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Am J Physiol Heart Circ Physiol 291: H114-H120, 2006. First published February 3, 2006; doi:10.1152/ajpheart.01116.2005
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Angiotensin II infusion restores stimulated angiogenesis in the skeletal muscle of rats on a high-salt diet

Matthew C. Petersen, Diane H. Munzenmaier, and Andrew S. Greene

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 21 October 2005 ; accepted in final form 20 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Elevated dietary salt intake has previously been demonstrated to have dramatic effects on microvascular structure and function. The purpose of this study was to determine whether a high-salt diet modulates physiological angiogenesis in skeletal muscle. Male Sprague-Dawley rats were placed on a control diet (0.4% NaCl by weight) or a high-salt diet (4.0% NaCl) before implantation of a chronic electrical stimulator. After seven consecutive days of unilateral hindlimb muscle stimulation, animals on control diets demonstrated a significant increase in microvessel density in the tibialis anterior muscle of the stimulated hindlimb relative to the contralateral control leg. High salt-fed rats demonstrated a complete inhibition of this angiogenic response, as well as a significant reduction in plasma ANG II levels compared with those of control animals. To investigate the role of ANG II suppression on the inhibitory effect of high-salt diets, a group of rats that were fed high salt were chronically infused with ANG II at a low dose. Maintenance of ANG II levels restored stimulated angiogenesis to control levels in animals fed a high-salt diet. Western blot analysis indicated that inhibition of angiogenesis in high salt-fed rats was not due to changes in VEGF or VEGF receptor type 1 protein expression in response to stimulation; however, the degree to which VEGF receptor 2 protein increased with stimulation was significantly lower in high salt-fed animals. This study demonstrates an inhibitory effect of high salt intake on stimulated angiogenesis and suggests a critical role for ANG II suppression in mediating this antiangiogenic effect.

vascular endothelial growth factor; electrical stimulation

HIGH DIETARY SALT INTAKE has long been known to impact the cardiovascular system. Classically, the deleterious effects of salt on the vasculature have been attributed to the development of hypertension in salt-sensitive animal models and in human patients (for review, see Ref. 39). However, a substantial body of evidence has suggested blood pressure-independent effects of high salt on vascular structure and function. Study of salt-loaded normotensive animals has been valuable in elucidating these effects.

Increased salt consumption has been demonstrated to mediate pressure-independent vascular remodeling at the level of large conduit arteries and smaller resistance vessels (39). Importantly, the structure of the microcirculation is also impacted, as inward eutrophic remodeling of small-order arterioles has been noted in salt-loaded normotensive rats (14). These structural changes have been associated with changes in microvascular function, including impaired myogenic responsiveness (29) as well as reductions in the dilatory response of resistance arterioles to various vasoactive stimuli (14). High dietary salt appears to have no effect on arteriolar density in skeletal muscle (7) but has been shown to stimulate significant rarefaction of capillaries in the cremaster muscle of rats after 4 wk of diet administration, even in the absence of the development of hypertension (16).

The effects of high salt on microvascular structure and function in the absence of hypertension have been largely attributed to the suppression of renin-angiotensin system (RAS) activity by inhibition of renal renin release. Maintenance of normal circulating levels of ANG II in high salt-fed animals by exogenous infusion has been associated with restoration of normal arteriolar reactivity responses (42) and prevention of high salt-induced capillary rarefaction (17).

ANG II has also been demonstrated to play a critical role in angiogenesis, or growth of new capillaries, in skeletal muscle. Chronic infusion of ANG II at a dose that has no effect on blood pressure has been demonstrated to cause significant capillary growth (24). In addition, inhibition of RAS activity with the use of pharmacological blockers of ANG II formation and blockade of the angiotensin type 1 (AT1) receptor significantly attenuated the angiogenic response of normotensive rats to exercise (2) and increased muscle activity induced by chronic electrical stimulation of muscle contraction (1). These studies are further supported by reports in mice that angiogenesis in ischemic muscle is dependent on ANG II acting on the AT1 receptor (12, 35).

Although significant evidence supports a role for ANG II in the angiogenic process, the hypothesis that elevated salt intake will inhibit physiological angiogenesis by suppressing ANG II remains largely untested. The purpose of this study was to directly examine the antiangiogenic effect of a high-salt diet (HSD) in working skeletal muscle and to determine the role of ANG II on this effect. Our results demonstrate that a HSD dramatically inhibits stimulated angiogenesis in muscle and further suggests a critical role for ANG II suppression in mediating the inhibitory effect of elevated salt on angiogenesis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal surgery. All animal protocols were approved by the Medical College of Wisconsin (MCW) Institutional Animal Care and Use Committee. Animals were housed and cared for in the MCW Animal Resource Center and were given food and water ad libitum. Thirty-three 7-wk-old male Sprague-Dawley rats were anesthetized with an intramuscular injection of a mixture of ketamine (100 mg/kg), xylazine (50 mg/kg), and acepromazine (2 mg/kg). Under aseptic conditions, polyethylene catheters were placed in the left carotid artery and left jugular vein, tunneled subcutaneously, and exteriorized at the back of the neck. The catheters were then passed through a flexible spring that was secured to the rat subcutaneously. The spring was attached to a swivel above the cage, allowing the animal full range of movement in the cage while protecting the catheters. Immediately after cannulation of the vessels, an incision was made over the thoracolumbar region, and a miniature battery-powered stimulator, previously designed and validated for chronic studies by our laboratory (19), was implanted subcutaneously and secured in place. A second incision was made in the skin and fascia covering the lateral side of the knee joint (over the region of the common peroneal nerve) of the right hindlimb. A pair of electrodes was guided under the skin from the stimulator and secured to the muscles surrounding the knee in close proximity to the common peroneal nerve with surgical suture. The skin over both incisions was sutured closed, and the rats were allowed to recover before the stimulation period was initiated.

Experimental protocols. After a 48-h recovery period, baseline blood pressure measurements were made. Mean arterial pressure (MAP) was measured for at least 2 h at the same time of day for one control day and 6 days of stimulation. MAP and heart rate were measured with Statham P23 ID pressure transducers connected to a four-channel BP display unit (Stemtech, Milwaukee, WI). The analog signal was low pass filtered at 100 Hz, sampled at 300 Hz, and processed with software of our own design. Acquired data were averaged in 1-min intervals throughout the measurement period.

On the day after baseline measurements, the implanted stimulator was activated by momentary closure of the magnetic reed switch with the use of a small handheld magnet. The stimulator produced electrically induced muscle contractions in the lower leg muscles by stimulating the common peroneal nerve with square-wave impulses of 0.3-ms duration, 10-Hz frequency, and 3-V potential (19). Contractions of the extensor digitorum longus (EDL) and tibialis anterior (TA) were automatically initiated at 8:00 AM each day and sustained for 8 h/day over a consecutive 7-day period. Blood pressure was measured for a minimum of 2 h each day throughout the stimulation period.

The animals were randomly assigned to three groups. All animals were fed normal rat chow until 1 wk before surgical protocols were carried out. The animals were then placed on control diet (CD, Dyets, 0.4% NaCl chow) or HSD (Dyets, 4.0% NaCl chow) for the duration of the protocol. The animals were continuously infused intravenously with ANG II or vehicle via the jugular catheter at a rate of 0.5 ml/h throughout the 7-day stimulation period. Animals in group 1 (CD, n = 11) were fed CD and infused continuously with sterile normal saline. Group 2 (HSD, n = 11) animals were fed a HSD and infused with sterile normal saline. Group 3 (HSD + ANG II, n = 11) animals were fed high salt and infused at a subpressor rate of ANG II (3 ng·kg–1·min–1), dissolved in sterile normal saline.

Tissue harvest and morphological analysis of vessel density. After 7 days of stimulation, the animals were euthanized by an overdose of pentobarbital sodium (Ovation Pharmaceuticals, Deerfield, IL), and the stimulated and contralateral unstimulated muscles were removed and weighed. The EDL muscle was placed in 1 ml ice-cold methanol and frozen at –80°C for tissue ANG II analysis. A 100-mg section was taken from the rostral portion of the TA muscle and frozen in liquid nitrogen for Western blot analysis. The remaining TA tissue was lightly fixed overnight in a 0.25% formalin solution. The muscles were sectioned with a microtome to a thickness of ~75 µm. The TA slices were then immersed in a solution of 30 µg/ml rhodamine-labeled Griffonia simplicifolia I lectin (33). After several rinses, slices were mounted on microscope slides in a water-soluble mounting medium (SP ACCU-MOUNT 280, Baxter Scientific, McGraw Park, IL).

Morphometric analysis of the scanned histochemical sections was performed as previously described (33). Sixty representative fields were selected for analysis from each TA muscle. Vessel-grid intersections have been demonstrated to provide an accurate and quantitative estimate of vessel density.

Western blot analysis to detect presence of VEGF and VEGF receptor protein. Western blot analysis was performed as previously described (2). Briefly, 100-mg TA muscle samples were homogenized, and the cytosolic protein fraction was suspended in 10 mM potassium phosphate buffer (pH 7.7) with 100 µM PMSF (Sigma). The membrane protein fraction was separated by centrifugation (100,000 g for 45 min) and suspended in a similar buffer (pH 7.25). For quantification of VEGF protein expression, 10 µg of protein (as determined by Bio-Rad DC protein assay kit, Bio-Rad, Hercules, CA) from the TA homogenate and 0.5 µg of human recombinant VEGF 165 standard (rVEGF165, Panvera, Madison, WI) was separated on a 12% denaturing SDS-polyacrylamide gel. VEGF receptor (VEGFR) protein levels were determined by electrophoresis of 15 µg of membrane protein on an 8% SDS gel. After transfer and overnight blocking was completed, blots were incubated with a monoclonal antibody derived from the human VEGF sequence (1:2,000 dilution, clone G143–850, Pharmingen, San Diego, CA) or polyclonal antibodies (1:1,000 dilution, Santa Cruz Biotechnologies, Santa Cruz, CA) against the VEGF type 1 receptor (VEGFR1, clone SC-316) and VEGF type 2 receptor (VEGFR2, clone SC-315). Washed blots were then incubated with a secondary antibody conjugated to horseradish peroxidase and visualized with the SuperSignal West Dura chemiluminescence substrate detection system (Pierce, Rockford, IL).

Plasma and tissue ANG II measurement. Arterial blood samples (1 ml) were drawn via the carotid arterial catheter into chilled tubes containing 50 µl/ml 0.125 M Na2EDTA, 0.025 M phenanthroline, and 0.5 mM neomycin sulfate. Samples were immediately centrifuged, and plasma was separated and frozen at –80°C until extracted. For tissue measurements, the muscles were homogenized and processed in large excess (~100 mg tissue/7 ml 100% methanol) at 4°C. Samples were centrifuged and stored at –80°C until extraction. ANG II levels in plasma and tissue extract were then measured by radioimmunoassay as previously described (34).

In vivo Matrigel angiogenesis assay. An additional 28 7-wk-old Sprague-Dawley rats were placed on CD or HSD 5 days before subcutaneous injection of 0.5 ml of ice-cold liquid growth factor-reduced Matrigel (BD Biosciences, Bedford, MA). Matrigel is a mixture of extracellular matrix proteins that at physiological temperatures forms a solid matrix favorable for vessel infiltration and angiogenesis (31). Before injection, a low dose of heparin (20 U/ml Matrigel), a critical cofactor for VEGF activity, was added to the Matrigel. In addition, 100 ng/ml of human rVEGF165 (Panvera) were added to some Matrigel vials. Angiogenesis was allowed for 1 wk before the Matrigel plugs were carefully harvested and homogenized in cell lysis buffer (Sigma). Angiogenesis was determined by Western blot analysis against the well-characterized endothelial cell protein marker platelet endothelial cell adhesion molecule (PECAM) (22). Matrigel homogenate (35 µg) was separated by electrophoresis on an 8% SDS gel as described above, transferred, and blotted with a polyclonal antibody against the rat PECAM protein, with cultured rat endothelial cells used as a positive control. The antibody was a gift from the laboratory of Dr. Peter Newman.

Data analysis and statistics. For each chronically stimulated muscle and its paired unstimulated counterpart, 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, relative VEGF expression, and muscle ANG II concentration of the stimulated muscle were compared with their unstimulated counterparts. All values are presented as means ± SE. The significance of differences in values measured between unstimulated and stimulated muscles within groups was evaluated by using a paired samples t-test. Comparisons between groups were made by one-way analysis of variance, followed by Tukey's post hoc test. Blood pressure measurements were evaluated with a repeated-measures analysis of variance, with treatment and time as variables. Significance was established at P < 0.05.


   RESULTS
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DISCUSSION
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Table 1 displays rat and muscle weights. There was no difference among groups in body weight at the start or completion of the study. Stimulation did not result in significant hypertrophy of the TA muscle in any group. A small but significant hypertrophy was demonstrated in the EDL muscle in both the CD and HSD + ANG II groups (P < 0.05) but was not observed in the HSD group.


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  		Table 1. BW and unstimulated and stimulated EDL and TA muscle weight-to-BW ratios

 
As presented in Fig. 1, 7 days of electrical stimulation induced a significant increase in microvessel density in the TA muscle of rats on a CD, from 130 ± 8.3 vessel-grid intersections in the unstimulated leg to 147 ± 7.9 in the stimulated limb (P < 0.05). This angiogenic response was completely inhibited by HSD consumption, because the vessel density in the unstimulated muscle (129 ± 6.8) was not different from that of the stimulated limb (132 ± 5.6). Angiogenesis was restored in HSD + ANG II animals, with vessel density increasing from 130 ± 7.1 in the unstimulated limb to 157 ± 10.2 in the stimulated muscle (P < 0.05).


Figure 1
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  		Fig. 1. Changes in tibialis anterior (TA) muscle microvessel density after 7 days of electrical stimulation in control rats consuming a control diet (CD, n = 8), rats fed a high-salt diet (HSD, n = 8), and high salt-fed rats infused with a low dose of ANG II (HSD + ANG II, n = 10). Unstimulated limb (open bars) and stimulated limb (filled bars) are shown. *P < 0.05, significant difference vs. respective unstimulated limb.

 

Figure 7
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  		Fig. 7. Representative Western blot and corresponding quantitative densitometry of several separate experiments comparing VEGF type 2 receptor (VEGFR2) protein levels in unstimulated (open bars) and stimulated (filled bars) TA muscles of rats on CD (n = 8), HSD (n = 10), and HSD + ANG II (n = 6). *P < 0.05, significant difference vs. corresponding unstimulated limb; #P < 0.05, significant difference vs. CD stimulated limb.

 
Plasma ANG II levels were measured to confirm that high-salt treatment suppressed circulating RAS activity (Fig. 2). As expected, HSD significantly reduced plasma ANG II levels from 12 ± 1.6 pg/ml of plasma in CD to 6.4 ± 1.2 pg/ml in HSD (P < 0.05). Chronic intravenous infusion of ANG II at a dose of 3 ng·kg–1·min–1 restored circulating ANG II in rats on high salt to 12 ± 2.7 pg/ml, which was not different from CD rats. At this dose, ANG II did not cause pressor effects, because MAP was not significantly elevated in HSD + ANG II rats relative to CD or HSD rats during the control and stimulation periods (Fig. 3). We have previously demonstrated that stimulation does not alter plasma ANG II levels (1).


Figure 2
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  		Fig. 2. Plasma ANG II levels after 7 days of stimulation in CD (n = 11), HSD (n = 10), and HSD + ANG II (n = 6). *P < 0.05, significant difference vs. control diet.

 

Figure 3
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  		Fig. 3. Mean arterial pressure over the course of a control day and 7-day stimulation and infusion period. CD ({blacksquare}), HSD ({blacktriangleup}), HSD + ANG II (bullet). *P < 0.05, significant difference vs. control diet.

 
Figure 4 displays the results of ANG II measurements performed in homogenized EDL muscle tissue. Tissue ANG II levels were consistently higher than levels observed in plasma. Electrical stimulation did not significantly alter tissue ANG II levels in CD (25 ± 3.0 pg ANG II/g muscle in the unstimulated limb to 28 ± 4.3 pg/g in the stimulated leg), HSD (30 ± 5.7 to 29 ± 4.8 pg/g), or HSD + ANG II (37 ± 4.3 to 33 ± 5.4 pg/g) rats. There was not a significant difference among groups in tissue ANG II levels in either the unstimulated or stimulated limb.


Figure 4
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  		Fig. 4. Tissue ANG II levels measured in extensor digitorum longus muscle homogenate harvested from CD (n = 10), HSD (n = 8), and HSD + ANG II (n = 8) after 7-day stimulation period. Unstimulated limb (open bars) and stimulated limb (filled bars) are shown.

 
VEGF has been previously shown to be an important angiogenic factor in skeletal muscle (1, 2). We performed Western blot analysis to verify whether the inhibition of angiogenesis in animals on high salt occurred with a decrease in expression of VEGF protein. Figure 5 shows a representative Western blot and the resulting quantitative densitometry of several blots, represented as a percentage of rVEGF165. VEGF protein levels significantly increased (P < 0.05) from the unstimulated to stimulated limb in CD (9 ± 0.8% to 98 ± 21%), HSD (18 ± 9.0% to 97 ± 20%), and HSD + ANG II (6 ± 2.2% to 54 ± 6.7%) rats. There was no difference among groups in VEGF expression in either the unstimulated or stimulated muscles.


Figure 5
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  		Fig. 5. Representative Western blot and corresponding quantitative densitometry of several separate experiments comparing VEGF protein levels in unstimulated (open bars) and stimulated (filled bars) TA muscles of rats on CD (n = 6), HSD (n = 6), and HSD + ANG II (n = 7). *P < 0.05, significant difference vs. corresponding unstimulated limb.

 
To determine whether differences in the angiogenic response may have been mediated by changes in the expression of the VEGFRs rather than the ligand itself, the levels of the two primary VEGFRs, VEGFR1 (also known as Flt-1) and VEGFR2 (also known as Flk-1 and KDR), were quantified by Western blot analysis. The expression of VEGFR1 decreased to a similar degree from the unstimulated to the stimulated TA muscle in the CD (150 ± 20% to 55 ± 12%, P < 0.05), the HSD (145 ± 9.0% to 56 ± 7.0%, P < 0.05), and the HSD + ANG II (157 ± 19% to 68 ± 16%, P < 0.05) groups. Stimulation resulted in a significant increase in VEGFR2 protein levels in CD (9.1 ± 5.2% to 224 ± 29%, P < 0.05), HSD (19 ± 8.4% to 126 ± 32%, P < 0.05), and HSD + ANG II (24 ± 6.7% to 204 ± 41%, P < 0.05) rats. The degree of change in VEGFR2 levels with stimulation was significantly decreased in the HSD group compared with the CD control group (P < 0.05); ANG II infusion restored VEGFR2 protein in the stimulated muscle of HSD + ANG II animals to a level similar to that of the CD group. Figures 6 and 7 display representative Western blots and the resulting quantitative densitometry of several blots for VEGFR1 and VEGFR2, respectively.


Figure 6
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  		Fig. 6. Representative Western blot and corresponding quantitative densitometry of several separate experiments comparing VEGF type 1 receptor (VEGFR1) protein levels in unstimulated (open bars) and stimulated (filled bars) TA muscles of rats on CD (n = 7), HSD (n = 6), and HSD + ANG II (n = 6). *P < 0.05, significant difference vs. corresponding unstimulated limb.

 
To more directly assess the role of HSD on VEGF-induced angiogenesis, Matrigel containing rVEGF165 was injected subcutaneously in the back of rats treated with CDs and HSDs. Angiogenesis was quantified by determining the expression of the endothelial cell marker PECAM in Matrigel homogenates by Western blot analysis. Heparin, a necessary cofactor for VEGF activity (31), was also added to the Matrigel solution. Figure 8 displays both a representative Western blot and the results of quantitative densitometry for several blots. Addition of rVEGF to Matrigel plugs in CD animals increased PECAM expression significantly compared with Matrigel containing heparin alone (107 ± 18% vs. 35 ± 15%, P < 0.05). However, in HSD animals, PECAM expression was not significantly different in Matrigel plugs with VEGF (50 ± 12%) compared with those with heparin alone (34 ± 19%, not significant). In addition, CD plugs containing VEGF demonstrated significantly greater endothelial cell content than those from HSD rats (P < 0.05).


Figure 8
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  		Fig. 8. Representative Western blot and corresponding quantitative densitometry of several separate experiments comparing platelet endothelial cell adhesion molecule (PECAM) protein levels in Matrigel plugs of rats on CD containing 20 U/ml of heparin alone (n = 6), plugs treated with heparin + 100 ng/ml VEGF (n = 8), and plugs from rats on HSD containing heparin (n = 6) and heparin + VEGF (n = 8). Heparin alone (open bars) and heparin + VEGF (filled bars). *P < 0.05, significant difference vs. corresponding heparin control; #P < 0.05, significant difference vs. HSD heparin + VEGF plug.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study suggest a critical and necessary role for circulating ANG II in mediating stimulated angiogenesis. A HSD suppressed plasma ANG II levels and inhibited the angiogenic response to 7 days of electrical stimulation. Infusion of ANG II completely restored physiological angiogenesis to a level similar to that of control animals on a CD. This study supports a number of findings demonstrating that a suppression of circulating ANG II inhibits angiogenesis and is the first to demonstrate the critical role of ANG II suppression in mediating the antiangiogenic effects of a HSD. In addition, this study suggests an important link between ANG II levels and expression of the VEGFR2, a key angiogenic molecule.

The role of ANG II in stimulating angiogenesis through activation of the AT1 receptor has been reported in a number of models. Inhibition of reparative angiogenesis after femoral ligation has been noted in mice treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (12), as well as specific AT1 receptor blockers (12, 40). In addition, the angiogenic response to limb ischemia has also been shown to be disrupted in AT1–/– mice (35) and exaggerated in AT2–/– mice (38), suggesting an inhibitory role of ANG II acting on the AT2 receptor type. ACE inhibition has been shown to augment hindlimb ischemia-induced angiogenesis in the rabbit; however, this effect was drug specific, because quinaprilat stimulated angiogenesis and captopril had no effect (13). Silvestre and colleagues (37) have also demonstrated that the ACE inhibitor perindopril stimulates reparative angiogenesis, possibly through a bradykinin-dependent mechanism. The divergent findings of various studies regarding the role of ACE inhibitors in angiogenesis may in part be explained by the unique properties of individual drugs, as well as the specific model of angiogenesis being studied and the length of drug treatment.

The opposing actions of the angiotensin receptor types in the skeletal muscle microcirculation (27) dictate that relative quantities of each subtype within a tissue determine the net effect of ANG II stimulation. The AT1 receptor has been demonstrated to be present in relatively high abundance in the skeletal muscle microvasculature, whereas the AT2 receptor is present in low concentrations (28). High salt has been demonstrated to alter the distribution of angiotensin receptors in the vasculature. Nora et al. (28) reported that in aortic tissue from rats fed high salt, the relative concentration of AT1 receptor protein was significantly decreased relative to low salt control animals, whereas AT2 protein was increased. Importantly, the receptor distribution was restored to normal in high salt-fed animals receiving chronic ANG II infusion (26). Hence, the effect of high salt consumption on angiogenesis may be explained in part by a reduction in the activation of the proangiogenic AT1 receptor as a result of decreases in the abundance of both ligand and receptor, as well as a relative increase in AT2 (e.g., antiangiogenic) activation.

Numerous studies (6, 40, 41) have suggested that ANG II may stimulate angiogenesis via AT1 receptor-mediated upregulation of VEGF, the most potent endothelial cell-specific mitogen identified, although Murakami et al. (25) failed to find a relationship between ANG II and VEGF in human patients with acute myocardial infarction. The lack of dependence of VEGF expression on circulating ANG II observed in the present study may be explained by the complex regulation of VEGF, which is primarily under the transcriptional control of hypoxia-inducible factor. Various studies have demonstrated regulation of VEGF expression by local hypoxia (9) and hemodynamic forces, such as shear stress (10). VEGF mRNA in ischemic muscle (20) and VEGF protein levels in normal muscle (1, 2, 23) have been demonstrated to increase in response to exercise and electrical stimulation in vivo. Although evidence suggests a contribution of ANG II in VEGF regulation, the presence of other important stimuli may overcome a lack of ANG II in animals fed a high-salt diet and stimulate significant VEGF production. Immunohistochemical analysis has indicated that the primary sources of VEGF in our model of stimulated muscle contraction are skeletal muscle myocytes (1), suggesting an important role for metabolic demand placed on the muscle in regulating VEGF expression. The finding that the functional hyperemic response to electrical stimulation is equivalent between CD and HSD animals (32) indirectly suggests that metabolic demand imposed by stimulation is similar among the groups and therefore may explain the high levels of VEGF observed in stimulated muscles of animals fed a high-salt diet despite low ANG II levels.

The critical role of VEGF signaling in the angiogenic pathway has been demonstrated by studies in which chronic injection of a VEGF-neutralizing antibody has completely eliminated angiogenesis in several models (1, 4, 40). However, the results of the current study suggest that upregulation of VEGF levels alone is insufficient to mediate angiogenesis in stimulated muscle, because animals on HSD demonstrate significant increases in expression of VEGF protein in stimulated muscles in the absence of an angiogenic response. Luo et al. (21) reported similar results in ischemic TA muscle, because significant increases in VEGF protein expression did not result in corresponding capillary growth. The results of the Matrigel assay conducted in the current study appear to suggest that a diet high in salt has a direct inhibitory effect on VEGF-induced angiogenesis. The observation that stimulated angiogenesis is restored in high salt-fed animals with chronic ANG II infusion seems to demonstrate a crucial role for ANG II signaling in angiogenesis, even in the presence of high levels of VEGF.

Several reports (18, 30, 36) in cultured cells have suggested that blockade of the RAS may inhibit angiogenesis by causing a reduction in the expression of the receptors of VEGF. These results would suggest that despite high levels of VEGF protein within a working muscle, in the condition of high salt consumption and subsequent suppression of ANG II, the actions of VEGF would be significantly attenuated due to reductions in the levels of the angiogenic receptors. This conclusion is supported by the findings of Gavin et al. (15), indicating that ACE inhibitor treatment inhibited the expression of VEGFR2 message after an acute exercise bout, without affecting the large increase in VEGF mRNA associated with exercise. In the current study, the degree of reduction in VEGFR1 protein levels in stimulated muscles was unaltered by HSD and ANG II maintenance. However, a significant decrease in the degree of upregulation of VEGFR2 protein with stimulation was observed in high salt-fed animals relative to the muscles of CD-fed rats. The relatively low levels of VEGFR2 protein in high salt-fed animals may in part explain the lack of angiogenesis induced by VEGF in the current study, because it has been reported that VEGFR2 mediates most of the angiogenic signaling of VEGF (30). This conclusion is supported by the findings of Milkiewicz and colleagues (23) that high levels of VEGF protein did not correspond to angiogenesis in the absence of significant VEGFR2 upregulation. The finding in the present study that VEGFR2 protein levels are restored in high-salt fed animals infused with ANG II supports previous reports in culture of a role for ANG II in the regulation of VEGFR2 expression and is among the first to support a relationship between ANG II and VEGFR2 expression in vivo.

In addition to a possible role for ANG II in mediating the angiogenic actions of VEGF through regulation of the expression of VEGF receptors, it is possible that the intracellular signaling cascades initiated by ANG II may activate critical pathways involved in maintaining susceptibility to VEGF-induced angiogenesis. An emerging literature has reported signaling pathways activated by ANG II that parallel those initiated by various cytokines and growth factors, including kinase-mediated pathways involved in the signaling of epidermal growth factor, insulin-like growth factor-1, and platelet derived growth factor (for review, see Ref. 11). It is possible that activation of one or several of these pathways by ANG II may modulate a protein or signaling pathway critical for maintaining susceptibility to the angiogenic signaling of VEGF. For instance, Zhao and colleagues (43) demonstrated that ANG II induced proliferation of vascular smooth muscle cells through a RAS/mitogen-activated protein kinase-dependent pathway that resulted in activation of the Elk-1 protein, a transcription factor that has been shown to be important for angiogenesis (5) and may impact VEGF signaling through an unidentified mediator. Further research is necessary to identify the critical target pathways that may be involved in the role of ANG II in maintaining susceptibility to angiogenic stimuli.

In summary, the present study indicates that high salt intake inhibits angiogenesis induced by electrical stimulation of skeletal muscle by suppressing circulating levels of ANG II. These findings provide further evidence for a deleterious effect of elevated dietary salt intake on microvascular structure and function independent of changes in blood pressure. In addition, the current study demonstrates that suppression of ANG II appears to inhibit VEGF-mediated angiogenesis by attenuating VEGFR2 expression, providing important insight into the mechanisms regulating the complex physiological process of angiogenesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-29587.


   ACKNOWLEDGMENTS
 
We thank Daniella Didier for expert technical assistance and Dr. Peter Newman of the Blood Research Institute in Milwaukee for kindly providing anti-PECAM antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Greene, Dept. of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (e-mail: agreene{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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  1. Amaral SL, Linderman JR, Morse MM, and Greene AS. Angiogenesis induced by electrical stimulation is mediated by angiotensin II and VEGF. Microcirculation 8: 57–67, 2001.[CrossRef][Web of Science][Medline]
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