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School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK
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Abstract |
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(Received 28 July 2003;
accepted after revision 3 November 2003)
Corresponding author P. D. Weinberg: School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK. Email: p.d.weinberg{at}reading.ac.uk
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Introduction |
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Our studies of the transport of macromolecules between plasma and the arterial wall suggest an explanation for these age-related patterns. Short-term (Sebkhi & Weinberg, 1996) and quasi-steady (Sebkhi & Weinberg, 1994a) uptake of labelled albumin by the wall were greater downstream than upstream of aortic branches in young rabbits, but showed the opposite trend in adult animals. The switch in pattern occurred at around 6 months (Sebkhi & Weinberg, 1994a), which is approximately the age of sexual maturation in this species (Berger et al. 1982). The spatial correlations are consistent with the view that the rate of uptake of circulating macromolecules by the arterial wall determines how rapidly atheromata develop at any particular site. The mature pattern of uptake has been shown to be determined in turn by endogenous nitric oxide (NO) synthesis (Forster & Weinberg, 1997) and by blood flow (Staughton et al. 2001a).
Hitherto, the age-related transport patterns have appeared robust: consistent results have been obtained in short-term and long-term experiments; in conscious animals or in situ perfused vessels; when measurements have been made along the branch midline or over a wider area; and when using two different detection systems (Sebkhi & Weinberg, 1994a, 1996; Forster & Weinberg, 1997; Ewins et al. 2002). However, more recent, unpublished experiments in our laboratory have sometimes given the downstream and sometimes the upstream pattern of uptake in mature rabbit aortas. The putative relevance of the original findings to a key human disease makes it important to account for this discrepancy. In the present study, we show that it can be explained by effects of time of day and rabbit strain. A preliminary report of some of the data has been published (Staughton & Weinberg, 1999).
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Methods |
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Experiments were conducted using 53 male New Zealand white (NZW) rabbits. The animals were individually housed at 18 ± 2°C on a 12-h light cycle (light on 07:00 to 19:00 h) and were maintained on a standard laboratory rabbit diet (TRB 9603; Harlan Teklad). All procedures complied with the Animals (Scientific Procedures) Act 1986 and with local regulations.
Strains, ages and times of day
Effects of time of day on permeability were investigated in NZW rabbits of the Interfauna strain, supplied by Harlan Interfauna (Huntingdon, Cambs., UK; n= 28); this strain has been used in all our previously published studies of transport (Sebkhi & Weinberg, 1994a, 1996; Forster & Weinberg, 1997; Staughton et al. 2001a; Ewins et al. 2002). Three groups of rabbits were used, having ages of 86 ± 7, 304 ± 53 and 710 ± 44 days (mean ±S.D., n= 10, 10 and 8, respectively), equivalent to approximately 3 months, 10 months and 2 years. Corresponding weights for the three groups were 2.52 ± 0.36, 3.61 ± 0.39 and 3.88 ± 0.51 kg. Within each group, half the animals were examined in the morning (10:30–11:00 h) and half in the afternoon (16:00–17:30 h) because retrospective examination of the earlier data suggested that experiments carried out at these times gave different results. Effects of the time of day on NO production and conduit artery tone were investigated in 10 additional animals of the same strain aged between 12 and 15 months.
Effects of strain on permeability, NO production and wall structure were investigated by using rabbits supplied by Murex Biotech (Dartford, Kent, UK; n= 7) and Highgate Farm (Market Rasen, Lincs., UK; n= 4). Preliminary studies showed a particularly large difference between these strains in the age at which transport switched from a downstream to an upstream pattern. Ages and weights of the rabbits are shown in Table 1. Experiments were conducted in the afternoon.
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Preparation of fluorescent tracer for transport experiments
Lissamine rhodamine B (sulphorhodamine B, CI 145100, Sigma) was purified, converted to its reactive sulphonyl chloride and conjugated with bovine serum albumin (BSA, fatty acid free, First Link UK Ltd) as previously described (Fothergill, 1964; Weinberg, 1988; Sebkhi & Weinberg, 1994a, 1996). The conjugate was purified of free dye by gel filtration (Sephadex G25, Pharmacia), eluting with Ringer's solution (9.0 g l–1 NaCl, 0.2 g l–1 KCl, 0.2 g l–1 Ca2Cl, 0.1 g l–1 NaHCO3) diluted 1 in 3 with water, frozen dropwise in liquid nitrogen, lyophilized and stored at –15°C.
Just prior to use, the tracer was reconstituted in water to one-third its eluted volume, and was thus concentrated and suspended in Ringer's solution. It was further purified of free dye by stirring with charcoal, and further concentrated with centrifuge filters (10-kDa cut-off, Sigma). After charcoal purification, >99.8% of the dye can be precipitated by trichloroacetic acid (Sebkhi & Weinberg, 1996).
In vivo transport studies
The protocols used are detailed elsewhere (Sebkhi & Weinberg, 1996; Ewins et al. 2002). Briefly, tracer (700 mg kg–1) was introduced into the circulation of conscious, unrestrained animals via the marginal ear vein. (This step was omitted in the autofluorescence experiments, which were otherwise identical.) Heparin (1000–2000 IU, I.V., Sigma) was administered 8 min later and an overdose of pentobarbitone (Euthatal, Rhone Merieux, 200 mg kg–1I.V.) was given after a further 2 min. In rapid succession, the thorax and abdomen were opened along the ventral midline, blood from the heart was collected into EDTA, and a cannula was tied into the aorta at the level of the diaphragm, via which the thoracic aorta was flushed for 30 s with saline and then fixed at physiological pressure with 10% formalin for 30 min. The vessel was excised and postfixed in 10% formalin for a further 24 h. The time elapsing between death and flushing the aorta averaged 3.0 ± 0.6 min (mean ±S.D.) and never exceeded 5 min, the period after which post mortem transport appears to affect results (Sebkhi & Weinberg, 1996). Studies using a model system have shown that formalin immobilizes the tracer effectively and does not stimulate tissue autofluorescence (Staughton et al. 2001b).
Plasma obtained from the blood sample was diluted with a gelatin solution, allowed to set and also fixed in 10% formalin, so that it could be processed in the same way as arterial tissue (Weinberg, 1988, 1989).
Measurement of tracer uptake
Uptake of the fluorescent tracer was assessed by digital imaging fluorescence microscopy as previously described (Weinberg et al. 1994; Staughton et al. 2001b). Briefly, small pieces of aorta, each containing one intercostal artery ostium (n= 7–12 per vessel), were embedded in epoxy resin (Epon 812 substitute) (Glauert, 1991) and six 2-µm-thick longitudinal sections were cut through the centre of the branch. In each section, the fluorescence from the aortic intima-media up to 345 µm (
1 branch diameter) upstream and downstream of the ostium was imaged and digitized by using an epifluorescence microscope with custom filters (Staughton et al. 2001b) in conjunction with a cooled CCD camera (Axiom Viper, 1536 x 1024-pixel resolution) and associated frame grabber.
Using custom software, offsets were subtracted, a flat-field correction applied and an average pixel intensity obtained for the area of interest (Weinberg et al. 1994). Appropriate autofluorescence values were subtracted from the experimental data. The difference in average pixel intensity between upstream and downstream regions in each section was expressed as a percentage of the mean value for both regions. (The upstream intensity was subtracted from the downstream one, so positive values indicate a downstream transport pattern and negative values indicate an upstream one.) The mean for both areas was also expressed as a percentage of the tracer concentration in plasma, the latter being obtained from examination of the gels.
Measurement of branch dimensions
To determine whether dimensions of the arterial wall were different in the Murex and Highgate strains, which might have accounted for effects of strain on permeability patterns, the thickness of the intima-media and the number of lamellar units were measured in those regions in which transport had been determined. Because intima-medial thickness varies locally, it was assessed at six points within each upstream or downstream region, and the six values were averaged to obtain a representative result. Thicknesses and lamellar numbers were determined for 18 branches from four Murex animals and 13 branches from three Highgate animals.
Measurement of NO synthesis
To assess whether differences in endogenous NO synthesis could account for any effects of time of day or strain on transport, plasma levels of nitrite and nitrate were measured by using the Griess reaction (Granger et al. 1996). To determine the effects of time of day, one blood sample was taken between 09:30 and 11:30 h and a second between 15:30 and 17:30 h in each of 10 Interfauna animals. To determine the effects of strain, blood samples were taken from all the mature Highgate and Murex animals.
A commercial colorimetric kit (Calbiochem) was used according to the manufacturer's instructions. In this kit, nitrate is converted to nitrite using an NADH-dependent nitrate reductase. Protein was removed from the samples prior to analysis by precipitation with NaOH and ZnSO4. Absorbance at 540 nm was converted to the concentration of nitrite plus nitrate using a linear calibration curve obtained for standard solutions. All assays were conducted in duplicate.
Measurement of conduit artery tone
Conduit artery tone was assessed from its influence on the peripheral pulse waveform. Nitrovasodilators and other substances affecting conduit artery tone lower the height of the dicrotic notch, relative to the overall amplitude of the peripheral pressure or volume pulse wave (relative height of the dicrotic notch, or RHDN –Klemsdal et al. 1994). This index was assessed between 09:30 and 11:30 h and between 16:00 and 17:30 h in each of the 10 Interfauna animals in which effects of time of day on NO production were also examined. (The technique had not been developed when the effects of strain were examined and was therefore not applied to the animals in that study.)
The methods and their validation have been detailed elsewhere (Weinberg et al. 2001). Briefly, the peripheral pulse wave was measured by using a reflectance photoplethysmograph (Biopac) placed on the shaved dorsal surface of one ear. The light emitter and detector of the sensor straddled the central artery, which lies close to the skin surface. To optimize signal quality, recordings were made under subdued lighting, the ear was manually supported in an upright position if necessary and the room temperature was kept at 
21°C. Sensor output was digitized using the AC-coupled input of an analog-to-digital converter (ADC-100, Pico Technology Ltd) and associated Picolog software. Representative notch heights and wave amplitudes were obtained from recordings, each of which comprised 4–8 s of sensor output, as previously described (Weinberg et al. 2001).
Statistics
ANOVA was used to assess differences in the slopes of regression lines, and analysis of covariance was used to test differences in their intercepts. Other analyses were conduced by Student's paired or unpaired t test (Armitage & Berry, 1987).
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Results |
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Morning and afternoon patterns of transport near branches are shown for rabbits of different ages in Fig. 1. Considering first the experiments conducted in the morning, uptake was greater downstream than upstream of branches in the 3-month-old group, but the opposite pattern was seen in the 10-month-old animals and this change was maintained in the 2-year-old group.
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Effect of time of day on mean uptake
The mean uptake in the upstream and downstream regions, expressed as a percentage of the tracer concentration in plasma, is shown for the three age groups and two times of day in Fig. 2. Mean intima-medial concentrations were of the order of 0.1% of those in plasma and showed no obvious trend with age, as in our previous studies. [There is a three-fold higher level of mean uptake shortly after weaning (Sebkhi & Weinberg, 1994a, 1996) but such ages were not examined in the present work.] There was no significant difference in mean uptake between the morning and afternoon experiments at 3 months (t= 1.15, P > 0.05), 10 months (t= 0.76, P > 0.05) or 2 years (t= 1.56, P > 0.05).
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There was no detectable effect of time of day on the plasma levels of NO2 plus NO3, measured in 10 rabbits aged between 12 and 15 months. Concentrations averaged 34.9 ± 8.4µmol l–1 (mean ±S.D.) in the morning and 34.9 ± 8.9µmol l–1 in the afternoon (t= 0, P > 0.05).
Effect of time of day on conduit artery tone
RHDNs for the same 10 rabbits averaged 0.46 ± 0.13 (mean ±S.D.) in the morning and 0.53 ± 0.16 in the afternoon, lower values indicating greater vasodilatation. The difference was not significant (t= 1.25, P > 0.05).
Effect of strain on the pattern of transport
The pattern of tracer uptake is shown as a function of rabbit strain and age in Fig. 3. For both the Murex and the Highgate strains, uptake was greater downstream than upstream of branches in the youngest animals and this difference decreased linearly and then reversed with age, consistent with our previously published data using Interfauna rabbits (Sebkhi & Weinberg, 1994a). For both strains, the effect of age on the pattern of transport was significant (F= 33.8, P P 0.005 for Murex; F= 99.5, P P 0.01 for Highgate). The age at which there was a switch from the downstream to the upstream pattern differed markedly between strains (F= 52.7, P P 0.001): the intercepts of the regression lines on the abscissa were 651 days for Murex rabbits and 1278 days for Highgate rabbits. The gradients of the regression lines, however, were not different (F= 0.44, P > 0.05).
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Mean uptake for the upstream and downstream regions combined is shown for the Murex and Highgate strains in Fig. 5. Tracer concentrations in the wall were of the order of 0.1% of plasma levels, as in Fig. 2. There was no change with age for either strain (t= 0.57, P > 0.05 for Murex; t= 0.17, P > 0.05 for Highgate) and no difference between the strains (t= 0.81, P > 0.05).
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The thickness of the intima-media and the number of lamellar units in upstream and downstream regions are shown for rabbits of the two strains in Table 1; none of the differences was significant (for thickness, t= 1.11 upstream and t= 1.30 downstream; for lamellar number, t= 2.24 upstream and t= 1.55 downstream; all P > 0.05).
Effect of strain on NO production
Plasma levels of NO2 plus NO3 for rabbits of the two strains are shown in Table 1. Values were higher on average in the Murex rabbits than in the Highgate rabbits but this difference was not statistically significant (t= 1.65, P > 0.05). The higher mean value for the Murex rabbits resulted largely from data obtained for the two animals aged 322 and 331 days, which had plasma levels of NO2 plus NO3 that were three-fold higher than the remaining rabbits. Apart from this anomaly, there was no trend with age in either strain. Values for the Murex animal of 697 days and the Highgate animal of 628 days were very similar (29.1 and 24.6 µmol l–1, respectively), whereas their transport patterns were nearly the most upstream and the most downstream detected for any animals in the study (averaging –16% and +91%, respectively).
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Discussion |
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Transport was measured by the techniques used in our previous studies. These techniques are considered reliable because they give results for the mean uptake and for variations in uptake that are consistent with data obtained by entirely different (e.g. radiotracer) methods (Sebkhi & Weinberg, 1994a, 1996; Weinberg et al. 1994). The tracer was based on albumin rather than the more atherogenic low-density lipoprotein because albumin is metabolized more slowly (Yedgar et al. 1983) and because measurement with adequate spatial resolution requires high concentrations of tracer in plasma. Where studies of transport of the two molecules can be compared, there appears to be good agreement between their patterns of uptake (Sebkhi & Weinberg, 1996). Uptake occurring over a period of 10 min was measured. After such short times, wall concentrations of tracer remain sufficiently low that influx into the wall predominates; efflux and the space available for tracer can be ignored (Sebkhi & Weinberg, 1996; Nielsen, 1996). Influx was assessed because its variation appears to be the main determinant of the age-related patterns of net uptake (Sebkhi & Weinberg, 1996).
Rabbit strain had a strong but highly specific influence on patterns of influx. In both strains that were examined in detail, there was a linear effect of age on the difference in uptake between downstream and upstream regions; the gradient of the regression line did not differ between the strains. Additionally, both strains showed an eventual switch from the downstream transport pattern to the upstream one, as in all our previous studies (Sebkhi & Weinberg, 1994a, 1996; Forster & Weinberg, 1997; Ewins et al. 2002). There was, however, a large difference between strains in the age at which this switch occurred. The transitional age, defined as the intercept of the regression line on the abscissa, was 651 days for Murex rabbits and 1278 days for Highgate rabbits. Further preliminary experiments (data not shown) have given a value of 675 days for Charles River rabbits, similar to that for the Murex rabbits. The difference between strains is consistent with the transitional age being under genetic control, although influences of early environment cannot be ruled out. The data also demonstrate, contrary to our original speculation (Sebkhi & Weinberg, 1994a), that the transition is not related to sexual maturation, because this occurs at around 6 months in all strains.
We recently reported that strain also affects the pattern of cholesterol-induced lesions in rabbits (Barnes & Weinberg, 2001). The distribution of such lesions had previously appeared inconsistent. Lesions developed downstream of aortic branches in mature rabbits from the first trial we conducted, but showed a more upstream pattern in a second trial (Barnes & Weinberg, 1999). In two further trials (Barnes & Weinberg, 2001), we tested the hypotheses that this disparity reflected a difference in the degree of impairment of the NO pathway, or that it reflected the precise age of the mature rabbits and the feeding protocol employed, which differed between the first two trials. No consistent effect of any of the controlled variables was observed, but a post hoc multivariate analysis of all four trials showed that the pattern of disease was associated with rabbit strain. This earlier result is not completely explained by the transport data from the present study (and the additional, preliminary data described in the preceding paragraph) because differences in lesion pattern were seen between strains that showed no difference in transport properties. This may have arisen because rabbits in the lesion studies were fed cholesterol, unlike those in the present work. Additional effects of strain on the response of arteries to hypercholesterolaemia might therefore have been involved. A further explanation is that transport properties were measured only along the ostial midline, whereas lesions were mapped all around the branch; in the lesion studies, differences between strains were often most apparent away from the midline.
Time of day also had a large influence on transport patterns. When transport was assessed in Interfauna rabbits in the morning, the transitional age was between 90 and 300 days. [Our previous data (Sebkhi & Weinberg, 1994a), which were also obtained with this strain and in the morning, gave a more precise estimate of 170 days]. For afternoon experiments, however, the transitional age was around 700 days. An even bigger difference might have been seen had more widely separated times been investigated; the times were chosen to investigate discrepancies in earlier experiments, some of which were conducted in the morning and some in the afternoon. Effects of time of day on vascular transport properties have rarely been reported before. We are aware of evidence for a circadian rhythm only in the permeability of rat cerebral arterioles to horseradish peroxidase (Mato et al. 1981).
A remarkable finding in both the strain and the time of day experiments was that despite large changes in the pattern of transport, the mean uptake in upstream and downstream regions combined did not change. Thus the differences in the pattern of transport reflect equal but opposite changes in uptake by upstream and downstream regions. This observation is consistent with previous results from our laboratory. We have been able to reverse the pattern of uptake by allowing animals to age (Sebkhi & Weinberg, 1994a, 1996), by administering a cholesterol-enhanced diet (Sebkhi & Weinberg, 1994b), by inhibiting NO synthesis (Forster & Weinberg, 1997) and by modifying flow (Staughton et al. 2001a). In all of these cases, there was no discernible change in mean uptake. The constancy of mean uptake is consistent with it being a controlled property of the wall.
A preliminary attempt was made to elucidate mechanisms underlying the differences in pattern. Wall dimensions were examined because they must change with maturation and may play a causal role; wall structure – including medial thickness (Caro et al. 1980) and the presence of elastic lamellae (Sims, 1989) – has direct effects on transport. (Structure was investigated only for rabbits from the strain experiments because the underlying architecture of the wall could not change with time of day.) No evidence consistent with a role of wall thickness or the number of lamellar units was obtained. First, there was no clear monotonic change in thickness or lamellar number with age in either strain. Second, the dimensions for the Highgate rabbits did not resemble those for Murex rabbits that were 500–600 days younger, contrary to the observations for transport.
The NO pathway was also examined, because previous work in in situ perfused aortas has shown that inhibition of NO synthesis reverses the adult pattern of transport but not the juvenile pattern (Forster & Weinberg, 1997). From this it is inferred that a change with age in NO synthesis (probably its flow-dependent synthesis; Staughton et al. 2001a) or activity may be involved in the switch in pattern. The Griess assay was used to measure plasma concentrations of NO2 plus NO3, the oxidation products of NO. These concentrations reflect constitutive NO synthesis by blood vessels but also synthesis of NO elsewhere in the body. In the time of day experiments we additionally employed a newly developed method for assessing conduit artery tone from the height of the dicrotic notch in the peripheral pulse wave. In rabbits (Klemsdal et al. 1994; Weinberg et al. 2001) and in humans (Chowienczyk et al. 1999) stimulating, inhibiting or mimicking NO synthesis with a range of pharmacological agents has a large effect on this index.
No evidence was obtained to support the view that differences in NO production cause the difference in transport properties between strains. There was no significant difference in NO production between strains, no monotonic change in NO with age in the mature rabbits of either strain, and no evidence that Highgate animals had an NO production resembling that of Murex animals at a younger age. Similarly, there was no significant difference in NO production or conduit artery tone between morning and afternoon experiments. The lack of difference with time of day was unexpected. Recent studies of flow-mediated dilatation of the human brachial artery, an NO-dependent phenomenon, unequivocally demonstrate a circadian rhythm, the response to flow being approximately twice as great at 16:00 or 17:00 h as at 08:00 h (Etsuda et al. 1999; Gaenzer et al. 2000). There is also evidence for a circadian rhythm in the NO pathway in rats (Witte et al. 1995; Keskil et al. 1996; Mastronardi et al. 2002). Importantly, this includes evidence obtained by analysing plasma concentrations of NO2 plus NO3. Our results suggest that, even though the mature transport pattern is NO-dependent, its attenuation in Highgate rabbits and during the afternoon is not due to an altered NO production or activity.
Finally, we consider the significance of the present observations. First, the data have re-established our original finding that there is a reversal in the pattern of transport near aortic branches with age. The recent failure to observe the upstream pattern in mature rabbits can be explained by the use of rabbits of different strains and the use of different times of day for experiments. Second, the data are important from a practical point of view. They show that in order to obtain consistent results when studying these phenomena, attention needs to be paid to the strain of rabbit and to the time of measurement. Third, differences between strains and with time of day provide a potentially useful method for investigating the mechanisms determining transport patterns, because they allow putative localizing factors to be compared between animals that show different patterns despite being of the same species and age. Investigation in the present study of the role of NO is one example; other possibilities would include examination of differential gene and protein expression. Fourth, if the phenomena apply to humans – as inferred for the juvenile and adult transport patterns themselves – there may be differences between populations in the transitional age for transport patterns and, by implication, in the transitional age for lesion patterns. We have previously suggested (Barnes & Weinberg, 2001) that this could account for discrepancies between studies of human disease. Furthermore, determinants of arterial transport properties might vary between human populations and with time of day. This would have implications for the development of anti-atherosclerotic interventions based on modifying transport.
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  R. D. Rudic, P. McNamara, D. Reilly, T. Grosser, A.-M. Curtis, T. S. Price, S. Panda, J. B. Hogenesch, and G. A. FitzGerald Bioinformatic Analysis of Circadian Gene Oscillation in Mouse Aorta Circulation, October 25, 2005; 112(17): 2716 - 2724. [Abstract] [Full Text] [PDF]
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