Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3365-3375
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3365-3375.)
© 1997 American Heart Association, Inc.
Effect of Atherosclerosis on Transmural Convection and Arterial Ultrastructure
Implications for Local Intravascular Drug Delivery
Ann L. Baldwin;
Lisa M. Wilson;
Irmina Gradus-Pizlo;
Robert Wilensky;
;
Keith March
From the Department of Physiology, University of Arizona, and B.W.
Zweifach Microcirculation Laboratories, Veterans Affairs Medical Center,
Tucson, (A.L.B., L.M.W.); Krannert Institute of Cardiology, Indianapolis,
Indiana (I.G.P., R.W., K.M.); and Veterans Affairs Medical Center,
Indianapolis, Indiana (K.M.).
Correspondence to Ann L. Baldwin, Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724. E-mail A160854{at}ccit.arizona.edu
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Abstract
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Abstract Local infusion of agents through perforated
catheters
may reduce neointimal formation following
vascular angioplasty.
Such treatment will succeed only if the drug is
retained within
the arterial intima long enough to promote
repair. Drugs will
be dispersed throughout the wall predominantly by
transmural
convection instead of diffusion if the Peclet number,
Pe=J(1-
f)/P,
is greater than unity, where J is the
transmural fluid flow
per unit surface area and
f and P
are the reflection and permeability
coefficients to the drug,
respectively. Although the targets
of local drug delivery will be
atherosclerotic vessels, little
is known about the transport properties
of these vessels. Accordingly,
we evaluated the effects of
hypercholesterolemia and
atherosclerosis
on J per unit pressure (hydraulic
conductance, L
p) and on ultrastructure
in femoral arteries.
Measurements were made at 30, 60, and 90
mm Hg in
anesthetized New Zealand white rabbits fed a normal
diet (n=6)
and after 3 weeks of lipid feeding (n=19).
Atherosclerosis
was induced in six lipid-fed animals by
air desiccation of a
femoral artery. Hydraulic conductance was
significantly greater
in vessels from
hypercholesterolemic than from normal animals
and
decreased with pressure only in hypercholesterolemic
arteries.
Atherosclerosis did not augment hydraulic
conductance compared
with hypercholesterolemia
alone. Electron microscopic examination
demonstrated damaged
endothelium in hypercholesterolemic
arteries
and both altered endothelium and less tightly
packed medial
tissue, compared with controls, in atherosclerotic
vessels,
at least at lower pressures. Peclet numbers for macromolecules
exceeded
unity for all three groups of arteries and reached 0.3 to 0.4
for
molecules as small as heparin. Thus, convection plays a dominant
role
in the distribution of macromolecular agents following local
delivery
and may result in their rapid transport to the adventitia in
the
femoral artery.
Key Words: rabbits artery, femoral hydraulic conductance hypercholesterolemia local drug delivery
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Introduction
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The
use of balloon angioplasty for nonsurgical
recanalization
of atherosclerotic arteries has been
associated with subsequent
remodeling and proliferation of smooth
muscle cells in the intima,
leading to restenosis of the artery
within a few months in about
30% of cases.
1 One
approach to reducing the incidence of restenosis
is local
administration of relatively high concentrations of
agents following
angioplasty. Such local delivery has been carried
out by intravascular
administration of agents from porous
catheters,
2 3 bioresorbable
gels,
4 or stents,
5 as well
as by perivascular
placement of matrices containing
drug.
6
Several studies have evaluated the intramural residence time of soluble
agents following delivery, including heparin (predominately eliminated
in less than 24 hours),3 methotrexate (65%
cleared within first 24 hours),7 and colchicine
(90% cleared within a few hours).8 The residence
time of agents delivered locally appears to play a significant role in
determining their bioactivity,9 and several
approaches have been developed to prolong residence time. These have
involved the incorporation of agents into polymeric matrices, which may
be macroscopic gels4 or
coatings,10 as well as microparticulates
appropriate for transcatheter
delivery.2
The residence time of agents delivered locally, as well as the
steady-state concentrations of compounds released from local stores,
may be strongly influenced by diffusive forces causing transport away
from the site of delivery, either deeper into the arterial
wall or back into the bloodstream, and by convective forces promoting
transport deeper into the wall. Moreover, these forces will determine
both the distribution of agent deposition in the vascular wall upon
catheter delivery and subsequent redistribution after delivery. Several
experimental studies have shown that transmural convection promotes
transport of macromolecules across the arterial wall, from
blood to tissue.11 12 13 14 15 16 However, other
authors17 have concluded that macromolecular
transport is dominated by diffusion unless the
endothelium is damaged. In addition, it has been
suggested that diffusion far outweighs convection in control of
transmural heparin transport in the normal artery and that convective
forces increase to only one-quarter the magnitude of diffusive forces
following endothelial injury.18
Thus, the effect of convection on local drug delivery has not been
resolved.
One way to assess the influence of convection on the effectiveness of
drug delivery is to calculate the ratio of convective to diffusive
solute transport for a given molecule. If this ratio exceeds unity,
solute transport is governed predominately by convection. The ratio of
imposed (convective) velocity of a molecule traveling through the
arterial wall to diffusive velocity is expressed by the
Peclet number,
Pe=J(1-
f)/P,19 where J
is the transmural flow per unit surface area, P is the permeability
coefficient of tissue to the solute, and
f is
the filtration reflection coefficient, which represents the
relative access that the solute molecules have to water transport
channels. If water and solute molecules have equal access to such
channels, that is, there is 100% coupling between solute and solvent,
then
f has a value of zero. If the solute has
no access to water transport channels, then
f
is equal to unity and there is no convective transport of solute.
Although measurements of hydraulic conductance,
Lp (J per unit transmural pressure), have
previously been made on healthy arteries,20 21 22 23 24
none have been performed on vessels from animals subjected to a
high-lipid diet, with or without the presence of atherosclerotic
lesions. Because the arteries to be treated to prevent
restenosis will inevitably be atherosclerotic, this study was
designed to determine whether this condition alters
Lp. Accordingly, Lp of
femoral arteries from rabbits fed normal or
hypercholesterolemic diets was measured to enable
calculation of Peclet numbers for large and small molecules. Lesions
had been induced in some of the femoral arteries from
hypercholesterolemic rabbits. The femoral artery was
chosen for these studies because it is a site of predilection for
obliterating vascular lesions and has been extensively studied with
respect to atherosclerosis as well as local drug
delivery.25 26 27
Control and hypercholesterolemic arteries, with and
without lesions, were examined by light and electron microscopy for the
purposes of obtaining structural information for determination of
Peclet numbers and of relating ultrastructural changes to alterations
in hydraulic conductance.
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Methods
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Induction of Focal Atherosclerosis
Experiments were performed on 18 male New Zealand White rabbits
(4.0
to 5.0 kg) anesthetized with ketamine (100 mg/kg)
and xylazine
(1.5 mg/kg IM). Focal femoral
atherosclerosis was induced unilaterally
as described
previously.
27 In brief, 1 cm below the inguinal
ligament,
a 3-cm cutdown over the femoral artery was made, and the
artery
was exposed. Lidocaine solution (2%) was dripped on the artery
to
prevent spasm. A 1.0- to 1.5-cm-long segment of the femoral
artery,
devoid of side branches, was isolated, and flow was
temporally
interrupted with silk ligatures. The arterial segment
was
then cannulated with a 27-gauge needle, vented distally,
and flushed
with saline to remove blood. Air was passed through
the
arterial segment for 8 minutes at 80 mm Hg
pressure to achieve
endothelial desiccation, after
which blood flow was reestablished.
An intramuscular injection of
Cefadyl (1 g) was routinely given.
The injured segment was marked by a
metal clip overlying the
muscle layer, and the wound was closed. The
rabbit was then
placed on a 2% cholesterol/5% peanut oil
diet (150 g/d) for
3 weeks.
An angiogram was taken 2 weeks after commencement of the diet to
confirm vessel patency and consequent suitability for bilateral
Lp measurements. Hydraulic conductance
measurements were made on all noninstrumented femoral arteries to
evaluate the effects of hypercholesterolemia
without atherosclerosis. Measurements of hydraulic
conductance were performed, as described below, 3 weeks after
commencement of the high-lipid diet. Control arteries (n=6) were
studied in rabbits fed a standard diet.
The Atherosclerotic Rabbit as a Model for Human
Angioplasty
Previous studies28 29 have demonstrated
that experimental atherosclerosis can be
consistently induced in the New Zealand White rabbit by
endothelial injury followed by a high
cholesterol (2% cholesterol/6% peanut oil)
diet. Our technique, originally described by LeVeen et
al,28 results in a mixed fibrocellular lesion.
Although the diet induces very high serum levels of
cholesterol (1146±238 mg%) and the
atheromatous lesions differ from advanced human
atherosclerosis in that necrosis, fibrosis, and
calcification are absent, there are many attractive features of this
model. These include the rapid and reliable development of stable
atherosclerotic lesions that can be assessed precisely by angiography,
with acute and chronic postangioplasty complications similar to those
documented after human angioplasty.
Surgical Procedure
The rabbits were anesthetized with pentobarbital sodium
(30 mg/kg IV). The surgical procedure was described in detail in a
previous publication.20 Briefly, the femoral
artery was exposed, and any branches were ligated close to the origin.
Heparin (1000 IU) was administered intravenously. During
the whole operative procedure, physiologically
buffered PBS, pH 7.4, warmed to 37°C and containing 4%BSA, was
applied continually to the adventitial surface of the artery to prevent
drying.
A length of femoral artery, from the inguinal ligament to the saphenous
bifurcation (about 2.5 to 3.0 cm), was cannulated. The
perfusate, which entered the vessel from a reservoir through
the proximal cannula, was identical to the suffusate except that it
contained 0.03% trypan blue dye. Dye was included so that leaks could
quickly be identified. Normally, no adventitial staining was observed.
Both proximal and distal cannulae were connected to pressure
transducers, with output recorded using a Gould chart recorder.
The inlet reservoir was positioned 60 cm above the cannulated femoral
artery, and the flow rate was adjusted, using a screw clamp, to flush
the blood from the segment. After the absence of leaks was established,
the muscles surrounding the femoral artery were retracted to form a
cavity, which was filled with the solution that had been used to keep
the vessel moist.
Measurement of Hydraulic Conductance
Arteries were initially preconditioned to reduce the hysteresis
effects resulting from viscoelasticity (ie, the tendency for the vessel
to return to a diameter greater than its initial value after
pressurization). This was achieved by lowering the proximal
arterial pressure to 25 mm Hg, then raising it to
100 mm Hg, three times while occluding the outlet
cannula.
Measurements of transmural fluid flux and external radius were then
made at 30, 60, and 90 mm Hg. To measure fluid flux, the
upstream reservoir was connected, via a stopcock, to a 2-m length of
nylon capillary tubing (0.06 cm inner diameter), a portion of which was
horizontally positioned adjacent to a meter rule. The downstream end of
the tubing was connected to the inlet cannula of the femoral artery. An
air bubble was injected into the capillary tubing, the bubble position
with reference to the meter rule was noted, and the pressure was raised
to the chosen value. The position of the bubble was monitored 1, 2, 4,
6, 8, 10, 15, 20, and 30 minutes after raising the pressure. The
external diameter and length of the cannulated segment of artery were
measured using a mechanical caliper (accurate to 0.1 mm) initially
and 6 and 30 minutes after pressurization. There was very little
tapering of the segment; nevertheless, the diameter was always measured
at the midpoint of the vessel length. All the tubing had been
previously coated with Sigmacote to prevent sticking of the
bubble.20
After the initial distension of the vessel, the air bubble was
confirmed to move at a constant rate, representative of
the rate of fluid filtration through the artery wall. The transmural
fluid flux was calculated for each pressure from the steady-state
velocity, v, of the air-bubble in the capillary tube using the
equation
where r
c is the radius of the capillary
tubing and SA is
the surface area of the arterial segment,
which was determined
from dimensional measurements assuming cylindrical
geometry.
L
p was calculated from the flux using
the equation
where
P
T is transmural pressure.
Light and Electron Microscopic Observations
After measurements of Lp had been
performed, two control arteries, five
hypercholesterolemic arteries, and three
atherosclerotic arteries were processed for light and electron
microscopy. Five additional control femoral arteries, one additional
hypercholesterolemic vessel, and six more
atherosclerotic vessels were included in the light microscopic study.
Some of these additional vessels were also examined by electron
microscopy. In total, the following vessels were fixed and sectioned
for electron microscopy: three control vessels (one at 30 mm
Hg and two at 60 mm Hg), six
hypercholesterolemic vessels (two at 30 mm
Hg, two at 60 mm Hg, and two at 90 mm Hg), and
eight atherosclerotic vessels (four at 30 mm Hg, three at
60 mm Hg, and one at 90 mm Hg). All the vessels
were perfusion-fixed, in situ, at room temperature for 2 hours at
pressures ranging from 30 to 90 mm Hg with phosphate-buffered
Karnovsky's fixative (pH 7.4) containing enough dextran 40 to match
the colloid osmotic pressure of plasma (25 mm Hg). Fixative
was also applied to the outside of the vessels. Each vessel was then
excised and cut into annular segments about 2 mm in length. The
segments were washed in 0.15 M phosphate buffer, postfixed in 1%
OsO4 for 2 hours, dehydrated in alcohols,
embedded in Spurrs resin, and sectioned for light and electron
microscopy. Sections from each vessel were examined by light microscopy
to determine medial thickness (average of eight measurements equally
spaced circumferentially around annulus), outer diameter (circumference
of external elastic lamella/
), and intimal thickness (intimal
area/circumference of internal elastic lamella). All measurements were
made using a stylus and Bioquant software. Ultrathin sections were
examined by electron microscopy to qualitatively assess the structural
integrity of the endothelium.
Statistics
To determine whether Lp varied between
groups (control, hypercholesterolemic, and
atherosclerotic) for a given pressure, Student t tests were
performed. Variation with pressure for a given group was evaluated
using paired Student t tests. If a t test gave a
P value of <.05, we considered the null hypothesis to
be rejected.
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Results
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Measurements of Hydraulic Conductance
Measurements of hydraulic conductance are shown in Table 1

.
Corresponding values from the
literature, measured in healthy
vessels, are shown in Table 2

. Hydraulic conductance was
significantly
greater in both hypercholesterolemic and
atherosclerotic vessels
than in control vessels at 30 mm Hg
and exhibited the same trend
at higher pressures. Hydraulic
conductances of hypercholesterolemic
and
atherosclerotic vessels were not significantly different
from each
other at any of the three pressures. In control arteries,
L
p did not vary significantly with pressure,
while in hypercholesterolemic
and in atherosclerotic
arteries, L
p decreased with increasing
pressure.
The values of L
p determined for control rabbit
femoral
arteries were two to three times higher than those previously
obtained
for rabbit aortas using the same technique (see references 20
and
21 in Table 2

). This difference can be accounted for by the
fact
that the aortic media is two to three times thicker than
that of the
femoral artery. Measured dimensions of the femoral
arteries are shown
in Table 3

. Assuming an aortic wall
thickness
of 120 µm, the product of L
p
and wall thickness, or the
Darcy constant, k, will be equal to
5x10
-10 cm
2/s
· mm
Hg
-1. This is close to the k
value of 6.9x10
-10
cm
2/s ·
mm
Hg
-1 calculated for control femoral arteries at
60 mm Hg
(Table 4

). We have
determined the L
p for rabbit aortas denuded
of
endothelium to be
10.19±1.91x10
-8 cm/s · mm
Hg
-1 (reference 21 in Table 2

), which gives a
Darcy constant of 12.2x10
-10
cm
2/s
· mm
Hg
-1, similar to the value obtained by Harrison
and
Massaro (reference 22 in Table 2

) for the porcine
endothelially
denuded aorta. These values for denuded
aorta are similar to
those obtained for the
hypercholesterolemic femoral arteries
at 30 and 60
mm Hg (Table 4

).
Measurements of Vessel Dimensions
As shown in Table 3
, the mean diameters of the
hypercholesterolemic and atherosclerotic vessels were
smaller than that of control vessels, but this difference was only
statistically significant for the latter. The mean medial thickness of
atherosclerotic vessels was significantly greater than that of control
and of hypercholesterolemic vessels. Intimal thickening
was eccentric and was seen only in atherosclerotic vessels.
Calculations of Peclet Numbers
In order to evaluate Peclet numbers using measured values of
Lp, compatible values for permeability and
reflection coefficients are required. Alternatively, Peclet numbers can
be expressed in terms of the Darcy constant, k, and the coefficient of
diffusivity, D, which is equal to the product of permeability
coefficient and wall thickness. By use of the parameters k
and D, the conductivity of arterial tissue to water and
solutes can be expressed independent of wall thickness. Because of the
absence of any measured values in the literature for P, D, and
f of the intact femoral artery, published
coefficients for the various single components of the
arterial wall (Table 2
) were used in a model based on the
structure of the wall as revealed by our microscopic studies. The
structure of arteries has been used effectively to predict diffusion
coefficients by Penn et al.31 In this previous
study, the total resistance to HRP movement into and through the intima
was calculated from measurements of HRP concentration in the intima and
media using photomicrographs of arterial sections.
Demonstration of the arterial ultrastructure was required
to exclude layers of medial elastic tissue from the measured areas. In
addition, Fry30 used light and electron
microscopy to check that his arterial transport model took
account of the structural details of the artery. Thus, in both cases,
knowledge of the structure of the artery was essential for
determination of permeability coefficients.
In our model, the arterial wall is considered as three
components in series: endothelium (endo), internal
elastic lamina (iel), and a media consisting of fenestrated layers of
smooth muscle cells and elastic tissue embedded in a matrix ofGAGs
(gag). In the femoral artery, unlike the aorta, there is only one
elastic lamina. This structure can be seen in Figs 1
, 2
, a,
3, a, and 5, a. Using this model, the
macromolecular permeability coefficient of the whole artery can be
estimated from the permeability coefficients of the individual
components as follows:
However, the goal
of this study is to predict the effect of
convective fluid flux
on the diffusion of solutes that are already
within the media;
thus, the permeability of the
endothelium will be neglected.
The following published
permeability values to either HRP (MW,
44 kd) or serum albumin
(MW, 67 kd) (Table 2

) were used in the
above equation (unfortunately,
the permeability coefficients
to the same macromolecule were not
available for both components):
Therefore,
The above permeability value was determined
under the assumption
that all transport was diffusive. Because
the HRP was injected in vivo
and could have been driven into
the artery wall by the transmural
pressure gradient, these values
of P may be overestimated. As shown in
Table 2

, Fry
32 gave
a value of
0.24x10
-9 cm
2/s for
D
iel to albumin, but he assumed
that the
internal elastic lamellar was just a thin part of a
homogeneous
media. For that reason, we shall use
0.24x10
-10 cm
2/s for
D
iel to macromolecules.

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Figure 1. Electron micrograph of transverse section through
control femoral artery. Note intact endothelium (E) and
closely packed smooth muscle cells (SM). This vessel was fixed at
30 mm Hg. Bar=5 µm.
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Figure 2. Electron micrographs of transverse sections through
femoral artery (a) and aorta (b) taken from a
hypercholesterolemic rabbit. No lesions were
mechanically induced in these vessels. Both vessels were fixed at
30 mm Hg. Note the absence of endothelium in
a (arrowhead) and the presence of intact endothelium in
b (arrowhead). Bar=5 µm.
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A value for Pgag was obtained from measurements
of the diffusion coefficient of serum albumin within GAGs, in
particular to HA. In cartilage interstitium, which consists largely of
a 5% to 10% HA solution, Dgag is
1.6x10-7 cm2/s (serum
albumin, reference 33). The exact GAG concentration is not
critical for our purposes, because Dgag is not
strongly dependent on concentration. For example,
Dgag for a 2.5% HA solution is
2.2x10-7 cm2/s (serum
albumin, reference 34).
where 1 is medial thickness (50 µm,
from Table 3

).
However, because the media does not consist
only of GAGs but
also contains smooth muscle cells and elastic
tissue, which exclude
macromolecules, this value for P
gag must
be
modified. In the femoral artery, measurements from our electron
micrographs
gave an average volume fraction of GAGs in the media of
30%.
The effect of interstitial volume fraction on the
diffusivity
of macromolecules in tissue has previously been
studied.
35 According to this study, a GAG volume
fraction of 30% should
reduce P
gag to about 22%
of the value that is has when no smooth
muscle cells or elastic tissue
are present.
Thus,
and
As shown in Table 2

, Fry
30 gave a value of
0.137x10
-7 cm
2/s for the
diffusion coefficient of albumin in the media,
but he did not
correct for the percentage space available for
diffusion. Bratzler and
Schwartz
36 obtained a value of 0.13
cm
2/s
in an excised rabbit aorta. Because the
aorta contracts longitudinally
on excision, this could explain the
difference from our calculated
value.
It can be seen that the GAGs are much more permeable to macromolecules
than are the endothelium or the internal elastic
lamina. Applying the published data values to our model, we calculate
that
and
The permeability coefficient of the rabbit aortic media to
albumin
has been calculated to be
80x10
-8 cm/s,
37 assuming
only diffusive
transport. However, in this model the media was assumed
to be
homogeneous.
Using our calculated value for Pwall and an
Lp value of 13x10-8
cm/s · mm Hg-1 determined for
control vessels at a pressure of 60 mm Hg, a Peclet number of
33(1-
f) was obtained. Therefore, the highest
possible value for Pe is 33. This assumes that there is total coupling
between convective fluid flux and macromolecular flux. There is very
little coupling between macromolecular and convective fluxes across the
endothelium.11 14 38 39
Measurements of
f for serum albumin
made in skeletal muscle capillaries, which possess
endothelial cells with tight junctions, similar to
those seen in arterial endothelium, give a
value for
f of 0.95. This indicates that the
rate of transendothelial albumin transport by
convection is only 5% of that of water. However, if we are considering
the effect of convective flux on molecules that have already crossed
the endothelium during the delivery process, then the
degree of coupling of solute and solvent may be different. There are no
measurements of arterial
f for
macromolecules in the literature. However, Fry32
made an "educated guess" regarding coupling of LDL and water across
the internal elastic layer from the intima to the media. He estimated a
value of 0.95 for muscular arteries, such as the femoral artery. Thus,
for molecules that are lodged between the endothelium
and the internal elastic lamella after delivery, we obtain a Peclet
number of 1.65, indicating that transport is still
convection-dominated. Molecules that had penetrated both the
endothelium and the internal elastic lamella during
delivery, would be more greatly affected by convection, because of
greater solute-solvent coupling, and the Peclet number would exceed
1.65.
With regard to hypercholesterolemic vessels, the value
for Lp is greater than for controls
(18x10-8 vs 13x10-8
cm/s · mm Hg-1). Also, our
electron micrographs demonstrate that the
hypercholesterolemic vessels sustained some degree of
endothelial damage (Fig 2
, a) but that the media was
similar to controls. Assuming that the permeability of the internal
elastic lamella and media are similar to that of controls and using a
medial thickness of 59 µm (Table 3
), then
Therefore,
and
The highest possible value for Pe is 45. With regard to lower
bounds for Pe, the value would be 2.3 if we assume that
f=0.95, as for control vessels. Therefore, for
arteries from hypercholesterolemic animals, Pe would
still be greater than unity, and thus macromolecular transport would be
convection-dominated.
With regard to atherosclerotic arteries, the value for
Lp is 16x10-8 cm/s
· mm Hg-1. Our light micrographs
showed that medial thickness for atherosclerotic vessels was about
twice that for controls (Table 3
). Therefore, we will use a value of
106 µm instead of 50 µm to calculate
Pgag. Although the intima is thickened in
atherosclerotic arteries, the thickening is highly eccentric and is
composed mainly of GAGs and cellular material. Our calculations show
that the GAGs do not appear to offer much resistance to macromolecular
transport, and unless the specific molecular agent is modified to
interact with the cells or matrix, the cellular material will not
impede transport. Therefore, the thickening of the intima will not be
included in the calculation of Pe. Insertion of the new value for
medial thickness into the equation for Pwall
leads to a value of 22.8x10-8 cm/s.
Dwall is equal to
23.9x10-10 cm2/s.
Using an Lp value of
16x10-8 cm/s · mm
Hg-1, we obtain
The highest possible value for Pe is 42, and the lowest is 2.1,
assuming that
f remains unaltered compared to
control vessels. It is probable that
f will be
reduced in atherosclerotic arteries, and, if so, the lower bound for Pe
would be higher than 2.1.
From the above estimations, it appears that transmural convection plays
a greater role in the transport of macromolecules through the femoral
artery wall than does diffusion and that this tendency is retained in
arteries from hypercholesterolemic and atherosclerotic
animals.
Because heparin (MW, 12 to 15 kd) had been considered as a drug to
reduce arterial restenosis and because values exist
in the literature for heparin for Pmedia of rat
aorta,18 which has a similar medial thickness to
the rabbit femoral artery, we determined possible values of Pe for
heparin:
Therefore,
for control vessels.
The highest possible value for Pe is 0.4. There are no available values
of
f for heparin. However, the published value
for Pendo for heparin is similar to that for
sucrose in skeletal muscle capillaries
(1.4x10-5 cm/s, reference 39), even though
sucrose has a much lower molecular weight than heparin (342 d).
Therefore, we will use the published value of
f for sucrose (0.11, reference 39) in the
equation for Pe. This approximation gives a lower bound for Pe of 0.36.
Therefore, for heparin, diffusion appears to play as great a role as
convection in arterial transport. However, convection will
have some influence on the distribution of small to medium-sized agents
throughout the arterial tissue.
Electron Microscopic Observations
Control Vessels
Control vessels showed intact endothelium, a thin
intima (up to about 2 µm thick), and closely packed,
cigar-shaped medial smooth muscle cells, which were aligned
circumferentially. The collagen fibers were arranged in groups, each
fiber within a given group having similar orientation. An electron
micrograph of a transverse section through a control aorta fixed at
30 mm Hg is shown in Fig 1
.
Hypercholesterolemic Vessels
The appearance of the hypercholesterolemic vessels
was different from that of the controls in one respect; in sections
from three of the six hypercholesterolemic vessels
prepared for electron microscopy, the endothelium was
largely missing, and in sections from two further vessels, the
endothelium was vacuolated or missing in places. This
endothelial damage was also demonstrated by the blue
staining of the intimal surface of hypercholesterolemic
vessels that was seen on their excision from the animals. The trypan
blue, which was included in the vessel perfusate, stains the
intima only if the endothelium is damaged or missing.
Control vessels did not show such staining. The appearance of the media
was similar to, or slightly less dense than, that of the controls. Fig 2
, a, shows an electron micrograph of a transverse section through a
hypercholesterolemic femoral artery in which the
endothelium is absent. A transverse section through the
aorta, prepared identically from the same animal, is shown for
comparison in Fig 2
, b, manifesting an intact, undamaged
endothelium. Sections from the femoral artery from the
same animal consistently showed sloughed or damaged
endothelium. The same contrast between femoral and
aortic endothelia was made in two other
hypercholesterolemic rabbits.
Atherosclerotic Vessels
In areas of atherosclerotic vessels distant from the actual
lesion, the tissue appearance was similar to that of
hypercholesterolemic arteries that had not undergone
air dessication, with an incomplete endothelium, thin
intima, and closely packed and highly organized medial tissue.
Endothelial cells were generally present overlying
lesions but were not in perfect condition. Often, on excision, these
vessels demonstrated relatively little intimal trypan blue staining
over the lesion but showed intense staining of the surrounding area.
Examples of the intimas of transverse sections through lesions in
vessels fixed at 30, 60, and 90 mm Hg are shown in Figs 3
, a,
4, a, and
5, a, respectively. The corresponding
medias are shown in Figs 3
, b, 4, b, and 5, b.

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|
Figure 3. Electron micrographs of a transverse section
through a mechanically induced lesion in a femoral artery of a
hypercholesterolemic rabbit showing intima (a) and
media (b). This vessel was fixed at 30 mm Hg. Note the
vacuolated endothelium (small arrow) and gap between
endothelial cells (arrowhead). The
endothelial cell cytoplasm is sparse in places (large
arrow). In b note the medial smooth muscle cells with cytoplasmic
pseudopodia (P). These cells are less tightly packed than seen in
control vessels. IC indicates intimal cells laden with fat globules
(lipid); F, fenestra in internal elastic lamina; and FG, fat globules
in medial smooth muscle cells. Bar=5 µm.
|
|
In lesions, the endothelial cells were vacuolated
(arrows, Figs 3
, a, 4, a, and 5, a), with some gaps between cells
(arrowhead, Fig 3
, a), and occasional sparse cytoplasm suggesting cell
rupture and release of cytoplasmic contents (large arrow, Fig 3
, a).
The intima reached up to 30 µm (Fig 4
, a), with significant
eccentricity as well as variability noted between vessels. Many of the
cells within the intima had the appearance of smooth muscle cells,
which may have migrated from the media through fenestrae in the
internal elastic lamina (Fig 3
, a). These cells were characterized by
an elongated shape and an abundance of cytoplasmic filaments and
plasmalemmal vesicles. Other intimal cells were present
that resembled "modified smooth muscle cells," characterized by
their ovoid shape and abundant rough-surfaced endoplasmic reticulum and
prominent Golgi apparatus.40 An
example of two such cells can be seen in Fig 4
, a, just beneath the
endothelial cell, indicated by an arrow. A large
fraction of the intimal cells of all lesions examined by electron
microscopy contained large fat globules (Figs 3
, a, 4, a, and 5, a).
Lipid-filled intimal cells have previously been noted in the femoral
arteries of rabbits intermittently fed a high-cholesterol
diet for 3 to 14 weeks.41 Fat globules were often
seen within smooth muscle cells in the adventitia, close to vasa
vasorum, suggesting that the processes responsible for the deposition
of blood-derived lipids within the intimal cells are also operating
across the walls of the vasa vasorum. Occasionally, fat globules were
found within smooth muscle cells within the central regions of the
media (see Fig 3
, b), but this was a rare occurrence. The intima of the
artery fixed at 90 mm Hg (Fig 5
, a) appeared to be more
densely packed (ie, less extracellular space per unit volume of tissue)
than the vessels fixed at 30 and 60 mm Hg (Figs 3
, a and 4,
a, respectively).
The appearance of the media within lesions was variable. In many
cases the extracellular space per unit volume of tissue appeared to
larger than that seen in controls and in nonlesion regions of
hypercholesterolemic vessels (see Figs 3
, b, and 4, b).
In these specimens the smooth muscle cells exhibited many pronounced
pseudopodia, in contrast to the smooth outlines displayed by the smooth
muscle cells in control preparations (Fig 1
). In the lesion from a
vessel fixed at 90 mm Hg, the media resembled that of control
vessels (Fig 5
, b). The smooth muscle cells within the media were quite
densely packed and showed few pseudopodia.
 |
Discussion
|
|---|
In summary, this study has shown that delivery of drugs may
be
significantly influenced by convective fluid flux through
the artery
wall, from the lumen to the adventitia. Convection-dependent
washout of
agents from the intima and inner media is expected
to occur. This
finding should be taken into account when designing
drug treatments to
reduce arterial restenosis. In this
atherosclerosis
model of the rabbit femoral artery,
diet-induced hypercholesterolemia
for a period
of 3 weeks caused an increase in hydraulic conductance
of the femoral
artery, regardless of whether lesions were initiated
by mechanical
damage of the endothelium. It might be expected
that
the occurrence of even early atherosclerosis in human
vessels
would favor increased convectional drug washout.
Our conclusion that macromolecular transport is affected by
convective fluid flux is in disagreement with the conclusion of Penn et
al.17 Those authors measured solute (HRP)
transport and obtained values for convective velocity by nonlinear
parameter estimation. The optimal values of the medial
parameters were those for which the model output provided
the best least-squares fit to the experimental data. The authors
assumed in their model that transendothelial transport
is dominated by diffusion. They stated that their estimations of
arterial transport properties had very poor precision
regarding animals with undamaged endothelium. However,
their estimation of P was close to other reported permeability
coefficients to macromolecules. For this reason, we used their
estimation of P in our calculations of Pe. In fact, it is their value
for Lp that is in disagreement with other
published data. In particular, their value of 10 cm/s ·
mm Hg-1 for the rat aorta is three orders
of magnitude smaller than the value that we measured in the femoral
artery. This deviation could explain their very low values of Pe. The
optimum experiment, that is, the measurement of macromolecular
transport properties and Lp of the same vessel,
remains to be performed.
Convective washout, and hence failure to maintain a high concentration
of drug in the arterial intima, is consistent with
the lack of success achieved so far by certain intramural treatment
regimens targeting restenosed arteries, such as for administration of
heparin by porous balloon catheter.27 The
limitation of convective washout needs to be addressed in order to
exploit the advantage of focal administration of drugs to an
atherosclerotic site. One possibility is to alter the physicochemical
characteristics of drugs that affect their
f
or P values. For example, an agent could be selected that rapidly binds
specifically to intimal smooth muscle cells and prevents their
proliferation. Delivery of the agent could also be via liposomes, which
would remain entrapped within the intima.42
Another interesting possibility is raised by recent studies that
suggest that intimal atherosclerotic lesions may be triggered by
occlusion or thrombosis of the adventitial vasa
vasorum.43 44 It is thought that the resulting
tissue hypoxia in the media may cause migration of the medial
smooth muscle cells toward the intima. In addition, mRNA for
platelet derived growth factor may be increased in the hypoxic
endothelial cells of the vasa vasorum, and the
platelet-derived growth factor may cause smooth muscle cell
proliferation. If this is the case, then it would be advantageous for
luminally administered drugs to be redistributed to the adventitia by
convective flux, since the adventitia would be the targeted site of
need. It is much easier to deliver drugs intraluminally and rely on
convection to rapidly propel them to the adventitia than it is to
administer drugs directly into the adventitia. Although drugs can be
delivered locally from polymers implanted in the adventitia, surgical
placement of drug-eluting matrices is impractical for most clinical
applications. Thus, it may be possible to exploit the role of
convection in transporting agents from the lumen to the adventitia in
preventing development of atherosclerotic plaques.
Ultrastructural evaluations provide further data confirming the basis
for the observation of hydraulic conductance. In
hypercholesterolemic vessels without lesions, our
ultrastructural studies indicate that hydraulic conductance is
increased because the endothelium is damaged or
missing. In the one case in which the endothelium was
intact, the Lp was close to control values. The
endothelium is known to present a considerable
barrier to transarterial water
transport.21 26 Vessels with focal lesions may
have demonstrated a high Lp for the same reason.
Although the endothelium was usually in place over the
lesions, it was frequently vacuolated or showed widened
interendothelial junctions. In addition, the
endothelium was often missing in regions adjacent to
the lesion that were included in the cannulated segment.
Our finding that hypercholesterolemia
alone caused endothelial denudation and damage in five
of the six femoral arteries examined was surprising. Other studies have
concentrated on the effect of
hypercholesterolemia on the structure of the
aorta and coronary arteries. Rabbits fed a 2%
cholesterol and 10% corn oil diet for 2 weeks did not show
endothelial damage of aortas.40
Rabbits fed 1% cholesterol for 10 weeks did show some
endothelial damage of coronary arteries in the
form of vacuolated cells and widened intercell
clefts.45 However, the damage was not as
extensive as we observed in most femoral arteries from
hypercholesterolemic animals in this study. Because we
did not observe endothelial damage in the aortas of our
hypercholesterolemic rabbits, it is possible that the
endothelium of femoral arteries is particularly
fragile. In fact, previous authors have shown that the femoral artery
is more susceptible than the carotid artery to
atherosclerosis.46 One of the
first signs of endothelial dysfunction during
hypercholesterolemia is the impaired ability of
arteries to undergo endothelial-dependent
relaxation.47 48 49 It is likely that
hypercholesterolemia reduces the synthesis
and/or availability of the endothelially derived
vasodilator, NO, because exposure of vessels to the NO substrate,
L-arginine, reverses this effect.50
In the femoral artery, if NO production is deliberately
impaired by perfusing the vessel with the NO inhibitor,
NG-nitro-L-arginine methyl ester, the
endothelium becomes structurally
damaged.51 This does not occur if similar
experiments are performed on the aorta. Thus, the
endothelial damage we observed in the femoral arteries
of hypercholesterolemic rabbits may be initiated by the
accompanying impaired release of NO.
The less dense media observed beneath lesions may also have contributed
to the higher Lp. Vessels with lesions always
showed similar Lps to
hypercholesterolemic arteries without lesions, even
though the intima and media were thicker in the former case. It is
possible that the effect of increased wall thickness on
Lp was canceled out by the effect of a less dense
media beneath lesions. As pressure was increased,
Lps of hypercholesterolemic
vessels, with and without lesions, decreased and approached control
values. This finding suggests that at high pressures the
endothelium becomes less important as a barrier to
transarterial water flux and that the media plays a more
dominant role.
The endothelium is also important because it can act as
a barrier to diffusion of material, for example, a locally delivered
drug, from the arterial wall into the bloodstream.
Immediately after angioplasty the affected region of the artery is
denuded of endothelium.52 53 54
Therefore, this would not be an optimal time to deliver drugs into the
arterial wall, intraluminally, to prevent
restenosis. Endothelial regeneration is usually
complete within a few weeks, and the endothelial cells
have completely matured within 3 months.52 This
would be a more appropriate time for drug delivery, because the intact
endothelium would reduce the rate of diffusion of
intimally delivered drug into the bloodstream and would reduce the
convective water flux from the lumen to the outer regions of the artery
wall. However, this type of approach would be successful only if the
drug delivery system itself did not damage the
endothelium.
To summarize, we made direct measurements of Lp
on normal hypercholesterolemic and atherosclerotic
arteries and used these data, together with information obtained from
our ultrastructural studies, to determine Peclet numbers for large and
small molecules. The ultrastructural component provides a structural
basis with which to interpret measurements of Lp.
This study demonstrates that convective flux may significantly affect
the distribution of small and large molecules within the artery wall in
normal and in atherosclerotic vessels. Both the local administration
and subsequent redistribution of drugs to lesions after angioplasty
will be affected. These measurements of hydraulic fluid flux provide a
framework for further studies to evaluate the possibility of
influencing drug residence time and distribution by modifying the
transport properties of the drug.
 |
Selected Abbreviations and Acronyms
|
|---|
| GAG |
= |
glycosaminoglycan |
| HA |
= |
hyaluronic acid |
| HRP |
= |
horseradish peroxidase |
| MW |
= |
molecular weight |
|
 |
Acknowledgments
|
|---|
This work was supported by Eli Lilly and Company, a Grant-in-Aid
from
the American Heart Association, Arizona Affiliate, and by National
Heart,
Lung, and Blood Institute grant HL-17421.
Received March 8, 1996;
accepted May 7, 1997.
 |
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