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Am J Physiol Heart Circ Physiol 287: H2300-H2308, 2004. First published July 1, 2004; doi:10.1152/ajpheart.00065.2004
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Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration

Oliver Baum,1,2 Luis Da Silva-Azevedo,2 Gregor Willerding,1 Achim Wöckel,1 Gerit Planitzer,1 Reinhart Gossrau,1 Axel R. Pries,2 and Andreas Zakrzewicz2

Departments of 1Anatomy and 2Physiology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, D-14195 Berlin-Dahlem, Germany

Submitted 26 January 2004 ; accepted in final form 23 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The increase of wall shear stress in capillaries by oral administration of the {alpha}1-adrenergic receptor antagonist prazosin induces angiogenesis in skeletal muscles. Because endothelial nitric oxide synthase (eNOS) is upregulated in response to elevated wall shear stress, we investigated the relevance of eNOS for prazosin-induced angiogenesis in skeletal muscles. Prazosin and/or the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) were given to C57BL/6 wild-type mice and eNOS-knockout mice for 14 days. The capillary-to-fiber (C/F) ratio and capillary density (CD; no. of capillaries/mm2) were determined in frozen sections from extensor digitorum longus (EDL) muscles of these mice. Immunoblotting was performed to quantify eNOS expression in endothelial cells isolated from skeletal muscles, whereas VEGF (after precipitation with heparin-agarose) and neuronal NOS (nNOS) concentrations were determined in EDL solubilizates. In EDL muscles of C57BL/6 mice treated for 14 days, the C/F ratio was 28% higher after prazosin administration and 11% higher after prazosin and L-NAME feeding, whereas the CD increased by 21 and 13%, respectively. The C/F ratio was highest after day 4 of prazosin treatment and decreased gradually to almost constant values after day 8. Prazosin administration led to elevation of eNOS expression. VEGF levels were lowest at day 4, whereas nNOS values decreased after day 8. In EDL muscles of eNOS-knockout mice, no significant changes in C/F ratio, CD, or VEGF and nNOS expression were observed in response to prazosin administration. Our data suggest that the presence of eNOS is essential for prazosin-induced angiogenesis in skeletal muscle, albeit other signaling molecules might partially compensate for or contribute to this angiogenic activity. Furthermore, subsequent remodeling of the capillary system accompanied by sequential downregulation of VEGF and nNOS in skeletal muscle fibers characterizes shear stress-dependent angiogenesis.

angiogenesis; skeletal muscle; endothelial nitric oxide synthase

ANGIOGENESIS IS THE PROCESS by which the microvascular system expands during organ growth and regeneration or in response to chronically altered metabolic demands of tissues (reviewed in Refs. 4, 13, 14, 48). The amplification of the vascular network occurs within a short time due to either abluminal outgrowth (sprouting) or intraluminal division (intussusceptive growth) of capillaries. Both angiogenic mechanisms comprise a specific, complex, and coordinated sequence of several cellular processes (e.g., endothelial cell proliferation and extracellular matrix degradation for sprouting; endothelial cell protrusions and smooth muscle cell recruitment for intussusceptive growth). Recent research revealed that both angiogenic mechanisms are induced and/or controlled by the interaction of specific angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietins, and others (17, 46). Most of these angiogenic factors represent soluble proteins that bind as autocrine and paracrine signaling molecules to transmembrane receptors that are present on endothelial cells. These factors are regulated in a tightly coordinated temporal and local manner to influence angiogenesis (17, 46). Hitherto it has been poorly understood how the qualitative and quantitative expression patterns of the angiogenic factors are established. Some evidence exists that information on tissue demands and the perfusion status of capillary networks might induce and coordinate the expression of angiogenic factors from different cells (48). Such proangiogenic information includes, e.g., hypoxia in the tissue or alterations in the hemodynamic forces, especially in the wall shear stress of the vasculature. Because hypoxia and wall shear stress signals can be transmitted by the gaseous signaling molecule nitric oxide (NO), it seems reasonable that the activity of NO-producing synthase (NOS) might contribute to the generation and/or transfer of such information and therefore modulate angiogenesis (reviewed in Refs. 9, 51).

NOS/NO systems might influence angiogenesis cascades either upstream or downstream of the actual angiogenic factors. An upstream effect of NOS/NO systems occurs if changes in the NO concentration directly influence (enhance or impair) the concentration of angiogenic factors and subsequently affect the extent of angiogenesis. On the other hand, NOS/NO systems might exert an effect downstream of angiogenic factors if NO is an integral component of a signal cascade initiated by the corresponding angiogenic factor. These two possibilities for NO to contribute to angiogenesis have been characterized as director (upstream) or actor (downstream) properties of NO (reviewed in Ref. 51).

Both downstream and upstream effects of NO on angiogenesis have been reported (e.g., Refs. 5, 8, 19, and 52 vs. 15, 49). However, most of these studies focused on the analysis of the expression patterns of angiogenic factors in cultured endothelial cells after exposure to NO donors or chemical inhibitors of NOS activity. Such approaches provide only a limited clue to the impact of NO on angiogenesis in vivo.

Because endothelial NOS (eNOS)-derived NO influences the establishment of vascular morphology and the hemodynamic environment in skeletal muscles at different levels (29, 30, 47), eNOS might have an impact on angiogenesis in this tissue. To prove this hypothesis, the angiogenic response in the extensor digitorum longus (EDL) muscle of eNOS-knockout mice and C57BL/6 wild-type mice was analyzed after treatment with the {alpha}1-adrenergic receptor antagonist prazosin for 14 days. This approach was selected for the following reasons: 1) oral administration of vasodilators to rodents is an established method for inducing angiogenesis in skeletal muscles (12). As a consequence of elevated wall shear stress, the capillary luminae are longitudinally split without basal membrane degradation in an intussusceptive growthlike process (16, 50). 2) Although NO apparently influences shear-stress driven angiogenesis in skeletal muscles (24), it is unknown which NOS form is involved in the angiogenic process. In skeletal muscle, two NOS forms are constitutively expressed (reviewed in Refs. 35, 42). Whereas eNOS is present in endothelial cells of capillaries and larger blood vessels (40), high concentrations of neuronal NOS (nNOS) are found in the sarcolemma of skeletal muscle fibers (22, 28). Analysis of EDL muscles of eNOS-knockout mice after prazosin administration should provide information about the relevance of this particular NOS form for shear stress-mediated angiogenesis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. For immunoblotting, the following commercially available primary antibodies were used: 1) polyclonal antibodies against an internal sequence (amino acids 1025–1203) of eNOS (NOS-3; obtained from BD Pharmingen; Heidelberg, Germany) in a 1:500 dilution; 2) goat polyclonal antibodies against platelet endothelial cell adhesion molecule-1 (PECAM-1; M-20; purchased from Santa Cruz Biotechnologies; Heidelberg, Germany) in a 1:500 dilution; 3) polyclonal antibodies against VEGF-A (A-20; obtained from Santa Cruz Biotechnologies) in a 1:500 dilution; and 4) polyclonal antibodies against the COOH-terminal amino acids 1409–1429 of the rat nNOS (NOS-1) sequence (Sigma; Munich, Germany) in a 1:2,000 dilution.

Animals and experimental procedures. In accordance with approvals obtained from the university and state authorities for animal welfare, this study was performed on C57BL/6 wild-type mice and eNOS-knockout mice, which were bred in our animal care facility under standard conditions. The eNOS-knockout strain was originally purchased from Jackson Laboratories (Bar Harbor, ME). For the experiments, 195 healthy mice weighing 25–30 g (3–5 mo old) were used. The mice were anesthetized with ketamine and killed by heart excision.

To dissolve prazosin, tap water was adjusted with HCl to pH 5.8 and heated to 60°C before addition of 50 mg/l ground prazosin powder (Sigma). This concentration has been shown to induce angiogenesis in rats (12, 25, 44). The inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME), which is specific for all NOS forms, was obtained from Sigma and freshly prepared in a concentration of 1 mg/ml dissolved in tap water as previously described (34) to be applied daily. Because a mouse drinks ~3 ml of water/day, 150 µg of prazosin and/or 3 mg of L-NAME, respectively, were required each day.

For determination of angiogenesis and its relation to NO availability, mice from each of the two mouse strains were assigned to four groups consisting of at least five animals each. For 14 days, one group was treated with prazosin dissolved in drinking water, whereas the control group received water without prazosin. A third group of mice was treated with a combination of prazosin and L-NAME, and a forth group was fed with L-NAME alone.

The time course of prazosin-induced angiogenesis was investigated in groups of C57BL/6 mice and eNOS-knockout mice (3 animals/group) treated with prazosin for 3, 4, 8, or 14 days compared with control animals that received tap water alone (0 days).

Sample processing. For histochemistry, at least one EDL muscle of each animal was mounted on a cork plate using Tissue-Tek, frozen in liquid nitrogen-cooled methylbutane, and stored in a closed plastic bag at –40°C until use. The contralateral EDL muscle was obtained for immunoblot analyses.

Endothelial cells from skeletal muscles of mice were isolated as described previously (11). Briefly, minced tissue of the EDL, tibialis anterior, and rectus femoris muscles were pooled and subjected to collagenase digestion and subsequent Griffonia simplicifolia (BS-1) lectin precipitation with paramagnetic beads to obtain a highly enriched endothelial cell fraction in the pellet.

Histochemistry. For nonspecific alkaline phosphatase (AP) histochemistry, 10-µm-thick cryosections of the EDL muscle (transversally cut from the middle part) were used. The histochemical reaction was performed with a medium that contained 0.3 mg of 5-Br-4-Cl-3-indoxyl phosphate (Bachem; Heidelberg, Germany) dissolved in 50 µl of dimethylformamide and 1 mg of tetranitroblue tetrazolium salt (Serva; Heidelberg, Germany) in 1 ml of 0.1 M Tris·HCl, pH 9.4. Chloroform-acetone-fixed cryosections (5 min at 4°C) were incubated with the reaction medium for 1 h at 37°C. The histochemical reaction was stopped by extensive washing. All cryosections were coverslipped in a 1:2 PBS-glycerol mixture.

Digital photographs of the EDL muscle sections were obtained using an Axioskop microscope with a x20 magnification objective (Zeiss, Oberkochen, Germany) connected to a Power Macintosh G3 computer equipped as described earlier (39). On paper prints of the images, we counted the AP-reactive capillaries and skeletal muscle fibers in a defined area with at least 200 skeletal muscle fibers to calculate the capillary-to-fiber (C/F) ratio and capillary density (CD). In each muscle, 3–5 fields were evaluated for statistical analysis.

Heparin-agarose precipitation for VEGF enrichment. Our first experiments revealed that skeletal muscle homogenates do not contain levels of VEGF sufficient for densitometric quantification of bands (data not shown). We therefore separated heparin-binding proteins from the homogenates by affinity precipitation on heparin-agarose beads according to the method of Annex et al. (3) before immunoblotting. EDL muscle samples were powdered in liquid nitrogen, homogenized in lysis buffer (10 mM Tris·HCl, pH 7.6, 100 mM NaCl, and 1% Triton X-100), and sonicated on ice three times for 5 s. Insoluble material was removed by centrifugation at 10,000 g for 15 min. Subsequently, 1 ml of extract that contained 500 µg of skeletal muscle protein was incubated with 25 µl of equilibrated heparin-agarose beads (Sigma) at 4°C for 1 h by end-over-end agitation. The beads were washed three times in buffer A (20 mM Tris·HCl, pH 7.4, and 0.4 M NaCl) by sedimentation at 5,000 g for 1 min. Heparin-binding proteins adsorbed to the agarose beads were eluted by incubating the final pellets in 25 µl of reducing Laemmli electrophoresis buffer at 65°C for 15 min in preparation for immunoblot analysis.

Immunoblot analysis. EDL muscle or endothelial cell fractions from several mouse skeletal muscles were homogenized in lysis buffer at 4°C. Insoluble material was removed by centrifugation at 20,000 g for 15 min. Equal amounts of solubilizate protein or pellets from heparin-agarose precipitation were subjected to SDS-PAGE (Bio-Rad; Munich, Germany) and transferred to nitrocellulose membrane filters. For nNOS, eNOS, and PECAM-1 immunoblotting, 7.5% gels were used, whereas for VEGF, immunoblotting was performed with recombinant VEGF (R&D Systems) in defined concentrations as standard on 15% gels.

Blot membranes were blocked with 5% (wt/vol) dry milk powder in washing buffer (0.1% vol/vol Tween 20 in PBS) and incubated first with primary antibodies overnight at 4°C and subsequently with a 1:5,000 dilution of horseradish peroxidase-conjugated secondary antibodies (Sigma) for 1 h. Unbound primary and secondary antibodies were removed by four incubations (5 min each) in washing buffer. Immunoblots were developed by chemiluminescence as described previously (6) using an ECL Detection Kit (Pierce-Perbio; Bonn, Germany) and visualized by exposure to Kodak XR-5 film for several time periods that varied between 30 s and 60 min. After development, the films were scanned for densitometric quantification as described previously (11).

For control of protein loading, eNOS immunoblots were stripped in 67 mM Tris·HCl, pH 6.8, 2% SDS (wt/vol), and 100 mM mercaptoethanol for 30 min at 56°C; intensively washed; blocked; and reprobed with anti-PECAM-1 antibodies.

Statistics. Data are expressed as means ± SD. To compare mean values, we tested for normal distribution using the {chi}2-test. For normally distributed values, the Student's t-test was used, otherwise the nonparametric Mann-Whitney U-test was applied. Contiguity was analyzed by Kendall's correlation test. To determine the extent of the correlation, this parameter was specified in addition to the error probability. A uniform significance level of P ≤ 0.05 was assumed for all statistical evaluations.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To induce angiogenesis in the EDL muscles of the hindlimb, C57BL/6 wild-type mice as well as eNOS-knockout mice were fed with prazosin for 14 days. Subsequently, the EDL muscles of these mice (and the corresponding prazosin-untreated mice) were transversally cryosectioned and subjected to AP histochemistry for the demonstration of capillaries in the endomysium surrounding the skeletal muscle fibers (Fig. 1).



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  		Fig. 1. Alkaline phosphatase (AP) histochemistry on cryosections of extensor digitorum longus (EDL) muscles derived from C57BL/6 mice (A and B) and endothelial nitric oxide synthase (eNOS)-knockout mice (C and D) without (A and C) and after 14 days of (B and D) prazosin administration. Histochemical reaction specifically demonstrates the presence of capillaries in the endomysium surrounding the skeletal muscle fibers. Bar, 50 µm.

 
The quotient between the number of AP-positive capillaries and the number of skeletal muscle fibers in a defined area represents the C/F ratio. As shown in Table 1, we found that the C/F ratio for EDL muscles of C57BL/6 mice increased by 28% (compared with untreated mice, which had a C/F ratio of 1.0) after administration of prazosin for 14 days. In eNOS-knockout mice, the basal C/F ratio (found in EDL muscles of untreated mice) was ~20% higher than in nonstimulated C57BL/6 mice and was not significantly changed after 14 days of prazosin treatment.


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  		Table 1. Capillary-to-fiber ratio and capillary density values of mice after 14 days of prazosin administration with or without L-NAME

 
The influence of NO on prazosin-induced changes in the C/F ratio of skeletal muscle was further investigated via chronic oral administration of L-NAME (which inhibits the enzymatic activity of all NOS forms) to mice. Additional groups of C57BL/6 mice and eNOS-knockout mice were treated for 14 days either with a combination of prazosin and L-NAME or with L-NAME only (Table 1). The C/F ratio was ~11% higher in EDL muscles of C57BL/6 mice that were treated simultaneously with both prazosin and L-NAME than untreated C57BL/6 mice, whereas eNOS-knockout mice showed no significant variations in C/F ratio in EDL muscles after the combined prazosin and L-NAME application. When mice of both investigated strains were treated for 14 days with L-NAME only, the C/F ratio in EDL muscles was similar to that of untreated mice.

Using the AP-reactive cryosections, we also determined the CD (no. of capillaries/mm2) in EDL muscles of C57BL/6 mice and eNOS-knockout mice after prazosin administration for 14 days (Table 1). In EDL muscles of C57BL/6 mice, the CD was ~21% higher for mice drinking prazosin-supplemented tap water for 14 days and ~13% higher after administration of L-NAME and prazosin when compared with control mice. L-NAME alone had no significant effect on the CD. In EDL muscles of eNOS-knockout mice, no significant variations in CD were detected either in the absence or presence of prazosin and/or L-NAME.

For characterization of the prazosin-induced angiogenic process, the development of the C/F ratio was monitored in EDL muscles of C57BL/6 and eNOS-knockout mice at selected time points during the 14-day prazosin treatment (Fig. 2). In C57BL/6 mice, the C/F ratio peaked after 4 days of prazosin stimulation (an elevation from 1.05 to 1.38) then decreased after 8 days (to 1.18) and remained in the same range after 14 days (at 1.21). In contrast, a comparable development in the C/F ratio (fast increase, decrease, and subsequent adjustment to constant value) was not seen in the EDL muscles of eNOS-knockout mice, where significant alterations in the C/F ratio were not detected.



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  		Fig. 2. Time course of the capillary-to-fiber (C/F) ratio in EDL muscles of C57BL/6 mice (C57) and eNOS-knockout mice (eNOS–/–) during 14 days of prazosin administration. Groups of three mice from both strains were treated with prazosin for time periods indicated. EDL muscles of the mice were prepared and subjected to AP histochemistry to demonstrate capillaries and skeletal muscle fibers on the transversally cut cryosections. C/F ratios were calculated and are presented as means ± SD. *P ≤ 0.05, values significantly different from controls of same strain not exposed to prazosin (day 0). #P ≤ 0.05, values of eNOS-knockout mice significantly different from C57BL/6 mice at same time point.

 
Because eNOS is upregulated due to elevated shear stress, we investigated whether prazosin treatment might influence eNOS expression. The analysis was performed on endothelial cell fractions isolated from skeletal muscles of C57BL/6 mice, because endothelial cells are directly exposed to shear stress and represent the cellular source where most eNOS is expressed in skeletal muscles. As shown in Fig. 3, eNOS concentrations were significantly higher at day 4 and decreased again at day 8 without reaching the concentration measured at day 0. At day 14, eNOS levels were again elevated.



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  		Fig. 3. Upregulation of eNOS in skeletal muscles of C57BL/6 mice in response to prazosin. Equal amounts of solubilized proteins from endothelial cells (20 µg), which were isolated from skeletal muscles of C57BL/6 mice treated with prazosin for 0, 4, 8, or 14 days were subjected to immunoblot analysis with antibodies against eNOS (A). Blot matrices were stripped and reincubated with antibodies against platelet endothelial cell adhesion molecule (PECAM)-1 for demonstration of protein loading (B). Densities of the eNOS and PECAM-1 bands on the films were quantified to be related to each time point investigated (C). Calculated ratios were defined as eNOS concentrations. Value of the control sample (0 days) was set as 100%. Values represent means ± SD; n = 3 independent immunoblots with samples isolated from one animal each. *P ≤ 0.05, values significantly different from controls not exposed to prazosin (day 0).

 
Homogenates were taken to determine the VEGF concentrations present in the EDL muscles during prazosin administration via immunoblotting. However, first attempts to directly demonstrate the presence of VEGF in these extracts failed: high background levels of numerous other proteins reacted with the A-20 antibody on the blotting matrices (data not shown). For quantification, we therefore used pellets obtained from EDL muscle homogenates by precipitation with heparin-agarose, which isolates all freely solubilized VEGF molecules from extracts as determined in preliminary experiments (L. Da Silva-Azevedo, unpublished observations). This enrichment procedure allows the use of large amounts (0.5 mg) of homogenate protein for the analysis with simultaneous reduction of the background. Applying this protocol, we first compared the VEGF concentrations in EDL muscles of nonstimulated C57BL/6 wild-type mice and eNOS-knockout mice. Comparing the density of the bands obtained in this experiment with those of coseparated recombinant VEGF, we found that eNOS-knockout mice expressed ~18% more VEGF in their EDL muscles than C57BL/6 mice (Fig. 4). During the 14-day period of prazosin administration, VEGF concentrations decreased until day 4 to increase again without reaching the basal values of EDL muscles from C57BL/6 mice. In contrast, only slight nonsignificant variations in VEGF concentration were detected in EDL muscles of eNOS-knockout mice.



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  		Fig. 4. Quantification of VEGF in EDL muscle homogenates of C57BL/6 mice and eNOS-knockout mice during 14 days of prazosin administration. Equal amounts of protein (500 µg) from EDL muscle homogenates of four nontreated C57BL/6 wild-type mice and four nontreated eNOS-knockout mice were subjected to heparin-agarose precipitation (A). Proteins eluted from heparin-agarose were electrophoretically separated on 15% SDS-polyacrylamide gels and subsequently subjected to immunoblot analysis with polyclonal anti-VEGF antibodies. As a standard, 20 ng of recombinant VEGF were coseparated. Densitometric analysis showed that EDL muscles of eNOS-knockout mice contained 18% more VEGF than EDL muscles of C57BL/6 mice. Protein (500 µg) from EDL muscle homogenates of C57BL/6 (B) and eNOS-knockout (C) mice that were treated with prazosin for the time periods indicated were introduced in heparin-agarose precipitation. Eluted proteins were subsequently immunoblotted with VEGF antibodies. Densitometric quantification of the 21-kDa VEGF band on the film collected from both mouse strains during 14 days of prazosin administration (D). Density value for VEGF in the control sample (0 days) was set as 100%. *P ≤ 0.05, significantly different from controls not exposed to prazosin (day 0). #P ≤ 0.05, eNOS-knockout mice significantly different from C57BL/6 mice at the same time point; n = 3 independent immunoblots with samples isolated from one animal each.

 
We also quantified the nNOS concentration in EDL muscle extracts from C57BL/6 mice and eNOS-knockout mice treated with prazosin for selected time periods (between 0 and 14 days) via densitometric analysis of immunoblots loaded with equal amounts of proteins. Because the densitometric comparison of the nNOS bands in C57BL/6 mice and eNOS-knockout mice revealed no significant differences in the nNOS double-band characteristics, both mouse strains express equal levels of nNOS under nonstimulated conditions (Fig. 5). Thus in Fig. 5, the scaling of the ordinate reflects the nNOS expression in both mouse strains. However, prazosin administration led to significant modulation of the nNOS concentration only in EDL muscles of C57BL/6 mice where the strength of the nNOS double band diminished continuously after 4 days of prazosin application to comprise ~57% after 14 days (compared to the basal value). In contrast, nNOS concentrations decreased to minimal levels of ~88% after 14 days of prazosin administration in EDL muscles of eNOS-knockout mice.



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  		Fig. 5. Quantification of neuronal NOS (nNOS) in EDL muscle homogenates of C57BL/6 mice and eNOS-knockout mice during 14 days of prazosin administration. EDL muscle homogenates (50 µg) from three C57BL/6 wild-type mice (C1, C2, and C3) and three eNOS-knockout mice (E1, E2, and E3) under basal conditions were subjected to immunoblot analysis with antibodies against nNOS (A). Densitometric analysis showed that the EDL muscles of C57BL/6 mice contained 3% (not significant) more nNOS than EDL muscles of eNOS-knockout mice. Protein (50 µg) from EDL homogenates of C57BL/6 (B) and eNOS-knockout (C) mice that were treated with prazosin for 0, 3, 4, 8, or 14 days were subjected to nNOS immunoblotting. Density of the 146/160-kDa nNOS double band was quantified (D). Value on the film for nNOS in the control sample (0 days) was set as 100%. *P ≤ 0.05, significantly different from controls not exposed to prazosin (day 0); #P ≤ 0.05, eNOS-knockout mice significantly different from C57BL/6 mice at the same time point; n = 3 independent immunoblots with samples isolated from one animal each.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that both C/F ratio and CD values were significantly increased in EDL muscles of C57BL/6 wild-type mice but not of eNOS-knockout mice in response to 14 days of prazosin administration. In general, the concomitant increase of the C/F ratio and the CD in skeletal muscle samples prepared at different time points indicates that the number of capillaries was enlarged in a defined area without change in the size of the skeletal muscle fibers. This allows the conclusion that angiogenesis occurred in the skeletal muscle of mice as it was previously shown for rats and rabbits (reviewed in Ref. 23). In summary, our data suggest that the 14-day administration of prazosin induced angiogenesis in EDL muscles of C57BL/6 mice but not eNOS-knockout mice. The influence of eNOS on both ischemia-driven (1, 38) and wound-healing-dependent (31) angiogenesis in knockout mice was reported earlier. In contrast, training-induced angiogenesis in ischemic skeletal muscle was not NO modulated (34). Thus the role of NO might vary depending on the nature of the angiogenic stimulus and the tissue involved.

In an earlier study (11), we showed that prazosin triggers the augmented production of VEGF in endothelial cells (which have thus been directly exposed to elevated shear stress) harvested from skeletal muscles of C57BL/6 mice but not eNOS-knockout mice. The combination of these data with the results presented here (no angiogenesis in eNOS-knockout mice) suggests that prazosin leads to a significant increase in VEGF concentration in endothelial cells and angiogenesis only if eNOS is present.

We propose the following molecular mechanism (Fig. 6): as a specific antagonist, prazosin binds to the {alpha}1-adrenergic receptors that are present on smooth muscle cells surrounding all larger blood vessels. The smooth muscle cells relax, which leads to vessel dilation and, consequently, elevation of blood flow in arterioles. Because the ensuing capillaries can only limitedly change their diameters in response to the increased blood flow, the velocity of flow and, as a consequence, the tangential shear stress, increase in these vessels. By a yet-unknown mechanosensor, the information about the altered hemodynamic is transduced into the endothelial cells, which then produce higher concentrations of angiogenic factors such as VEGF. These factors might then trigger as autocrine and/or paracrine mediators the cellular processes necessary for the generation of new functional capillaries. In the case that angiogenesis is induced by prazosin treatment, these cellular processes comprise pericyte and endothelial cell activation without significant proliferation as well as generation of intraluminal protrusions (16). However, increased wall shear stress might also lead to upregulation of eNOS concentration and activity in vascular endothelial cells (18) as was shown in white but not red skeletal muscle in an exercise-training model (33). We suggest that the eNOS increase we observed (and the resulting higher availability of NO) due to the prazosin administration has an impact on the augmented synthesis and/or secretion of VEGF in endothelial cells, which is then responsible for the initiation and control of angiogenesis. Because the VEGF increase does not occur in eNOS-knockout mice, eNOS-derived NO produced in endothelial cells represents an obligate upstream signal for shear stress-induced, VEGF-dependent angiogenesis in skeletal muscle. Furthermore, eNOS that is possibly present in mitochondria of skeletal muscle fibers (35, 42) might also participate in the angiogenic response. However, we cannot exclude the possibility that other NO-independent signaling pathways exist, because prazosin induced significant angiogenesis in C57BL/6 wild-type mice despite chemical NOS inhibition by L-NAME. Consistently, NO-independent mechanisms might also contribute to the 21% higher C/F ratio observed in EDL muscles of eNOS-knockout mice (compared with C57BL/6 wild-type mice). It seems reasonable that the 18% higher level of VEGF measured in these mice maintains this higher C/F ratio, but other angiogenic factors (e.g., FGF-2) might also be involved. Interestingly, there was no further induction of VEGF in response to elevated wall shear stress in eNOS-knockout mice that could depend on their higher basal VEGF concentration.



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  		Fig. 6. Schematic summary of the effects in response to prazosin administration in C57BL/6 and eNOS-knockout mice. Application of prazosin leads to elevation of the wall shear stress in capillaries of skeletal muscles in both mouse strains. In C57BL/6 mice but not eNOS-knockout mice, VEGF expression in endothelial cells exposed to higher shear stress was subsequently increased as reported earlier (10), and angiogenesis was induced as shown here. Thus the presence of eNOS is an obligate prerequisite for shear stress-induced, VEGF-dependent angiogenesis in skeletal muscles but might be partially compensated by other signaling systems, e.g., after acute chemical NOS blockade with N{omega}-nitro-L-arginine methyl ester (L-NAME). Excessive angiogenesis might lead to luxury perfusion and hyperoxia accompanied by subsequent suppression of VEGF and nNOS in the skeletal muscle fibers and vascular remodeling (e.g., capillary pruning). Interrupted lines and white arrows indicate suggested relationships. For further details, see DISCUSSION.

 
An upstream effect of NO on the expression of angiogenic factors in skeletal muscle was suggested by Benoit et al. (7), who observed a 50% increase in VEGF mRNA but no significant change in FGF-2 mRNA in rat gastrocnemius muscle after a 1-h administration of the NO donor sodium nitroprusside. Furthermore, it was also reported that the contraction-induced upregulation of VEGF mRNA was attenuated in the presence of NOS inhibitors, whereas the expression of FGF-2 and transforming growth factor-{beta} mRNA was not affected (20). These studies, however, measured only changes in mRNA levels in response to acute chemical NO exposure or NOS blockade and did not analyze angiogenesis in vivo under chronic conditions.

Thus far we cannot specify whether a direct or indirect mechanism is responsible for the observed upstream effect of eNOS on prazosin-dependent angiogenesis in skeletal muscle. A direct influence would occur if NO influences the VEGF concentration (at least) at the transcriptional level (20, 27). An indirect effect would be achieved if eNOS-derived NO influences endothelial cell apoptosis, which is important for vascular remodeling in response to hindlimb ischemia (10). An indirect upstream effect would also exist if a higher eNOS activity increases the prazosin-induced relaxation of the vascular smooth muscle cells and, by that, synergistically enhances the wall shear stress in capillaries of skeletal muscles. Thus eNOS would be necessary for the establishment of a hemodynamic environment appropriate to trigger the expression of the factors responsible for shear stress-dependent angiogenesis in skeletal muscles (e.g., to exceed a threshold of shear stress).

Besides the upstream effect of eNOS described here, an additional downstream effect of eNOS-derived NO on VEGF-dependent angiogenesis in skeletal muscle cannot be excluded. Such an effect would be characterized by the impact of VEGF availability on the concentration of NO, which then modulates the strength of cellular processes accompanied with angiogenesis. Two studies describe such a downstream effect of NO on angiogenesis in skeletal muscle. Murohara et al. (38) observed that exogenously supplied VEGF induced capillary proliferation in ischemic skeletal muscles of C57BL/6 mice but not eNOS-knockout mice, which suggests that VEGF could not rescue the eNOS-derived NO deficiency important for angiogenesis. Correspondingly, NO was found to be necessary for the increase of VEGF-A121- and FGF-2-inducible blood flow in skeletal muscle as a prerequisite for arteriolar remodeling (47).

In the present study, a three-step angiogenic reaction was detected in EDL muscles of C57BL/6 mice that were treated with prazosin for 0, 3, 4, 8, and 14 days. In the first step (after 4 days of prazosin application), the number of AP-positive capillaries increased; in the second step, the number decreased again. In the third step (after 8 days of prazosin application), the C/F ratio appeared to be balanced at a constant value that was higher than the basal C/F ratio. We suggest that prazosin first induced excessive capillary generation in EDL muscles of C57BL/6 mice. Unneeded capillaries regressed to finally lead to a higher CD than observant under nonstimulated conditions. This time course of prazosin-induced angiogenesis was not observed in EDL muscles of eNOS-knockout mice.

Prazosin application leads to the expansion of the capillary system by modulation of the hemodynamic environment (flow rate, shear stress) in skeletal muscle (12). It seems reasonable that the prazosin effect on the vasculature is lowered subsequently by mechanisms of hemodynamic counterregulation after a few days, which then leads to the reduction of the initial angiogenic stimulus and degradation of superfluous vessels (pruning). This obstruction of irregularly perfused capillaries could occur until a CD is established that corresponds to the strength of the stimulus of the newly adjusted hemodynamic force (vascular remodeling). Some evidence in the literature supports this suggestion. Ichioka et al. (25) observed that wall shear stress values returned to initial levels after 9 days of prazosin application in rat ear chambers, which is indicative of short-term counterregulation mechanisms. The readjustment of wall shear stress may thus be the cause for vascular remodeling. Linderman et al. (32) found that the CD in extensor muscles decreased rapidly after the chronic electrical stimulation of the rat hindlimb was stopped and the stimulus for capillary growth (very probably increased blood flow) was removed. It has also been shown (36) that the ultrastructure of endothelial cells reflects directly their activation level, which can be modulated by alterations in hemodynamic forces. The suggestion that angiogenesis can be accomplished by angioadaptive vascular remodeling is additionally supported by the observation that prazosin-induced angiogenesis is realized by internal division of capillaries (16); this is similar to intussusceptive growth, which occurs within a few hours (13). In contrast with our results, Milkiewicz et al. (37) reported that the C/F ratio in rat EDL muscles continuously increased during 2 wk after both chronic electrical stimulation and prazosin administration (in similar concentrations to those we applied). It is not yet clear why the time course of angiogenesis detected in Milkiewicz's study (37) differed from that described here. The differences might depend on the way that prazosin was dissolved in tap water, or they might reflect species-specific variations.

Shear stress is a strong modulator of eNOS concentration in endothelial cells (18). Thus the kinetics of eNOS expression observed in our study in EDL muscles of C57BL/6 mice supports our concept that the hemodynamic environment is not stably established within skeletal muscles during prazosin administration. We suggest that initial increases of blood flow and shear stress are responsible for the strong induction of eNOS (as well as for the angiogenic growth of the capillary system). Subsequent capillary pruning apparently goes along with a slight eNOS downregulation, whereas the final vascular remodeling step is accompanied by a second eNOS increase. Additional experiments (e.g., studies applying intravital microscopy) should be performed to clarify the relationship between shear stress and eNOS expression in mouse skeletal muscles.

As mentioned above, earlier we also reported (11) on VEGF upregulation in endothelial cells in response to prazosin. By performing quantitative immunoblot analysis, we now find that the VEGF concentration in homogenates decreases until 4 days of prazosin application only to increase afterward. Thus the time course of the C/F ratio in C57BL/6 mice is inversely related to the VEGF level that is detectable in total tissue extracts. Because VEGF is produced in high concentrations by skeletal muscle fibers (2, 3, 38), the amount of VEGF in total muscle extracts mainly reflects its expression in skeletal muscle fibers. We suggest that prazosin-dependent increases in shear stress are initially so strong that they induce excessive angiogenesis; this, together with the flow increase, is responsible for the establishment of a luxury perfusion in the skeletal muscles (see Fig. 6). Furthermore, increased NOS levels (and, consequently, higher NO availability) as initially observed in response to the prazosin stimulus might also inhibit mitochondrial cytochrome oxidase and thereby lower oxygen consumption in the tissue (35, 42). Both processes (luxury perfusion and inhibition of mitochondrial activity) would lead to a reduction in VEGF expression, because increased intracellular PO2 lowers the level and activity of the VEGF regulator protein hypoxia-induced factor-1{alpha} in skeletal muscle fibers (45). Thus skeletal muscle fibers and capillary endothelial cells represent two VEGF sources that respond in different ways to the prazosin stimulus. The VEGF concentration in skeletal muscle fibers might reflect the extent of the oxygen supply, whereas shear stress is the VEGF regulator in endothelial cells (11).

The nNOS concentration in EDL muscles of C57BL/6 mice decreased concomitantly with the C/F ratio after 4 days of prazosin administration. Thus nNOS is apparently not regulated if the angiogenic proliferation of the capillary system occurs. In general, the nNOS concentration in skeletal muscle fibers is lowered if a surplus of oxygen or nutrients is available and is increased if the supply is catabolically consumed (reviewed in Ref. 26). We therefore suggest that the luxury perfusion first established in skeletal muscle fibers as consequence of excessive angiogenesis induced by prazosin leads to suppression of nNOS. This nNOS downregulation might be mediated by the preceding VEGF reduction and may contribute to the vascular remodeling identified in this study as the second step in the systemic response to prazosin. Additional research is necessary to identify the actual molecular mechanism involved in the nNOS decrease.

In skeletal muscle, NO availability and angiogenesis are clearly linked to one another. Moreover, the metabolic state of the skeletal muscle fibers is also involved in the regulation of growth and density of the microvascular system as was shown for myoglobin-deficient knockout mice (21). Therefore, it seems reasonable that the successful assembly of an intact vascular network depends on the balanced cooperation of NO, angiogenesis, and metabolism of skeletal muscle fibers with reciprocal interactions (43). This demands that the concentrations and expression patterns of these factors and/or parameters are locally controlled. Consequently, disturbed localization patterns might lead to the development of malfunctioning blood vessels (capillaries) as was observed (41) for VEGF in skeletal muscle, and result in the formation of unstable hemorrhagic blood vessels.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Pr 271/5-4, Za 184/1-4, and Za 184/3-2) and the Freie Universität Berlin (Forschergruppenschwerpunkt Angiogenese, Teilprojekte 2, 3, and 7).


   ACKNOWLEDGMENTS
 
The skillful technical support of Martina Gutsmann and Heidrun Richter is gratefully acknowledged. The authors thank Dr. Olga Hudlicka (Birmingham) as well as Dr. Valentin Djonov and Dr. Martin Flück (Bern) for critical discussions and help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Baum, Dept. of Anatomy and Physiology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Königin-Luise-Str. 15, D-14195 Berlin-Dahlem, Germany (E-mail: olibaum{at}zedat.fu-berlin.de)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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