Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior (TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.
- gene expression
the growth of new blood vessels plays a critical role in normal physiological processes but is also central to the progression of many diseases. Angiogenesis is involved in several disorders such as cancer, diabetic retinopathy, macular degeneration, and endometriosis (15). In an attempt to control pathological angiogenesis, a growing interest in better understanding prosurvival and prodeath signals has emerged (7, 8, 26, 34). Each endothelial cell within a vessel wall is exposed to a combination of prosurvival and prodeath signals. The sum of these signals determines whether the cell remains viable or undergoes apoptosis (10).
Our laboratory has demonstrated that physiological, pharmacological, or genetic manipulation of the renin-angiotensin system (RAS) has an important impact on both the basal number of microcirculatory blood vessels and the ability of tissues to undergo angiogenesis induced by exercise (3) or electrical stimulation (2). In previous studies (4), we demonstrated that transfer of a region of chromosome 13 containing the renin gene from Dahl R into Dahl S rats restores both plasma renin activity (PRA) and the angiogenesis response to electrical stimulation. Similar results were observed in SS-13BN/Mcwi rats produced by the transfer of the chromosome 13 from the BN/Mcwi rat into the SS/JrHsdMcwi rat genetic background. Although transfer of the chromosome 13 in the SS-13BN/Mcwi rats restored angiogenesis when the rats were fed a low-salt diet, high-salt feeding significantly inhibited the ability of these rats to undergo angiogenesis in response to electrical stimulation. These chromosomal transfer rats provide an outstanding experimental control in which to study the effect of salt intake because of the genetic similarity between the SS-13BN/Mcwi and the SS/JrHsdMcwi rats despite the extreme difference in the observed angiogenic phenotype. Although these studies suggest a role for the RAS in skeletal muscle angiogenesis, the mechanisms underlying the interaction between salt intake, the RAS, and the regulation of the capillary growth process are not totally understood. We hypothesized that under the condition of high salt intake the balance between death and survival factors is switched to favor endothelial cell apoptosis. Because a myriad of factors influence the balance between life and death in angiogenic endothelial cells, preliminary experiments were performed using microarray to examine genetic changes and gene expression profiles that might correlate to the angiogenic response in skeletal muscle after electrical stimulation in animals fed low- and high-salt diets. On the basis of the microarray results, we performed a series of studies in which we directly tested the hypothesis that angiogenesis is inhibited by high-salt diet through an upregulation of proapoptotic pathways.
MATERIALS AND METHODS
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. A consomic rat strain (SS-13BN/Mcwi) derived from BN/Mcw rats and Dahl-SS/JrHsdMcwi rats were used in these studies; the origin of this strain was described previously (12). All rats for all protocols were prepared as follows. Consomic SS-13BN/Mcwi rats were placed on a high (4% NaCl)- or low (0.4% NaCl)-salt diet 2 days before surgery and maintained throughout the entire experiment. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were electrically stimulated for 8 h/day for 7 consecutive days as previously described (30). The contralateral leg was used as a control. All animals were euthanized 7 days after the onset of stimulation. The numbers of animals for each group are indicated in Figs. 2–7.
Plasma renin activity.
After 7 days of electrical stimulation an arterial blood sample was obtained and PRA was measured as previously described (38).
Tissue harvest and morphological analysis of vessel density.
After 7 days of stimulation, the animals were euthanized by an overdose of Beuthanasia solution (Sigma, St. Louis, MO) and the stimulated and contralateral unstimulated TA muscles were removed and weighed. A 100-mg section was taken from each TA muscle and immediately frozen in liquid nitrogen for RNA isolation. The remaining TA was lightly fixed overnight in 0.25% formalin solution and sectioned longitudinally. TA sections were stained with lectin, and vessel density was determined as previously described (21, 38).
Additionally, frozen 8-μm TA sections were stained for CD31, an endothelial cell marker, with an FITC-labeled secondary antibody. Capillaries and myofibers were counted in 10 microscopic fields (×40 magnification), and capillary density was expressed as capillary-to-myofiber ratio (36).
Construction of cDNA microarrays of known rat genes.
Microarrays containing 1,751 named cDNA clones were constructed as described previously (29), using 1,687 rat gene cDNA clones purchased from Research Genetics and 64 rat additional genes cloned by our group. This array has been shown to contain ∼80% of the known rat genes (11).
cDNA labeling and microarray hybridization.
Total RNA was isolated from TA muscle with TRIzol (Invitrogen-Life Technologies, Carlsbad, CA). Fifty micrograms of total RNA was reverse-transcribed to cDNA in a reaction primed by two micrograms of oligo(dT)12–18 as described previously (29). The gene expression profiles from stimulated TA muscle from SS-13BN/Mcwi rats on a low-salt diet were compared with profiles from unstimulated TA muscle from the same rat, and gene expression profiles from stimulated TA muscle of SS-13BN/Mcwi rats on the high-salt diet were compared with profiles from unstimulated TA muscle from the same rat. For each comparison, one muscle sample was labeled with Cy3 and the other was labeled with Cy5. The two samples were pooled after labeling and hybridized to a microarray. To control for dye variations, these two samples were labeled again with opposite dyes and hybridized to a second microarray.
Data normalization and identification of differentially expressed genes.
Data were normalized and analyzed with methods described previously (11, 28). A gene was considered differentially expressed only if the averaged, log-transformed, and normalized ratio of the gene was beyond mean ± 2SD of the entire set of ratios from that comparison and if the raw data for that gene passed the quality selection process (29) and yielded ratios in at least five of the six paired “flip dye” hybridizations in that comparison.
Immunofluorescence for CD31/PECAM-1 (endothelial cells) and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (apoptotic cells).
Frozen muscle tissues were sectioned (8 μm), mounted on positively charged slides, air dried for 30 min, and fixed in cold acetone for 5 min, 1:1 acetone-chloroform (vol/vol) for 5 min, and acetone for 5 min. Samples were washed three times with PBS, pH 7.2 and double stained with CD31, and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) was performed as previously described (44). The slides were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes) to quantify the total nuclei and to minimize fluorescence bleaching. Immunofluorescence microscopy was performed with a ×40 objective. Endothelial cells were identified by red fluorescence (CD31), total cell number was detected by blue fluorescence (DAPI DNA staining), and apoptosis was detected by green fluorescence (TUNEL). Apoptotic endothelial cells were detected by colocalized red and green (displayed as yellow) fluorescence. Quantification of apoptotic endothelial cells was expressed as an average of the ratio of number of apoptotic endothelial cells per square millimeter to total number of cells (DAPI staining) or total number of endothelial cells (CD31 staining) per square millimeter at ×40 magnification.
To determine whether endothelial cells in skeletal muscle underwent apoptosis after electrical stimulation, the number of apoptotic cells (TUNEL positive) was divided by total cell number (DAPI staining) to determine the percentage of apoptotic cells in the muscle tissue. To determine the percentage of apoptotic endothelial cells, the number of TUNEL-CD31-positive cells (apoptotic endothelial cells) was normalized to the total endothelial cell number (CD31 staining).
Western blot for Bcl-2 and Bax.
One hundred milligrams of TA muscle was homogenized in 0.1 M potassium buffer, pH 7.7, containing 0.1 mM PMSF. Protein was separated in denaturing SDS-15% polyacrylamide gel (30 μg/lane) and then blotted onto a nitrocellulose membrane. Membranes were incubated with a rabbit polyclonal antibody for Bax (dilution 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and Bcl-2 (dilution 1:1,000; BD Pharmingen, San Diego, CA) for 2 h at room temperature and after serial washes (5 × 3 min in Tris-buffered saline-Tween 20) with the secondary antibody (anti-rabbit IgG, 1:3,000) for 1 h at room temperature. Bax and Bcl-2 proteins were detected by chemiluminescence (Pierce, Rockford, IL) followed by autoradiography.
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) or as capillary-to-fiber ratio. For each experimental group, the measured vessel density of the stimulated muscle was compared with its unstimulated counterpart. All values are presented as means ± SE. The significance of differences in values measured in the same animal was evaluated with a two-factor ANOVA (diet × stimulation) with repeated measures on one factor (stimulation). To evaluate the significance of differences in vessel density between stimulated and unstimulated limbs, a one-factor ANOVA was performed. Significant differences were further investigated with Tukey's post hoc test.
Effects of dietary salt intake on PRA.
The high-salt diet dramatically suppressed (87.5%) PRA compared with the low-salt diet (Fig. 1).
Microvascular density changes in TA muscle induced by electrical stimulation.
As previously shown by our laboratory (4), 7 days of electrical stimulation does not induce angiogenesis in Dahl SS/JrHsdMcwi rats fed either low- or high-salt diets. However, the transfer of chromosome 13, containing a functioning renin gene, from Brown Norway rats to Dahl-S rats (to generate strain SS-13BN/Mcwi) effectively restores PRA and the angiogenic response in rats fed a low-salt diet (4). In SS-13BN/Mcwi rats fed a low-salt diet, electrical stimulation caused a significant 16% increase in vessel density (P = 0.0001) measured in longitudinal sections from the stimulated TA muscle compared with unstimulated control (Fig. 2). When these animals were placed on a high-salt diet, the angiogenic response to electrical stimulation was attenuated to a 9% increase (Fig. 2). Electrical stimulation also significantly increased the capillary-to-fiber ratio in the stimulated TA muscle of rats fed a low-salt diet compared with the unstimulated limb (P = 0.003; Fig. 3, A, C, E). Electrical stimulation did not induce a significant increase in capillary-to-fiber ratio when rats were fed a high-salt diet (Fig. 3, B, D, E).
Classification of genes with altered expression in skeletal muscle after electrical stimulation in rats under low- and high-salt diets.
Gene expression profiles from stimulated and unstimulated TA muscles were compared between rats fed either low- or high-salt diet. The sets of genes whose expression was altered after stimulation were identified by a conservative statistical analysis as described previously (11, 28). The genes that were differentially regulated by electrical stimulation are shown in Tables 1 and 2 and Fig. 4. These genes were categorized based on their reported functions. Seven major categories, including genes related to signal transduction, transcription processing, cell growth, survival, cell adhesion, migration, cell cycle arrest, and apoptosis were used in the analysis. Many of the genes on the array are either not well characterized in terms of function or have multiple functions. For cases in which multiple functions were assigned, we carefully reviewed the literature and selected the most relevant category for each gene.
In rats fed a low-salt diet, 21 genes were upregulated in TA muscle after stimulation and none was downregulated. Most of these genes (48%) were related to cell proliferation or to adhesion and migration events that are intimately related to the angiogenic process (Table 1 and Fig. 4). In contrast, in rats fed a high-salt diet, 84 genes changed expression pattern after stimulation. Among those genes whose expression changed after stimulation, 48 (57%) were upregulated and 36 (43%) were downregulated (Table 2 and Fig. 4). Interestingly, 25% of the upregulated genes are known to function in apoptosis and/or cell cycle arrest. Of the downregulated genes, 14% are related to cell survival, among them VEGF and A-RAF, which regulate endothelial cell survival. On the basis of these preliminary findings we hypothesized that endothelial cell apoptosis would be increased in the stimulated hindlimb of animals fed a high-salt diet compared with those fed a low-salt diet. To test this hypothesis we therefore performed additional assays to evaluate the level of endothelial cell apoptosis in TA muscle after electrical stimulation and its contribution to the angiogenesis response in SS-13BN/Mcwi rats fed a high- or low-salt diet.
Apoptotic nuclei detected by in situ DNA end labeling.
The number of TUNEL- and CD31-stained cells in TA muscle was not significantly increased after stimulation in the low-salt group (Fig. 5, A and B). On the other hand, a substantial increase in TUNEL-CD31-positive staining was observed in the stimulated TA of the high-salt group compared with unstimulated muscle (P = 0.038; Fig. 5, D and E). The triple staining (TUNEL, CD31, DAPI) revealed that TUNEL-positive nuclei were coincident with DAPI chromatin staining (Fig. 5, C and F).
The percentage of total apoptotic cells in the unstimulated leg was ∼4.6% and 3.6% in high- and low-salt groups, respectively (Fig. 6A), and only a very small portion of these cells (0.4–0.6%) were endothelial cells (Fig. 6B). Neither the percentage of all apoptotic cells nor the percentage of apoptotic endothelial cells (Fig. 6, A and B) changed significantly in the stimulated hindlimb of rats fed a low-salt diet. However, in the stimulated hindlimb of rats fed a high-salt diet, the percentage of apoptotic cells increased by 35% compared with the unstimulated limb (P = 0.025; Fig. 6A), as visualized by the increase in green fluorescence indicated in Fig. 3, D and E. Many of these cells were TUNEL-CD31 positive, indicating a substantial increase (P = 0.038) in endothelial cell death (∼4-fold) under these conditions (Fig. 6, B and C). The numbers of nonendothelial TUNEL-positive cells were not significantly changed in the stimulated vs. unstimulated hindlimb in rats fed a high-salt diet (Fig. 6D).
After electrical stimulation the total number of cells (determined by DAPI staining) was significantly (P < 0.05) increased in the stimulated hindlimb of both high- and low-salt groups (Table 3). As expected, electrical stimulation promoted a significant increase (P = 0.002), of approximately twofold, in the number of endothelial cells (Table 3 and Fig. 5, A and B) in the stimulated hindlimb of the low-salt group compared with unstimulated muscle from the low-salt group. In contrast, this increase was not significant in animals fed a high-salt diet. It is important to note that the increase in endothelial cell apoptosis caused by stimulation was approximately twofold greater (P = 0.025) in the high-salt group than the low-salt group, and this correlates with the differences found in vessel density response between these groups.
Bcl-2 and Bax expression.
To further understand the mechanisms involved in stimulation of apoptosis in the TA muscle after electrical stimulation, we examined the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins by Western blotting. The levels of Bax were significantly (P < 0.05) increased in the stimulated limb compared with unstimulated TA for rats fed both diets (Fig. 7A). The level of Bcl-2 expression was significantly attenuated only in the stimulated TA of the high-salt group compared with the unstimulated muscle (Fig. 7B; 487 ± 12.6 vs. 357 ± 53.7 arbitrary units, P < 0.05). The Bcl-2-to-Bax ratio, which is an important regulator of apoptosis (41), was significantly reduced in the stimulated compared with unstimulated TA muscle of the high-salt group (Fig. 7C; 1.01 ± 0.13 vs. 0.48 ± 0.06 arbitrary units, P < 0.05), confirming the TUNEL studies and indicating an activation of the mitochondrial cell death pathways under these conditions.
Angiogenesis is a complex and multistep process that involves programmed dedifferentiation of preexisting endothelial cells, followed by endothelial cell proliferation, migration, and tissue invasion (10). In the current study electrical stimulation promoted a dramatic angiogenesis response in the stimulated hindlimb of SS-13BN/Mcwi rats fed a low-salt diet. Preliminary data from a gene expression analysis of the TA muscle by microarray showed that genes involved in cell adhesion, proliferation, and migration formed the largest class of genes affected by stimulation when rats were fed a low-salt diet. When animals were placed on a high-salt diet the angiogenic response was attenuated and many genes involved in apoptotic pathways were upregulated.
A high-salt diet is associated with many pathological alterations in organ function that are independent of blood pressure effect (5); among these endothelial dysfunction and vascular injury (42) may have a great impact in the angiogenesis process. Studies have indicated that endothelial cell apoptosis has an important role in the integrity and function of the endothelium and may contribute to the pathogenesis of a variety of human diseases (46).
Previous studies from our laboratory (2, 3) demonstrated that the physiological inhibition of RAS with a high-salt diet or pharmacological inhibition with an angiotensin-converting enzyme inhibitor and ANG II type 1 receptor blocker significantly attenuates angiogenesis in skeletal muscle after electrical stimulation or exercise. The mechanisms responsible for these responses still need to be clarified. ANG II can stimulate angiogenesis in vivo and endothelial cell proliferation and migration in vitro (6, 14, 23, 27, 32, 43). Our data suggest that RAS activation through a low-salt diet could increase the expression of a set of genes that will ultimately result in an improved angiogenic response in stimulated TA muscle. When RAS was inhibited with a high-salt diet, the angiogenic response induced by electrical stimulation was abolished and a switch in gene expression from a proangiogenesis to a proapoptosis profile was observed. A number of genes involved in apoptosis and/or cell cycle arrest such as class III Fc-γ receptor, cyclophilin B, annexin III, transferrin, cytochrome-c oxidase subunit VIa, cystatin C, thymosin β-10, antiproliferative factor (BTG1), serine protease, S-100-related protein, cell surface glycoprotein CD44, and cytosolic phosphoprotein (p19) were found to be upregulated in the stimulated muscle under high-salt diet conditions. In addition, genes related to endothelial cell survival, such as A-Raf and VEGF, were downregulated after stimulation. These results indicate that activation of genes related to cell death or downregulation of survival genes could have a great impact on endothelial cell proliferation and/or survival and consequently impair the neovascularization when the diet is high in salt. In support of this hypothesis, we showed that in the TA muscle of rats fed a high-salt diet, but not of those fed a low-salt diet, the number of TUNEL-CD31-positive cells increased significantly after stimulation and that the increased level of apoptosis was associated with a dramatic attenuation in vessel density determined by direct vessel counting and also the capillary-to-fiber ratio. Previous studies showed that inhibition of endothelial cell apoptosis with caspase inhibitors impaired the early stages of in vitro angiogenesis and blocked VEGF-dependent vascular formation in vivo (40). Recently, it was demonstrated that retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy is markedly attenuated in Bcl-2−/− mice (45). Pathological angiogenesis, such as the persistence of fetal ocular vasculature, can also be observed in mice deficient in proapoptotic proteins, Bax, Bak (22), and P53 (37). In light of this and other evidence, the inhibition of endothelial survival factors, or the activation of endothelial cell death machinery, is considered an attractive strategy in the inhibition of pathological angiogenesis, especially in clinical trials for cancer treatment (39).
Angiogenesis is controlled by a variety of factors. The balance between proangiogenic and prodeath signals regulates not only neovessel formation but also microvessel persistence. Although apoptosis of endothelial cells is important for the regulation of physiological and pathological angiogenesis (15), little is known about whether ANG II plays an anti- or a proapoptotic role, especially in microvascular endothelial cells (Fig. 8). \. Salt intake is one important physiological factor that regulates renin secretion and consequently ANG II levels. Our data support a recent study (35) that indicates that ANG II plays a critical antiapoptotic role in vascular endothelial cells by interfering with mechanisms involving phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation and survival pathways. ANG II has been reported to increase VEGF expression in endothelial cells (18), and sodium intake may exert its effects through this pathway. The microarray experiments further support this hypothesis, showing a downregulation of VEGF expression in the stimulated leg of rats fed a high-salt diet.
In vitro, VEGF inhibits the apoptosis of endothelial cells cultured either as monolayers or as capillary-like structures (33, 1, 31). VEGF may impose its survival function through multiple mechanisms, including activation of the PI3-kinase/AKT pathway (20, 17), activation of the nitric oxide pathway (9), and induction of Bcl-2 and A1 (19). After stimulation in our experiments, Bcl-2 was significantly attenuated in the TA muscle of the high-salt group and Bax expression was equally increased in the stimulated TA muscle of both groups. However, the Bcl-2-to-Bax ratio, an important indicator of apoptosis, was significantly suppressed only in the stimulated limbs of rats fed a high-salt diet, indicating an activation of the death program. Thus, under high-salt conditions, suppression of the Bcl-2-to-Bax ratio may lead to Bax-dependent mitochondrial membrane permeability changes with a loss of the membrane potential and cytochrome c and other proapoptotic protein release. On the other hand, it is important to note that the Bcl-2-to-Bax ratio tended to be lower in the stimulated TA muscle of the low-salt group compared with unstimulated TA. Under these conditions there was also some increment in the level of TUNEL-CD31-positive cells.
Studies have shown that in most adult blood vessels endothelial cells survive for prolonged periods and apoptosis is difficult to detect (16, 13). However, during angiogenesis apoptosis is activated and plays an important role in removing damaged or immature endothelial cells to allow for vascular remodeling. As soon as endothelial cells incorporate into vascular structures they appear to become more resistant to apoptosis (13, 25). Although a very low level (<0.5%) of endothelial cell apoptosis was detected in unstimulated TA muscles, our results indicate that the exaggerated endothelial cell apoptosis observed in the stimulated hindlimb of the high-salt group may impair the angiogenic response and/or accelerate the vessel regression process. Apoptosis is also augmented during the final phase of angiogenesis, a process termed vascular pruning, in which endothelial cells that have failed to mature are eliminated (24). Previous studies in our laboratory (30) demonstrated that electrical stimulation progressively increases vessel density in the TA and EDL muscles, peaking at 7 days after stimulation and subsequently decreasing to control levels by 14 days after stimulation. In light of these previous studies, it is possible that the vessel regression process is accelerated when animals are fed a high-salt diet and high levels of endothelial cell apoptosis may be detected during this transitional period. However, additional experiments are necessary to prove this hypothesis.
In summary, this study indicates that apoptotic pathways are activated in skeletal muscle after stimulation in SS-13BN/Mcwi rats fed a high-salt diet, and this activation is associated with an increased number of apoptotic endothelial cells and a suppressed angiogenic response to electrical stimulation. The exacerbation in endothelial cell death resulting from a high-salt diet may represent one important mechanism for the suppression of the angiogenic response after electrical stimulation.
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-29587 and NHLBI Contract N01-HV-28182.
The authors thank Elizabeth Berdan and Christine Puza for expert technical assistance and Peigang Li for data analysis assistance.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: A. S. Greene, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail:).
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