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* Baxter Laboratory in Genetic Pharmacology, Departments of Molecular Pharmacology and of Microbiology and Immunology;
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California;
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
1Correspondence: Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5175, USA. E-mail: hblau{at}stanford.edu
SPECIFIC AIMS
Vascular endothelial growth factor (VEGF) expression requires exceedingly tight regulation during development, as either reduced expression after the targeted deletion of just one allele or modest overexpression lead to early embryonic lethality. Despite the great interest in harnessing the therapeutic potential of VEGF for the treatment of patients with ischemic conditions, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and clinical trials of VEGF delivery have been disappointing. This is apparently due to the limitations of current approaches to delivering the VEGF gene or protein to the adult organisms which, unlike transgenic models, are inherently unable to provide uniform VEGF distribution. We have developed a novel approach to systematically study the effects of local (microenvironmental) VEGF concentrations in ischemic muscle: monoclonal populations of VEGF-expressing myoblasts that, following implantation, fuse with the muscle fibers to provide uniform VEGF distribution in vivo. Using this technique, we investigated the effects of VEGF concentration on functional recovery from ischemia, toxicities, and long-term stability and morphology/architecture of induced vessel growth.
PRINCIPAL FINDINGS
1. Control over local (microenvironmental) distribution improves the efficacy of VEGF delivery in ischemia without increasing total dose
We generated monoclonal populations of myoblasts that express equal amounts of VEGF "per cell." First, a polyclonal population expressing VEGF164 was generated by retroviral transduction, from which single cells were isolated and expanded to obtain a set of monoclonal populations. 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. All populations also expressed the LacZ reporter. Hind limb ischemia was induced in mice by surgical transection of the superficial femoral artery, and 8 million myoblasts were implanted into the distal thigh muscles: either control myoblasts, the parental polyclonal VEGF-expressing population or one of the monoclonal VEGF-expressing populations or vehicle.
Fourteen days after surgery and cell implantation, the control groups injected with vehicle or control myoblasts showed strongly reduced blood flow (microsphere method) in the ischemic leg compared with the contralateral nonischemic leg (54.6±6.8% and 55.3±13.1%; Fig. 1
). The parental polyclonal VEGF population increased blood flow only moderately (74.1±8.6%, P <0.05 vs. controls). Improvement was substantially greater in mice that received the 100% VEGF clones, 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) (Fig. 1)
. Ischemia-induced muscle damage (histology) was reduced only in mice injected with the 100% clone. Importantly, functional improvement was seen despite the fact that the 100% clone produced similar total amounts of VEGF to the parental polyclonal population in vitro and in vivo. Furthermore, delivery of the 100% clone was less toxic, as hemangiomas were avoided and leakage was not increased. These findings show that, by providing uniform VEGF distribution at the microenvironmental level, therapeutic effects can be markedly improved and toxicities avoided compared with nonuniform distribution.
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2. A lower and an upper threshold level of microenvironmental VEGF concentrations determine a therapeutic window
The monoclonal populations of VEGF-expressing myoblasts showed markedly different effects in the ischemic leg. Surprisingly, blood flow was not improved in mice having received either low (10% clone: 54.7±17.5%) or high microenvironmental VEGF doses (180% clone: 69.8±18.9%) (Fig. 1)
. These findings demonstrate the existence of a therapeutic window determined by microenvironmental VEGF concentrations and indicate that expression levels need to be tightly controlled to be functionally efficacious in the ischemic muscle of the adult organism.
3. VEGF has biphasic effects on vessel number and diameter
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 by immunofluorescence. Myoblast engraftment (ßbeta;-galactosidase expression) was distributed along the needle tracks and comprised 
20% of the cross-sectional muscle area with no difference between the different populations. Vessel growth was tightly localized around the muscle fibers that expressed VEGF. Vessel length density was increased in all groups injected with VEGF-expressing myoblasts. Surprisingly, the effect was not dose dependent but peaked in muscles implanted with the 10% clones and was not further increased by even highest VEGF levels.
In contrast, vessel diameter was gradually increased by VEGF in a dose-dependent manner with no discernable upper limit over the 30-fold dose range investigated. The parental polyclonal VEGF myoblasts induced a mixture of small and large vessels, including dilated glomeruloid bodies, aberrant structures that subsequently grow into hemangiomas. In contrast, each clonal population uniformly induced vessels of distinct morphology. Surprisingly, the 10% clone, found to induce a peak increase in vessel number, did not also increase vessel size, but the 100% and 180% VEGF clones significantly increased vessel diameter in a dose-dependent manner. However, whereas the vessels induced by the 100% clone were still normal in morphology, the 180% clone induced irregularly shaped, preangiomatous glomeruloid bodies. The 325% clone induced large hemangiomas visible by microangiography.
4. Control over microenvironmental distribution promotes collateral growth
The growth of collaterals from preexisting arteries is required for blood flow increase in ischemia. Collaterals were quantified on cryosections of the proximal adductor muscles by costaining for endothelial cells (PECAM) and vascular smooth muscle cells (
-SMA). The number of collaterals 
30 µm diameter was increased in mice injected with 100% VEGF clones compared with the controls (P < 0.05), whereas no significant collateral growth was seen with the parental polyclonal population or with any of the other clones. Thus, collateral growth was only seen in the group injected with the 100% clone, matching the improved blood flow and reduced tissue damage.
5. Vessels induced by optimized VEGF delivery are pericyte-covered and persist long-term
Vessels induced by short-term VEGF expression lack pericytes, are unstable and prone to regression. On the other hand, sustained VEGF expression can induce hemangiomas, 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. In the area of cell engraftment (ßbeta;-galactosidase), vessel density was still clearly increased. Remarkably, no glomeruloid bodies or hemangiomas were found despite long-term constitutive VEGF expression. We examined whether long-term persistence of vessels correlated with pericyte coverage by costaining for endothelial cells (PECAM) and pericytes (NG2). Vessels induced by the 100% clone displayed tightly associated pericyte coverage, indicating that long-term stability was a result of adequate maturation of the newly induced vessels. In contrast, vessels induced by the parental polyclonal VEGF population had abnormal coverage with pericytes that appeared detached and expressed smooth-muscle-actin, ultimately leading to vascular aberrancies and hemangiomas. The present findings show that VEGF expression that is prolonged, but at a controlled and uniform level, by the 100% clone in ischemic muscle yields mature, pericyte-covered vessels that persist presumably lifelong.
CONCLUSIONS AND SIGNIFICANCE
The most important and clinically relevant finding of this study is 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 and efficiently rescues blood flow, while reliably avoiding aberrant angiogenesis and hemangiomas. 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 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.
Importantly, vessels induced by the 100% clone were pericyte-covered and persisted more than 15 mo as a result of constitutive VEGF expression. Remarkably, implantation of 8 million cells of the 100% clone did not induce hemangiomas, whereas this form of aberrant vessel growth was invariably induced by the polyclonal VEGF population even if very few cells were injected. Hence, if microenvironmental VEGF concentrations are kept below the upper 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 the potential to induce long-term stable vessels without aberrancies provided that microenvironmental VEGF levels are controlled.
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. Blood flow increase and reduced ischemic tissue damage provided by the 100% vascular endothelial growth factor clone correlated with specific vascular adaptations: growth of pericyte-covered vessels that were enlarged but regularly shaped vessels without glomeruloid bodies and collateral growth. 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 was increased without enlargement (10% clone). These findings demonstrate the existence of a lower threshold level of VEGF concentration for the induction of functional vessel growth. Contrary to what might have been expected, implantation of the 180% clone, secreting still higher levels of VEGF per cell, did not further improve blood flow or reduce tissue damage compared with the 100% clone. Hence, the beneficial effects seen with the 100% VEGF clone were lost at higher doses, marking an upper threshold of microenvironmental VEGF concentrations (Fig. 2
).
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The results of this study help explain the apparent lack of a manageable therapeutic window for VEGF in reports from experimental and clinical studies, as gene vectors or protein delivery cannot provide controlled microenvironmental VEGF distribution in vivo. In conclusion, this study revealed that control over the microenvironmental levels of VEGF delivery in ischemia makes the achievement of long-term functional benefit without significant toxicity possible and suggests that therapeutic strategies incorporating this biological concept have the potential to fully reap VEGF’s therapeutic power.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6568fje
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