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

Department of Microbiology and Immunology, Stanford University, Palo Alto, California, United States
The FASEB Journal (Impact Factor: 5.04). 01/2007; 20(14):2657-9. DOI: 10.1096/fj.06-6568fje
Source: PubMed


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.

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    • "Despite its importance, to the best of our knowledge, there are no published reports in the literature describing the detailed vascular anatomy of the murine hindlimb. The majority of papers using murine ischemia models have paid attention only to the medial-superficial aspect of the thigh and have not considered the circulation of the whole hindlimb[4], [9], [11], [13], [15]–[26]. Since the thigh has great muscles surrounding the femur, the arteries previously described are not sufficient to feed the entire hindlimb. "
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    ABSTRACT: Appropriate ischemia models are required for successful studies of therapeutic angiogenesis. While collateral routes are known to be present within the innate vasculature, there are no reports describing the detailed vascular anatomy of the murine hindlimb. In addition, differences in the descriptions of anatomical names and locations in the literature impede understanding of the circulation and the design of hindlimb ischemia models. To understand better the collateral circulation in the whole hindlimb, clarification of all the feeding arteries of the hindlimb is required. The aim of this study is to reveal the detailed arterial anatomy and collateral routes in murine hindlimb to enable the appropriate design of therapeutic angiogenesis studies and to facilitate understanding of the circulation in ischemia models. Arterial anatomy in the murine hindlimb was investigated by contrast-enhanced X-ray imaging and surgical dissection. The observed anatomy is shown in photographic images and in a schema. Previously unnoticed but relatively large arteries were observed in deep, cranial and lateral parts of the thigh. The data indicates that there are three collateral routes through the medial thigh, quadriceps femoris, and the biceps femoris muscles. Furthermore, anatomical variations were found at the origins of the three feeding arteries. The detailed arterial anatomy of murine hindlimb and collateral routes deduced from the anatomy are described. Limitations on designs of ischemia models in view of anatomical variations are proposed. These observations will contribute to the development of animal studies of therapeutic angiogenesis using murine hindlimb ischemia models.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "Even though MSCs have immunosuppressant properties, these cells also produce growth factors and vessel dilators such as VEGF and nitric oxide, respectively [11,50], to promote angiogenesis and tissue repairing. Excess VEGF and nitric oxide in a tissue is known to be capable of forming unstable and malfunctioning vessels, which can lead to vessel disruption and edema [51,52]. The choice of an adequate moment for cell injection into ischemic tissue for therapy is therefore one of the important points to be considered in cell therapy. "
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    ABSTRACT: BALB/c and C57/BL6 mice have different abilities to recover from ischemia. C57/BL6 mice display increased vessel collateralization and vascular endothelial growth factor (VEGF) expression with a consequent rapid recovery from ischemia compared to BALB/c mice. Mesenchymal stem cells (MSCs) are one of the main cells that contribute to the recovery from ischemia because, among their biological activities, they produce several pro-angiogenic paracrine factors and differentiate into endothelial cells. The objective of this study was to evaluate whether the MSCs of these two mouse strains have different inductive capacities for recovering ischemic limbs. MSCs from these two strains were obtained from the bone marrow, purified and characterized before being used for in vivo experiments. Limb ischemia was surgically induced in BALB/c mice, and MSCs were injected on the fifth day. The evolution of limb necrosis was evaluated over the subsequent month. Muscle strength was assessed on the thirtieth day after the injection, and then the animals were sacrificed to determine the muscle mass and perform histological analyses to detect cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. The MSCs from both strains promoted high level of angiogenesis similarly, resulting in good recovery from ischemia. However, BALB/c MSCs promoted more muscle regeneration (57%) than C57/BL6 MSCs (44%), which was reflected in the increased muscle strength (0.79 N versus 0.45 N). The different genetic background of MSCs from BALB/c and C57/BL6 was not a relevant factor in promoting angiogenesis of limb ischemia, because both cells showed a similar angiogenic activity. These cells also showed a potential myogenic effect, but the stronger effect promoted by BALB/c MSCs indicates that the different genetic background of MSCs was more relevant in myogenesis than angiogesis.
    Full-text · Article · Jul 2013 · Stem Cell Research & Therapy
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    • "However, the relative merits of the angiogenic factors currently in use continue to be the subject of much debate. In addition, vascular endothelial growth factor (VEGF), the most common angiogenic factor in clinical tests, can lead to undesirable consequences such as hemangiomas and atherosclerotic lesions if expressed at too high a level in animal models [2], [3], [4], [5], [6], [7] even if VEGF concentrations become too high in extremely localized regions on a microscopic level [8], [9]. Members of the FGF family have shown intriguing possibilities [10], [11], but therapeutic angiogenesis has yet to become a clearly beneficial clinical tool. "
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    ABSTRACT: Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.
    Full-text · Article · Apr 2013 · PLoS ONE
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