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Three-dimensional arrangement of the vasa vasorum in explanted segments of the aged human great saphenous veinScanning electron microscopy and three-dimensional morphometry of vascular corrosion casts.

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THE ANATOMICAL RECORD PART A 281A:1372–1382 (2004)
Three-Dimensional Arrangement of the
Vasa Vasorum in Explanted Segments
of the Aged Human Great Saphenous
Vein: Scanning Electron Microscopy
and Three-Dimensional Morphometry
of Vascular Corrosion Casts
ALOIS LAMETSCHWANDTNER,1* BERND MINNICH,1 DAVID KACHLIK,2
MAREK SETINA,3 AND JOSEF STINGL2
1
Vascular and Muscle Research Group, Department of Organismic Biology,
University of Salzburg, Salzburg, Austria
2
Department of Anatomy, Third Medical Faculty of Charles University,
Prague, Czech Republic
3
Department of Cardiovascular Surgery, Regional Hospital, Ceske Budejovice,
Czech Republic
ABSTRACT
The vasa vasorum of skeletonized and nonskeletonized segments of five human great saphenous veins (GSVs),
harvested during coronary bypass grafting, were cannulated, rinsed, and injected (casted) with the polymerizing resin
Mercox-Cl-2B. After removal of the dry vascular tissue, the casts were examined using scanning electron microscopy.
Stereopaired images (tilt angle, 6°) were taken, imported into a 3D morphometry system, and the 3D architecture of the
vasa vasorum (arterial and venous vasa as well as capillaries) was studied qualitatively and quantitatively in terms of
vasa diameters, intervascular and interbranching distances, and branching angles. Diameters of parent (d0) and large
(d1) and small (d2) daughter vessels of arterial and venous bifurcations served to calculate asymmetry ratios (␣) and
area ratios (␤). Additionally, deviations of bifurcations and branching angles from optimal branches were calculated for
selected arterial vasa. The arrangement of the vasa vasorum closely followed the longitudinally oriented connective
tissue fibers in the adventitia and the circularly arranged smooth muscle cell layers within the outer layers of the
media. Venous vasa by far outnumbered arterial vasa. Vasa vasorum changed their course several times in acute angles
and revealed numerous circular constrictions, kinks, and outpouchings. Due to their spatial arrangement, the vasa
vasorum are prone to tolerate vessel wall distension generated by acute increases in blood pressure or stretching of the
vessel without severe impact on vessel functions. Preliminary comparisons of data from the bifurcations of cast arterial
vasa vasorum, with calculated optimal bifurcations, do not yet give clear insights into the optimality principle(s)
governing the design of arterial vasa vasorum bifurcations of the human GSVs. © 2004 Wiley-Liss, Inc.
Key words: man; saphenous vein; vasa vasorum; vascular casts; scanning electron microscopy; 3D
morphometry
The vessels within the wall of larger blood vessels are
known as the vasa vasorum. In normal blood vessels with
less than 29 layers of vascular smooth muscle cells
(VSMCs) or lamellar units (Wolinsky and Glagov, 1967),
vasa vasorum lie in the adventitia. They supply oxygen
and nutrients to the outer layers of the vessels; blood
circulating in the vessel lumen supplies the inner wall
areas. Vasa vasorum also provide an exchange area for
delivery to and absorption of substances from the surrounding wall tissue (Williams and Heistad, 1996).
In blood vessels with more than 29 lamellar units, adventitial vasa vasorum penetrate into the outer layers of
©
2004 WILEY-LISS, INC.
*Correspondence to: Alois Lametschwandtner, Organismic Biologie, Vascular and Muscle Res. Group, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. Fax: 43-662-80445698. E-mail: alois.lametschwandtner@sbg.ac.at
Received 16 February 2004; Accepted 25 May 2004
DOI 10.1002/ar.a.20098
Published online 14 October 2004 in Wiley InterScience
(www.interscience.wiley.com).
VASA VASORUM IN HUMAN GREAT SAPHENOUS VEIN
the media (Wolinsky and Glagov, 1967). Vasa vasorum
either abut against the proper vessel lumen (vasa vasorum interna), against branches of the vessel, or against
arteries next to the vessel (vasa vasorum externa)
(Schönenberger and Müller, 1960; Gössl et al., 2003). In
the stem of the human great saphenous vein (GSV), only
vasa vasorum externa are present, whose stems penetrate
the GSV every 5 to 15 mm (Kachlik et al., 2002). In
arteries, the vasa vasorum drain into concomitant nearby
veins (Gössl et al., 2003); in GSVs, they drain into
branches of the vein proper or into concomitant veins
(Kachlik et al., 2002).
Under altered conditions, the vasa vasorum contribute
to the neovascularization of atherosclerotic lesions
(plaques) and neointima formation. A direct relationship
between the severity of atherosclerosis and the degree of
adventitial vasa vasorum is established (Kumamoto et al.,
1995). Moreover, hypercholesterolemia has been shown to
enhance coronary vasa vasorum adventitial neovascularization (Kwon et al., 1998) and the coexistence of hypercholesterolemia and hypertension is reported to impair
adventitial vasa vasorum formation, resulting in insufficient compensation for hypoxia in the thickened media
(Kai et al., 2002). A blockade of angiogenesis of vasa vasorum is therefore considered a promising new strategy to
inhibit vessel restenosis (Fuchs et al., 2001).
Recently, it was shown that in an experimental model
(common carotid artery of a rabbit) upregulation of hypoxia-induced factor-1␣ (HIF-1␣) and vascular endothelial
growth factor (VEGF) expression drive angiogenesis of the
vasa vasorum (Bayer et al., 2002) and that formation of
adventitial vasa vasorum is induced through the HIF-1␣/
VEGF/Ets-1 (transcription factor) pathway in hypertension (Kuwahara et al., 2002). It is assumed that blockade
of this pathway is a possible means to prevent restenosis
of grafted or stented vessels.
Although there is a long-lasting interest in the vasa
vasorum, the fine distribution and the three-dimensional
arrangement of the entire vasa vasorum bed within the
wall of blood vessels still remain to be elucidated in more
details.
A promising new technique, microcomputed tomography (␮-CT) (Jorgensen et al., 1998), enables the study of
the spatial arrangement of the vasa vasorum in large
specimens (Lerman and Ritman, 1999; Bentley et al.,
2002; Wan et al., 2002; Gössl et al., 2003), even in its
frozen state (Kantor et al., 2002). In conjunction with
semiautomated tree analysis software, it can provide
quantitative geometrical data not yet available by other
techniques (Gössl et al., 2003). Regrettably, due to the
voxel width of ⬃ 20 ␮m used in current ␮-CT, vasa vasorum with a diameter smaller than 40 ␮m cannot be visualized. Thus, ␮-CT images lack the entire capillary bed
and pre- and postcapillary vasa vasorum (Kwon et al.,
1998; Gössl et al., 2003). In contrast, scanning electron
microscopy (SEM) of vascular corrosion casts (Murakami,
1971; Lametschwandtner et al., 1984; Aharinejad and
Lametschwandtner, 1992) has a sufficiently high power of
resolution to visualize the entire vasa vasorum circulatory
bed from the feeding arterial stem vessels throughout the
capillaries to the draining venous vasa vasorum. The
great depth of focus of the SEM, and the possibility to take
stereopaired images for subsequent 3D morphometry
(Malkusch et al., 1995; Minnich et al., 1999, 2002), enables
the measurement of parameters in the casted vasa vaso-
1373
rum required to calculate flow and pressure gradients
within the vascular network. Additionally, it can provide
the vascular parameters needed to define which optimality principle(s) (Murray, 1926a, 1926b; Gössl et al., 2003)
are applicable to the vasa vasorum of the aged human
GSV.
As yet, only the vasa vasorum of the rabbit common
carotid artery have been studied using SEM of vascular
casts in an experimental model (Bayer et al., 2002). The
present study is the first to analyze the vasa vasorum
system in a vein. The human GSV was chosen because it
is frequently used as a coronary bypass graft. It is also
known that this vein has a high incidence of stenosis that
dramatically impairs graft patency.
In the current study, SEM of vascular corrosion casts
(Murakami, 1971) in combination with 3D morphometry
(Minnich et al., 1999) provides detailed qualitative insights into the 3D arrangement of the vasa vasorum, as
well as quantitative data on vasa diameters, intervascular and interbranching distances, and branching angles. From these data, it is possible to calculate branching indexes of arterial, capillary, and venular
bifurcations (mergings). These data can then be used to
calculate optimum vessel diameters and optimum
branching angles to find out which of the four optimality
principles (minimum lumen volume, minimum pumping
power, minimum lumen surface, minimum endothelial
shear force) (Murray, 1926a, 1926b) is achieved in the
vasa vasorum network.
MATERIALS AND METHODS
Vascular Casting
Five explanted segments of human GSV were taken
during their dissection for coronary bypass grafts in five
patients who granted written consent and who were aged
67–72 years (three males, two females; risk factors, triplevessel disease, hypertension, previous myocardial infarction, and/or diabetes mellitus). The segments (lengths,
3–5 cm; external diameter, 3– 4 mm) were transported to
the laboratory in ice-cooled heparinized saline (1,000
I.E./ml 0.9 sodium chloride) and casted within 6 –10 hr
after harvesting. GSV segments were subsequently submerged in saline at room temperature (20°C) and a feeding artery was dissected from the surrounding tissue under a dissecting microscope. It was then cannulated using
a mechanical micromanipulator (WPI Instruments, Berlin, Germany) with a fine-tipped glass cannula (outer diameter, 80 –120 ␮m) and rinsed with warm (37°C) saline.
Then 4 ml of prepolymerized resin (Mercox-Cl-2B; Ladd
Research, Burlington, VA) was gently mixed with 1 ml of
monomeric methylmethacrylic acid containing 62.5 mg
paste MA (accelerator) and injected using an automatic
infusion pump with a flow rate of 7 ml/hr. The injection
stopped when the efflux from the open-cut vasa vasorum
of the vascular segment became highly viscous. The specimens were then left at room temperature (20°C) for 30
min to allow polymerization of the injected resin and then
transferred into a water bath at 60°C to temper overnight.
The following morning, the specimens were transferred to
a solution of 7.5% potassium hydroxide (40°C) to remove
all organic material. After maceration, the remaining
plastic vascular casts were rinsed several times with distilled water, then submerged for 5–15 min in 5% formic
acid. After further rinses with distilled water, the specimens were frozen (in distilled water). Ice-embedded casts
1374
LAMETSCHWANDTNER ET AL.
were then freeze-dried (the ice is sublimated and dry
vascular casts remain), mounted on specimen stubs, sputtered with gold, and examined in Stereoscan 250 SEM
(Cambridge, U.K.) at an accelerating voltage of 10 kV. For
further details on cast preparation, see Aharinejad and
Lametschwandtner (1992).
for an optimality principle based on minimizing lumen
surface and total shear force on endothelial tissue (Zamir,
1988a).
Finally, the deviation of diameters of daughter vessels
and branching angles of several consecutive bifurcations
from optimal values were calculated according to Equation 7:
Quantification
For quantification of the vasa vasorum, stereopaired
images (tilt angle, 6°) were taken with the SEM at an
accelerating voltage of 10 kV, a working distance of 10
mm, and magnifications ranging from 20⫻ to 500⫻.
Images were imported into the 3D morphometry system
(ComServ, Minnich and Muska OEG, Salzburg, Austria)
and the following measures were taken: diameters, intervascular and interbranching distances, and branching angles. Data were used to calculate the following:
branching indexes ␣ (⫽ asymmetry ratios; d2/d1), diameter ratios of daughter vessels (d1, d2) to parent vessel
(d0), and area ratios ␤ of branches to parent vessel
[(d12 ⫹ d22)/d02].
For testing the optimality of consecutive arterial and
capillary branchings and venous mergings, the two
branching diameters d1 (large branch) and d2 (small
branch) were normalized in terms of the diameter of the
parent vessel (d0) at each bifurcation (merging). Optimally, the diameters of the two branches at a bifurcation
would be (Zamir, 1988a, 1988b; Gössl et al., 2003):
1
d1
⫽
d0 (1 ⫹ ␣3)1/3
(1)
d2
␣
⫽
d0 (1 ⫹ ␣3)1/3
(2)
To find out the optimality principle upon which the
bifurcations (mergings) of the vasa vasorum of the aged
human GSVs are based, the following formulas proposed
by Zamir (1988a, 1988b) and Gössl et al. (2003) were used:
cos ␪1 ⫽
(1 ⫹ ␣3)4/3 ⫹ 1 ⫺ ␣4
2(1 ⫹ ␣3)2/3
(3)
cos ␪2 ⫽
(1 ⫹ ␣3)4/3 ⫺ 1 ⫹ ␣4
2␣2(1 ⫹ ␣3)2/3
(4)
for an optimality principle based on minimizing lumen
volume and pumping power, respectively, and
cos ␪1 ⫽
(1 ⫹ ␣3)2/3 ⫹ 1 ⫺ ␣2
2(1 ⫹ ␣3)1/3
(5)
cos ␪2 ⫽
(1 ⫹ ␣3)2/3 ⫺ 1 ⫹ ␣2
2␣(1 ⫹ ␣3)1/3
(6)
Deviation from optimal (%)
⫽
Value(s) gained using data from cast vessels
⫻ 100
Value(s) gained using Equations 1–6
(7)
RESULTS
Qualitative Analysis
The casting of the five segments resulted in three reasonably good vascular casts of different sizes based on the
areas supplied by the cannulated arteries and the length
of the segments available. Gross inspection of the casts
under the dissecting microscope showed that the wall of
the GSV contained a rich supply of the vasa vasorum. The
vasa ran predominantly parallel with the longitudinal
axis of the vein. SEM of resin casts at low magnification
confirmed this finding and demonstrated that venous vasa
by far outnumbered arterial vasa (Figs. 1– 4). The latter
generally had smaller diameters and could be differentiated from the venous vasa by their round profiles and the
characteristically long endothelial cell nuclei imprints,
orientated parallel with the vessel axis (Fig. 2). Feeding
arterial vasa ran in close association with the draining
venous vasa (Figs. 2– 4). Diameters of feeding arteries
were approximately 50 ␮m; those of their venous partners
were approximately 100 ␮m. After infiltrating the external adventitial layers, the feeding arteries generally followed a longitudinal course (Figs. 2– 4). According to the
terminology proposed by Schönenberger and Müller
(1960), these vessels were termed first-order arteries (1A).
These arteries generated side branches, termed secondorder arteries (2A). Second-order arteries ran in a circular
formation (Figs. 3 and 4), then became longitudinal and
were termed third-order arteries (3A). These branched
several times before they changed into capillaries (Figs.
5– 8). Locally, 2As followed a corkscrew-like course (Figs. 6
and 7) and finally formed capillaries that were arranged in
a longitudinal fashion and ran parallel (Fig. 7). These
capillaries had (luminal) diameters ranging from 7 to 15
␮m (number of measurements, n ⫽ 22). Intercapillary
distances ranged from 12 to 114 ␮m (n ⫽ 15). After average lengths of 45 to 320 ␮m (n ⫽ 12), the capillaries
gradually changed into postcapillary venules. Depending
on their location, they were termed fourth- (4V) or thirdorder venules (3V), which either followed a circular (4V) or
longitudinal (3V) path (Fig. 8). Locally, arteries became
narrow in places with adhering plastic strips imitating
myocytes and/or pericytes (Fig. 9). Venules of descending
order not only changed from a circumferential course into
a longitudinal course (e.g., from 4V to 3V respectively from
2
V to 1V), but often also changed from a deeper layer into
a more superficial layer (Fig. 10). In venous vasa vasorum,
tight constrictions were often found (Figs. 11 and 12).
Locally, small capillary loops were squeezed between
venules, thereby overbridging the venules (Fig. 12). To fit
into the narrow intervenular space between two larger
VASA VASORUM IN HUMAN GREAT SAPHENOUS VEIN
1375
Fig. 1. Vascular corrosion cast (VCC) of a segment of the proximal
portion of the human GSV revealing the vasa vasorum from an adventitial
view. Note the prevailing longitudinal arrangement of the vasa. A, artery;
V, vein. Arrows mark the feeding and/or draining vessels infiltrating the
GSV every 0.5–1.5 cm.
Fig. 2. VCC of the proximal segment of the GSV. Adventitial view.
Longitudinal axis of GSV: left to right. Hierarchy of feeding arterial and
draining venous vasa (terminology introduced by Schönenberger and
Müller, 1960). Vasa that have a longitudinal arrangement are designed
orders 1, 3 and 5 (1A, 3A, 5A for arterial vasa and 1V, 3V, 5V for venous
vasa) and orders 2, 4, and 6 (2A, 4A, 6A and 2V, 4V, 6V ) for vasa with a
circular arrangement. Note the characteristic close association of sup-
plying artery and draining vein and the difference in vessel calibers.
Asterisks mark extravasations.
Fig. 3. Branching pattern and caliber of feeding artery and draining
vein penetrating the adventitia of the GSV every 5–15 mm. Detail from
Figure 1 (boxed area). Note that the main branch of the feeding artery
(asterisk) does not supply the GSV but the adjacent fatty tissue. Arrows
indicate direction of blood flow.
Fig. 4. Longitudinal first-order artery (1A), circular second-order artery (2A), longitudinal 3A, and circular 4A. Note that the corresponding
veins (1V to 3V) have a larger diameter than the arteries. Arrows indicate
direction of blood flow. E, evasate.
venules, anastomoses followed a hairpin-like course with
the two loops close beside each other (Fig. 12). In the
external media, venous vasa vasorum with diameters
ranging from 24 to 40 ␮m had a circular arrangement
occupying empty spaces where vascular smooth muscle
cells formerly resided. Capillaries ran a longitudinal
1376
LAMETSCHWANDTNER ET AL.
Fig. 5. Branching pattern and course of third-order arteries (3A) and
second- and third-order veins (2V and 3V). C, capillary. Arrows mark
direction of blood flow.
Fig. 6. First-order artery (1A) branching off a second-order artery (2A) in
an acute angle. Adventitial view. Note the first-order vein (1V) running
parallel is much larger than the artery. Capillaries drain either into secondorder (2V) or third-order veins (3V). Arrows indicate direction of blood flow.
Fig. 7. Terminal portion of a circular second-order artery (2A) giving
rise to capillaries running parallel in a longitudinal arrangement. Detail
from Figure 6 (boxed area). Arrows mark direction of blood flow.
Fig. 8. Transition of a third-order artery (3A) into a capillary and its
merging into a third-order vein (3V). Arrows mark direction of blood flow.
course with intercapillary distances from 12 to 43 ␮m
(Figs. 13–16). Spacing of circular 4V was around 140 ␮m
(Fig. 13).
The innermost layer of the vasa vasorum facing the luminal side consisted mostly of capillaries with draining veins at
various points (Fig. 14). A close-up view revealed that capillaries formed rectangular or squared meshes with occasionally plastified pericytes (Hodde, 1981; Castenholz et al.,
1982) sticking on them (Figs. 14 and 15). Plastified means
that the pericytes by a hitherto controversially disputed process have been permeated by the casting medium, which
polymerized and prevented their structure from digestion
(removal) by the maceration solution (7.5% KOH). Postcapillary venules that drained capillaries (e.g., 4V in Fig. 14) ran
a circular course over a short distance only to turn into a
longitudinal one (e.g., 3V in Fig. 14). Transverse section casts
VASA VASORUM IN HUMAN GREAT SAPHENOUS VEIN
1377
Fig. 9. Plastified myocyte/pericyte (P) embracing and constricting an
artery.
Fig. 10. Pattern and course of the adventitial vasa vasorum. Overview. Note the rather large venous anastomoses between longitudinal
running first-order veins (1V) formed by circular running second-order
(2V) or third-order veins (3V) that run transversely. Note also the spacing
of the origin of second-order arteries (arrowheads). 1A and 2A denote
first- and second-order arteries. Arrowheads mark origin of secondorder arteries. Arrows indicate direction of blood flow.
Fig. 11. Drainage of longitudinal postcapillary venules (pv) into fifth(5V), fourth- (4V), and third-order veins (3V). Note the local indentations on
3
V (arrowheads). Cl, capillary loop. Arrows mark direction of blood flow.
Fig. 12. Third-order veins (3V) with deep indentations (arrowheads).
Capillaries interposed between the veins loop and form an anastomosis
between the veins. Arrows indicate direction of blood flow.
clearly revealed that terminal arterial vasa extended toward
the innermost layer of the vasa vasorum where they capillarized (Fig. 15). The spacing of the vasa vasorum can be
seen in Figure 16. Branched structures were found on capillaries facing the luminal side (Fig. 16). On higher magnification, these structures clearly identified themselves as plas-
tified pericytes with their processes embracing the capillary
(Figs. 16 and 17).
Quantitative Analysis
Three-dimensional morphometry on GSV vasa vasorum
revealed that bifurcation indexes (␣) ranged from 0.4 to
1378
LAMETSCHWANDTNER ET AL.
Fig. 13. Arrangement and course of the venous vasa vasorum (5V to
V). Note the transition of the longitudinal fifth-order vein (5V) into the
circular fourth-order vein (4V) and the transition into the third-order vein
(3V). At the transition sites, the veins loop and ascend (5V to 4V) or
descend (4V to 3V). Note the parallel arrangement of the capillaries and
the space below (asterisks) devoid of vessels. Arrows mark direction of
blood flow. pv, postcapillary venule.
Fig. 14. Vasa vasorum facing the lumen. Luminal aspect. Capillaries
form a rectangular meshwork and drain into circular or longitudinal veins.
Arrows indicate direction of blood flow.
Fig. 15. Spatial arrangement of the vasa vasorum. Transverse-sectioned vascular cast. Note the transition of the terminal artery into the
capillaries of the innermost layer. Arrows mark the direction of blood
flow.
Fig. 16. Spacing of the vasa vasorum as shown in a transversesectioned GSV vascular cast. Note the plastified pericytes (arrowheads)
embracing luminal capillaries. Asterisks mark transverse-cut vessels.
Arrows indicate direction of blood flow.
0.9 in arterioles, 0.7 to 1.0 in capillaries, and 0.4 to 1.0 in
venules. Interestingly, in venular bifurcations (mergings),
the ratio of diameters of large (d1) and small daughter
vessels (d2) to the diameters of their parent vessels (d0),
i.e., d1/d0 respectively d2/d0, was the same (mean, 0.6). In
arterioles, this ratio was 0.9 (mean). While larger daugh-
ter vessels showed the same diameter ratio to the parent
vessels in both arterioles and venules (mean, 0.6), this
ratio was different in smaller daughter vessels (mean, 0.9
vs. 0.6; Table 1).
Analysis of the preliminary data from eight consecutive
bifurcations in a single arterial vas using Equations 1– 6
3
VASA VASORUM IN HUMAN GREAT SAPHENOUS VEIN
Fig. 17. Plastified pericyte embracing a capillary. Arrowheads mark
lateral processes of the pericyte. Note the internal structure of the resin
cast exposed by the cutting (asterisk).
revealed a maximum deviation from the theoretical optimum of 20% in branch diameters; a deviation of 301.3% in
branching angles ␪1 if for an optimality principle of minimum vessel volume and minimum pumping power is
tested; a deviation of 138.8% if for an optimality principle
of minimum surface and minimum drag force is tested; a
deviation of ⫺81.2% in branching angles ␪2 if for an optimality principle of minimum vessel volume and minimum
pumping power is tested; and a deviation of 84.2% if for an
optimality principle of minimum surface and minimum
drag force is tested (Table 2).
DISCUSSION
Previously unpublished studies by the authors have
shown that the vascular bed of organs and tissues can be
successfully cast with polymerizing resins for up to 12 hr
after excision. The results from this study confirm that a
delicate vascular system such as the vasa vasorum of the
human GSV can also be cast within this period.
Since the injections were carried out via a single perforating artery, the size and quality of the vascular casts
varied between injections depending on the area of the
wall supplied by the artery. Because the feeding arteries
were rather small in diameter (⬍ 100 ␮m), the resin was
initially injected retrogradely via the much larger draining vein. Venous injections, however, failed in all cases;
only the larger venous vasa vasorum filled. Capillaries
failed to fill. Injection was therefore continued orthogradely via one perforating artery, exposed, and cannulated under the dissecting microscope.
Recently, SEM of resin corrosion casts was used in an
experimental rabbit model to give an overview of the
amount of neovascularization of the vasa vasorum occurring between the rabbit carotid artery cuffed by a segment
of its contralateral partner (Bayer et al., 2002). This study
1379
gave no detailed account of the 3D arrangement of the
vasa vasorum within the wall of the artery. However, the
technique proved valuable as it allowed the authors to
demonstrate that some of the newly formed vasa vasorum
branched off the lumen of the carotid artery and showed
the characteristic arterial endothelial cell nuclei imprint
patterns first described on the surface of vascular casts by
Miodonski et al. (1976).
The present study allowed, for the first time, a thorough
analysis of the entire microvascular bed and its 3D arrangement of the vasa vasorum of a blood vessel in general and of the human GSV in particular. The present
work has provided the first quantitative data on vasa
parameters and the general geometry of the vasa vasorum
network within the wall of a vein. Previous work used
careful dissections and/or India ink injections to visualize
the vascular patterns. The experienced anatomist can positively identify all types of vessels in India ink-injected
specimens by careful light microscopical inspection
(Kachlik et al., 2002). However, only SEM of microvascular corrosion casts allows reliable information to be gained
on the hierarchy of vessels, their exact branching patterns
and branching angles, as well as the relationships between parent and daughter vessels in arterial branchings
and in venous mergings.
As the vascular corrosion casts were devoid of any tissue, no direct statement can be made regarding the proximity of the capillaries to the lumen of the saphenous vein.
According to our previous findings, the proximity is between 250 and 660 ␮m to the lumen of the saphenous vein
(Kachlik et al., 2002).
If these results are compared with those of Bayer et al.
(2002), who were the first to demonstrate the adventitial
vasa vasorum network of the rabbit common carotid artery, it should be noted that the studies deal with anatomically and physiologically different kinds of blood vessels
(artery versus vein), different species (rabbit vs. human),
differently sized vessels (1 vs. 4 mm), different aged vessels (months vs. 69 –73 years), different casting conditions
(in vivo casting with vessel under normal pressure vs. ex
vivo with vessel under zero intravasal pressure), and different injection routes (common carotid artery vs. single
feeding artery of the vasa vasorum). Both studies, however, clearly show the rich vascularization of the vessel
walls by the vasa vasorum [compare Fig. 5A in Bayer et al.
(2002) with Fig. 1 here].
Interestingly, the adventitial vasa vasorum are randomly arranged in the rabbit common carotid artery,
while those in the human saphenous vein follow a clear
longitudinal orientation (Fig. 1). In the experimental
model, the newly formed vasa vasorum are located between the intact common carotid artery and the cuff
formed by a segment of the contralateral vessel. This area
was occupied by connective tissue that had no obvious
characteristic arrangement that could serve as a support
structure for the newly grown vessels. In the wall of the
saphenous vein, the vasa vasorum, however, followed the
course of the longitudinal connective tissue fibers and the
circular vascular smooth muscle cells in the outer media
layers.
Moreover, in the human great saphenous vein, the typical mammalian pattern of blood supply with a small
artery running closely aside a much larger vein (clearly
seen in Fig. 1) is found, whereas in the cuffed rabbit
common carotid, a large undulating artery arose from the
1380
LAMETSCHWANDTNER ET AL.
TABLE 1. Ranges and means of branching indexes ⌬, ratios of diameters of larger daughter vessel (d1) and
smaller daughter vessel (d2), area ratios ␤, sum of branching angles between daughter vessels (Q1 ⴙ Q2) of
selected arterial, capillary and venular vasa vasorum of the GSV*
Arterioles
Range
Mean
Capillaries
Range
Mean
Venules
Range
Mean
␣
d1/d0
d2/d0
␤
Q1 ⫹ Q2
Interbranching
distance (␮m)
0.4–0.9
0.7
0.7–1.0
0.9
0.4–0.9
0.6
0.8–1.7
1.2
90–150
119
44–131
88
0.7–1.0
0.8
0.7–1.0
0.9
0.6–0.9
0.7
0.8–1.7
1.3
77–154
116
14–117
39
0.4–1.0
0.7
0.3–1.0
0.6
0.4–0.9
0.6
0.6–1.6
1.0
57–199
134
14–111
60
*Note that (merging) venules are similar in dimension while in arterial bifurcations these ratios are different.
TABLE 2. Characterization of eight consecutive branchings in a single arterial vasa vasorum.
Branching level
d0 (␮m)
1
28.52
2
29.07
3
19.97
4
18.47
5
15.51
6
15.16
7
10.63
8
11.80
d1 (␮m)
d2 (␮m)
d1/d0 measured
d1/d0 optimal
Deviation from
optimal (%)
d2/d0 measured
d2/d0 optimal
Deviation from
optimal (%)
␣
␪1 (°) measuredb
␪1 optimalb
␪1 optimalc
Deviation from
optimal (%)b,c
␪2 (°) measured
␪2 optimalb
␪2 optimalc
Deviation from
optimal (%)
28.15
26.06
0.99
0.83
23.57
20.69
0.81
0.84
17.96
12.72
0.90
0.91
17.34
16.23
0.94
0.82
18.63
14.46
15.07
11.72
0.99
0.88
11.11
7.75
11.35
8.44
0.96
0.89
19.2
0.91
0.76
⫺1.2
0.71
0.73
1.1
0.64
0.63
19.5
0.88
0.76
d
12.5
0.77
0.68
d
0.63
19.7
0.93
49.08
34.10
48.21
⫺2.7
0.88
29.86
32.23
46.33
1.6
0.71
75.55
23.37
38.87
15.8
0.94
40.32
34.58
48.60
0.78
69.93
26.82
42.00
13.2
0.78
67.14
26.89
42.07
0.70
91.63
22.83
38.37
9.1
0.74
62.53
25.17
40.52
43.9/1.8
31.67
40.86
53.65
⫺3.1/⫺35.5
13.39
43.20
55.48
223.3/94.4
9.84
52.29
62.39
16.6/⫺17.0
23.08
40.38
53.27
160.8/66.5
20.11
48.52
59.55
149.6/59.5
23.23
48.43
59.48
301.3/138.8
53.52
52.90
62.85
148.4/54.3
26.75
50.30
60.90
1.8/⫺14.8
⫺46.8/⫺56.1
a
0.88
a
0.68
d
⫺22.5/⫺41.0 ⫺69.0/⫺79.0 ⫺81.2/⫹84.2 ⫺42.8/⫺56.7 ⫺58.6/⫺66.2 ⫺52.0/⫺60.9
a
0.91
a
d
7.9
0.72
0.66
a
Atypical branching with one daughter vessel larger than the parent vessel.
Principle of minimum vessel volume and minimum pumping power.
c
Principle of minimum surface and minimum drag force (Zamir, 1988a, 1988b).
d
No calculation done because ofa.
b
lumen of the carotid artery with no vein aside [see Fig. 5B
in Bayer et al. (2002)]. The vein ran at some distance away
from the artery and was only slightly wider. This difference most likely resulted from the experimental situation
(cuffing) with subsequent neovascularization within the
space between the cuff and the native vessel.
A comparison with the findings gained by ␮-CT (Kwon
et al., 1998; Wilson et al., 2002; Gössl et al., 2003) clearly
shows that ␮-CT lacks all capillaries and pre- and postcapillary vessels smaller than ⬃ 40 ␮m. Here, SEM of
corrosion casts is superior to the previous technique, as it
enables the detailed study of each microcirculatory unit of
the vasa vasorum and thus allows the origin and merging
of individual vessels to be clearly defined. As vascular
casts made from Mercox-CL-2B tend to be very brittle,
superficial vessels hiding vessels underneath can be removed with the help of a fine tipped insect pin and thus
visualization of successive layers of vessels becomes possible. For more details on how to dissect microvascular
corrosion casts, see Aharinejad and Lametschwandtner
(1992). One of the advantages of ␮-CT, namely, the visualization of the vascular bed within a rather large specimen, can also be gained by removing individual vascular
layers until the entire cast is analyzed.
As all tissue was removed during maceration, individual vasa vasorum territories with defined layers of the
wall of the saphenous vein can be attributed only by
correlation with light microscopy of tissue sections done
previously (Kachlik et al., 2002). A partial maceration of
resin-injected specimens would be possible (Castenholz,
1983), but would allow the correlation of exposed cast
blood vessels with the tissue surface only, and deeper
layers would be still obscured from investigation.
Because of its richness, the vasa vasorum are a unique
source of neoangiogenesis (via sprouting) under pathological conditions (e.g., varicosis, intimal hyperplasia, atherosclerosis). We hypothesize that the sites that are most
likely to give origin to vascular sprouts are the areas
where venules change their course at an acute angle. Here
the venous endothelium will be exposed to a higher shear
VASA VASORUM IN HUMAN GREAT SAPHENOUS VEIN
rate that in turn will initiate vessel sprouting. The lack of
sprouts in our specimens is most likely due to the age and
health of the specimens from which the major part was
used for coronary bypass surgery. With respect to the
kinks of the venous vasa vasorum casts in the current
study, these structures can be attributed to the age of the
donors rather than to pathological changes of the vasa
vasorum.
If the given spatial arrangement of the vasa vasorum is
considered, it can be concluded that the GSV is probably
less vulnerable to dimensional changes of the vessel wall
emerging from changing intravasal volumes and/or pressures resulting from muscular contractions or stretching
of the vein. The corkscrew-like course of circular-running
second-order arteries (2A) in the current specimens most
likely resulted from the lack of intraluminal pressure during the casting procedure. On the other hand, this course
enables a distension of the wall of the vein without severely impairing blood flow of the vein.
Optimality in Vasa Vasorum
Quantitative analysis of the longitudinally first-order
arteries already revealed that the diameters of the main
stem of these vessels remained almost the same over
several branching levels and thus most likely serves as a
distributing vessel (Zamir, 1988a, 1988b). The external
vasa vasorum of the porcine coronary artery is also reported to have a distributing function (Gössl et al., 2003).
Normalized ratios of branch diameters that ranged from
0.81 to 0.99 (d1/d0) respectively 0.64 to 0.91 (d2/d0) in six
consecutive bifurcations in a single first-order arteriole in
the GSV casts (Table 2) support a delivering function of
this vessel. A delivering function of arteries is indicated by
a significant decrease of vessel diameters at each bifurcation level. In the porcine coronary artery, the internal
vasa vasorum are reported to have a delivering function
(Gössl et al., 2003).
Interestingly, branching angles varied to a much
greater degree around theoretical optima than diameters
of daughter vessels. These preliminary data corroborate
the findings of Gössl et al. (2003), where the diameters in
the arterial vasa vasorum were scattered around much
closer to theoretical optima. This scattering of branching
angles has been reported in the vasculature in general
(Zamir and Brown, 1982). More data, however, are needed
to evaluate these findings statistically.
ACKNOWLEDGMENTS
The authors thank Dr. W.D. Krautgartner for technical
support in SEM, Ms. Synnöve Tholo for assistance of the
SEM work, Mr. Andreas Zankl for image processing, Ms.
Birgit Peukert and Ms. Gudrun Thallinger for 3D data
acquisition, and T.R. Koll for programming Equations 1-6,
making calculations much easier. This work was supported by grants from the Charles University Prague (GA
UK 57/2002/C to D.K. and J.S.) and the Stiftungs- und
Förderungsgesellschaft der Paris-Lodron-Universität Salzburg (to A.L.).
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