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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1361-1368.)
© 1997 American Heart Association, Inc.

Articles

Changes With Age in the Influence of Endogenous Nitric Oxide on Transport Properties of the Rabbit Aortic Wall Near Branches

Beverley A. Forster; ; Peter D. Weinberg

From the School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom.

Correspondence to Dr P.D. Weinberg, School of Animal and Microbial Sciences, University of Reading, Whiteknights PO Box 228, Reading RG6 6AJ, United Kingdom. E-mail p.d.weinberg{at}reading.ac.uk.

	Abstract

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Abstract Uptake of circulating albumin by the aortic wall is greater downstream than upstream of branches in immature rabbits, but the opposite pattern occurs in mature animals. We investigated the role of NO in determining these variations. Descending thoracic aortas of rabbits were cannulated using techniques that avoid depressurization, overstretching, and excessive fluid dynamic stresses at the endothelial surface. They were perfused in situ at a constant pressure and flow rate with oxygenated, protein-containing physiological buffer, with or without N-monomethyl-L-arginine, an inhibitor of NO synthesis. Aortas were fixed 7 to 8 minutes after the addition of rhodamine-labeled albumin to this perfusate, and uptake of the tracer near intercostal ostia was measured by digital imaging fluorescence microscopy of sections through the wall. Despite the absence of pulsatile flow, blood cells, and many plasma components, patterns of transport in control experiments were the same as those occurring in vivo; uptake was greatest downstream of ostia in immature vessels and upstream in mature ones, although mean uptake was higher than previously reported. In the presence of the inhibitor, mean uptake in immature arteries was elevated threefold and the maximum tracer concentration occurred deeper in the wall, but there was no change in the fractional difference between regions. Conversely, the reverse of the control pattern of transport was observed in mature arteries exposed to the inhibitor, but there was no change in mean uptake. The reversal was almost entirely prevented by adding excess L-arginine to the perfusate and was largely stereospecific. Endogenous NO thus appears to determine the mature pattern of transport near branches and helps to maintain the barrier function of the immature wall.

Key Words: age • arterial branches • arterial permeability • endothelium-derived relaxing factor • nitric oxide

	Introduction

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Transport of macromolecules between plasma and arterial tissue may play a critical role in atherogenesis. Transport near branches has received particular attention because large variations in the prevalence of lesions occur within these regions. Quasi-steady-state uptake of circulating albumin by the aortic wall is greater downstream than upstream of intercostal branch ostia in young rabbits,¹ but the opposite distribution is seen in mature animals.² The rate at which albumin enters the wall varies in the same way and probably accounts for the quasi–steady-state phenomena.³ These patterns of transport correlate with the distribution of lipid deposits in the human aortic wall. Lesions occur most frequently downstream of intercostal ostia in fetuses, neonates, and infants,⁴ but upstream in adult vessels.⁵ Hence similar variations in transport properties of the wall may occur in human aortas and determine the location of lesions. The mechanisms that underlie these variations in transport properties have not been established. However, the normal adult pattern of influx reverts to the juvenile pattern in mature rabbit aortas briefly exposed to tracer in the absence of blood pressure and flow.³ A similar switch in quasi-steady-state uptake can be induced, at least transiently, by feeding mature rabbits with a cholesterol-enhanced diet.⁶

The synthesis or biological activity of endothelium-derived relaxing factor, putatively NO, can be influenced by age,^{7 8 9} blood flow,^{10 11} and hyperlipidemia.^{12 13} Furthermore, NO can modify transport properties of blood vessel walls, although the direction of change is controversial.^{14 15 16 17 18} In this preliminary study, we investigated whether variations in NO synthesis or activity can explain the age-dependent patterns of transport observed near rabbit aortic branches. Uptake of rhodamine-labeled albumin was assessed by quantitative fluorescence microscopy of sections through the wall.¹⁹ To avoid indirect effects of NO on transport that could be mediated in vivo by alteration of blood pressure, aortic flow, or interactions of blood cells with the vessel wall, its uptake was investigated in aortas that were perfused in situ with physiological buffer. Short-term transport was examined since variations in rates of influx appear to determine steady-state patterns. Inhibition of NO synthesis caused an increase in uptake around branches of young rabbits and a switch from the adult to the juvenile pattern of transport in mature animals.

	Methods

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Tracer and Animals
Bovine serum albumin (improved standard grade, Applied Protein Products) was labeled with sulforhodamine B (Lissamine rhodamine, C.I. 45100, Sigma Chemical Co) and purified of free dye using established procedures.^{3 20} The tracer has physical, chemical, and biological properties similar to those of unlabeled albumin and is stable in vitro and in vivo.^{1 21 22 23 24}

The aortas of 27 male New Zealand White rabbits were used to determine the uptake of tracer or levels of tissue autofluorescence. Rabbits of two ages were studied, young (78 to 107 days old) and old (294 to 437 days old). These age groups lie either side of the transitional age (130 to 210 days old) at which the pattern of quasi-steady-state uptake reverses in vivo, and both are older than the age (approximately 50 days old) at which there is a threefold drop in the mean uptake around branches.² Weights were, respectively, 1.8 to 3.6 kg and 3.4 to 4.6 kg; a small overlap between the groups was apparent. All animals were fed a standard laboratory diet (200 g/d, Special Diet Services) and tap water ad libitum.

Surgical Procedures
Cannulation techniques were designed to ensure that the aorta was not depressurized or overpressurized at any stage of the experiment, that it was exposed to blood or oxygenated, protein-containing buffer throughout, and that fluid dynamic shear stresses at the blood–wall interface never reached levels known to damage the endothelium. Depressurization was avoided by cannulating the descending thoracic aorta at its distal end and perfusing it in the retrograde direction before inserting the proximal cannula, which permitted anterograde flow.

After anesthesia with pentobarbital (Sagatal, Rhone Merieux, 30 to 40 mg/kg IV) and lidocaine (Xylocaine, Astra, 10 to 20 mg IC and SC), the trachea was intubated, and the animal was ventilated with air (Small Animal Ventilator, Harvard) at a frequency of 50 breaths per minute. In a few early experiments, pentobarbital anesthesia was preceded by administration of acetylpromazine (Acepromazine, C-Vet, 0.1 mg/kg IM) and fentanyl fluanisone (Hypnorm, Janssen, 0.1 mg/kg fentanyl citrate and 0.3 mg/kg fluanisone IM). The abdomen was opened along the midline, with additional pentobarbital (25 mg/kg IV) being administered as required, and the viscera were deflected. The phrenic arteries were clamped, and the diaphragm was cut from the sternum to the aorta. The sternum was then split along its length.

Ligatures were loosely placed around the aorta at the level of the diaphragm, around the proximal descending aorta, and around the inferior vena cava close to its junction with the hepatic vein. The animal was given heparin (grade 1A, Sigma, 1000 IU IV), and the ligatures around the vena cava and the lower thoracic aorta were closed, leaving an intact circulation in the upper body and head. The latter closure was conducted over a period of approximately 1 minute so that baroreflex compensation for the increase in resistance could prevent the occurrence of an excessive blood pressure.

The vena cava was clamped 1 cm cranial to its point of ligation. A closed cannula (6FG or 8FG, Portex), oriented toward the heart, was tied into the vena cava between these points and the clamp was released. Similarly, the aorta was clamped approximately 1 cm cranial to its point of closure at the level of the diaphragm. A cannula (8FG, Portex), oriented toward the heart, filled to its tip with perfusate, and attached to a reservoir 100 cm above the vessel, was tied into the aorta between these points. The perfusate was Holman's buffer (concentrations in mmol/L: NaCl 120; KCl 5; CaCl₂ 2.5; NaH₂PO₄ 1.3; NaHCO₃ 25; sucrose 10, and D-glucose 11.1; pH 7.4) containing 10 mg/mL unlabeled albumin and equilibrated with 95% O₂ and 5% CO₂. The clamp was removed, the heart was stopped by intravenous and intracardiac administration of excess pentobarbital, and ventilation was discontinued. Buffer from the reservoir then perfused the upper body and head at near-physiological pressure but with retrograde aortic flow. The cannula in the vena cava was opened to allow egress of blood and buffer. In most experiments, a second drain (14G infusion set, Vygon) was inserted directly into the right ventricle to prevent tissue edema and effusions into the thoracic cavity.

To permit anterograde rather than retrograde aortic flow, the ligature around the proximal descending aorta was closed, a clamp was temporarily applied 1 cm caudal to the ligature, and a cannula (8FG, Portex) was tied into place between these points. This cannula, oriented in the direction of in vivo aortic flow, was attached in series to a bubble trap, prepressurized Windkessel damping chamber, heat exchanger (to give a temperature of 37°C to 38°C at the cannula tip), peristaltic pump (Watson Marlow), and a reservoir of the buffer described above. The pump was switched on, causing a switch to anterograde flow without loss of pressure.

Measurement of Intercostal Flow and Aortic Pressure
Perfusate draining from the downstream aortic cannula was directed to the reservoir supplying the peristaltic pump. This gave a closed circulation apart from fluid lost via the intercostal arteries. The rate of loss was obtained by monitoring the volume in the reservoir. There was uncertainty about the precise number of intercostal arteries being perfused in each preparation since some ostia may have been obscured by the cannulas. The number was constant during each experiment, and total intercostal flow rates could therefore indicate fractional changes in flow arising from changes in perfusate composition. However, meaningful comparisons could not be made between groups of experimental animals.

During anterograde flow, aortic pressure was continuously monitored with a transducer (Sensym) attached to the analog-to-digital convertor (maximum frequency, 25 Hz) of a microcomputer (BBC model B, Acorn). It was controlled by altering the height of the aortic drain and by adjusting a screw clamp on the outflow tube. Fig 1 illustrates the final configuration, reached approximately 1.5 hours after initial anesthesia.

View larger version (43K): [in this window] [in a new window]   Figure 1. Diagram of the perfusion system. I, II, and III indicate reservoirs for the three different perfusates; P, peristaltic pump; H, heat exchanger; W, Windkessel damping chamber; B, bubble trap; A, in situ perfused thoracic aorta (drainage of the intercostal circulation via the vena cava and right ventricle); T, pressure transducer; and C, screw clamp.

Perfusion Protocol
Aortas were perfused at a constant rate of 150 mL/min with the following sequence of solutions: (1) 20 to 30 minutes with the buffer described above; (2) 15 to 25 minutes with the same solution as a control or the same solution plus (a) 10 µmol/L L-NMMA (Calbiochem-Novabiochem), (b) 10 µmol/L D-NMMA (Calbiochem-Novabiochem), or (c) 10 µmol/L L-NMMA and 3 mmol/L L-arginine (Sigma); and (3) 7 to 8 minutes with the solution used as the second perfusate but in which approximately 75% of the unlabeled albumin had been replaced by an equal amount of tracer. The variations in timing reflect the variable rate of loss of perfusate from the system.

The peristaltic pump was then stopped and the aorta was flushed for approximately 30 seconds via the upstream cannula with 0.15 mol/L NaCl at a pressure of 120 cm water. The vessel was then perfusion fixed for 20 minutes with formal sublimate (6% wt/vol HgCl₂ and 2% vol/vol formalin in water) in the same way. This fixative rapidly immobilizes proteins without giving rise to intense autofluorescence.²⁵ The downstream cannula was closed after approximately 1 minute to reduce the volume of fixative required. The vessel was excised and postfixed in 10% vol/vol formalin for 48 hours.

Tissue Processing and Quantitative Fluorescence Microscopy
Regions of aortic wall containing intercostal branches were embedded in epoxy resin and 2-µm-thick longitudinal sections were cut through the center of the ostia. A total of 136 branches were examined; those close to the points of cannulation were not used. Fluorescence from the sections was excited with an epifluorescence microscope (577±7-nm excitation filter, 590-nm dichroic mirror, 610±10-nm barrier filter, Omega optical), and digital images of the emission from regions upstream and downstream of the branches were obtained with an intensified CCD camera, video digitizer, and microcomputer as previously described.¹⁹ Areas up to 370 µm (approximately one branch diameter) from the ostial lip were included.

Images were corrected to remove offsets and the effects of spatial and temporal biases in excitation or detection efficiency. The response of the system is uniform to within ±5%, and corrected intensities are proportional to tracer concentration over at least a 1000-fold range after correction.¹⁹

Image Analysis
To allow comparison with the different indices of uptake used in previous studies, images were analyzed in two ways. First, a mean tracer concentration was calculated for each column of pixels, from the endothelium to the medial–adventitial border. These means were then averaged along the wall within the upstream or downstream region. This procedure gives equal weight to intimal–medial tracer levels at all points along the wall, irrespective of wall thickness. Concentrations were expressed as a percentage of those in the perfusate.^{2 19}

Second, a series of lines parallel to the endothelium was constructed within the wall of the upstream or downstream region as previously described.³ The spacing between lines was equivalent to 2.88 µm before magnification. Data from grid lines that penetrated into the adventitia were discarded. A total quantity of tracer and a mean concentration, expressed as a percentage of the concentration in the perfusate, were calculated for each depthwise slice of the wall. Quantities were used to calculate a normalized mass transfer coefficient, defined as the quantity of tracer within a volume of wall divided by the product of the tracer concentration in perfusate, the area of endothelium overlying that volume, and the duration of exposure to tracer. For short periods of uptake and low concentrations of tracer in the wall, these coefficients indicate the permeability of the wall to diffusive or convective entry.³ They were calculated for tracer quantities integrated over a range of depths into the wall. These procedures were applied to 72 branches from a subset of animals comprising 4 young and all the old control rabbits, and equal numbers of animals treated with L-NMMA.

Autofluorescence intensities, obtained from arterial tissue that had not been exposed to tracer, were subtracted from all experimental values. Because autofluorescence intensities vary significantly with age,³ intensities from an animal in the same age group were used.

To measure levels of fluorescence from tracer in the perfusate, a sample of each perfusate was diluted 1/100 with 12.5% wt/vol gelatin (type B, Sigma) in PBS (0.15 mol/L, pH 7.4; Sigma). The solution was allowed to set, fixed in formal sublimate for 20 minutes, and then postfixed, embedded, sectioned, and examined in the same way as arterial tissue.²⁶ To improve the accuracy of these calibration procedures, the values obtained were averaged across all the experiments in which the same batch of tracer had been used. This approach was applicable to the present study, but not to earlier in vivo investigations, because all arteries were exposed to the same concentration of labeled albumin.

Statistics
Means were compared using unpaired t tests. Values of n indicate the number of perfusion experiments.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Perfusate Pressure, Flow, and Fluorescence Intensity
Table 1 shows aortic pressure in the different experimental groups, averaged for the three stages of anterograde perfusion. Variation between groups was small, and within each group the pressures observed before and after the changes in perfusate composition were similar. There were no significant differences associated with age, experimental condition within each age group, or changes in perfusate composition (P=NS for all, n=3 to 6 for each value), indicating that pressure was adequately controlled. Pressures were approximately the same as mean systemic blood pressure in vivo for these age groups.²⁷

View this table: [in this window] [in a new window]   Table 1. Maintenance of Pressure During the Different Stages of Perfusion

Intercostal flow rates were determined at intervals during each experiment. The timing of these measurements was normalized in Fig 2 to allow for minor variations that occurred between experiments in the duration of the different phases of perfusion. The initial fluctuations in flow rate resulted, at least in part, from the adjustments made to establish a suitable perfusion pressure but may also be related to other transient effects. For example, there appears to be a consistent initial rise that may reflect washout of blood from the intercostal circulation.

View larger version (18K): [in this window] [in a new window]   Figure 2. Loss of perfusate via the intercostal arteries. Times were adjusted for the minor differences in the duration of perfusion that occurred between experiments. L-Arginine and/or its analogues were added to the buffer at 25 minutes (omitted in controls), and tracer was added at 45 minutes. a, young control (n=6, flow rates were not obtained in one experiment); b, young L-NMMA (n=6); c, old control (n=3); d, old L-NMMA (n=3); e, old D-NMMA (n=3); f, old L-NMMA plus L-arginine (n=3).

Following these fluctuations there was a prolonged fall in flow rate in all six groups of animals. We speculate that this was caused by a progressive vasoconstriction of the intercostal circulation resulting from the gradual washout of the pentobarbital administered before perfusion began. Pentobarbital is known to depress contractile activity of vascular smooth muscle, possibly as a result of its interference with calcium movement or translocation.²⁸ The introduction of L-NMMA did not appear to be associated with any additional decrease in flow rate. At least for the control and L-NMMA groups of each age, flow rates reached a steady level before the introduction of tracer and were not affected by the switch from unlabeled to labeled albumin in the perfusate. This is consistent with previous observations that the tracer is not vasoactive.²⁹

Table 2 shows the intensity of tracer fluorescence in perfusate calibration gels, averaged for each type of perfusate. Mean intensities varied by <5% between groups (P=NS for all, n=3 to 9), indicating that arginine and its derivatives had no influence on tracer emission.

View this table: [in this window] [in a new window]   Table 2. Effect of Perfusate Composition on Tracer Fluorescence

Effects of Perfusate Composition on Differences in Transport Between Upstream and Downstream Regions
For each section, the mean intimal–medial tracer concentration in the upstream region was subtracted from the equivalent downstream value and the difference was expressed as a percentage of the mean concentration in both regions. The differences were averaged for all sections (n=2 to 7) from a branch and then for all branches (n=3 to 8) studied within each animal. The mean and SEM of the latter averages are shown for each group of rabbits in Fig 3.

View larger version (17K): [in this window] [in a new window]   Figure 3. Histogram showing the difference between intimal–medial tracer concentrations in downstream and upstream regions, expressed as a percentage of the mean concentration in both regions. A value above zero occurs if concentrations are greater downstream, and a value below zero occurs if they are greater upstream. Values are mean±SEM, n=3 to 7 animals (12 to 31 branches).

Tracer concentrations were substantially greater downstream of branches than upstream in young control animals, but a smaller difference in the opposite direction was seen in the old control animals. Addition of L-NMMA to the perfusate did not significantly change the difference between regions observed in young animals (P>.8, n=6 to 7), but in older animals the pattern of transport was reversed (P<.01, n=3). Not only did uptake become higher downstream of branches, but also the difference between regions was significantly greater than in the young control group (P<.02, n=3 to 7).

At least part of the reversal observed in mature animals was due to stereospecific effects of L-NMMA since D-NMMA had a much smaller influence (P=.02, n=3). D-NMMA, however, did give rise to a transport pattern that was significantly different from controls (P<.01, n=3). Small but seemingly inhibitory effects of D-NMMA or N^G-nitro-D-arginine methyl ester on NO synthesis were discernible in several previous studies.^{17 30 31} These effects may reflect contamination of some batches with vasoactive substances. The transient increase in intercostal flow rate observed on administration of D-NMMA (Fig 2) is also consistent with the presence of vasoactive contaminants.

The effect of L-NMMA on the mature pattern of transport was almost abolished by including excess L-arginine in the perfusate. The difference between regions in this group of animals was significantly different from that seen in the L-NMMA group (P<.01, n=3); the difference from the value seen in controls also appeared to be significant (.05>P>.02, n=3), but this marginal probability should be treated with caution given the number of t tests conducted in the present study.

Results obtained when mass transfer coefficients were analyzed in the same way are shown in Table 3. Differences between regions were calculated after integrating tracer accumulation over depths of 10, 20, 30, and 40 lines into the wall, equivalent to 29, 58, 86, and 115 µm from the endothelial surface, respectively. The trends were the same as those observed in the concentration data and were essentially independent of the depth of wall examined.

View this table: [in this window] [in a new window]   Table 3. Effect of Inhibiting NO Synthesis on Mass Transfer Coefficients in Young and Old Rabbits

Effects of Perfusate Composition on Mean Uptake
The mean intimal–medial tracer concentration for both the upstream and downstream regions was calculated for each section, and these values were then combined as described above for the differences between regions. The mean and SEM for each group of animals are shown in Fig 4. In young animals, adding L-NMMA to the perfusate produced a large increase in the mean tracer concentration compared with the control value (P<.01, n=6 to 7), but in older animals it did not cause a significant change (P.6, n=3). The lack of change seen in older animals appeared to reflect the net result of an increase in concentration downstream of the branch and a decrease upstream; concentrations in the L-NMMA group were, respectively, 194% and 67% of the control values for these regions, although neither change was statistically significant (P>.02, n=3 for both). The same lack of effect on mean uptake in older animals was observed for D-NMMA (P>.8, n=3) and for L-NMMA plus excess L-arginine (P>.2, n=3).

View larger version (17K): [in this window] [in a new window]   Figure 4. Histogram showing the average intimal–medial tracer concentration for upstream and downstream regions combined, expressed as a percentage of the concentration in perfusate. Values are mean±SEM, n=3 to 7 animals (12 to 31 branches).

Similar trends were seen when mean uptake was expressed in terms of mass transfer coefficients (Table 3). For all depth criteria, L-NMMA caused a large increase in the coefficients observed in immature vessels but had no effect in mature vessels.

Effects of Perfusate Composition on Transmural Concentration Profiles
Profiles of tracer concentration across the aortic wall are shown for young and old rabbits in Fig 5. In young animals, L-NMMA increased uptake by approximately the same proportion in upstream and downstream regions, consistent with the large change in mean uptake and the insignificant change in the difference between regions described above. The shapes of the profiles also changed. Concentrations were approximately constant over a substantial fraction of the outer media in controls but decreased continuously throughout this region in animals treated with L-NMMA. Furthermore, in the latter group, particularly in the downstream region, the highest concentrations appeared to occur in the inner media rather than in the intima.

View larger version (18K): [in this window] [in a new window]   Figure 5. Profiles showing tracer concentrations, expressed as a percentage of the concentration in perfusate, at different depths from the luminal surface. Top panels are for young animals; and bottom panels, for old animals. a indicates upstream; and b, downstream. Circles indicate control; and squares, L-NMMA. Symbols and heavy lines indicate the mean value; and fine lines, ±SEM. n=3 to 4 animals (12 to 21 branches).

In old animals, perfusion with L-NMMA appeared to increase uptake in the downstream region and to decrease it in the upstream region, as already noted, consistent with the small effect on mean transport and the large effect on its pattern described above. In both the control and L-NMMA groups, tracer concentrations were approximately constant over the outer media, and the depth at which the highest concentrations occurred was greater in the control groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Studies of in vivo transport properties of the rabbit aortic wall have established that short-term and quasi-steady-state uptake of albumin are greater downstream than upstream of intercostal ostia in young animals and that this difference decreases and then reverses with age.^{2 3} The same species, arterial branches, tracer, and measurement techniques were used in the present study of short-term uptake, but transport was assessed in vessels perfused in situ with a steady flow of physiological buffer. Despite this difference, the patterns of transport observed in the control groups were the same as those found in vivo. Intimal–medial tracer concentrations and mass transfer coefficients were greater downstream than upstream of branches in young rabbits and the opposite pattern was seen in mature vessels. However, the mean uptake was approximately twofold to fivefold greater than in vivo.³

Because the normal immature and mature patterns were observed in the perfused vessels, their occurrence in vivo cannot depend, at least in the short term, on interactions between blood cells and the vessel wall, on cyclic changes in blood pressure or flow, or on components of plasma other than those present in the buffer. Conversely, the absence of any one of these factors could have given rise to the elevated mean uptake seen in the present study. Macromolecule transport is more than tripled in capillaries perfused with albumin-containing buffer rather than with plasma,³² an effect that can be largely reversed by adding orosomucoid, a circulating glycoprotein, to the buffer.³³

In immature aortas, L-NMMA increased uptake approximately threefold in both upstream and downstream areas, leading to a significant change in the mean uptake but no alteration of the fractional difference between regions. Endogenous NO thus seems to play a role in maintaining the barrier function of the wall near branches of these vessels. Such regions have atypical transport properties,^{34 35 36 37} but the phenomenon may occur more widely. Similar effects on transport in the microvasculature have been observed in some earlier studies.^{14 16 18} Additionally, Cardona-Sanclemente and Born³⁸ showed that inhibition of NO synthesis in vivo increases the uptake of tyramine cellobiose-labeled LDL by rat aorta. Our results support their conjecture that this reflects an alteration of the transport properties of the wall rather than an effect of the increased blood pressure or a change in lipoprotein catabolism. Clearly, an alteration of this type could account, at least in part, for the inhibitory influence of NO synthesis on aortic lipid deposition in hypercholesterolemic rabbits.^{39 40 41}

Different results were obtained in mature aortas. Inhibition of NO synthesis reversed the pattern of transport in these vessels, giving rise to a tracer distribution resembling that seen in weanlings. In this respect, the effects of L-NMMA are identical to the changes in quasi-steady-state uptake transiently induced by a cholesterol-enhanced diet.⁶ It is therefore plausible that the diet-induced changes reflect a modification of the NO pathway by hypercholesterolemia. A reversed pattern has also been observed in mature rabbit aortas that were inadvertently exposed to tracer for several minutes in the absence of blood pressure and flow.³ Consequently, it seems likely that it is the shear-dependent release of NO that determines the mature pattern of transport.

L-NMMA did not significantly increase the mean uptake of tracer in mature vessels. The changing pattern appeared to reflect an increase in transport downstream of the branch and a decrease in the upstream area. Measurements of mean uptake are less reliable than measurements of fractional differences between regions, since the latter involve comparisons only within sections, while the former require an additional calibration procedure. A small change in mean uptake could have been hidden by these inaccuracies. However, other evidence exists that changes in the pattern of transport tend to occur without alteration of the mean. For example, when immature patterns of short-term and quasi-steady-state uptake spontaneously switch to the mature patterns with age, there is no accompanying change in mean uptake.^{2 3} Additionally, when the mature pattern of quasi-steady-state transport is reversed in vivo by a cholesterol-enhanced diet,⁶ mean uptake remains constant (Sebkhi and Weinberg, unpublished data, 1993).

The experiments were designed to restrict the number of indirect ways in which inhibition of NO synthesis could influence transport. Several factors that were circumvented by using a buffer-perfused vessel are listed above. Additionally, a relatively inert, albumin-based tracer was used as an indicator of macromolecular transport properties to avoid effects caused by altered metabolism.⁴² Two potential indirect causes of change remained; neither the aortic diameter nor the intercostal flow rate could be kept constant. Theoretically, NO-dependent changes in either could have affected transport by modifying shear rates at the luminal surface. However, previous studies have shown that changes in aortic diameter produced by inhibiting the basal production of NO are small⁴³ or nonexistent,¹⁷ while L-NMMA did not measurably influence intercostal flow rates in the present study. Although small changes could have occurred in these properties, resulting in some alteration of transport, they seem unlikely to account for a reversal in the pattern of transport and a threefold increase in mean uptake. Direct influences of NO on the wall are the most plausible explanation for these trends.

The data do not distinguish between effects of NO on endothelium and effects on smooth muscle. NO affects transport in exchange vessels devoid of smooth muscle,^{14 15 16 18} and such changes must be mediated by modification of the endothelium. Although relaxation of the endothelial cytoskeleton by NO is expected to close intercellular junctions, NO has been reported to increase transport in exchange vessels^{15 18} as well as to reduce it.^{14 16 18} In arteries, relaxation of smooth muscle cells by endogenous NO or exogenous nitrovasodilators can additionally result in the media becoming more porous to water¹⁷ and macromolecules,⁴⁴ and this in turn could enhance influx, efflux, and distribution volumes. Short-term transport was examined in the present study, but the elevated mean uptake raised tracer concentrations to a level at which rates of efflux and distribution volumes, as well as rates of influx, might have exerted some influence. Because of these complexities, it is not possible to deduce which mechanisms were involved. The maximum tracer concentration occurred at a greater depth in the wall of young animals perfused with L-NMMA than in controls, consistent with endogenous NO limiting the transendothelial entry of albumin and facilitating its egress across the media. However, NO appeared to have the opposite effect in older animals. The greatest similarity in transmural concentration profiles and, by implication, in the balance of endothelial and medial properties was seen between the young control and old L-NMMA groups.

Two changes in transport, one concerned with mean uptake and the other with the difference between regions, were produced by L-NMMA, but they occurred at different ages. The two types of change could be explained by age- and location-dependent alteration of the effects of NO on endothelium or smooth muscle. Alternatively, changes in the synthesis of NO could be important. One possibility arises because NO is likely to be synthesized by both the luminal endothelium and by endothelia in the vasa vasorum. The latter, which would not be exposed to variations in shear stress associated with the branch, may be more important in younger animals. A second possibility is that basal synthesis is dominant in immature aortas but shear-dependent synthesis becomes more prominent in mature vessels. Further studies are required to distinguish between these potential causes.

	Selected Abbreviations and Acronyms

 

D-NMMA = N-monomethyl-D-arginine L-NMMA = N-monomethyl-L-arginine

	Acknowledgments

 
The excellent technical help of J.P. Richards and the generous assistance of Dr T.M. Griffith and D. Edwards are gratefully acknowledged. B.A.F. was supported by a studentship from the Research Endowment Trust Fund of the University of Reading.

Received June 10, 1996; accepted October 10, 1996.

	References

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