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1 Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid; and 2 Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The aim of the present study was to determine whether the adventitia of large arteries modulates vascular function. We developed a method to obtain functional vascular rings devoid of adventitia. Carotid and iliac arteries from 3-mo-old Sprague-Dawley rats were denuded from adventitia after treatment with collagenase followed by gentle peeling. Adventitia removal and integrity of the media was demonstrated by optical and confocal microscopy. Arterial rings with or without adventitia and with or without endothelium were mounted in an organ bath for isometric tension recording. Responses to 75 mM KCl or norepinephrine (0.1 nM-1 µM) were significantly reduced in segments without adventitia. Acetylcholine-induced relaxation (0.1 µM-0.1 mM) was enhanced in arteries without adventitia, whereas sodium nitroprusside-induced responses were not modified. These results demonstrate that the combination of stripping with a previous collagenase treatment allows us to obtain functional rings devoid of adventitia and that this layer plays a role in contractile capacity and in endothelium-modulated responses.
vascular layers; carotid arteries; confocal microscopy; endothelium
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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BLOOD VESSEL WALL is composed of three layers: adventitia, media, and intima. The media, formed by smooth muscle cells (SMC) and elastic lamella, is responsible for vasomotor tone, which is modulated by contractile and relaxant factors released by the endothelium (10) and by the adventitia.
The now well-known influence of the endothelium on vascular function has been mainly assessed in the last two decades. Before, it was considered mainly a barrier between the media and the circulating blood. It was not until the development of a successful and easy method to remove the endothelium that the role of this layer on vasomotor function could be extensively studied (5).
Adventitia is a very complex layer formed by different types of fibers, cells, and nerve endings. It is a structural support for the media, and its main physiological role known, until now, is that mediated by innervation (11). In pathological conditions, where vascular function is altered, structural and biochemical changes are observed in the adventitia (for a review, see Ref. 7). In a model of neointima formation, the adventitia remodels, increasing in thickness and changing the phenotype of adventitia fibroblasts to myofibroblasts (12, 13). Similarly, adventitia remodeling has been reported in arteries from hypertensive rats, where increases in thickness and adventitia cell number were observed (2). In addition, biochemical changes have been described in the adventitia during septic shock, where the marked elevation in nitric oxide contributing to cardiovascular failure seemed to be due to the induction of inducible nitric oxide synthase recently shown in this vascular layer (14).
In view of these structural and biochemical alterations of the adventitia, the question is: does adventitia influence vascular function and how? To answer this question, it is necessary to develop a method to remove the adventitia completely, without medial functional damage.
Previous attempts to remove adventitia by stripping have been made (1, 3, 8, 9). However, these methods do not remove adventitia completely. Kemler et al. (8) compared different methods of adventitia stripping in conduit arteries and demonstrated, with histological methods, that stripping alone did not result in complete removal of this layer. Moreover, the above-mentioned studies do not assess both structurally and functionally possible medial damage after adventitia removal.
This study has two aims. The first was to develop a simple method for removing the adventitia layer of conduit vessels that 1) allows for complete adventitia removal, 2) preserves medial function of vascular rings, and 3) is reproducible. The second was to analyze whether the adventitia contributes to vascular tone by means other than innervation. The establishment of this method would open a new field of research similar to the way endothelium removal enabled determination of the functional contribution of the intima.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Three-month-old rats (Harlan Sprague Dawley under specific pathogen-free conditions; n = 25; weight 350-400 g) were used. All experimental procedures were approved by the Institutional Animal Care and Use Committee according to the guidelines for ethical care of experimental animals of the European Community.
Rats were anesthetized with pentobarbital sodium (50 mg/kg) and bled by cardiac puncture. Carotid and iliac arteries were carefully isolated, placed in oxygenated physiological salt solution (PSS), and cleaned of blood and perivascular fat. PSS was composed of the following: 115 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.01 mM EDTA, 11 mM glucose, 0.8 µM dexamethasone, and 5 µM indomethacin.
Adventitia removal.
Arteries were placed in a shaking bath at 37°C for 15 min in PSS
containing 2 mg/ml collagenase type II (chlostridiopeptidase A; EC
3.4.24.3). Thereafter, vessels were immediately rinsed and placed for
10 min in PSS at 4°C. The arteries were then fixed with pins at both
ends to a Sylgard-based dissecting dish containing cold PSS, and the
adventitia was carefully removed by gentle peeling with two pairs of
fine forceps under a dissecting microscope. Control arteries with
adventitia (+A) were submitted to the same steps and temperature
changes as arteries without adventitia (
A). This protocol was
established after testing several incubation times with collagenase and
cold PSS. In some arteries (both +A and A), the endothelium was
removed (E). This was performed by gentle scraping with a cotton
thread through the vessel lumen. In A segments, endothelium removal
was always performed after incubation in collagenase; otherwise, the
media would be seriously damaged.
Histology. Optical and confocal microscopy were used to assess the degree of adventitia removal and to determine histologically possible SMC and external elastic lamina damage.
The degree of adventitia removal was tested at different time points after incubation with collagenase (5, 15, or 30 min). After adventitia peeling, the segments were visualized mounted on a slide with a Zeiss Axiovert 25 inverted microscope with a ×5 objective. This protocol allowed us to determine the optimal time of incubation with collagenase. The degree of arterial damage was tested with confocal microscopy. +A orA segments of carotid artery (not used for functional experiments)
were fixed in 4% formaldehyde solution for 2 h. Thereafter, the
segments were incubated for 15 min in a PSS solution containing 0.1 µM propidium iodide (PI), a nonpermeable fluorescent nuclear dye, and
washed three times in PSS. Rings of ~50 µm were cut and mounted on
slides for visualization with a laser scanning confocal microscope
(model MRC 1024, Bio-Rad) coupled to a Nikon microscope with a ×20 air
objective [numerical aperture 0.45]. PI stains nuclei of all vascular
cells (2) that can be visualized using the 488/615-nm line
of the microscope. Elastic lamella were visualized simultaneously with
the 488/515-nm line due to elastine autofluorescence at this wavelength.
To determine the degree of SMC and external elastic lamella damage
after adventitia removal, longitudinal sections of intact and
adventitia-denuded rat carotid arteries were incubated without fixation
in PI-containing PSS solution following the above-described protocol.
The sections were mounted on slides with the adventitia side up and
visualized with a ×20 air objective at the above-mentioned wavelengths. To determine the degree of SMC and internal elastic lamina
damage after endothelium removal, the same protocol was performed, and
the arteries were visualized with the endothelium side up with a ×20
air objective.
Functional studies.
To determine vascular function, 3-mm-long carotid artery rings were
suspended on two intraluminal parallel wires, introduced in an organ
bath containing PSS, and connected to a Piodem strain gauge for
isometric tension recording. A set of experiments was first performed
to establish the optimal resting tension of rat +A and A carotid
arteries. Thus arterial rings were submitted to different tensions
between 0.5 and 2 g. This tension was readjusted every 15 min
during a 90-min equilibration period. After the equilibration period,
the vessels were exposed to 75 mM KCl to check their contractility.
E).
Both +A and A carotid and iliac artery segments were submitted to
transmural nerve stimulation (TNS; 200 mA, 0.2 ms, 30 s at
0-32 Hz) (6) in addition to the previous protocol.
Analysis of data. Contractions are expressed as the percentage of contraction produced by 75 mM KCl. Relaxations are expressed as the percentage of contraction induced by 0.1 µM NE. The half-maximal effective concentration (EC50) was calculated choosing as maximal response (Emax) the maximum value of the control curve (+A/+E). Statistical significance was analyzed by one-way ANOVA followed by the Newman-Keuls test or Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Assessment of adventitia removal.
Adventitia removal was confirmed histologically by optical microscopy.
Denudation was dependent on the collagenase incubation time (Fig.
1A). Incubation periods in
collagenase solution shorter than 15 min did not allow an adequate and
complete removal of the adventitia. Incubation periods longer than 15 min produced artery rupture on peeling (Fig. 1A) and
impaired vascular function. In addition, periods in cold PSS shorter or
longer than 10 min made the process of peeling difficult, inducing
medial damage (results not shown).
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A rings,
adventitia had been completely removed, as shown by the lack of red
fluorescence in the outer part of the ring.
In addition, confocal microscopy was useful for analyzing vascular
damage. Adventitia removal does not damage external elastic lamella and
SMC underneath, as shown by confocal images of unfixed arteries (Fig.
1C, left). Only dead or broken cells exhibit
staining with PI. The degree of cell death was similar, or even
smaller, to that produced by mechanical endothelium denudation (Fig.
1C, right).
Adventitia removal was confirmed functionally by analyzing the response
of iliac arteries to TNS. TNS-induced frequency-dependent contractions
in iliac arteries were abolished in A arteries (Fig. 2). TNS produced no modification of
vascular tone on carotid artery segments.
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Effect of adventitia removal on responses to vasoconstrictor
agents.
Responses to 75 mM KCl were analyzed in carotid arteries in tensions
ranging 0.5-2.0 g. Arteries (+A) elicited a similar maximal contraction at resting tension values between 1 and 2 g. Arteries (A) showed a maximal contraction at 1.5 g and the contractile response was significantly reduced at 2 g (Fig.
3A). An
optimal resting tension of 1.5 g was chosen for subsequent
experiments.
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A
arteries. KCl-induced contractions were significantly lower in A
arteries at all concentrations studied (Fig. 3B).
To analyze whether the reduction in response to KCl was due to
enzymatic treatment, several segments were incubated with collagenase without removing the adventitia afterward. This procedure did not
modify KCl-induced contractions (Fig. 3C). To test whether manipulation was responsible for the reduction of contractile capacity,
gentle stretching of the artery simulating peeling of the adventitia
was performed. This procedure did not modify KCl-induced contractions
either (Fig. 3C).
In adventitia-denuded vessels with intact endothelium, NE-induced
contractions (0.1 nM-1 µM) were significantly lower in efficacy (Emax) than in control rings (Fig.
4A; Table
1). After endothelium denudation, NE
significantly enhanced efficacy both in +A and A arteries (Fig.
4B; Table 1). Endothelium removal abolished the differences
in NE contractions between +A and A arteries (Fig. 4, A
and B; Table 1).
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Effect of adventitia removal on responses to vasodilator agents.
Concentration-response curves to ACh were performed in +A and A
vessels precontracted with 0.1 µM NE. In the presence of endothelium,
ACh elicited a concentration-dependent relaxation in carotid arteries
with no differences in efficacy (Emax) between +A and A arteries (Fig. 5A;
Table 1). There was a significant reduction of the EC50
value after removal of the adventitia (Table 1). The
concentration-response curve to SNP (0.1 nM-10 µM) was similar in
both groups of vessels (Fig. 5B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We developed an enzymatic method for removing the adventitia layer of conduit vessels that 1) allows complete adventitia removal, 2) preserves medial function of vascular rings, and 3) is reproducible in different vessels. The present work also demonstrates that removal of the adventitia reduces contractile capacity and potentiates endothelium function in rat carotid arteries.
The adventitia is a very complex layer formed by different types of fibers, cells, and nerve endings. A well-known physiological role of the adventitia is that mediated by innervation (11). To study vascular reactivity in vessels lacking adventitia, it was necessary for us to exclude possible interferences of innervation on vascular function. We have, therefore, chosen the rat common carotid artery as an artery lacking functional innervation, and we compared the results with a well-innervated vessel, like the rat iliac artery (6).
Kemler et al. (8) compared different methods of adventitia stripping in conduit arteries and demonstrated histologically that stripping alone did not result in a complete removal of this layer. In the present work, the combination of stripping with a previous collagenase treatment allowed complete removal of the adventitia, as confirmed histologically by optical and confocal microscopy in carotid arteries and functionally by the abolishment of the response to TNS (but not to KCl) in iliac arteries.
Because sympathetic fibers are harbored within the adventitia, removal of this layer would interrupt sympathetic modulation of vascular tone. In fact, in densely innervated iliac arteries, TNS-induced frequency-dependent contractions were abolished after removal of the adventitia (6). We suggest that this approach may serve as an easy confirmation of adventitia removal in innervated vessels.
Carotid arteries exhibited diminished contractions to KCl after removal
of the adventitia. One possibility was that removal of the adventitia
might induce a shift of the optimal resting tension. This was excluded
because we set the vessels at 1.5 g, which was the optimal resting
tension for both +A and A vessels. However, we found that in +A
segments the optimal contraction was maintained at 2 g, whereas in
A segments contraction dropped at this tension. This might be due to
a change in mechanical properties of the vessels after adventitia
removal. A second possible explanation for the reduction in
contractility in A vessels is that manipulation and/or collagenase
treatment could be impairing contractile function, although there
seemed to be no histological damage. However, neither incubation with
collagenase nor stretching of the vessels modified responses to KCl.
Another indication that the smooth muscle layer was well preserved
after adventitia removal was the fact that the relaxation to SNP was
not different between +A and A vessels. These results suggest that
manipulation and collagenase treatment for adventitia removal do not
impair smooth muscle functionality. We suggest that differences in
contractility observed in adventitia-denuded rings might be due to a
possible influence of the adventitia on vascular function and has to be
further studied.
The possible interrelationship between endothelium and adventitia on
vascular function was analyzed using +E and E arteries in addition to
adventitia removal. Sensitivity to ACh was higher in A carotid
arteries, suggesting first, that collagenase treatment did not affect
the endothelium layer and second, that endothelium-dependent relaxations might be affected by the presence of adventitia. In addition, in A/+E segments, the concentration-response curve to NE
was reduced in Emax. Removal of the endothelium
significantly increased responses to NE, as previously described for
several vasoconstrictors in numerous vascular beds after endothelium
removal (10) in both +A and A carotid arteries. It is
interesting to note that endothelium removal abolished the differences
in Emax observed between +A and A arteries.
These results suggest that in the absence of adventitia, endothelium
function is potentiated. In fact, it has been suggested that
adventitia-derived superoxide anions might shorten the half-life of
nitric oxide (4).
In conclusion, we developed an enzymatic method for removing the adventitia layer of conduit vessels, which is reproducible in different vessels and preserves the contractile and relaxant function of the smooth muscle layer. This method will allow to study in more detail the influence of this layer on vascular function.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. C. Fernández Criado for the care of the animals and to P. Arnaiz Sánchez for technical assistance.
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FOOTNOTES |
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This work was supported by Fondo de Investigaciones Sanitarias Grant 98/0736.
Address for reprint requests and other correspondence: M. C. González, Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 2, 28029 Madrid, Spain (E-mail: m.c.gonzalez{at}uam.es)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 October 2000; accepted in final form 29 January 2001.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Arnal, JF,
Battle T,
Rasetti C,
Challah M,
Costerousse O,
Vicaut E,
Michel JB,
and
Alhenc-Gelas F.
ACE in three tunicae of rat aorta: expression in smooth muscle and effect of renovascular hypertension.
Am J Physiol Heart Circ Physiol
267:
H1777-H1784,
1994
2.
Arribas, SM,
Hillier C,
González C,
McGrory S,
Dominiczak AF,
and
McGrath JC.
Cellular aspects of vascular remodeling in hypertension revealed by confocal microscopy.
Hypertension
30:
1455-1464,
1997
3.
Battle, T,
Arnal JF,
Challah M,
and
Michel JB.
Selective isolation of rat aortic wall layers and their cell types in culture. Application to converting enzyme activity measurement.
Tissue Cell
26:
943-955,
1994[ISI][Medline].
4.
Di Wang, H,
Hope S,
Du Y,
Quinn MT,
Cayatte A,
Pagano PJ,
and
Cohen RA.
Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II-induced hypertension.
Hypertension
33:
1225-1232,
1999
5.
Furchgott, RF,
and
Zawadzki JV.
The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.
Nature
228:
373-376,
1980.
6.
González, C,
Fernández A,
Martin C,
Moncada S,
and
Estrada C.
Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.
Biochem Biophys Res Commun
186:
150-156,
1992[ISI][Medline].
7.
Gutterman, DD.
Adventitia-dependent influences on vascular function.
Am J Physiol Heart Circ Physiol
277:
H1265-H1272,
1999
8.
Kemler, MA,
Kolkman WFA,
Slootweg PJ,
and
Kon M.
Adventitial stripping does not strip the adventitia.
Plast Reconstr Surg
99:
1626-1631,
1997[ISI][Medline].
9.
Lohman, R,
Siemionow M,
Rockwell WB,
and
Lister GD.
Acute adverse effects of blunt adventitial stripping.
Ann Plast Surg
35:
60-65,
1995[ISI][Medline].
10.
Moncada, S,
Palmer RMJ,
and
Higgs EA.
Nitric oxide: physiology, pathophysiology and pharmacology.
Pharmacol Rev
43:
109-142,
1991[ISI][Medline].
11.
Rhodin, JAG
Architecture of the vessel wall.
In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Soc, 1980, sect. 2, vol. II, chapt. 1, p. 1-31.
12.
Shi, Y,
Pieniek A,
Fard A,
O'Brien J,
Mannion JD,
and
Zalewski A.
Adventitial remodeling after coronary arterial injury.
Circulation
93:
340-348,
1996
13.
Shi, Y,
O'Brien J,
Fard A,
Mannion JD,
Wang D,
and
Zalewski A.
Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries.
Circulation
94:
1655-1664,
1996
14.
Zhang, H,
Du Yue,
Cohen RA,
Chobanian AV,
and
Brecher P.
Adventitia as a source of inducible nitric oxide synthase in the rat aorta.
Am J Hypertens
12:
467-475,
1999[ISI][Medline].
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