Scientific Contributions

Renin Gene Transfer Restores Angiogenesis and Vascular Endothelial Growth Factor Expression in Dahl S Rats

Sandra L. Amaral, Richard J. Roman, Andrew S. Greene
https://doi.org/10.1161/01.HYP.37.2.386
Hypertension. 2001;37:386-390
Originally published February 1, 2001

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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/ren RR group) restores renin secretory responses. In the present study, we compared the angiogenic responses to electrical stimulation in the SS and S/ren RR 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/ren RR rats fed a 0.4% or 4% salt diet were surgically prepared by chronic implantation of an electrical stimulator. Another group of S/ren RR 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/ren RR 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.

The 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 VEGF-neutralizing 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⇓.

Figure 1.
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

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 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 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 μg/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

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 μL of sample to inhibit converting enzyme and protease activity. After generation, the Ang I concentration was determined by radioimmunoassay.11

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 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.

View this table:
Table 1.

Body Weights and EDL and TA Muscle Weight–to–BW Ratios

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.
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).

Figure 3.
Figure 3.

Top, Western Blot of VEGF in TA. For each sample, 50 μg 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).

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 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 13 14 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 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.

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.

  • Received October 24, 2000.
  • Revision received November 30, 2000.
  • Accepted December 18, 2000.

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  • Renin Gene Transfer Restores Angiogenesis and Vascular Endothelial Growth Factor Expression in Dahl S Rats
    Sandra L. Amaral, Richard J. Roman and Andrew S. Greene
    Hypertension. 2001;37:386-390, originally published February 1, 2001
    https://doi.org/10.1161/01.HYP.37.2.386
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