Despite its promise for treating ischemic diseases (for a review, see ref. 33
), VEGF can induce the growth of aberrant blood vessels and hemangiomas (5
), and this could limit its therapeutic utility. In the present study we addressed the question of whether VEGF can be administered in a way that favors normal angiogenesis and, if so, what the optimal conditions for achieving this goal are. Our experiments provided evidence for three new findings: (a) constitutive long-term VEGF gene expression in adult muscle can induce stable, normal angiogenesis when the dosage is precisely controlled; (b) VEGF effects depend on microenvironmental, and not total, VEGF dosage, as averaging of different levels over an area of muscle does not occur; and (c) a discrete threshold in VEGF dosage exists below which normal, stable capillaries are induced and above which hemangioma growth occurs, with no stable intermediate phenotypes. When produced under optimal conditions, VEGF promoted growth of capillaries of uniform size that were not leaky at 28 days, were stable for at least 3.5 months, and were independent of continued VEGF expression. Implantation of even small numbers of myoblasts with high expression of VEGF resulted in growth of aberrant vascular structures, demonstrating that extremely localized levels of VEGF determine the morphology of the new vessels.
With homogeneous expression by clones, an increase in the dose up to about 70 ng/106
cells/day was accompanied by a gradual increase in the diameter of the new vessels, but without the formation of pathological structures. At doses of about 100 ng/106
cells/day and above, however, bulbous glomeruloid bodies appeared early and full-fledged hemangiomas invariably developed over time. This dosage interval (between 70 and 100 ng/106
cells/day) appears to represent a threshold level between VEGF-induced angiogenesis and “hemangioma-genesis” (34
). A possible explanation is that such threshold could be determined by the balance between the local concentrations of proangiogenic and vessel stabilizing factors. Below the threshold, sufficient amounts of endogenous vessel maturation factors, either pre-existing or induced by the delivered VEGF itself, may be present to produce normal vessel assembly and stabilization. Above the threshold, VEGF production presumably tips the balance toward exaggerated proangiogenic activity and unregulated growth of aberrant blood vessels and hemangiomas. In support of this possibility, we found that the phenotype of mural cells associated with the different kinds of vessels differed dramatically. Pericytes with a phenotype resembling that of pericytes on normal muscle capillaries (NG2-positive/α-SMA-negative) were in fact scarce on aberrant structures induced by VEGF levels above the threshold and in their place were mural cells immunoreactive for α-SMA. A similar switch in expression of α-SMA by aberrant pericytes has been described recently in the abnormal vessels of the RIP-Tag2 genetic model of pancreatic islet tumors (35
). Interestingly, this phenotypic transition correlated with tumor progression: no pericytes expressed α-SMA on capillaries in normal pancreatic islets; some pericytes expressed α-SMA on vessels in hyperplastic premalignant islets; and virtually all vessels in advanced cancers had α-SMA-positive aberrant pericytes. Our results show that the appearance of mural cells with a similar phenotype on vessels induced by VEGF levels above the threshold correlates with aberrant vascular morphology, progressive growth to hemangiomas (Figure ), and continued dependence on VEGF (Figure ). On the other hand, the blood vessels induced by VEGF levels below the threshold displayed capillary-like morphology, were tightly associated with NG2-positive/α-SMA-negative pericytes, did not regress or change morphology over at least 3.5 months, and became independent of continued VEGF expression by 3.5 weeks; that is, they were truly stabilized. These observations are consistent with previous evidence for a paracrine/juxtacrine inhibition of endothelial cell proliferation by pericytes in vitro (36
) and during development (38
), pointing to a possible role of the balance between VEGF-induced angiogenesis and pericyte-mediated vascular maturation in determining the observed threshold between the induction of stable capillaries and unstable hemangiomas.
Upon injection into host muscle, most myoblasts die, while some proliferate and fuse either to pre-existing fibers or among themselves (26
). In a clinical study of Duchenne muscular dystrophy, the number of donor nuclei stably participating in hybrid fibers was found to be about 10% of the injected myoblasts (39
). In mice, the number of hybrid fibers remained constant from 5 days to 6 months after implantation (26
) and quantification of reporter gene expression (LacZ) correlated tightly with the number of transgenic fibers (40
). Although a determination of the precise amount of VEGF produced by myoblasts after integration in the injected muscle is impossible, we have recently shown that after myoblast fusion, VEGF protein remains tightly localized around the secreting fibers in vivo, creating a steep VEGF concentration gradient to which neighboring cells respond (32
). The VEGF164
isoform delivered binds heparin (41
) and is likely to localize around each VEGF-producing cell. This concentration gradient would explain why the different vascular effects depend on microenvironmental, rather than total (or averaged), VEGF levels produced across a broad region of muscle.
The results presented here have clear implications for clinical applications of VEGF gene delivery aimed at promoting therapeutic angiogenesis. Currently utilized delivery methods, such as injection of constitutively expressing VEGF plasmids or adenovirus, carry an inherent potential for heterogeneous VEGF production within treated areas, depending for example on the number of plasmid copies or adenovirus particles taken up by individual muscle fibers. In light of the data we present here, simply controlling VEGF dose by altering the total amount of vector delivered will not regulate microenvironmental VEGF levels, and this may impose significant limits on the effective dose achieved. In fact, to be safe, even the foci of highest expression must be below the threshold level, which implies that most areas are likely to have much lower microenvironmental VEGF than they might safely achieve otherwise. Consequently, room for increased efficacy is wasted (43
). The development and delivery of expression vectors that can be regulated and that can appropriately tailor levels of VEGF expression could be useful to circumvent this (46
). Alternatively, controlled-release polymers may provide a solution. These represent a diverse class of biomaterials that allow the homogeneous delivery of one or even two factors simultaneously (47
). The chemical properties of the polymer scaffold can be engineered to control precisely both the rate and the time over which a certain dose of recombinant protein is released in vivo, so that treatment strategies can be modified as new insights emerge about the basic biology of angiogenesis. Thus, this method of delivery potentially holds great promise for achieving control over the microenvironmental levels of angiogenic factors and their homogeneous distribution.
Growth of hemangiomas has not been a major issue in patient trials to date, mainly because methods of gene transfer have been relatively inefficient or have led to only short-term expression of VEGF; a hemangioma appearing in one patient treated with a VEGF-expressing plasmid disappeared as recombinant VEGF expression was lost over time (5
). It should be noted that we found the range of VEGF produced from implanted cells that induce normal neovascularization without signs of aberrant growth to be quite wide, from approximately 5 ng/106
cells/day to at least 70 ng/106
cells/day in culture. Also, such vessels correctly matured, stabilized, and were VEGF independent by 3.5 weeks, showing the potential for indefinite persistence after discontinuation of treatment. This bodes well for the achievement of a manageable therapeutic window in clinical applications. However, for successful treatment of a wide variety of patients, it would be useful to be able to take advantage of the full range of microenvironmental therapeutic VEGF levels within the safe window. This window will need to be determined in ischemic conditions and for different delivery methods, as the amounts of VEGF produced by myoblasts in vitro cannot be directly extrapolated to the microenvironmental concentrations achieved in vivo. Angiogenesis is a complex process and, although coregulation of more than one factor is being actively investigated (20
), our results demonstrate that when expressed in appropriate microenvironmental amounts, even the long-term delivery of a single factor, VEGF, can safely induce growth of stable vessels with normal morphology and function. While it is a technical challenge, the development of delivery methods that can control the level of VEGF delivered in a homogeneous fashion could significantly improve the efficacy and flexibility of VEGF-based therapeutic angiogenesis.