Renin Gene Transfer Restores Angiogenesis and Vascular
Endothelial Growth Factor Expression in Dahl S Rats
Sandra L. Amaral, Richard J. Roman, Andrew S. Greene
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Abstract—In a previous study, we demonstrated that Dahl S rats (SS group) have low plasma renin activity, whereas
transfer of a region of chromosome 13 containing the renin gene from Dahl R onto a congenic strain of Dahl
SS/Jr/Hsd/MCW rats (S/renRR group) restores renin secretory responses. In the present study, we compared the
angiogenic responses to electrical stimulation in the SS and S/renRR groups to explore the hypotheses that the
renin-angiotensin system is involved in vascular endothelial growth factor (VEGF) expression and angiogenesis in
skeletal muscle. Congenic SS and S/renRR rats fed a 0.4% or 4% salt diet were surgically prepared by chronic
implantation of an electrical stimulator. Another group of S/renRR rats was treated with lisinopril 2 days before the
surgery and throughout the stimulation protocol. The right tibialis anterior (TA) and extensor digitorum longus (EDL)
were stimulated for 8 hours per day for 7 days. The contralateral muscles served as controls. Western blot analysis was
performed to identify VEGF protein expression in these muscles. Electrical stimulation produced no change in vessel
density of the SS group fed a 0.4% salt diet (change 5.50% and 8.14% for EDL and TA, respectively). Transfer of a
region containing the renin gene restored the angiogenic response (change 16% and 30% for EDL and TA, respectively)
despite a significantly higher blood pressure. Blockade of the renin-angiotensin system by lisinopril or high salt restored
the responses observed in the SS group fed a low salt diet. In addition, increases in VEGF expression to electrical
stimulation were observed only in the S/renRR group fed a low salt diet. These results suggest that renin gene transfer
restores angiogenesis and VEGF expression in the skeletal muscle of Dahl S rats. (Hypertension. 2001;37[part 2]:386390.)
Key Words: blood vessels 䡲 muscle, skeletal 䡲 growth substances 䡲 renin-angiotensin system 䡲 chromosome 13
T
he renin-angiotensin system (RAS) plays an important
physiological role in the regulation of arterial pressure
and body fluid volume. Angiotensin II (Ang II) has also been
implicated in the formation of new vessels in vitro1 and in the
physiological angiogenesis induced by exercise2 or electrical
stimulation.3 Although results from our laboratory have
demonstrated that continuous infusion of Ang II increases
vessel density in the skeletal muscle of rats,4 the role of Ang
II in angiogenesis is still controversial. ACE inhibitors have
been reported to either attenuate the angiogenesis3,4 or increase the vessel density.5 More recently, we have shown that
the RAS can also modulate the angiogenic response to
electrical stimulation in the skeletal muscle of rats, and we
have proposed a pathway involving Ang II and vascular
endothelial growth factor (VEGF) expression.3
VEGF, a 45-kDa homodimeric glycoprotein mitogen, has
been considered to be an important regulator of angiogenesis,
which increases vascular permeability and promotes endothelial proliferation.6 Results from our laboratory have supported
the hypothesis that VEGF is an important regulator of the
angiogenesis induced by electrical stimulation, inasmuch as
the angiogenic response was inhibited by a VEGFneutralizing antibody.3
In our laboratory,7 we have derived a congenic strain of
Dahl SS/Jr/Hsd/MCW (SS) rats, in which 10 cm of a region
of chromosome 13 containing the renin gene of Dahl R rats
was transferred onto the Dahl S genetic background (S/renRR).
We have previously demonstrated that although the SS rat is
unable to modulate renin activity in response to alterations in
sodium intake, transfer of the resistant renin gene confers
normal salt sensitivity to the S/renRR rat.7 These congenic rats
provide a model to evaluate the importance of the modulation
of the RAS in VEGF expression and angiogenesis in skeletal
muscle. In the present study, we have used the chromosome
13 congenic rat to test the hypothesis that VEGF expression
and physiological angiogenesis in skeletal muscle induced by
electrical stimulation can be modulated by the RAS. A map of
chromosome 13 is shown in Figure 1.
Methods
Animal Surgery and Experimental Protocol
The Medical College of Wisconsin (MCW) Institutional Animal
Care and Use Committee approved all animal protocols. Animals
were housed and cared for in the MCW Animal Resource Center and
were given food and water ad libitum. Experiments were performed
Received October 24, 2000; first decision November 30, 2000; revision accepted December 18, 2000.
From the Department of Physiology, Medical College of Wisconsin, Milwaukee.
Correspondence to Andrew S. Greene, PhD, Medical College of Wisconsin, 8701 Watertown Plank Rd, PO Box 26509, Milwaukee, WI 53226-0509.
E-mail [email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
386
Amaral et al
VEGF Expression in Chromosome 13 Congenic Rats
387
was taken from TA muscle and immediately frozen in liquid nitrogen
for Western analysis. The remaining TA and the EDL were lightly
fixed overnight in 0.25% formalin solution and sectioned.8 From
every animal, sections of EDL and TA were immersed in a solution
of 25 ␮g/mL rhodamine-labeled Griffonia simplicifolia I lectin
(Sigma). After a 2-hour exposure to G simplicifolia I lectin, the
muscles were rinsed and mounted on microscope slides.9 The
sections were visualized by using a video fluorescent microscope
system with epi-illumination (Olympus ULWD CD Plan, ⫻20
objective). Ten representative fields were selected for study from
each muscle section. Images were digitized and quantified with an
automated computer vessel counting as previously described.10
Plasma Renin Activity
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The method used for measurements of plasma renin activity (PRA)
was previously described by Rieder et al.11 Arterial blood was
collected in tubes containing K3EDTA and immediately centrifuged
at 1500g and 4°C. The samples were thawed on ice, and neomycin
sulfate (0.1%), phenylmethylsulfonyl fluoride (0.25%), and maleic
anhydrate (0.2 mol/L) were added to 50 ␮L of sample to inhibit
converting enzyme and protease activity. After generation, the Ang
I concentration was determined by radioimmunoassay.11
Figure 1. Map of rat chromosome 13 illustrating location of
polymerase chain reaction markers used for genotyping of congenic S/renSS rats. All markers used were polymorphic in SS/Jr
and SR/Jr rats, and both the S/renRR and S/renSS strains were
homozygous for the SS/Jr allele at all the markers shown
except for the renin marker. Therefore, the segment transferred
from the Dahl R onto Dahl SS/Jr/Hsd/MCW rats lies between
markers D13N1 and Syt2 and is ⬇10 cM in length.7
on SS rats and S/renRR rats, a congenic strain in which 10 cm of
chromosome 13 spanning the region of the renin gene has been
transferred onto the Dahl S background by backcross breeding.
Forty-five male SS and S/renRR rats, aged 8 to 9 weeks, were
anesthetized with an intramuscular injection of a mixture of ketamine
(100 mg/kg) and acepromazine (2 mg/kg). Under aseptic conditions,
a subcutaneous incision was made over the thoracolumbar region,
and the rats received a miniature battery-powered stimulator that was
previously designed and validated for chronic studies by our laboratory.8 After a 24-hour recovery period, the ankle flexor muscles
(tibialis anterior [TA] and extensor digitorum longus [EDL]) were
stimulated by electrodes localized in the vicinity of the common
peroneal nerve, with square-wave impulses of 0.3-ms duration,
10-Hz frequency, and 3-V potential.8 The contractions of the EDL
and TA were sustained for 8 hours per day over a period of 7
consecutive days. The rats were subdivided into the following
groups: experiment 1, 6 SS rats fed 0.4% NaCl throughout the entire
stimulation protocol (SS-LS group); experiment 2, 6 S/renRR rats fed
0.4% NaCl throughout the entire stimulation protocol (S/renRR-LS
group); experiment 3, 7 S/renRR rats fed 4% NaCl throughout the
entire stimulation protocol (S/renRR-HS group); and experiment 4, 6
S/renRR rats fed 0.4% NaCl and given lisinopril (100 mg/kg per day)
in their drinking water 2 days before the surgery and throughout the
entire stimulation protocol (S/renRR-LSLIS group). Additionally, 6
SS-LS, 6 S/renRR-LS, 5 S/renRR-HS, and 3 S/renRR-LSLIS animals
underwent the implantation surgery, but the stimulators were never
turned on, and the animals were euthanized 7 days later (sham
group).
The effectiveness of chronic inhibition of ACE by oral lisinopril
was determined by an intravenous bolus of angiotensin I (Ang I, 0.1
mL at 1 ng/mL IV).3
Tissue Harvest and Morphological Analysis of
Vessel Density
After 7 days of stimulation, the animals were euthanized by an
overdose of Beuthanasia solution (Sigma Chemical Co), and the
stimulated and contralateral unstimulated muscles were removed,
rinsed in physiological salt solution, and weighed. A 100-mg section
Western Blot Analysis to Detect the Presence of
VEGF Protein
The 100 mg of TA was homogenized, and the protein was suspended
in 0.1 mol/L potassium buffer (KPO4). Five micrograms of protein
(as determined by protein assay kit, Bio-Rad) from each muscle and
a tumor cell line, known to express VEGF at high levels (C6,
American Type Culture Collection, 107-CCL), were separated on a
12% denaturing polyacrylamide gel. The gels were transferred to a
nitrocellulose membrane, which was blocked for 2 hours in 5%
nonfat dry milk diluted in Tris-buffered saline (50 mmol/L Tris and
750 mmol/L NaCl, pH 8) with 0.08% Tween 20 (Bio-Rad). The blots
were then incubated with a polyclonal antibody to a peptide derived
from the human VEGF sequence (1:1000 dilution, clone G143-850,
Pharmingen) for 2 hours at room temperature. Washed blots were
then incubated with goat anti-mouse secondary antibody at a dilution
of 1:1000 for 1 hour at room temperature and then subjected to a
SuperSignal West Dura chemiluminescence substrate (Pierce) detection system. Membranes were exposed to x-ray film (Fuji Medical)
for 30 seconds and developed by using a Kodak M35 X-Omat
processor. For the quantitative VEGF analysis, film was always
exposed for a period of time that ensured that all signals were within
the linear range of the detection of the film. The VEGF band
intensity was quantified by using a Scion Image Beta 4.02 for
Windows (Scion Corp), and values were expressed as a percentage
of the C6 tumor cell standard.
Data Analysis and Statistics
For each muscle, the vessel counts of all the selected fields were
averaged to a single-vessel density. Vessel density was expressed in
terms of mean number of vessel-grid intersections per microscope
field (0.224 mm2). For each experimental group, the measured vessel
density of the stimulated muscle was compared with its unstimulated
counterpart. All values are presented as mean⫾SEM. The significance of differences in values measured in the same animal was
evaluated by a 2-factor ANOVA (diet⫻stimulation) with repeated
measures on 1 factor (stimulation). To evaluate the significance of
differences in vessel density between stimulated and unstimulated
sides, a 1-way ANOVA was performed. Significant differences were
further investigated by a post hoc test (Tukey).
Results
The Table summarizes the average of body weight and EDL
and TA muscle weight/body weight ratios of all groups
analyzed. All rats were approximately the same age (8 to 9
weeks) at the start of the experiments, and 7 days of
stimulation did not change the body weight, except for the
388
Hypertension
February 2001 Part II
Body Weights and EDL and TA Muscle Weight–to–BW Ratios
BW, g
Groups
Start Weight
End Weight
U (n⫽6)
268.33⫾7.65
283.00⫾7.65
S (n⫽6)
䡠䡠䡠
EDL/BW
TA/BW
SS-LS
䡠䡠䡠
0.044⫾0.01 0.194⫾0.01
0.043⫾0.01 0.195⫾0.01
S/renRR-LS
U (n⫽6)
S (n⫽6)
320.67⫾7.65† 330.83⫾7.65†
䡠䡠䡠
䡠䡠䡠
0.042⫾0.01 0.204⫾0.01
0.042⫾0.01 0.206⫾0.01
S/renRR-HS
U (n⫽7)
S (n⫽7)
320.29⫾7.08† 345.43⫾7.08*† 0.043⫾0.01 0.194⫾0.03
䡠䡠䡠
䡠䡠䡠
0.041⫾0.01 0.189⫾0.03
S/renRR-LSLIS
U (n⫽6)
S (n⫽6)
319.33⫾7.65† 328.83⫾7.65†
0.039⫾0.01 0.189⫾0.01
0.039⫾0.01 0.185⫾0.01
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䡠䡠䡠
䡠䡠䡠
Values are mean⫾SEM. BW indicates body weight; U, unstimulated; S,
stimulated; and n, number of muscles in each group. Muscle weights are given
in mg/kg units.
*P⬍0.05 vs before stimulation; †P⬍0.05 vs S/renSS-LS.
S/renRR-HS group. The difference found between SS-LS and
other groups at the beginning of the stimulation period was
maintained up to the end. After 7 days of stimulation, there
were no differences in the muscle weights when they were
normalized by the body weight.
The mean arterial pressure of the S/renRR-LS group was
significantly higher than that of the SS-LS group
(148.67⫾1.12 versus 113.3⫾2.25 mm Hg, respectively).
When the high salt diet was introduced to the S/renRR animals
(S/renRR-HS), the mean arterial pressure increased to
172.0⫾4.78 mm Hg. The blockade of ACE in S/renRR-LS
rats, by lisinopril, effectively lowered the mean arterial
pressure to 100.76⫾7.46 mm Hg (S/renRR-LSLIS). The control
value of PRA in the SS-LS group was (1.69⫾0.50 ng Ang
I/mL per hour), and this value was not affected by increasing
dietary sodium intake (1.27⫾0.52 ng Ang I/mL per hour for
the SS-HS group). Transfer of the region containing the renin
gene restored the PRA responses. The renin activity was high
in the S/renRR-LS group (4.59⫾1.51 ng Ang I/mL per hour)
and was suppressed by 72% with a high salt diet (1.29⫾0.22
ng Ang I/mL per hour for S/renRR-HS).
In contrast to previous findings in other strains of rats,3,8 7
days of electrical stimulation resulted in no increase in vessel
density in either the TA (Figure 2, top left) or EDL (Figure 2,
top right) of SS rats on a low salt diet. Transfer of the region
of chromosome 13 containing the normally functioning renin
gene from the R rat onto the S background (S/renRR) restored
the angiogenic response to electrical stimulation in both TA
(Figure 2, top left) and EDL (Figure 2, top right). In the
S/renRR group, the suppression of RAS by high salt or
blockade of the RAS by lisinopril restored the antiangiogenic
phenotype observed in the SS rats in both muscles (Figure 2).
Neither sodium intake nor ACE blockade had any effect on
vessel density in the unstimulated muscles of either rat strain.
Vessel density analysis on sham-operated animals showed
that stimulation alone had no effect on vessel density.
Figure 2. Top, Changes in vessel density of TA (left) and EDL
(right) in SS-LS (n⫽6), S/renRR-LS (n⫽6), S/renRR-HS (n⫽7), and
S/renRR-LSLIS (n⫽6) groups after 7 days of electrical stimulation.
Bottom, Percentage of increment of vessel density from stimulated to unstimulated side in TA (left) and EDL (right). *P⬍0.05
vs unstimulated side (top) and vs SS-LS group (bottom).
Because we have previously shown that the angiogenesis
induced by electrical stimulation is largely mediated by
VEGF, Western blot analysis was performed to detect the
presence of VEGF protein in stimulated and control TA
muscles of congenic rats (Figure 3, top). A tumor cell line,
known to be rich in VEGF protein, was used as a positive
control. Figure 3 (top) shows that the intensity of the bands
was relatively high for tumor cells (VEGF, lane 1) and
stimulated S/renRR-LS rats (VEGF, lane 5). In contrast, the
contralateral unstimulated muscle had very faint band intensity (lane 4). Treatment with high salt diet and lisinopril did
not alter the intensity of the bands of unstimulated muscles
(lanes 2, 4, and 8) and also blocked the presence of VEGF
protein in rats stimulated for 7 days (lanes 7 and 9).
Quantitative densitometry was used to compare the responses
of VEGF after 7 days of stimulation in all of the animals. As
shown in Figure 3 (bottom), VEGF protein levels were
significantly increased by stimulation in only the S/renRR-LS
group (P⬍0.05).
Discussion
The present study demonstrates for the first time that the
transfer of a region of chromosome 13 containing the renin
gene from Dahl R rats onto the Dahl S genetic background
restores renin secretory responses and the angiogenic and
VEGF responses to electrical stimulation.
We have previously transferred in our laboratory7 10 cM of
a region of chromosome 13 containing the renin gene from
the Dahl R rat onto the Dahl S background and derived a
congenic strain of Dahl SS/Jr/HsD/MCW rat (S/renRR). Results from the present study confirmed that in SS rats, PRA is
lower and unresponsive to known physiological stimuli of the
RAS. In contrast, the congenic S/renRR group exhibited the
expected changes in PRA. In the present study, we investigated the effects of electrical stimulation on VEGF protein
Amaral et al
VEGF Expression in Chromosome 13 Congenic Rats
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Figure 3. Top, Western Blot of VEGF in TA. For each sample,
50 ␮g of total protein was loaded. Lanes are as follows: lane 1,
positive control (tumor cells C6); lanes 2, 4, 6, and 8, muscles
from unstimulated sides (U) of SS-LS, S/renRR-LS, S/renRR-HS,
and S/renRR-LSLIS groups, respectively; and lanes 3, 5, 7, and 9,
stimulated skeletal muscle (S) of SS-LS, S/renRR-LS, S/renRR-HS,
and S/renRR-LSLIS groups, respectively. The expression of VEGF
induced by electrical stimulation was observed in only the
S/renRR-LS group. Bottom, Quantitative densitometry of VEGF
protein in all groups of rats: S/renSS-LS (SS-LS, n⫽4),
S/renRR-LS (n⫽4), S/renRR-HS (n⫽4), and S/renRR-LSLIS (n⫽3).
Values are expressed as percentage of control (tumor cells).
expression and angiogenesis in these rats by use of 2 different
designs, physiological and pharmacological manipulations of
the RAS.
Physiological angiogenesis in skeletal muscle as a result of
electrical stimulation3,8,12–15 and physical exercise16,17 has
been extensively studied. However, the mechanisms responsible for angiogenesis in adult animals remain unclear.
Angiogenesis in adult skeletal muscle does not necessarily
follow the standard description: capillary growth may occur
without the breakage of the basement membrane and without
proliferation of endothelial cells, whereas proliferation may
precede and not follow migration.14 Several growth factors
have been postulated to play an important role in the
vasculogenesis and differentiation of the cells in the vascular
wall, including smooth muscle cells. Among them, VEGF has
been considered to be a potent angiogenic factor and a
specific mitogen for endothelial cells.6,18 Our previous results3 support the hypothesis that VEGF plays an important
role in regulating the angiogenesis induced by electrical
stimulation, inasmuch as the angiogenic response was inhibited by treatment with VEGF-neutralizing antibody.
Increases in VEGF have been found after electrical stimulation and exercise. Breen et al19 and, more recently, Gavin
et al20 have demonstrated that 1 hour of exercise promotes a
3- to 4-fold increase in VEGF mRNA, and Hang et al21 have
shown a 6-fold increase in VEGF mRNA in TA and EDL
after 4 days of electrical stimulation. Although these authors
389
have shown increases in VEGF mRNA expression, ours is the
first study to document increases in VEGF protein and vessel
density in the same animals.
The regulation of VEGF expression is not completely
understood. VEGF can be induced by several factors, such as
adenosine,22 cytokines,6 hypoxia,6,23 NO,15,20 growth factors
(such as platelet-derived growth factor and basic fibroblast
growth factor),24 and Ang II.1,25 In the present study, we
speculated on the involvement of the RAS in angiogenesis
and VEGF protein expression in electrically stimulated skeletal muscle. The hypothesis that Ang II can induce VEGF has
been shown in cell culture. Chua et al1 and Pupilli et al26 have
demonstrated increases in VEGF mRNA in response to Ang
II and have shown that this response was mediated by Ang II
type 1 (AT1) receptors. Previous data from our laboratory
have confirmed this hypothesis also in vivo and have demonstrated that Ang II, when infused continuously in conscious
rats, produces increases in the vessel density of the cremaster
muscle and that this action also is mediated by the AT1
receptor.4 More recently, we have demonstrated one pathway
involving AT1 receptors of Ang II and VEGF protein expression in electrically stimulated angiogenesis in skeletal muscle.3 Taken together, these results suggest that the RAS may
be involved in the regulation of VEGF protein in vivo as well
as angiogenesis.
Results from the present study have confirmed the hypothesis that the RAS has a role in angiogenesis and that VEGF
expression implicates a defect in the renin gene as the
causative factor for impaired angiogenesis in Dahl S rats.
After 7 days of electrical stimulation, both increases in VEGF
expression and angiogenesis in skeletal muscle were absent in
SS rats. In contrast, the transfer of 10 cM of chromosome 13,
containing the renin gene (S/renRR), restored the angiogenic
response in EDL (16% increase) and TA (30% increase),
despite significant higher blood pressure in the S/renRR group.
To confirm the importance of the RAS in VEGF expression
and angiogenesis in these strains, we used pharmacological
suppression and pharmacological inhibition. Although the
angiogenic response induced by electrical stimulation was
still present in the TA of the S/renRR-HS group, we observed
that suppression of the RAS by a high salt diet significantly
attenuated the increases in vessel density when we compared
the differences between the stimulated and unstimulated side
of the S/renRR-LS and S/renRR-HS groups. The blockade of
RAS by lisinopril abolished this response. On the other hand,
both suppression and blockade of RAS by high salt diet and
lisinopril, respectively, restored the responses observed in the
EDL of the SS group. These effects were likely due to
inhibition of the RAS, although the possibility of other
angiogenic factors arising from electrical stimulation cannot
be completely eliminated.
We have shown previously2 that inhibition of ACE effectively blocked the angiogenesis induced by a short-term
exercise program, and more recently, Amaral et al3 have
suggested that the VEGF expression and angiogenesis, induced by electrical stimulation, could be modulated by Ang
II. In these studies, a causative link between VEGF expression and vessel growth was made through the use of a
VEGF-neutralizing antibody.
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February 2001 Part II
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These findings regarding Ang II have important implications for the role of RAS in the normal physiology of blood
vessels in vivo. Although Ang II has been classically defined
as an endocrine substance acting on blood pressure regulation, many tissues express endogenous RAS activity, implying that locally generated Ang II is involved in autocrine/
paracrine regulatory mechanisms. Blood vessels contain all
components of RAS27; thus, Ang II could act as an autocrine
hormone to regulate VEGF as well as vascular permeability
factor, allowing the vascular permeability factor to act as a
paracrine hormone to regulate the permeability of the overlying endothelium.28 Although we did not measure the tissue
Ang II in these animals, previous studies using the same
model have suggested that the circulating RAS is not responsible for the electrically induced increases in vessel density.
In summary, our data showed that VEGF expression and
angiogenesis in skeletal muscle were absent in the SS-LS
group and reestablished in the S/renRR-LS group. We have
also shown that the suppression of RAS completely blocked
the VEGF expression and attenuated the angiogenesis. These
results are consistent with the hypothesis that genetic or
pharmacological manipulations of the RAS may contribute to
the angiogenesis and VEGF protein expression in skeletal
muscle induced by electrical stimulation.
Acknowledgments
This work was supported by National Institutes of Health grant
HL-29587. Dr Amaral was supported by a fellowship from FAPESP
(Fundacão de Amparo a pesquisa do Estado de São Paulo) 1998/
13772-0. The authors thank Jennifer Clark, Lisa Henderson, Camille
Torres, and Michael R. Kloehn for expert technical assistance.
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Sandra L. Amaral, Richard J. Roman and Andrew S. Greene
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Hypertension. 2001;37:386-390
doi: 10.1161/01.HYP.37.2.386
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