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Published as doi: 10.1096/fj.06-6568fje.
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(The FASEB Journal. 2006;20:2657-2659.)
© 2006 FASEB

Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia

Georges von Degenfeld*,{ddagger}, Andrea Banfi*,§, Matthew L. Springer*,||, Roger A. Wagner{dagger}, Johannes Jacobi{dagger}, Clare R. Ozawa*, Milton J. Merchant*, John P. Cooke{dagger} and Helen M. Blau*,1

* Baxter Laboratory in Genetic Pharmacology, Departments of Molecular Pharmacology and of Microbiology and Immunology;

{dagger} Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, USA;

{ddagger} Cardiovascular Research, Bayer Healthcare, Wuppertal, Germany;

§ Institute for Surgical Research and Hospital Management, Department of Research and Department of Surgery, University Hospital, Basel, Switzerland; and

|| Division of Cardiology, University of California, San Francisco, California, USA

1Correspondence: Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5175, USA. E-mail: hblau{at}stanford.edu

ABSTRACT

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.—von Degenfeld, G., Banfi, A., Springer, M. L., Wagner, R. A., Jacobi, J., Ozawa, C. R., Merchant, M. J., Cooke, J. P., Blau, H. M. Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia.


Key Words: angiogenesis • gene therapy • cell transplantation

DELIVERY OF VASCULAR ENDOTHELIAL growth factor-A (VEGF) in patients with ischemic heart disease or peripheral artery disease is a promising approach but has not yet provided clear evidence of therapeutic efficacy (1) . The mixed clinical results have been corroborated by recent experimental studies in animal models of coronary and peripheral ischemia that have failed to observe sustained improvements in blood flow following VEGF delivery (2 3 4 5 6 7) . Increasing evidence suggests that the lack of robust and lasting effects is due to difficulties in achieving an appropriate dosage of VEGF, a growth factor with an apparently narrow therapeutic window (8 , 9) .

Indeed, VEGF expression requires exceedingly tight regulation during development, as both reduced expression following the targeted deletion of just one allele (10 , 11) and modest overexpression (12) lead to early embryonic lethality. In adult animals, VEGF has been found to induce pathological, glomeruloid vascular proliferation using diverse gene delivery vectors in different tissues (7 , 9 , 13 , 14) . When VEGF is constitutively delivered long term, vascular proliferation is unabated and results in hemangiomas (15) . Moreover, VEGF-induced vascular leakage can cause severe edema (16 , 17) , which has been linked to limb loss (3 , 18) and even death (19) in different animal models. Appropriate VEGF dosage may be critical to inducing therapeutic improvement while avoiding deleterious effects.

We recently showed that in normal, nonischemic muscle, the "local" (microenvironmental), concentration, rather than the "total" dose, of VEGF164 determined whether its delivery induced the formation of hemangiomas or capillaries of normal appearance (9) . However, the functional consequences were not investigated, and it is not known whether microenvironmental VEGF concentrations are critical for the achievement of therapeutic effects in the clinically relevant pathology of peripheral ischemia. In this study, we investigated the dose-dependent mechanism of VEGF-induced functional vessel growth in ischemia by exploiting the unique advantages of a cell-based delivery system that provides uniform VEGF levels in vivo: homogeneous monoclonal populations of genetically engineered myoblasts derived from a single cell that express equal amounts of VEGF per cell. We found that the parental polyclonal population of VEGF-expressing myoblasts induced a mixture of normal capillaries and glomeruloid vascular structures that increased blood flow only modestly. In contrast, the same total VEGF dose delivered in a microenvironmentally controlled manner by monoclonal populations derived from a single cell achieved full recovery of blood flow to nonischemic levels, without increasing vascular leakage and while avoiding completely the appearance of aberrant vasculature or hemangioma growth, even after 15 mo of constitutive expression. Our approach revealed a biphasic dose-dependent effect of VEGF: whereas the lowest concentration already induced the maximal increase in new vessel number, vessel diameter enlarged continuously with higher VEGF doses without apparent upper limit. Vessel size and shape, rather than vessel number, correlated with therapeutic improvement in blood flow and collateral growth.

In conclusion, this study revealed a pathophysiological mechanism by which microenvironmental levels of VEGF improve functional benefit long term with reduced toxicity, findings that have implications in designing clinical trials of therapeutic angiogenesis.

MATERIALS AND METHODS

Myoblast culture
Primary myoblasts expressing LacZ were infected using MFG-vascular endothelial growth factor164 retrovirus, and cultured as described previously (20) . Single cells isolated by flow cytometry were expanded into monoclonal populations, and VEGF secretion was monitored periodically by ELISA (R&D Systems, Minneapolis, MN, USA) (9) . The four clonal populations secrete ~10%, 100%, 180%, and 325% of the average VEGF levels of the parental polyclonal population per cell in vitro (9) .

Surgical induction of hind limb ischemia
Male C.B-17-SCID mice (16–20 wk old) were obtained from the Stanford University Department of Comparative Medicine and treated according to the guidelines of the Stanford Administrative Panel on Laboratory Animal Care. Anesthesia was induced with 0.01 mg Avertin and maintained by Metoxifane inhalation. Unilateral hind limb ischemia was surgically induced by ligation and transection of the medial portion of the right superficial femoral artery distal to the deep femoral artery origin. Muscle blood velocimetry was assessed over 2 min before and after surgery using a calibrated laser Doppler-probe (Perimed-PF3/Perisoft-software, Perimed, Järfälla, Sweden) positioned on the distal adductor muscle using a three-dimensional micromanipulator stage, as described by Jacobi et al. (21) . In preliminary studies, laser Doppler velocimetry manifested greater variability than microsphere measurements but reliably identified severe hypoperfusion. Specifically, postoperative blood flow values that were generally less than 40% of the contralateral leg correlated well when determined by laser Doppler or measured by microspheres in all 20 animals studied with both techniques. Accordingly, laser Doppler was used to document postoperative ischemia after surgery. Mice were randomized after surgery to receive 8 x 106 myoblasts suspended in 0.5% BSA/PBS (108cells/ml) or vehicle injected into the distal thigh muscles (adductor and quadriceps femoris groups) (8 injections/106 cells each).

Microsphere measurement of blood flow and microangiography
Fourteen days after surgery, blood flow was measured using fluorescent microspheres as described previously (21) . After median thoracotomy, 2 x 105 15-µm diameter red-fluorescent microspheres (Molecular Probes, Eugene, OR, USA) were continuously injected over 60 s into the beating left ventricle. Perfusion fixation was performed in four mice/group: the heart was cannulated and perfused at 110 mmHg with Tris-HCl buffer containing Na+, Ca2+, Mg2+, and 0.1% adenosine (2 min), followed by 1.5% formaldehyde (2 min) (22) . The muscle group of the thigh was excised, cut in midthigh, weighed, embedded in OCT-compound, and snap frozen. Kidneys were reference organs for equilateral microsphere distribution. Microspheres were individually counted by direct fluorescence microscopy on 100-µm cryosections from the entire samples, a method that avoids difficulties associated with fluorescence recovery and background fluorescence in tissue homogenates (23 , 24) . An additional advantage is that neighboring thin cryosections can be obtained for histology and immunofluorescence analysis matching microsphere counts from the same muscle (21) . Microsphere counts, normalized for muscle weight, from the right, ischemic leg were normalized to the contralateral, nonischemic leg. Microangiography was performed in some animals as described (21) . Following cannulation of the abdominal aorta, barium-sulfate solution (0.5 mg/ml) was injected and images acquired using a Faxitron radiography system (Faxitron, Wheeling, IL, USA). To determine capillary perfusion in vivo, fluorescein-isothiocyanate-labeled tomato lectin (Vector Laboratories, Burlingame, CA, USA) was injected intravenously (i.v.) and allowed to circulate for 2 h before perfusion fixation (n=2/group) using a modified published protocol (25) .

Vascular leakage
Vascular leakage was assessed 4 days after surgery and cell implantation using a modified described protocol (n=3/group) (9) . Evans Blue (J. T. Baker, Phillipsburg, NJ, USA) was injected i.v. (30 µg/g body wt). Four hours later, mice were perfused (1% paraformaldehyde/0.05 M citric acid, pH 3.5), muscle samples from the distal thigh were harvested, Evans Blue was extracted in formamide at 55°C overnight, quantified with a spectrophotometer at 610 nm, and normalized to tissue wet wt.

Immunofluorescence and histomorphometry
For immunofluorescent staining, 10-µm sections were fixed in 1.6% formaldehyde, and blocked using 2% normal goat serum, 0.5% casein, and 0.3% Triton-X-100 (1 h). Slides were incubated (1 h) with rat monoclonal antibody (mAb) against CD31/PECAM-1 (PharMingen, San Diego, CA, USA; 1:100 dilution), mouse mAb against {alpha}-SMA (ICN-Biomedicals, Aurora, OH, USA, 1:400), rabbit antibody (Ab) against laminin (Chemicon, Temecula, CA, USA, 1:200) or rabbit Ab against ß-galactosidase (Eppendorff-5-Prime, Westbury, NY, USA, 1:200). Secondary antibodies conjugated with Alexa-fluorophores (Molecular Probes) and Hoechst-33258 nuclear stain were used. Adjacent sections were stained with H&E or X-gal. Slides were analyzed using a Zeiss Axioplan microscope.

Vessel growth was differentially quantified for length and size. For vessel length density, three images of cryosections of the quadriceps femoris and adductor muscles stained for PECAM were randomly acquired (20x objective). Centerlines of vessels were manually drawn using a modified technique, as previously described (9) , quantified using calibrated OpenlabTM image analysis software (Improvision, Lexington, MA), and normalized to the number of muscle fibers. Vessel sizes were measured on 3 randomly chosen images of the quadriceps femoris muscle (20x objective) with a standardized grid overlay, using a modified protocol described previously (9 , 18 , 26) . The diameter of each vessel (if any) in the center of randomly selected squares was measured using calibrated software. Muscle regions expressing LacZ following fusion with the injected myoblasts were compared with remote LacZ-negative regions. Collateral vessels ≥30 µm in diameter were identified on cross sections of the proximal adductor muscle by costaining for PECAM and {alpha}-SMA and quantified as described previously (21 , 27) .

Damaged muscle was defined as either inflammation (mononucleated cell infiltrates) or necrosis ("ghost fibers" lacking nuclei) (28) . Areas were manually drawn on digital images (5x objective) and quantified using calibrated software (3 legs/group).

Implantation of myoblasts into the ear muscle and whole-mount preparation
5 x 105 myoblasts in 5 µl 0.5% BSA/PBS were implanted into the posterior auricular muscle (9) . In vivo intravascular staining was performed 4 wk after cell implantation by i.v. injection of biotinylated tomato lectin (Vector Laboratories), as described previously (9 , 19) . Ears were removed, bisected in the cartilage plane, and stained with X-gal and avidin-biotin complex (ABC)/diaminobenzidine histochemistry (Vector Laboratories), dehydrated through an alcohol series, and cleared with toluene. Vessel diameters and length densities around LacZ-positive muscle fibers were measured on images of ear whole-mounts. The centerline of blood vessels (brown lectin stain) and LacZ-expressing muscle fibers were manually traced and quantified on digital photomicrographs (20x objective) overlaid with a computer-generated square lattice using calibrated software for vessel length density (vessel length/muscle fiber length) and branching (branching points/100 µm muscle fiber length) on 3 fields/ear (3 ears/group). The diameter of each vessel in the center of randomly selected squares was measured (30 measurements/ear; 3 ears/group).

Statistics
Data are presented as mean ± SEMs. Differences between groups with respect to vessel length, diameter, blood flow, collateral score, VEGF quantity, and leakage were assessed using ANOVA and Student’s t test; P < 0.05 was considered statistically significant and P < 0.01 highly significant [Statistical Packages for the Social Sciences-13.0, statistical Packages for the Social Sciences (SPSS) Inc., Chicago, IL, USA].

RESULTS

Implantation into the ischemic hind limb of clonal myoblast populations uniformly expressing different levels of VEGF
To study the microenvironmental dose effects of VEGF164 in ischemia, we generated monoclonal populations of myoblasts that express equal amounts of VEGF per cell. Primary murine myoblasts expressing LacZ were infected with MFG-vascular endothelial growth factor164 retrovirus. From the resulting polyclonal population of VEGF-expressing myoblasts, single cells were isolated by flow cytometry and expanded into monoclonal populations as described (9) . Compared with the parental polyclonal myoblast population producing on average 61 ± 5 ng/106 cells/24 h VEGF in vitro, each of the four derived clonal populations secreted different VEGF amounts per cell: "10% clone": 6 ± 1, "100% clone": 55 ± 5, "180% clone": 104 ± 8, and "325% clone": 191 ± 15.

To induce hind limb ischemia, the right superficial femoral artery was surgically ligated and transected. Laser-Doppler velocimetry performed after surgery showed marked blood flow reduction below 40% of preoperative levels in each individual mouse without differences between treatment groups (not shown). Fourteen days later, regional blood flow was determined by injection of fluorescent microspheres, a well-characterized method considered as the gold standard, as it measures the effective perfusion of the microvasculature that correlates well with metabolic and functional measures of ischemia (16 , 21 , 29 30 31) .

The area of myoblast engraftment (X-Gal staining) was distributed along the needle tracks and comprised ~20% of the cross-sectional muscle area (example shown in Fig. 6G ). No difference was seen with regard to the extent and distribution of cell engraftment or fusion with endogenous muscle fibers along the needle tracks between the parental polyclonal population and any of the monoclonal populations 14 days after implantation. Furthermore, no tumorigenicity was seen with any myoblast population, including the clones. Finally, we quantified the amount of VEGF expressed following implantation of the parental polyclonal population and the 100% clone into the ischemic muscle in order to rule out VEGF-mediated toxicities to the myoblasts expressing "high" amounts of VEGF in vivo. As predicted from the in vitro expression levels, VEGF expression following implantation of the 100% clone into the muscle was similar to that of the parental polyclonal population (see below). In conclusion, there was no evidence for differences in cell survival, distribution or VEGF production between the monoclonal populations, and compared with the parental polyclonal population from which they had been derived.


Figure 1
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  		Figure 1. Microenvironmental concentrations of vascular endothelial growth factor (VEGF) determine blood flow improvement in ischemia. Regional blood flow was measured in the ischemic leg 14 days after surgical induction of hind limb ischemia using microspheres (n=9/group). Blood flow in ischemic legs (expressed as percentage of the nonischemic contralateral leg) was severely reduced in mice injected with vehicle or control myoblasts. Implantation of the parental polyclonal population of VEGF-expressing myoblasts improved blood flow only modestly (*P<0.05 vs. controls). Diverse effects were induced by the clonal populations uniformly expressing distinct levels of VEGF "per cell". In particular, the 100% clone markedly improved blood flow in the ischemic leg to a level comparable with the contralateral nonischemic leg (**P<0.01 vs. controls; P<0.05 vs. polyclonal VEGF myoblasts). In contrast, neither the 10% nor the 180% clone improved blood flow significantly, although the latter showed a positive trend


Figure 2
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  		Figure 2. Despite improved blood flow by uniform VEGF delivery, vascular leakage is not increased. A) "Total" VEGF protein concentration in ischemic muscle was increased 3 days after ischemia induction, reflecting endogenous up-regulation (ELISA). VEGF levels were increased after implantation of VEGF-expressing myoblasts; however, there was no difference between the parental polyclonal population and the 100% clone confirming in vitro levels. B) Vascular leakage measured by Evans Blue extravasation was increased 4 days after ischemia induction. Implantation of VEGF-expressing myoblasts further increased leakage, but no difference was found between the parental polyclonal population and the 100% clone. #P < 0.01 vs. nonischemic muscle; *P < 0.01 vs. ischemic control groups (n=3/group).


Figure 3
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  		Figure 3. Microenvironmental VEGF levels determine size of angiogenic vessels. Cryosections were stained for endothelial cells (PECAM/CD31, red). Areas of myoblast engraftment were identified by X-gal staining of neighboring sections. A) Implantation of control myoblasts did not induce vessel growth or alter vessel morphology. B) The parental polyclonal VEGF population induced a mixture of small capillaries and dilated structures. C) The 10% VEGF clone induced a robust, localized angiogenic response, consisting of a dense network of regularly shaped capillaries that appeared to wrap around the muscle fibers (some muscle fibers indicated by asterisk for better clarity). D) The 100% clone did not further increase vessel length, but the induced vessels were now larger in size yet regularly shaped. E) The 180% clone induced even larger vessels displaying irregular morphology with focally dilated segments. F) Large hemangiomas induced within 14 days by the 325% clone were apparent by angiography. G–H) Two bars represent each group: left bar: nonischemic leg without cell injection; right bar: ischemic leg injected with vehicle or cells as indicated. G) Vessel length density was increased in the ischemic legs compared to the nonischemic legs in the controls, reflecting endogenous angiogenesis in ischemia. Control myoblasts had no additional effect. In contrast, all VEGF clones induced similar increases in vascular length, indicating that longitudinal growth was not dependent on VEGF concentrations (*P<0.05 vs. ischemic controls). H) Controls and 10% VEGF clone did not increase vascular diameter, but the 100% and, to a greater extent, 180% VEGF clone induced gradual vessel enlargement without apparent upper limit *P < 0.05, **P < 0.01 vs. controls. Scale bar (A–D): 50 µm.


Figure 4
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  		Figure 4. Vessel size, but not number, is dependent on microenvironmental VEGF concentration. A–D) Whole-mount preparation of the ear 28 days after myoblast injection revealed clearly distinct morphologies of induced vessels. Perfused vascular lumen: brown lectin staining; engrafted myoblasts: bluish green X-gal staining. Scale bar: 50 µm. E–G) Morphometry shows that vessel number and branching were maximally increased by the 10% clone, without further increase with ascending VEGF doses. In contrast, vessels were gradually enlarged with increasing microenvironmental VEGF concentrations: unlike the 10% clone, the 100% clone induced the enlargement of vessels that were, however, still normal in appearance; in contrast, the 180% clone induced vessels that were substantially enlarged and glomeruloid bodies. *P < 0.05 vs. controls.


Figure 5
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  		Figure 5. The 100%-vascular endothelial growth factor clone induces collateral growth. A) Collateral growth, determined by costaining for PECAM/CD31 (endothelial cells) and {alpha}-SMA (vascular smooth muscle cells) of sections of the proximal adductor muscle, was significantly increased only by the 100% VEGF clone, and showed a trend toward increase in the parental polyclonal population and the 180% clone. B, C) Microangiograms corroborated the quantitative collateral score, showing increased growth of collaterals originating from large feeder vessels, crossing the thigh and connecting to the vasculature of the lower leg in mice implanted with the 100% clone by comparison with control myoblasts. Numerous collaterals display the typical, corkscrew-like morphology of growing collaterals.


Figure 6
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  		Figure 6. A–C) Vessels induced by the 100% VEGF clone are covered by pericytes. On cryosections from muscle harvested 14 days after induction of ischemia and implantation of the 100% VEGF clone, regions of myoblast engraftment were identified (ß-galactosidase staining, not shown) and stained for endothelial cells (PECAM/CD31, red) (A) and pericytes (NG2, green) (B). C) The pericytes were tightly associated with the endothelial cells, indicating maturation of the angiogenic vessels. D–F) Vessels induced by the 100% VEGF clone are perfused in vivo. FITC-lectin was injected into mice 14 days after ischemia induction and implantation of the 100% clone. On cryosections, vessels were stained for PECAM/CD31 (red) (D), and perfused vessels identified by green fluorescence (E), appearing yellow in the merged image (F). Most VEGF-induced vessels are perfused similarly to the surrounding region. Two unperfused vessels are indicated (arrowheads). Scale bar: 100 µm. G, H) Vessels induced by the 100% VEGF clone persist over more than 15 mo. G) X-gal staining clearly reveal transgenic muscle fibers to which the injected myoblasts have fused in a typical arrangement along the needle track (from lower right to upper left corner of the panels), expressing the ß-galactosidase reporter after more than 15 mo. H) PECAM/CD31 staining (red) shows increased vessel density in the area of myoblast engraftment. Scale bar: 100 µm.

Control over microenvironmental distribution improves the efficacy of VEGF delivery in ischemia without increasing leakage
To determine the effects of VEGF dosage, mice were randomized after surgery to be injected either with vehicle only, LacZ-expressing control myoblasts, the parental polyclonal VEGF-expressing myoblast population, or one of the monoclonal VEGF-expressing populations (6 groups, n=9/group). Fourteen days after surgery and cell implantation, the control group injected with vehicle showed markedly reduced perfusion in the ischemic leg compared with the contralateral leg (54.6±6.8%), showing that the surgical procedure provided sustained blood flow reduction (Fig. 1 ). Injection of control myoblasts did not improve blood flow (55.3±13.1%). Mice implanted with the parental polyclonal VEGF population exhibited only moderately increased blood flow (74.1±8.6%, P<0.05 vs. controls). Improvement was substantially greater in mice that received the 100% vascular endothelial growth factor clone, in which blood flow was similar to the contralateral, nonischemic leg (96.8±10.5%, P<0.01 vs. controls; P<0.05 vs. polyclonal VEGF population). By contrast, blood flow was neither improved in mice having received low (10% clone: 54.7±17.5%) nor high microenvironmental VEGF doses (180% clone: 69.8±18.9%), although the latter showed a positive trend.

Differences in blood flow were accompanied by various degrees of ischemic muscle damage, quantified on H&E-stained muscle sections, according to previously described criteria (28) . Muscle damage was smaller in mice treated with the 100% vascular endothelial growth factor clone compared with all other groups (P<0.05; Table 1 ). Furthermore, the incidence of foot gangrene appeared lower in mice injected with the 100% VEGF clone (Table 1) . Thus, although the 100% clone expresses the same total VEGF dose as the parental polyclonal population, they have much greater positive effects on perfusion and salvage of ischemic tissue.


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  		Table 1. Blood flow improvement made with VEGF

We compared the amounts of VEGF expressed by the 100% clone and the parental polyclonal population in vivo. Both populations express similar amounts of VEGF in vitro. The aim of the in vivo quantification was to rule out increased cell survival or proliferation of the 100% clone, which might have occurred as a result of cell transformation in the process of clonal expansion from a single cell. Average VEGF levels were measured in muscles harvested 3 days after ischemia induction and cell injection (ELISA). Endogenous VEGF was up-regulated in ischemia (control myoblasts: 25±2 ng/mg protein; vehicle: 26±2) compared with nonischemic muscle (7±1) (Fig. 2 A). The VEGF myoblasts strongly increased total VEGF content, but levels were not different for the parental polyclonal population and the 100% clone (polyclonal: 129±22 ng/mg protein; 100% clone: 128±47). Hence, although microenvironmental distribution of VEGF concentrations in muscle cannot be directly measured, the finding that both VEGF populations expressed similar total amounts of VEGF implies that the greater antiischemic effects provided by the 100% clone was due to a more favorable, uniform local distribution and not to increase survival or proliferation of the clonally expanded population.

We determined whether functional improvements induced by the 100% clone came at the expense of adverse effects. Vascular leakage is a hallmark of VEGF effects that leads to potentially harmful tissue edema (3 , 19) . Leakage peaks 4 days after intramuscular (i.m.) implantation of polyclonal VEGF myoblasts before decreasing to baseline (9) (D. M. McDonald, UCSF, personal communication). Leakage was quantified 4 days after surgery and myoblast injection by i.v. injection of Evans Blue. Both VEGF populations induced similar amounts of leakage in ischemic muscle (Fig. 2B ) that were higher than that seen in the ischemic control legs, resulting from endogenous VEGF up-regulation. Hence, increased therapeutic effects provided by the 100% VEGF clonecompared with the parental polyclonal population did not come at the expense of increased leakage.

Vessel number increase peaks at the lowest VEGF dose
To determine the structural features underlying the distinct functional effects elicited by different microenvironmental VEGF doses, we examined the morphology, length, and diameter of vessel growth 14 days after ischemia induction and cell injection. Cryosections from ischemic muscle were stained for endothelial cells (platelet endothelial cell adhesion molecule-1 PECAM/CD31). Vessel growth was seen exclusively tightly localized around the muscle fibers that expressed VEGF. Vessel length density was assessed by drawing a mask in randomly selected fields and normalized to muscle fiber counts to avoid distortions caused by ischemia-induced muscle shrinkage. Vessel length density was increased in the ischemic muscles of all groups compared with the nonischemic contralateral leg, reflecting endogenous angiogenesis. Implantation of control myoblasts had no further effect (Fig. 3 A, G). In contrast, vessel length density was increased in all groups injected with VEGF-myoblasts (Fig. 3B-G ). Surprisingly, the effect was not dose-dependent: vessel length density peaked already in ischemic muscles implanted with the 10% clone and was not further increased by even the highest VEGF levels.

Although vessel density can be reliably quantified on two-dimensional cryosections, it is impossible to distinguish between longitudinal growth or tortuousity of preexisting vessels and truly increased number of vessels resulting from sprouting or intussusception. The clones were therefore studied in the ear muscle, which is amenable to three-dimensional vessel analysis. Samples were analyzed 4 wk after cell implantation because myoblast fusion and angiogenesis are delayed in the ear compared with leg muscles (9) . In vivo blood vessel staining was performed by i.v. injection of biotinylated lectin (9 , 19) . Following ABC/diaminobenzidine staining, branching patterns and segment length between branches were analyzed in detail by quantifying a manually drawn mask overlay on whole-mount images of the vasculature around LacZ-expressing muscle fibers. The 10% clone induced a peak increase in the number of branching points and vessel length (Fig. 4 B, E, F) and no further increase was seen with the 100% and 180% clones (Fig. 4C-F ). VEGF-induced vessels were tortuous, indicating longitudinal growth, but this effect was balanced by an equivalent increase in branch points. Thus, the calculated average length of vessel segments between branch points was unchanged between the controls and all three VEGF clones. Therefore, the data from the ear muscle whole-mounts confirmed the findings from the ischemic leg muscles and showed that the VEGF clones truly increased vessel number.

Microenvironmental VEGF concentrations increase vessel diameter in a dose-dependent manner
Vessel size was measured on randomly selected fields of immunofluorescent-stained muscle cryosections using a standardized grid overlay and calibrated software. In contrast to vessel length, vessel diameter was increased by VEGF in a dose-dependent manner with no discernible upper limit over the 30-fold dose range investigated. Ischemia itself increased capillary diameter slightly, and no additional enlargement was induced by the control myoblasts (Fig. 3H ). The parental polyclonal VEGF myoblasts induced a mixture of small and large vessels, including focally dilated glomeruloid bodies, aberrant structures known to subsequently grow into hemangiomas (9 , 14) . In contrast, each clonal population uniformly induced vessels possessing distinct morphology. Surprisingly, the 10% clone, found to induce peak increase in vessel number, did not also increase vessel size (Fig. 3C, H ). In contrast, the 100% and 180% VEGF clone significantly increased vessel diameter in a dose-dependent manner (Fig. 3D-E, H ). However, whereas the vessels induced by the 100% clone appeared normal in morphology, those induced by the 180% clone were irregularly shaped. The 325% clone induced the growth of large hemangiomas readily visible by microangiography (Fig. 3F ) and were not further studied.

Vessel morphology was further analyzed in the ear muscle to identify aberrant structures (9) . VEGF-induced vascular enlargement increased progressively in a dose-dependent manner, confirming the findings in ischemic muscle: whereas the 10% clone did not increase vessel size, vessels induced by the 100% clone were substantially enlarged yet regularly shaped and those induced by the 180% clone were not only larger on average but included the appearance of aberrant glomeruloid bodies (Fig. 4) .

Optimized uniform VEGF delivery stimulates collateral growth
Arteriogenesis, the growth of collaterals from preexisting arteries, is required for blood flow increase in ischemia (1 , 32) . Collaterals were quantified on cryosections of the adductor muscles proximal of the site of cell injection by costaining for endothelial cells (PECAM/CD31) and vascular smooth muscle cells ({alpha} -SMA). The number of collaterals ≥ 30 µm in diameter was increased in mice injected with 100% VEGF clone compared with the controls, and a trend toward enlargement was seen with the 180% clone and the parental polyclonal population (Fig. 5 A). Quantitative analyses were corroborated by microangiograms that showed more numerous arteries in the thigh area of legs implanted with the 100% clone (Fig. 5B ). Thus, the increase in blood flow induced by the 100% clone was accompanied by proximal collateral growth.

Vessels induced by optimized VEGF delivery are perfused in vivo and persist more than 15 mo
To confirm that vessels induced by the 100% VEGF clone were perfused in vivo, fluorescent lectin was injected i.v. Muscle sections costained for PECAM showed that most vessels induced by the 100% clone were perfused similarly to the surrounding vessels (Fig. 6 D–F). In accordance with blood flow improvement (microspheres), these findings demonstrate that the vessels induced by the 100% clone were able to recruit blood flow.

Vessels induced by transient VEGF expression lack pericytes, are unstable, and are prone to regression (13 , 14) . On the other hand, sustained VEGF expression can induce hemangioma growth, posing a challenge for safe and therapeutic VEGF delivery. To address this issue, we harvested muscles 66 wk after ischemia induction and implantation of the 100% clone. Sustained ß-galactosidase expression over more than 15 mo was found in muscle fibers to which the injected myoblasts had fused. In the area of cell engraftment, increased vessel density was still readily visible (Fig. 6G-H ). Remarkably, no evidence of glomeruloid bodies or hemangioma growth was found despite long-term survival of cells constitutively expressing VEGF. We examined whether long-term persistence of vessels induced by the 100% clone correlated with pericyte coverage by costaining muscle sections obtained 14 days after ischemia induction and cell implantation for endothelial cells (PECAM/CD31) and pericytes (NG2). Vessels induced by the 100% clone displayed tightly associated pericyte coverage (Fig. 6A-C ), indicating that long-term stability was a result of adequate maturation of the newly induced vessels. In contrast, the vessels induced by the parental polyclonal VEGF population showed abnormal coverage with pericytes that appeared detached from the growing vessels and expressed smooth-muscle-actin (not shown), ultimately leading to the growth of glomeruloid bodies and hemangiomas. The present findings show that prolonged, but uniform VEGF expression by the 100% clone in ischemic muscle yields mature, pericyte-covered vessels that persist for more than 15 mo, approximately half of the life span of a mouse, without regressing or becoming aberrant.

DISCUSSION

Uniform VEGF distribution within a microenvironmental dose window determines blood flow improvement in ischemia
The most important and clinically relevant finding of this study is that that controlling the microenvironmental concentration of VEGF, without changing total dose, defines a dose window in which constitutive, long-term VEGF gene expression in ischemic muscle predictably induces stable and functional vessel growth, efficiently rescues blood flow to nonischemic levels, while avoiding any aberrant angiogenesis and hemangioma growth. These findings show clearly in a therapeutic setting that, because of the pathophysiological mechanism of VEGF-induced vessel growth, average total doses across muscle tissue are not the critical determinant of efficacy and safety and that local concentrations determine functional outcome. This study is, to our best knowledge, the first to show that optimizing the microenvironmental distribution is critical to improving both efficacy and safety of VEGF in ischemia, without reducing or increasing total dose.

Because no available technique allows the direct quantification of microenvironmental VEGF concentrations in tissue, we used a cell-based method to examine the dose-dependent effects in ischemia (9) . In the polyclonal population, the amount of VEGF expressed per cell is variable, because the numbers of retroviral DNA copies and their integration sites into the host genome is unpredictable. By contrast, a monoclonal population derived from a single cell expresses a given amount of VEGF per cell. Remarkably, we find in the ischemic hind limb that the uniformity of VEGF secretion critically influences both vascular structure and function. Blood flow was improved by the 100% VEGF clone compared with the parental polyclonal population, reaching a level similar to the nonischemic leg, and ischemic tissue damage was reduced. In contrast, neither low (10%) nor high (180%) microenvironmental VEGF concentrations improved function, demonstrating the previously unrecognized existence of a lower and an upper threshold VEGF concentration for the induction of beneficial effects in ischemia.

Enhanced efficacy of the 100% clone compared with the parental polyclonal population was not due to a higher total VEGF dose, which is similar between both populations in vitro and in vivo, showing that microenvironmental factors are critical. Importantly, the vessels induced by the 100% clone were pericyte-covered and persisted more than 15 mo, to our best knowledge, the longest reported persistence of vessels induced by exogenous VEGF delivery, and possibly indefinitely, as a result of constitutive VEGF expression. On the other hand, implantation of the 100% clone did not induce hemangioma formation, a form of aberrant vessel growth invariably induced by the polyclonal VEGF population, regardless of how few cells were injected (9) . The successful avoidance of hemangiomas is remarkable in that 8 million cells were injected in the present study, whereas as few as 35,000 cells of the parental polyclonal population had previously been shown to induce aberrant vessel growth (9) . Hence, if microenvironmental VEGF concentrations are kept below a threshold level, the total VEGF dose in ischemia can be dramatically increased without reintroducing the risk of hemangioma growth. These findings show that constitutive expression of VEGF has, indeed, the potential to induce long-term stable vessels without giving rise to aberrancies provided that microenvironmental VEGF levels are controlled within a therapeutic dose window.

Morphological characteristics of vessel growth that increases blood flow in ischemia
The methodology used here offered the opportunity to specifically "engineer" a set of distinct vessel types and to systematically correlate their morphology with function in ischemia. Vascular adaptations associated with blood flow increase induced by the 100% VEGF clone included the local induction of enlarged, pericyte-covered and regularly shaped vessels without glomeruloid bodies and the induction of distant collateral growth. Recently, the ability of VEGF to remodel the entire vascular tree, including collaterals, arterioles, and capillaries has been established (33) . The precise physiological and pharmacological prerequisites for growth induction in different vascular segments are still incompletely understood, although shear stress is a likely factor,

A remarkable and unexpected finding was the biphasic effect of increasing VEGF doses on the number of induced vessels (already maximal at the lowest level) and their diameter (which instead increased with VEGF dose). The combined increase in vessel size and number was required to achieve blood flow improvement (100% clone), whereas no effect was seen if vessel number alone were increased without enlargement (10% clone). These findings indicate that vessel diameter is equally or more important than vessel number. Although increased vessel number alone may alleviate the ischemia burden by facilitating exchange between blood and tissue, blood flow improvement is required for true recovery. These findings challenge the utility of analyzing vessel density, a common measure to evaluate angiogenic efficacy in experimental ischemia models, suggesting that vessel size should be routinely measured as well to improve our understanding of how efficacious vessel growth can be induced.

Contrary to what might have been expected, implantation of the 180% clone, secreting higher still levels of VEGF per cell, did not further improve blood flow or reduce tissue damage compared with the 100% clone. Indeed, neither blood flow nor collateral growth were significantly increased compared with the controls. Hence, the beneficial effects induced by the 100% VEGF clone were lost at higher doses, indicating the existence of an upper limit of microenvironmental VEGF concentrations for the induction of therapeutic effects. Intriguingly, the same VEGF concentrations marked the transition from the induction of morphologically normal vasculature (100% clone) to that of aberrant, glomeruloid bodies with insufficient and abnormal pericyte coverage (180% clone) that later grow into hemangiomas. In a previous report, VEGF delivery in a manner that induced hemangioma-like structures also failed to improve blood flow in ischemic myocardium (7) . It is unclear whether the association between aberrant vessels and the loss of functional improvement in ischemia is a coincidence, or indicates a causal relationship. However, striking parallels exist of the concept of transient normalization of tumor vasculature that occurs after treatment with VEGF inhibitors (34) . Tumor vasculature is frequently abnormally dilated due to excessive proangiogenic stimulation. Therapeutic VEGF inhibition prunes such abnormalities, transiently resulting in a vasculature that is smaller in diameter and closer to normal, resulting in increased tumor blood supply and oxygenation in a seemingly paradoxical manner (34) . The present study indicates that similar rules apply to angiogenesis in ischemia and that excessive proangiogenic stimulation induces dilated and glomeruloid vasculature (180% clone) that abolishes the functional benefit seen at lower VEGF doses. This imbalance is completely avoided if the microenvironmental VEGF concentration is uniformly maintained below a threshold level (100% clone). It is currently not fully understood, which biological mechanisms underlie the "decision" of a vessel exposed to angiogenic stimuli to grow in length, form a new branch or enlarge, and how these factors can be used to improve therapeutic efficacy and safety. Factors downstream of VEGF might act to stabilize (e.g., Angiopoietin-1) or further destabilize the angiogenic vessels (e.g., proteases). Spatial gradients of VEGF have been proposed to induce angiogenic sprouting by guiding the filopodia-mediated migration of endothelial tip cells (35) . Furthermore, pericytes, which reside on microvessels and contact several endothelial cells with their long processes, could modulate control vessel sprouting and branching (36 , 37) .

The results of this study help explain the apparent lack of a manageable therapeutic window for VEGF dosage in reports from experimental and clinical studies. In fact, gene vectors (e.g., adenovirus or plasmid DNA) or protein delivery cannot control the distribution of microenvironmental VEGF levels in vivo. For example, adenoviral VEGF delivery results in a mixture of different vessel types, most of which are unstable and regress (14) . These results pose a challenge for the development of future therapeutic angiogenesis strategies. In conclusion, this study revealed that, although increasing the total amount of VEGF in the ischemic muscle is a prerequisite for the induction of angiogenic effects, control over the microenvironmental levels of VEGF delivery is required for the achievement of long-term functional benefit without significant toxicity, suggesting that therapeutic strategies incorporating this biological concept have the potential to fully harvest VEGF’s therapeutic power.

ACKNOWLEDGMENTS

We thank Tim Kovachy and Chris Xu for technical assistance. This work was supported by a grant from the Deutsche-Forschungsgemeinschaft to G.v.D. (DE 740/1-1), and American Heart Association Scientist Development Grant to A.B. (0430312N) and by NIH grants AG009521, HL065572, HD018179, AG020961, AG024987, and the Baxter Foundation to H.M.B.

Received for publication May 29, 2006. Accepted for publication August 7, 2006.

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