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Capillary regression in vascular endothelial growth factor-deficient skeletal muscle

¹ Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
² Department of Molecular Oncology, Genentech, Incorporated, South San Francisco, California

	ABSTRACT

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Skeletal muscle angiogenesis is an important physiological adaptation to increased metabolic demand, possibly dependent on vascular endothelial growth factor (VEGF), the increased expression of which is a known early response to exercise. To test the hypothesis that VEGF is essential to muscle capillary maintenance, we evaluated the consequences of targeted skeletal muscle inhibition of VEGF expression in postnatal, cage-confined VEGFloxP(+/+) mice. To delete VEGF, cre recombinase expression was accomplished using direct intramuscular injection of a recombinant adeno-associated cre recombinase expressing viral vector. Four weeks postinfection, VEGF-inactivated regions revealed 64% decreases in capillary density and capillary-to-fiber ratio. Substantial apoptosis was also observed in VEGF-depleted regions. There was no evidence of rescue at 8 wk, with a persistent 67% reduction in capillary-to-fiber ratio and a 69% decrease in capillary density. These data implicate VEGF as an essential survival factor for muscle capillarity and also demonstrate insufficient VEGF-dependent signaling leads to apoptosis in mouse skeletal muscle.

muscle; apoptosis; capillaries; peripheral vascular disease; growth factors/cytokines

	INTRODUCTION

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SKELETAL MUSCLE DYNAMICALLY adapts to activity level or environmental limitations by altering structural and functional determinants of oxidative metabolism (21, 30). A key component of this adaptive response is the number of capillaries perfusing each muscle fiber, which increases with repeated activity. Increased capillarity allows for greater O₂ availability and exchange of metabolites between the vasculature and myocytes. While skeletal muscle angiogenesis is an important physiological response to exercise in healthy individuals, impaired exercise capacity in patients with chronic renal failure, heart disease, or chronic obstructive pulmonary disease may in part be due to a reduction in the number of capillaries delivering oxygen and nutrients to the skeletal muscle (12, 22, 36).

Multiple signals generated during exercise, including mechanical events associated with muscle contraction and reduced muscle intracellular oxygen tension, have the potential to regulate vascular endothelial growth factor (VEGF) gene expression (6, 28, 29). VEGF is a well-known, potent angiogenic factor, which also functions as a vascular permeability and endothelial cell survival factor (13). We have recently demonstrated that a 1-h exercise session in untrained rats or humans leads to elevated gastrocnemius VEGF mRNA levels (6, 28). Furthermore, exercise-induced angiogenesis may be attenuated by neutralizing antibodies, which block soluble VEGF function (2). Thus VEGF has been proposed to be a critical factor for regulating the dynamic and temporal microvessel remodeling which occurs in skeletal muscle. However, an attempt to use traditional knockout of all three VEGF isoforms, VEGF₁₂₀, VEGF₁₆₈, VEGF₁₈₈, or its receptors (Flk-1 and Flt-1) results in embryonic lethality (14, 15, 33). Conditional knockout of VEGF₁₆₈ and VEGF₁₈₈ isoforms using a Cre-loxP-mediated strategy to remove VEGF exon 6, allowing only expression of mouse VEGF₁₂₀, has been reported to result in severe ischemic cardiomyopathy and a progressive reduction in skeletal muscle capillarity in the small percentage of mice that survived through postnatal day six (9). However, studies in other organ systems have suggested that lowering VEGF levels by either transcriptional or receptor blockade mechanisms leads to apoptosis of the capillary endothelial cells and eventual capillary attrition (1, 3, 23). In the present study, all three VEGF isoforms were specifically inactivated in skeletal muscle through the viral delivery of cre recombinase to muscle fiber regions of mice containing a floxed VEGF gene resulting in deletion of exon 3. The effect of lowering skeletal muscle VEGF was determined by correlating local VEGF levels with changes in capillary density, capillary-to-fiber ratio, and presence of apoptotic cells.

	MATERIALS AND METHODS

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Animals.
This study was approved by the University of California, San Diego, Animal Subjects Committee. Eight control (C57BL/6) and eight transgenic VEGFloxP mice, all aged 2–3 mo, were used for this study. VEGFloxP transgenic mice were provided by Dr. Napoleone Ferrara (Genentech, South San Francisco, CA), and details of the genetic engineering have previously been published (16).

Recombinant AAV/Cre construction and delivery.
AAV Helper-Free system (Stratagene, La Jolla, CA) was used for AAV/Cre production (31, 32, 37). The cre recombinase gene along with CMV promoter and SV80 poly-A sequence were inserted into the NotI site of pAAV-LacZ plasmid (Stratagene) to form a pAAV-Cre plasmid (19). This new, pAAV-Cre plasmid carries the gene cassette between two AAV2 LTRs in order to allow recombinant AAV to be assembled in strain 293 packaging cells. Recombinant AAV/Cre virus was produced by cotransfection of pAAV-Cre, pAAV-RC (expressing AAV2 rep/cap) and pHelper (expressing ADV E2A/E8), into strain 293 cells by calcium phosphate transfection (CalPhos Mammalian Transfection Kit; BD Sciences Clontech, Palo Alto, CA). Cells were collected and lysed using a freeze-thaw method. AAV/Cre recombinant virus titer was determined by hybridization of [³²-P]dCTP oligolabeled pAAV-Cre plasmid (Prime-It II random primer labeling kit, Stratagene) to serial dilutions of slot blotted viral supernatant and pAAV-Cre plasmid. To deliver AAV/Cre, mice were anesthetized with pentobarbital (40–60 mg/kg), and the midbelly of the gastrocnemius was injected with 5–10 µl (5 x 10⁹ total AAV/Cre particles) of viral supernatant. Four animals in each group were killed at 4 wk postinfection and another four at 8 wk postinfection for histological studies.

Immunohistochemistry.
At collection time, mice were euthanized with pentobarbital (65 mg/kg ip). The gastrocnemius muscle was frozen in isopentane and 10-µm sections were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS), rinsed with PBS, and blocked with 10% goat serum/0.4% Triton X-100 in PBS. Cre recombinase and VEGF were detected by incubation with cre recombinase-specific (1:1,000) (Novagen, Madison, WI) or VEGF-specific (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in PBS/18% goat serum at 4°C overnight. Slides were subsequently incubated with biotinylated secondary antibody followed by Alexa Fluor 488-conjugated streptavidin (Molecular Probes, Eugene, OR) and viewed by fluorescent microscopy.

Apoptosis.
TUNEL-positive apoptotic nuclei were detected in serial frozen sections using an in situ cell death detection kit (POD; Roche Diagnostic, Indianapolis, IN) and viewed by light microscopy.

Capillary measurements.
Frozen 10-µm serial cross sections were prepared and stained using the sensitive alkaline phosphatase detection method, which stains muscle fibers yellow and capillary endothelial cells blue (18). Sections were first presoaked in precooled (–20°C) 1:1 mixture of acetone and chloroform and allowed to dry at room temperature. Slides were transferred to an incubation mixture containing 0.08% gly-pro-8-methoxy-ß-naphtylamide and 0.034% fast blue in 0.1 M phosphate buffer, pH 7.5, for 60 min at 37°C, air dried overnight, and mounted. Sections were viewed by light microscopy and digitally imaged (each rectangular image being 1.118 mm x 0.86 mm). MATLAB 18.3 computer software was used to perform morphometric measurements. The total number of capillaries around each fiber (NCAF) was determined throughout entire cross-sectional regions of contiguous cre recombinase-expressing fibers and a surrounding region of noninfected fibers of an equal cross-sectional area. This circumference of noninfected fibers, surrounding the contiguous region of infected fibers, was chosen to avoid bias and minimize selection of a heterogeneous region. We analyzed 30–70 individual fibers from either AAV infected or noninfected regions. Fiber area was measured as the total sum of the area of all the fibers within a definable region divided by the number of fibers contained within the defined cross-sectional area. Detection of capillary number by alkaline phosphatase staining was also confirmed by immunohistochemical detection with antibodies specific for von Willebrand factor (Santa Cruz Biotechnology).

	RESULTS

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AAV/Cre-infected fibers in both control C57BL and VEGFloxP transgenic mice were confined to a contiguous region of 30–70 individual fibers as detected by immunohistochemical staining with an antibody specific for Cre recombinase (Figs. 1A–4A). Cre-positive fibers revealed a clear absence of VEGF expression in VEGFloxP(+/+) mice (Figs. 1B and 2B) but not in wild-type mice (Fig. 3B and 4B). AAV/Cre-infected regions were also assessed for changes in capillarity by staining for alkaline phosphatase-positive vascular structures (Fig. 1C–4C) (18). In VEGFloxP(+/+) mice, AAV/Cre-infected regions revealed substantial capillary regression, which was accompanied by the widespread appearance of TUNEL-positive apoptotic myocytes and capillary endothelial cells (Figs. 1D and 2D). In AAV/Cre-infected fiber regions, 13.4- and 12.3-fold increases in the number of apoptotic nuclei per square millimeter were measured compared with noninfected regions at 4 and 8 wk, respectively (Table 1). In wild-type mice infected with the AAV/Cre, changes in localized VEGF expression, capillarity, or apoptotic cells were not observed (Figs. 3, B–D, and 4, B–D, and Table 1). No inflammatory cells were identified in transfected fibers of control or VEGF loxP(+/+) mice at either 4 or 8 wk.

View larger version (80K): [in this window] [in a new window]   Fig. 1. Four weeks postinfection of VEGFloxP(+/+) mice with AAV/Cre reveal capillary regression. Gastrocnemii from VEGFloxP(+/+) transgenic mice were infected with 5 x 109 adeno-associated virus-expressing Cre recombinase (AAV/Cre) particles, intramuscularly. Four weeks later, infected fibers from VEGFloxP(+/+) transgenic mice were identified by nuclear immunostaining of serial cross sections with a Cre recombinase antibody (green) (A) and revealed lower levels of VEGF at the myocyte periphery (green) (B), a loss of capillaries detected by alkaline phosphatase staining (purple) (C), and TUNEL-positive apoptotic cells (brown) (D). VEGF, vascular endothelial growth factor.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Eight-week AAV/Cre-infected C57BL mice display normal capillary parameters and apoptosis. Gastrocnemii from C57BL mice were infected with 5 x 109 AAV/Cre particles, intramuscularly. Eight weeks later, infected fibers were identified by nuclear immunostaining of serial cross sections with a Cre recombinase antibody (green) (A) and revealed unchanged levels of VEGF at the myocyte periphery (green) (B), capillarity (purple) (C), and TUNEL-positive apoptotic cells (brown) (D).

View larger version (84K): [in this window] [in a new window]   Fig. 2. Eight-week AAV/Cre-infected VEGFloxP(+/+) mice show evidence of long-term effects on skeletal muscle capillarity. Gastrocnemii from VEGFloxP(+/+) transgenic mice were infected with 5 x 109 AAV/Cre particles, intramuscularly. Eight weeks later, infected fibers from VEGFloxP(+/+) transgenic mice were identified by nuclear immunostaining of serial cross sections with a Cre recombinase antibody (green) (A) and revealed lower levels of VEGF at the myocyte periphery (green) (B), a loss of capillaries detected by alkaline phosphatase staining (purple) (C), and TUNEL-positive apoptotic cells (brown) (D).

View larger version (91K): [in this window] [in a new window]   Fig. 3. Four-week AAV/Cre-infected C57BL mice infected do not display altered VEGF levels or capillarity. Gastrocnemii from control, C57BL mice were also infected with 5 x 109 AAV/Cre particles, intramuscularly. Four weeks later, infected fibers were identified by nuclear immunostaining of serial cross sections with a Cre recombinase antibody (green) (A) and did not reveal lower levels of VEGF at the myocyte periphery (green) (B), loss of capillaries detected by alkaline phosphatase staining (purple) (C), or increased TUNEL-positive apoptotic cells (brown) (D). Bar equals 100 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Quantitation of capillary parameters in low vs. normal VEGF expressing muscle regions. Morphometric confirmation of decreased capillarity in VEGFloxP(+/+) gastrocnemius fibers infected with AAV/Cre. Four (4W) and eight weeks (8W) postinfection with AAV/Cre, capillary number, number of fibers, and fiber surface area were measured in VEGFloxP(+/+) and C57/BL (wild type) in a contiguous area of infected fibers (+) and surrounding, noninfected (–) fiber regions of an equivalent area. From these determinants, the capillary/fiber ratio (number of capillaries/fiber) (A) and the capillary density (capillary number/fiber cross-sectional area) (B) were calculated. Error bars are SE; n = 4–6 mice. Student’s t-test was used to estimate significant differences between noninfected (–) and infected (+) regions: *P < 0.01.

	DISCUSSION

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Choice of targeted Cre-loxP method to ablate muscle VEGF.
To experimentally determine VEGF function in skeletal muscle, a Cre-loxP-mediated strategy of gene inactivation was used to deplete available VEGF throughout skeletal muscle fiber regions. This was accomplished by local injection of a recombinant cre recombinase adeno-associated viral vector directly into mouse gastrocnemii of VEGFloxP(+/+) transgenic mice (31). Subsequent cre recombinase expression resulted in conditional deletion of mouse VEGF exon 3, which is flanked by loxP sites in this transgenic mouse strain (16), and prevented expression of all three VEGF isoforms, VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈. This experimental approach allows a degree of temporal and spatial control over inactivation of any gene surrounded by bacteriophage P1 loxP sequences (32), and makes functional analysis of skeletal muscle VEGF in postnatal mice possible. This is an important consideration since knockout of embryonic VEGF is lethal. Furthermore, complicating developmental effects and potential long-term compensatory pathways were avoided by choosing to study regional cre recombinase delivery to mature, postnatal mice as opposed to using a muscle-specific promoter (i.e., muscle creatine kinase promoter) to drive cre recombinase expression in skeletal muscle throughout embryonic development. Another advantage of this experimental approach was that AAV/Cre-infected fibers were confined to a definable contiguous region of muscle fibers. Infected fibers could be localized by immunohistochemical detection of cre expression, and the identical fibers could be tested for changes in VEGF level, capillarity, and apoptotic progression. Areas of diminished capillarization and reduced VEGF expression closely overlaid that of cre expression, suggesting that there was little or no availability of VEGF from surrounding normal regions or via the blood supply to maintain muscle structure in the infected fibers. Overall, this is a powerful tool for studying the local effects of VEGF in skeletal muscle fibers within their native extracellular and cellular environment.

VEGF as an essential capillary survival factor.
The effects of VEGF inactivation were observed within 4 wk of a one-time AAV/Cre delivery and suggest that critical VEGF levels must be maintained to preserve muscle capillary-to-fiber structural organization. These observed changes in capillarity persisted for 8 wk without improvement, indicating that sufficient time had not occurred for potential compensatory pathways to become activated or biologically effective during this time frame. We suggest on this basis that adult mouse skeletal muscle may not be able to invoke alternative rescue pathways, via redundant angiogenic factors such as bFGF, TGF-ß, or angiopoietins. This finding highlights the importance of critical VEGF levels in the myocyte to transduce normal activity signals (such as shear stress, muscle contraction, gravitational forces, hypoxia) so as to preserve capillary number.

Potential mechanism of capillary regression.
One mechanism to destabilize capillary structure is to shift the balance of pro- and anti-angiogenic factors. In our analysis, inactivating the VEGF gene abolished approximately two-thirds of the capillaries in an AAV/Cre-infected region. Thus the remaining third of the capillaries appear to be refractory or VEGF-independent vessels. Studies in which VEGF expression was conditionally turned on and then off in the heart and liver suggest that the duration of elevated VEGF expression (greater than 2 wk) is a critical turning point for establishing VEGF-independent, refractory vessels (11). Furthermore, in tumor and retina model systems, capillaries are thought to remodel into VEGF-independent vessels as the endothelial cell layer is surrounded by pericytes or smooth muscle cells (4). However, mouse skeletal muscles exist as several diverse types based on their make up of varying proportions of glycolytic and oxidative fiber types (5, 8, 10). Signals stemming from these diverse skeletal myocytes, rather than pericytes or smooth muscles cells, are likely to regulate VEGF-dependent changes in the endothelial cell. Such a range of myocyte-endothelial interactions may allow an additional level of regulation over capillary formation or regression dependent on skeletal muscle fiber composition. For example, Lloyd and colleagues (24) have reported that ligated white gastrocnemius muscle responds to exercise training by increasing capillarity. In contrast, the same exercise stimulus results in a blunted angiogenic response in red gastrocnemius (38). Interestingly, VEGF as well as angiopoietin 1 and angiopoietin 2 were reported to be at a lower expression levels in white gastrocnemius (composed primarily of glycolytic fibers) compared with the red gastrocnemius (composed predominantly of oxidative fibers). Angiopoietins 1 and 2 antagonistically bind to the same endothelial receptor, Tie-2, and are reported to have opposing functions (26). Angiopoietin 1 functions to stabilize capillaries, while angiopoietin 2 has a dual role. In the presence of VEGF, angiopoietin 2 augments angiogenesis, whereas in the absence of VEGF it promotes capillary destabilization (20, 25). Furthermore, differences in the ratio of angiopoietin 2 to angiopoietin 1 transcripts are also present in white and red gastrocnemii of sedentary rats. The transcript ratio of angiopoietin 2 to angiopoietin 1 in white gastrocnemius has been reported to be 0.87, whereas the ratio in red gastrocnemius is only 0.4 (24). Thus lowering the already limited amount of VEGF in white gastrocnemius could potentially shift the balance of angiopoietin 2 to VEGF proportionally more than it would in red gastrocnemius. Red gastrocnemius contains higher VEGF levels and a lower ratio of angiopoietin 2 to angiopoietin 1, which would favor capillary stabilization. The balance and overall levels of angiogenic regulators in glycolytic white gastrocnemii may contribute to a muscle type design that is more sensitive to changes in VEGF and able to dynamically regulate capillarity with modest changes in angiogenic-dependent stimuli. The data collected from our mouse skeletal muscle targeted knockout model of VEGF gene inactivation is from a mixed region of gastrocnemius, composed of a heterogeneous population of glycolytic and oxidative fibers (mostly type IIa and IIb fibers and a small proportion of type I fibers) with presumably varying amounts VEGF transcript levels similar to the rat (5, 7). Thus it is reasonable to speculate that a portion of the capillary population may be refractory to depletion of VEGF.

Future VEGF based skeletal muscle therapy.
Currently, several animal studies and clinical trials are ongoing to test the possibility of delivering VEGF to ischemic limbs. Viral vectors expressing various VEGF isoforms have shown promise toward improving oxygen delivery and blood flow to impaired muscles (27, 34). The present study not only supports the rationale behind the use of VEGF to restore oxygen supply in capillary-depleted skeletal muscle of patients with renal failure, heart disease, or peripheral ischemia but also demonstrates the importance of critical VEGF levels for maintaining capillarity in otherwise healthy individuals. Understanding the mechanism of action of VEGF in normal and compromised skeletal muscle will allow greater insight into therapeutic strategies for patients with impaired exercise capacity secondary to many of the chronic illness occurring in older, sedentary populations.

	GRANTS

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This work was funded by National Institutes of Health Grant 18PO1-HL-17731-28.

	ACKNOWLEDGMENTS

 
We thank Harrieth Wagner, Li Cui, and Odile Mathieu-Costello for technical assistance in preparing histological sections.

	FOOTNOTES

 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: K. Tang, UCSD Dept. of Medicine 0623A, 9500 Gilman Drive, La Jolla, CA 92093-0623 (E-mail: ktang{at}ucsd.edu).

	REFERENCES

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1. Alon T, Hemo I, Itin A, Pe’er J, Stone J, and Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1: 1024–1028, 1995.[CrossRef][Web of Science][Medline]
2. Amaral SL, Papanek PE, and Greene AS. Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol Heart Circ Physiol 281: H1163–H1169, 2001.[Abstract/Free Full Text]
3. Benjamin LE, Golijanin D, Itin A, Pode D, and Keshet E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 103: 159–165, 1999.[Web of Science][Medline]
4. Benjamin LE, Hemo I, and Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125: 1591–1598, 1998.[Abstract]
5. Brasseur JE, Curtis RL, Mellender JW, Rimm AA, Melvin JL, and Sulaiman AR. Systematic distribution of muscle fiber types in the medial gastrocnemius of the laboratory mouse: a morphometric analysis. Anat Rec 218: 396–401, 1987.[CrossRef][Medline]
6. Breen EC, Johnson EC, Wagner H, Tseng HM, Sung LA, and Wagner PD. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol 81: 355–361, 1996.[Abstract/Free Full Text]
7. Brutsaert TD, Gavin TP, Fu Z, Breen EC, Tang K, Mathieu-Costello O, and Wagner PD. Regional differences in expression of VEGF mRNA in rat gastrocnemius following 1 hr exercise or electrical stimulation. BMC Physiol 2: 8, 2002.[CrossRef][Medline]
8. Carlson CJ, Booth FW, and Gordon SE. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol Regul Integr Comp Physiol 277: R601–R606, 1999.[Abstract/Free Full Text]
9. Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D’Amore PA, and Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat Med 5: 495–502, 1999.[CrossRef][Web of Science][Medline]
10. Couplan E, Gelly C, Goubern M, Fleury C, Quesson B, Silberberg M, Thiaudiere E, Mateo P, Lonchampt M, Levens N, De Montrion C, Ortmann S, Klaus S, Gonzalez-Barroso MD, Cassard-Doulcier AM, Ricquier D, Bigard AX, Diolez P, and Bouillaud F. High level of uncoupling protein 1 expression in muscle of transgenic mice selectively affects muscles at rest and decreases their IIb fiber content. J Biol Chem 277: 43079–43088, 2002.[Abstract/Free Full Text]
11. Dor Y, Djonov V, Abramovitch R, Itin A, Fishman GI, Carmeliet P, Goelman G, and Keshet E. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 21: 1939–1947, 2002.[CrossRef][Web of Science][Medline]
12. Duscha BD, Kraus WE, Keteyian SJ, Sullivan MJ, Green HJ, Schachat FH, Pippen AM, Brawner CA, Blank JM, and Annex BH. Capillary density of skeletal muscle: a contributing mechanism for exercise intolerance in class II–III chronic heart failure independent of other peripheral alterations. J Am Coll Cardiol 33: 1956–1963, 1999.[Abstract/Free Full Text]
13. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 280: C1358–C1366, 2001.[Abstract/Free Full Text]
14. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, and Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380: 439–442, 1996.[CrossRef][Medline]
15. Fong GH, Rossant J, Gertsenstein M, and Breitman ML. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376: 66–70, 1995.[CrossRef][Medline]
16. Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, and Ferrara N. VEGF is required for growth and survival in neonatal mice. Development 126: 1149–1159, 1999.[Abstract]
17. Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, and Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273: 30336–30343, 1998.[Abstract/Free Full Text]
18. Grim M and Carlson BM. Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study. J Histochem Cytochem 38: 1907–1912, 1990.[Abstract]
19. Gu H, Marth JD, Orban PC, Mossmann H, and Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103–106, 1994.[Abstract/Free Full Text]
20. Holash J, Wiegand S, and Yancopoulos G. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 18: 5356–5362, 1999.[CrossRef][Web of Science][Medline]
21. Hudlicka O, Brown M, and Egginton S. Angiogenesis in skeletal and cardiac muscle. Physiol Rev 72: 369–417, 1992.[Free Full Text]
22. Jobin J, Maltais F, Doyon JF, LeBlanc P, Simard PM, Simard AA, and Simard C. Chronic obstructive pulmonary disease: capillarity and fiber-type characteristics of skeletal muscle. J Cardiopulm Rehabil 18: 432–437, 1998.[CrossRef][Medline]
23. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, and Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 106: 1311–1319, 2000.[Web of Science][Medline]
24. Lloyd PG, Prior BM, Yang HT, and Terjung RL. Angiogenic growth factor expression in rat skeletal muscle in response to exercise training. Am J Physiol Heart Circ Physiol 284: H1668–H1678, 2003. First published January 23, 2003; 10.1152/ajpheart.00743.2002.[Abstract/Free Full Text]
25. Lobov I, Brooks P, and Lang R. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci USA 99: 11205–11210, 2002.[Abstract/Free Full Text]
26. Maisonpierre P, Suri C, Jones P, Bartunkova S, Wiegand S, Radziejewski C, Compton D, McClain J, Aldrich T, Papdaopoulos N, Daly T, Davis S, Sato T, and Yancopoulos G. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277: 55–60, 1997.[Abstract/Free Full Text]
27. Rajagopalan S, Shah M, Luciano A, Crystal R, and Nabel E. Adenovirus-mediated gene transfer of VEGF(121) improves lower-extremity endothelial functions and flow reserve. Circulation 104: 753–755, 2001.[Abstract/Free Full Text]
28. Richardson RS, Wagner H, Mudaliar SR, Henry R, Noyszewski EA, and Wagner PD. Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol Heart Circ Physiol 277: H2247–H2252, 1999.[Abstract/Free Full Text]
29. Roca J, Gavin TP, Jordan M, Siafakas N, Wagner H, Benoit H, Breen E, and Wagner PD. Angiogenic growth factor mRNA responses to passive and contraction-induced hyperperfusion in skeletal muscle. J Appl Physiol 85: 1142–1149, 1998.[Abstract/Free Full Text]
30. Saltin B and Gollnick P. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology, Skeletal Muscle. Bethesda, MD: American Physiological Society, 1983, p. 555–632.
31. Samulski RJ, Chang LS, and Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 63: 3822–3828, 1989.[Abstract/Free Full Text]
32. Sauer B and Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 85: 5166–5170, 1988.[Abstract/Free Full Text]
33. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, and Schuh AC. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376: 62–66, 1995.[CrossRef][Medline]
34. Shimpo M, U, Maeda Y, Takahashi M, Moiyashita H, Urabe M, Mizukami H, Urabe M, Kume A, Takizawa T, Shibuya M, Ozawa K, and Shimada K. AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model. Cardiovasc Res 53: 993–1001, 2002.[Abstract/Free Full Text]
35. Takahashi A, Kureishi Y, Yang J, Luo Z, Guo K, Mudhopadhyay D, Ivashchenko Y, Branellec D, and Walsh K. Myogenic Akt signaling regulates blood vessel recruitment during myofiber growth. Mol Cell Biol 22: 4803–4814, 2002.[Abstract/Free Full Text]
36. Wagner PD, Masanes F, Wagner H, Sala E, Miro O, Campistol JM, Marrades RM, Casademont J, Torregrosa V, and Roca J. Muscle angiogenic growth factor gene responses to exercise in chronic renal failure. Am J Physiol Regul Integr Comp Physiol 281: R539–R546, 2001.[Abstract/Free Full Text]
37. Xiao X, Li J, and Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72: 2224–2232, 1998.[Abstract/Free Full Text]
38. Yang H, Ogilvie R, and Terjung R. Exercise training enhances basic fibroblast growth factor-induced collateral blood flow. Am J Physiol Heart Circ Physiol 274: H2053–H2061, 1998.[Abstract/Free Full Text]

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				I. M. Olfert, R. A. Howlett, K. Tang, N. D. Dalton, Y. Gu, K. L. Peterson, P. D. Wagner, and E. C. Breen Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice J. Physiol., April 15, 2009; 587(8): 1755 - 1767. [Abstract] [Full Text] [PDF]

				A. R. Cuddihy, S. Ge, J. Zhu, J. Jang, A. Chidgey, G. Thurston, R. Boyd, and G. M. Crooks VEGF-mediated cross-talk within the neonatal murine thymus Blood, March 19, 2009; 113(12): 2723 - 2731. [Abstract] [Full Text] [PDF]

				K. A. Zwetsloot, L. M. Westerkamp, B. F. Holmes, and T. P. Gavin AMPK regulates basal skeletal muscle capillarization and VEGF expression, but is not necessary for the angiogenic response to exercise J. Physiol., December 15, 2008; 586(24): 6021 - 6035. [Abstract] [Full Text] [PDF]

				P. D. Wagner The biology of oxygen Eur. Respir. J., April 1, 2008; 31(4): 887 - 890. [Abstract] [Full Text] [PDF]

				T. P. Gavin, R. S. Ruster, J. A. Carrithers, K. A. Zwetsloot, R. M. Kraus, C. A. Evans, D. J. Knapp, J. L. Drew, J. S. McCartney, J. P. Garry, et al. No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men J. Physiol., November 15, 2007; 585(1): 231 - 239. [Abstract] [Full Text] [PDF]

				S. Sakao, L. Taraseviciene-Stewart, C. D. Cool, Y. Tada, Y. Kasahara, K. Kurosu, N. Tanabe, Y. Takiguchi, K. Tatsumi, T. Kuriyama, et al. VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells FASEB J, November 1, 2007; 21(13): 3640 - 3652. [Abstract] [Full Text] [PDF]

				F. Suhr, K. Brixius, M. de Marees, B. Bolck, H. Kleinoder, S. Achtzehn, W. Bloch, and J. Mester Effects of short-term vibration and hypoxia during high-intensity cycling exercise on circulating levels of angiogenic regulators in humans J Appl Physiol, August 1, 2007; 103(2): 474 - 483. [Abstract] [Full Text] [PDF]

				Y. Tan, H. Shao, D. Eton, Z. Yang, L. Alonso-Diaz, H. Zhang, A. Schulick, A. S. Livingstone, and H. Yu Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor Cardiovasc Res, March 1, 2007; 73(4): 823 - 832. [Abstract] [Full Text] [PDF]

				L. Taraseviciene-Stewart, I. S. Douglas, P. S. Nana-Sinkam, J. D. Lee, R. M. Tuder, M. R. Nicolls, and N. F. Voelkel Is Alveolar Destruction and Emphysema in Chronic Obstructive Pulmonary Disease an Immune Disease? Proceedings of the ATS, November 1, 2006; 3(8): 687 - 690. [Abstract] [Full Text] [PDF]

				N. F. Voelkel, R. W. Vandivier, and R. M. Tuder Vascular endothelial growth factor in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L209 - L221. [Abstract] [Full Text] [PDF]

				N. A. Ryan, K. A. Zwetsloot, L. M. Westerkamp, R. C. Hickner, W. E. Pofahl, and T. P. Gavin Lower skeletal muscle capillarization and VEGF expression in aged vs. young men J Appl Physiol, January 1, 2006; 100(1): 178 - 185. [Abstract] [Full Text] [PDF]

				A. N. Croley, K. A. Zwetsloot, L. M. Westerkamp, N. A. Ryan, A. M. Pendergast, R. C. Hickner, W. E. Pofahl, and T. P. Gavin Lower capillarization, VEGF protein, and VEGF mRNA response to acute exercise in the vastus lateralis muscle of aged vs. young women J Appl Physiol, November 1, 2005; 99(5): 1872 - 1879. [Abstract] [Full Text] [PDF]

				M. Liang and B. Ventura Physiological genomics in PG and beyond: July to September 2005 Physiol Genomics, October 17, 2005; 23(2): 119 - 124. [Full Text] [PDF]

				T. H. Adair Growth regulation of the vascular system: an emerging role for adenosine Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R283 - R296. [Abstract] [Full Text] [PDF]

				T. Gustafsson, H. Ameln, H. Fischer, C. J. Sundberg, J. A. Timmons, and E. Jansson VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise J Appl Physiol, June 1, 2005; 98(6): 2137 - 2146. [Abstract] [Full Text] [PDF]

				A. Wagatsuma, H. Tamaki, and F. Ogita Capillary supply and gene expression of angiogenesis-related factors in murine skeletal muscle following denervation Exp Physiol, May 1, 2005; 90(3): 403 - 409. [Abstract] [Full Text] [PDF]

				N. Ouchi, R. Shibata, and K. Walsh AMP-Activated Protein Kinase Signaling Stimulates VEGF Expression and Angiogenesis in Skeletal Muscle Circ. Res., April 29, 2005; 96(8): 838 - 846. [Abstract] [Full Text] [PDF]

				P. G. Lloyd, B. M. Prior, H. Li, H. T. Yang, and R. L. Terjung VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H759 - H768. [Abstract] [Full Text] [PDF]

				T. P. Gavin, H. W. Stallings III, K. A. Zwetsloot, L. M. Westerkamp, N. A. Ryan, R. A. Moore, W. E. Pofahl, and R. C. Hickner Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans J Appl Physiol, January 1, 2005; 98(1): 315 - 321. [Abstract] [Full Text] [PDF]

				R. E. Waters, S. Rotevatn, P. Li, B. H. Annex, and Z. Yan Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1342 - C1348. [Abstract] [Full Text] [PDF]

				T. H. Adair An Emerging Role for Adenosine in Angiogenesis Hypertension, November 1, 2004; 44(5): 618 - 620. [Full Text] [PDF]

				K. Tang, H. B. Rossiter, P. D. Wagner, and E. C. Breen Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice J Appl Physiol, October 1, 2004; 97(4): 1559 - 1566. [Abstract] [Full Text] [PDF]

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