Microvessel changes in hypertension measured by Griffonia simplicifolia I lectin.

A S Greene, J H Lombard, A W Cowley, Jr and F M Hansen-Smith

Hypertension. 1990;15:779-783
doi: 10.1161/01.HYP.15.6.779
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 779

Microvessel Changes in Hypertension

Measured by Griffonia simplicifolia I Lectin
Andrew S. Greene, Julian H. Lombard, Allen W. Cowley Jr., and Fay M. Hansen-Smith

Commonly used methods for assessing reductions in microvascular density (rarefaction) in

hypertension detect only perfused microvessels. In the present study, samples of cremaster and
spinotrapezius muscles were taken from rats with chronic (4-week) reduced renal mass
hypertension and normotensive sham-operated control rats, as well as from 12-week-old
spontaneously hypertensive rats and their normotensive Wistar-Kyoto control strain. Mean
arterial pressure was 149±8 mm Hg in the rats with reduced renal mass hypertension, 114±7
mm Hg in sham-operated rats, 177±9 mm Hg in spontaneously hypertensive rats, and 95±4
mm Hg in Wistar-Kyoto rats. Muscle samples were incubated with rhodamine-labeled Griffonia
simplicifolia I lectin, which identifies both perfused and nonperfused microvessels. Microvas-
cular density was assessed by counting intersections with a 20-/um grid. Microvessel density
was significantly reduced in cremaster muscles of both spontaneously hypertensive and reduced
renal mass hypertensive rats, and in the spinotrapezius muscle of spontaneously hypertensive
rats, compared with their respective normotensive controls. Further studies in the reduced
renal mass rats on low salt diets indicated that lectin binding was also decreased as salt intake
was increased, independent of blood pressure. This change was not due to an alteration in
lectin-binding affinity. These studies indicate that lectin binding can be a useful tool for
assessing microvessel density that does not depend on the perfusion state of the vessels and that
rarefaction due to hypertension is not evenly distributed in all vascular beds. These results also
provide evidence that dietary salt intake alone can influence microvessel density, as measured
by the lectin technique. {Hypertension 1990;15:779-783)

C onsiderable evidence has been accumulated

to indicate that both changes in vessel cali-
ber and vessel number can contribute to the
increased vascular resistance in hypertension.1-4 The
degree to which variations in microvessel density
alone contribute to the increase in total peripheral
resistance in hypertension has been debated.1-2*5'6
Several studies have suggested that decreases in
microvascular density occur in skeletal muscle in
hypertension,2-5-8 whereas others have shown no
change.9-10 In most of these studies, microvessel
density has been estimated either from intravital
measurements or from injection methods. Both of
these techniques, however, rely on the perfusion
status of the vessel at the moment of observation.
Studies in spontaneously hypertensive rats (SHR)5
From the Department of Physiology (A.S.G., J.H.L., A.W.C.),
Medical College of Wisconsin, Milwaukee, Wisconsin, and the
Department of Biological Sciences (F.M.H.-S.), OaJdand Univer-
sity, Rochester, Michigan.
Supported by grants HL-29587 and HL-37374 from the National
Institutes of Health and grant 890822 from the American Heart
Association.
Address for reprints: Andrew S. Greene, Department of Phys-
iology, Medical College of Wisconsin, 8701 Watertown Plank
Road, Milwaukee, WI 53226.
and in one-kidney, one clip renal hypertensive rats
indicate that active closure of arterioles precedes
anatomic rarefaction.6 Finally, cremasteric arterioles
of rats in the early phases of reduced renal mass
(RRM) hypertension are actively constricted and
exhibit active closure and a high degree of vaso-
motion.11 In these situations, an accurate measure of
true anatomic vascular density might be difficult due
to active constriction and a high incidence of com-
plete closure.
In this study, we have evaluated a histological
method for the determination of microvessel density
based on the specific binding of Griffonia simplicifolia
I (GS-I) lectin to microvascular structures. GS-I
lectin, or its B4 isomer, binds selectively to basement
membranes of microvessels,12 allowing for excellent
visualization of the microvasculature.13-15 The pres-
ent studies used rhodamine-labeled GS-I lectin to
stain muscle whole mounts, which were then evalu-
ated using computer fluorescence microscopy.
The objective of these experiments was to compare
the lectin method with previously obtained results
using vascular filling techniques in SHR.and RRM
hypertension. These comparisons were then used to
evaluate the degree of anatomic rarefaction observed
in the RRM model of hypertension.

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780 Hypertension Vol 15, No 6, Part 2, June 1990
FIGURE 1. Photomicrograph of a cremaster muscle labeled with fluorescent Griffonia simplicifolia /
lectin. Note that binding was confined to vessels less than 20 ym in diameter.
Methods
Renal Mass Reduction
Male Sprague-Dawley rats were subjected to a
75% reduction in renal mass by a two-stage surgical
procedure as previously described.16 The rats were 6
weeks old and 180-190 g in weight at the time of the
initial surgery. Age-matched normotensive control
rats underwent a sham operation (SOC) in which the
kidneys were exposed, cleared of perirenal fat, and
returned to the abdominal cavity. The rats were
randomly assigned to one of the experimental groups
herein described.
Experimental Groups
Three tofivedays after the final reduction in renal
mass, the rats in the RRM and SOC groups were
placed on either a high salt rat chow containing 4%
NaCl or a low salt chow containing 0.4% NaCl for 4
weeks. One group of normal age-matched rats was
placed on standard (0.8% NaCl) rat chow and
studied after 4 weeks to verify that structural
changes in the rats in the RRM group were the
result of the renal mass reduction-salt loading
procedure. All rats were allowed to drink water ad
libitum. SHR and normotensive Wistar-Kyoto
(WKY) rats were purchased at 12 weeks of age
from Harlan Labs (Madison, Wisconsin) and were
studied within 1 week of their arrival.
Hemodynamic Measurements
Rats were anesthetized with sodium pentobarbital
and placed on a heated table. A catheter was placed
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in the left carotid artery and connected to a pressure
transducer (Statham P23AC, Statham Instr. Div.,
Gould Inc., Oxnard, California) for the measurement
of blood pressure and heart rate.
Histochemical Studies
Immediately after measurement of hemodynamic
parameters, samples of the cremaster and spinotra-
pezius muscles were removed with a trephine and
immersed in 30 /i-g/ml rhodamine-labeled GS-I lectin
for 30 minutes to define the microvascular bed.13
After exposure of the tissue to the lectin, the muscle
was rinsed thoroughly in physiological salt solution
(PSS) and mounted on a microscope slide with a
water-soluble mountant. Samples were studied as
whole mounts by using a video fluorescent micro-
scope system with epi-illumination.
Figure 1 shows a photomicrograph of a cremaster
muscle labeled with rhodamine GS-I lectin. Micro-
vascular density was measured by counting the inter-
sections of fluorescently labeled microvessels with a
20-/Am computer-generated grid overlaying the
microscope field observed at x 300. Vessels of order
3 and below were visible after lectin treatment. No
attempt was made to classify vessels by order. Two
slides of each muscle were studied. Five fields from
each slide were randomly selected and counted. The
results from the 10 fields were averaged to give a
single density for each muscle.
Lectin-Binding Affinity
In a final series of experiments, muscle samples
were removed from normal Sprague-Dawley rats to
 Greene et al Rarefaction in Chronic Hypertension 781

TABUB 1. Hemodynamic Measurements

Group MAP HR
RRM
0.4% 104±13.7 314±20.3 7
4% 131±13.1*t 355 ±11.2* 14
soc
0.4% 105±4.2 303±10.1 7
4% 114±5.5 365 ±15.7* 17 Cramastar
Normal
0.8% 108±9.9 337±19 9
SHR 177±8.9* 351±9.9* 17
WKY 95±4.12 295 ±12.3 17
Values are mean±SEM. MAP, mean arterial pressure; HR,
heart rate; RRM, reduced renal mass; SOC, sham-operated
control; SHR, spontaneously hypertensive rats; WKY, Wistar- SHR WKY
Kyoto; LS, low salt diet; HS, high salt diet.
•Indicates significantly different from LS control rats. FIGURE 2. Bar graphs showing reduction of microvessel
tlndicates significantly different from HS SOC rats. density in spinotrapezius muscle (upper panel) and cremaster
^Indicates significantly different from WKY rats. muscle (lower panel) in spontaneously hypertensive rats
(SHR) (open bars) and Wistar-Kyoto (WKY) rats (solid bars),
evaluate the effect of sodium chloride concentration indicates significant differences from WKY rats.
on lectin-binding affinity. The muscle was homoge-
nized, and the homogenate was centrifuged. The
supernatant was discarded, and the pellet was resus- those of normotensive WKY control rats (Figure 2).
pended in PSS with sodium concentrations varying Vessel density was reduced by 14.1% in SHR cre-
between 135 and 155 meq/1. GS-I lectin was then master and 13.8% in SHR spinotrapezius muscles.
added to the suspension at a final concentration of 15 Vessel density was reduced by 22.8% in the cremas-
/xg/ml and incubated for 30 minutes at room temper- ter muscle of hypertensive rats in the salt-loaded
ature. The suspension was centrifuged, and the RRM group, relative to the normotensive SOC rats on
supernatant was saved. The pellet was then washed a high salt diet. Microvessel density in the spinotrape-
with three rinses of PSS, and the suspension was zius muscle, however, was not significantly different in
returned to its initial volume. Rhodamine-lectin con- the rats in the RRM and SOC groups on a high salt
centration in both the saved supernatant and the diet. Vessel densities in rats in the SOC group were
suspension was measured spectrophotometrically, not different from those measured in normal age-
matched Sprague-Dawley rats fed normal rat chow.
and a free/bound ratio was computed. These results are summarized in Figure 3.
Statistical Analysis
All results were expressed as the mean±SEM. Dif- 90
ferences between groups were analyzed by a two-factor
analysis of variance with no repeated measures. Signif-
80
icant differences between individual means were deter-
mined using Duncan's new multiple range test. Values
oip <0.05 were considered significant. 70

Results
Hemodynamic Parameters
BO
As previously reported, blood pressure was ele-
vated both in rats in the RRM group on high salt
diets16 and in SHR rats on normal chow.4 All other 80

groups of rats had normal blood pressures. Heart

rates were higher in rats on a high salt diet than the 70
respective control rats on a low salt diet although an "8 _L
elevation in blood pressure did not occur in some of
these groups. Heart rate and blood pressure for all
BO 1 RRM SOC
groups of rats are summarized in Table 1. FIGURE 3. Bar graphs showing microvessel density in
spinotrapezius muscle (upper panel) and cremaster (lower
Microvascular Density Changes panel) of hypertensive rats in the reduced renal mass (RRM)
Microvascular density, as demonstrated by the group (open bars) and normotensive sham-operated control
lectin technique, was reduced in both the cremaster (SOC) rats (solid bars). *Indicates significant differences from
and spinotrapezius muscles of the SHR, relative to SOC rats.

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782 Hypertension Vol 15, No 6, Part 2, June 1990
•3
HS LS
FIGURE 4. Bar graphs showing effect of salt on apparent
vessel density in normotensive sham-operated control rats.
Upper panel: Data from spinotrapezius muscle. Lower panel:
Data from cremaster muscle. Solid bars represent rats on low
salt diets (LS), and open bars represent rats on high salt diets
(HS). indicates significant difference from low salt group.
Although the lectin technique demonstrated vessel
rarefaction in both the SHR and the rats in the RRM
group, salt intake also had a significant effect on the
estimated vessel density, which was independent of
blood pressure. Figure 4 summarizes the microvessel
densities for rats in the SOC group on low and high
salt diets. Salt intake alone decreased measured
vessel densities by 16.5% in the cremaster muscle but
had no effect on the spinotrapezius muscle.
Lectin-Binding Affinity
The reduction in measured vessel density in rats on
a high salt diet was clearly not due to a direct effect
of sodium chloride on lectin binding. Increasing
sodium chloride concentration in five steps between
135 and 155 meq/1 in muscle homogenates did not
decrease the binding of the lectin molecule as mea-
sured by the free/bound ratio. Therefore, the affinity
of lectin binding to microvascular structures in mus-
cle homogenates was not affected by sodium chloride
concentration per se.
Discussion
The techniques that have been previously used to
measure microvascular rarefaction in hypertensive
animals are limited because of their reliance on
vessel perfusion to deliver a dye or afillingcompound
(India ink, silicone rubber compounds, or various
resins) to the microcirculation. In hypertension,
alterations in the vasculature can cause inhomoge-
neous tissue perfusion,1 increased vascular tone,6'11
excessive vasomotion,11 or vessel degeneration.2-7-8
Therefore, methods that depend on the perfusion
status of the vessel might not be suitable for quanti-
tation of vessel density.
The results of the present study indicate that the use
of a specific marker such as the GS-I lectin molecule,
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Low Salt Hlf h Salt tOf h Salt
3h»TTi or PHU Slum RRM
FIGURE 5. Bar graphs of summary of data indicating addi-
tive effect of salt and blood pressure on vessel density in
spinotrapezius muscle (upper panel) and cremaster muscle
(lower panel). Left bar is combined data from rats in low salt
sham-operated control (SOC) and low salt reduced renal mass
(RRM) groups. Rats in low salt SOC and low salt RRM groups
(left bar) as well as rats in high salt SOC group (middle bar)
were normotensive. Rats in high salt RRM group (right bar)
were hypertensive, indicates significantly different from low
salt. ^Indicates a significant difference from high salt sham.
which does not depend on the perfusion status of the
vessel, is valuable when assessing microvascular rar-
efaction. With this technique we have demonstrated
significant rarefaction in the cremaster muscle of hyper-
tensive rats in the RRM group and in SHR. Previous
studies using less specific techniques have measured
rarefaction of a similar magnitude in cremaster muscles
of SHR2 and RRM16 rats.
In the present study, we could not demonstrate
rarefaction in the spinotrapezius muscle of rats in the
RRM group. This heterogeneous distribution of rar-
efaction in the rats in the RRM model might be due
to a redistribution of arteriolar resistance resulting
from nonuniform closure of microvessels, which
probably proceeds to the anatomic rarefaction. This
nonuniformity could be due to differences in sympa-
thetic innervation or local autoregulatory responses
in these vascular beds.
Rarefaction in the microcirculation is reportedly
confined to the smallest vessel orders.2-5-7-11-16 Many of
thefillingtechniques that have been previously used do
not allow differentiation between vessel orders or pro-
vide any insight into structural integrity of the filled
vessels. Although the specific target molecule for lectin
binding is unknown, it does appear that the molecule
binds specifically with a-D-galactopyranosyl groups
associated with the capillary basal lamina12-14 and is
thus a specific and sensitive probe for a normal capil-
lary. In a previous study, we demonstrated that the
GS-I lectin probe binds strongly to capillaries and small
arterioles with a progressive decrease influorescenceof
the postcapillary venules and small veins. Minimal

binding was observed in vessels with diameters greater
than 20 ^m.13
In the present study, we observed a significant effect
of sodium intake on the apparent vessel density in the
cremaster muscle of both hypertensive rats in the RRM
group and normotensive rats in the SOC group. Vessel
density changes in the cremaster muscle of hyperten-
sive rats in the RRM group are in good agreement with
our previous studies using vascular filling techniques.16
The present experiments suggest that a high salt diet
alone might cause a loss of microvessels despite a
normal blood pressure. One change that is known to
occur when salt intake is elevated is a remodeling of
collagen in the vascular basement membranes.17 Our
recent ultrastructural studies suggest that high salt
intake might indeed haw effects on the morphology of
microvessels and the integrity of their basement
membranes.15 This loss of basement membrane integ-
rity might be an early stage of vessel degradation that
proceeds to anatomic rarefaction.
Another possibility that needs to be considered is
that alterations in vascular basement membranes
could lead to a loss of lectin-binding sites that would
result in the decrease in apparent vessel density
observed with high salt intake. Our in vitro studies on
lectin binding did not show any dependence of
lectin-binding affinity on sodium concentration.
Additionally, comparison studies using both the GS-I
lectin and alkaline phosphatase methods have shown
that GS-I lectin reveals significantly more capillaries
in animals on normal salt diets13 and that the binding
of the GS-I molecule is less affected by age and
capillary neoformation activity in adult muscle than
the alkaline phosphatase method.14 Thus, it appears
that the specific lectin binding to microvessels is quite
stable under a variety of physiological conditions.
Although the measured vessel density in the cre-
master muscle of rats in the RRM and SOC groups
was influenced by the sodium intake, in these exper-
iments, this effect was apparently additive to the
effect of an increase in blood pressure. Figure 5
summarizes our data and indicates that the effect of
salt, like the rarefaction itself, appears to be confined
to the cremaster muscle and suggests that both a high
salt diet and hypertension contribute to the loss of
blood vessels in the rats in the RRM group.
We have used the GS-I lectin technique to dem-
onstrate microvessel rarefaction equivalent to that
reported in other studies.7 We have confirmed the
presence of rarefaction in rats with chronic hyperten-
sion both with vascular filling16 and ultrastructural
studies.15 Additionally, SHR and WKY rats on nor-
mal salt chow exhibited large differences in vessel
density by the lectin technique. This reduction of
vessel density demonstrated by the lectin technique
in SHR was independent of salt intake. Thus, it
appears that the GS-I lectin technique allows a
specific and perfusion-independent assessment of
changes in the microvasculature that result from
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Greene et al Rarefaction in Chronic Hypertension 783
hypertension and might provide a valuable tool in
understanding the early changes in microvessel mor-
phology that lead to rarefaction.
Acknowledgments
We thank Ann Kosmatka and Luellen Lougee for
their dedicated technical assistance.
References
1. Greene AS, Tonellato PJ, Lui J, Lombard JH, Cowley AW Jr:
Microvascular rarefaction and tissue vascular resistance in
hypertension. Am J Physio! 1989;256:H126-H131
2. Prewitt RL, Chen IIH, Dowell R: Development of microvas-
cular rarefaction in the spontaneously hypertensive rat. Am J
Physiol 1982;243:H243-H251
3. Short DS, Thomson AD: The arteries of the small intestine in
systemic hypertension. / Pathol Bacterial 1959;78:321-334
4. Lombard JH, Hess ME, Stekiel WJ: Neural and local control
of arterioles in SHR. Hypertension 1984;4:530-535
5. Chen IIH, Prewitt RL, Dowell RF: Microvascular rarefaction
in spontaneously hypertensive rat cremaster muscle. Am J
Physiol 1981;241:H306-H310
6. Prewitt RL, Chen IIH, Dowell RF: Microvascular alterations
in the one-kidney, one-clip renal hypertensive rat.,4m J Physiol
1984;246:H728-H732
7. Hutchins PM, Darnell MS: Observation of a decreased num-
ber of small arterioles in spontaneously hypertensive rats. Ore
Res 1974;34-35(suppl I):I-161-I-165
8. Henrich HL, Romen W, Heimgartner W, Hartung E, Baumer
F: Capillary rarefaction characteristic of the skeletal muscle of
hypertensive patients. Klin Wochenschr 1988;66:54-60
9. Gray SD: Lack of capillary rarification in skeletal muscle of
spontaneously hypertensive rats, in Rascher W, Clough D,
Ganten D (eds): Hypertensive Mechanisms: The Spontaneously
Hypertensive Rat as a Model to Study Human Hypertension.
Stuttgart, FK Schattauer Verlag, 1982, pp 204-207
10. Engelson ET, Schmid-Schonbein GW, Zweifach BW: The
microvasculature in skeletal muscle: II. Arteriolar network
anatomy in normotensive and spontaneously hypertensive
rats. Microvasc Res 1986;31:356-374
11. Lombard JH, Greene AS, Cowley AW, Liard JF: Microcircu-
lation in rats with volume-expanded hypertension, in Hansson
L, Omae T (eds): Mechanisms in Hypertension: New Aspects in
Hemodynamics. New York, Raven Press Publishers, 1989, pp
11-20
12. Peters BP, Goldstein II: The use of fluorescein-conjugated
Bandeiraca simplicifolia B4-isolectin as a histochemical reagent
for the detection of a-D-galactopyranosyl groups: Their occur-
rence in basement membranes. Exp Cell Res 1979;12O:321-324
13. Hansen-Smith FM, Watson L, Lu DY, Goldstein I: Griffonia
simplicifolia I: Fluorescent tracer for microcirculatory vessels
in non-perfused thin muscles and sectioned muscle. Microvasc
Res 1988;36:199-215
14. Hansen-Smith FM, Watson L, Joswiak GR: Postnatal changes
in capillary density of rat sternomastoid muscle. Am J Physiol
1989;257(//eart Ore Physiol 26):H344-H347
15. Hansen-Smith F, Greene AS, Cowley AW Jr, Lombard JH:
Structural changes during micTovascular rarefaction in chronic
hypertension. Hypertension 1990;15:922-928
16. Lombard JH, Hinojosa-Laborde C, Cowley AW Jr: Hemody-
namics and microcirculatory alterations in reduced renal mass
hypertension. Hypertension 1989;13:128-138
17. Hazema F, Ooshima A, Tanaka T, Tomimoto K, Okamoto K:
Vascular lesions in the various substrains of spontaneously
hypertensive rats and the effects of chronic salt ingestion. Jpn
Ore J 1975;39:7-22
KEY WORDS • blood pressure • blood flow • microcirculation
• histology • fluorescent indicators • vascular tissue • capillaries
• rarefaction • reduced renal mass hypertension