperoxia treatment (3 weeks) in our study compared with other protocols that included 10 weeks of continuous 28% oxygen [4], 14 weeks of 100% oxygen (2 h daily for 5 days a week) [5], 8 weeks of 40% oxygen (5 h daily for 5 days a week) [10] and 10 weeks of hyperbaric oxygen using 100% oxygen at 2.5 atm (2 h daily for 5 days a week) [6]. A novel finding of the present study was the observed positive remodeling resulting from hyperoxia, although the mechanism(s) for this is unclear. We conclude that intermittent repetitive hypoxia strongly promotes vascular disease both in normal and damaged arteries. The mechanism for this is unclear but appears indepen-

dent of lipid accumulation and oxidation. Research into the direct effects of hypoxia on VSMC and endothelial cells in vivo may further the understanding of this process. Although our results on the effects of hyperoxia are not consistent with previous work, this may be limited by the relatively short duration of our study aimed at assessing early changes in the arterial wall. Overall, our findings support the ‘anoxemia theory’ but not the ‘oxidative modification theory’ of atherosclerosis. They lend further evidence that OSA is likely to have direct pro-atherogenic effects, independent of and in addition to the co-existing risk factors.

Acknowledgments We thank Dr. D. Sullivan (Royal Prince Alfred Hospital) for his assistance. This work was supported by a grant from the National Health & Medical Research Council of Australia (NH&MRC) to Dr. Stocker. Dr. Lau was supported by a NH&MRC post-graduate scholarship, Dr. Celermajer by the Medical Foundation Sydney University and Dr. Stocker by a NH&MRC Senior Principal Research Fellowship.

References [1] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–26. [2] Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24. [3] Stocker R, Keaney Jr JF. The role of oxidative modifications in atherosclerosis. Physiol Rev 2004;84:1381–478. [4] Kjeldsen K, Astrup P, Wanstrup J. Reversal of rabbit atheromatosis by hyperoxia. J Atheroscler Res 1969;10:173–8. [5] Vesselinovitch D, Wissler RW, Fisher-Dzoga K, et al. Regression of atherosclerosis in rabbits. I. Treatment with low-fat diet, hyperoxia and hypolipidemic agents. Atherosclerosis 1974;19:259–75. [6] Kudchodkar BJ, Wilson J, Lacko A, et al. Hyperbaric oxygen reduces the progression and accelerates the regression of atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol 2000;20:1637–43. [7] Hueper WC. The anoxemia theory. Arch Pathol 1944;38:173–81. [8] Kjeldsen K, Wanstrup J, Astrup P. Enhancing influence of arterial hypoxia on the development of atheromatosis in cholesterol-fed rabbits. J Atheroscler Res 1968;8:835–45. [9] Helin P, Lorenzen I. Arteriosclerosis in rabbit aorta induced by systemic hypoxia. Biochemical and morphologic studies. Angiology 1969;20:1–12. [10] Okamoto R, Hatani M, Tsukitani M, et al. The effect of oxygen on the development of atherosclerosis in WHHL rabbits. Atherosclerosis 1983;47:47–53. [11] He J, Kryger MH, Zorick FJ, et al. Mortality and apnea index in obstructive sleep apnea. Experience in 385 male patients. Chest 1988;94:9–14. [12] Partinen M, Guilleminault C. Daytime sleepiness and vascular morbidity at seven-year follow-up in obstructive sleep apnoea patients. Chest 1990;97:27–32. [13] Hung J, Whitford EG, Parsons RW, et al. Association of sleep apnoea with myocardial infarction in men. Lancet 1990;336:261–4. [14] Levinson PD, McGarvey ST, Carlisle CC, et al. Adiposity and cardiovascular risk factors in men with obstructive sleep apnea. Chest 1993;103:1336–42.

A.K. Lau et al. / Atherosclerosis 185 (2006) 254–263 [15] Lau AK, Leichtweis SB, Hume P, et al. Probucol promotes functional reendothelialization in balloon-injured rabbit aortas. Circulation 2003;107:2031–6. [16] Chaufour X, Issa F, Sullivan C, et al. A fully-automated environmental chamber for examination of long-term effects of intermittent hypoxia on medium-size animals. Jpn J Physiol 1999;49:207–11. ¨ [17] Witting PK, Pettersson K, Ostlund-Lindqvist A-M, et al. Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits. J Clin Invest 1999;104:213–20. [18] Myasnikov AL. Influence of some factors on development of experimental cholesterol atherosclerosis. Circulation 1958;17:99–113. [19] Terada LS, Guidot DM, Leff JA, et al. Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity. Proc Natl Acad Sci USA 1992;89:3362–6. [20] Kourembanas S, Marsden PA, McQuillan LP, et al. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 1991;88:1054–7. [21] Holden WE, McCall E. Hypoxia-induced contractions of porcine pulmonary artery strips depend on intact endothelium. Exp Lung Res 1984;7:101–12. [22] Santilli SM, Fiegel VD, Aldridge DE, et al. Rabbit aortic endothelial cell hypoxia induces secretion of transforming growth factor beta and augments macrophage adhesion in vitro. Ann Vasc Surg 1991;5:429–38.

263

[23] Durmowicz AG, Badesch DB, Parks WC, et al. Hypoxia-induced inhibition of tropoelastin synthesis by neonatal calf pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol 1991;5: 464–9. [24] Bhat GB, Tinsley SB, Tolson JK, et al. Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury. J Cell Physiol 1992;151:228–38. [25] Cowan DB, Jones M, Garcia LM, et al. Hypoxia and stretch regulate intercellular communication in vascular smooth muscle cells through reactive oxygen species formation. Arterioscler Thromb Vasc Biol 2003;23:1754–60. [26] Goyal P, Weissmann N, Grimminger F, et al. Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species. Free Radic Biol Med 2004;36:1279–88. [27] Rydberg EK, Krettek A, Ullstrom C, et al. Hypoxia increases LDL oxidation and expression of 15-lipoxygenase-2 in human macrophages. Arterioscler Thromb Vasc Biol 2004;24:2040–5. [28] Letters JM, Witting PK, Christison JK, et al. Changes to lipids and antioxidants in plasma and aortae of apoE-deficient mice. J Lipid Res 1999;40:1104–12. [29] Jones R, Zapol WM, Reid L. Pulmonary artery remodeling and pulmonary hypertension after exposure to hyperoxia for 7 days. A morphometric and hemodynamic study. Am J Pathol 1984;117:273– 85.