In Vitro Bioactivity
All VEGF isoforms, including the cell-associated forms, are active as EC mitogens by signaling through VEGFR2. Recombinant VEGF189 and VEGF206 are unable to stimulate endothelial cell mitogenesis, because of protein folding that masks regions necessary for receptor binding. However, ECM derived from cells expressing VEGF189 or VEGF206 induces EC to proliferate, demonstrating that VEGF trapped in the ECM is an active mitogen.
In Vivo Bioactivity: Developmental
The first definitive demonstration that the various VEGF isoforms serve distinct functions in vivo was provided by analysis of mice that were engineered to express single VEGF isoforms . Earlier analysis of VEGF isoform expression in adult tissues indicated that heart and lung expressed the highest levels of VEGF188. Consistent with this observation, mice that express only the soluble VEGF 120 are born at a reduced frequency (indicating embryonic lethality), and those that develop to term die rapidly after birth as a result of defective pulmonary and/or cardiac development. Analysis of vessel density in a limited number of mice that lived up to two weeks revealed that whereas vessel density in wild-type mice increased threefold within a week following birth, there was no increase in vessel density in VEGF 120/120 over the same time frame. These results demonstrate that the isoforms are in fact functionally distinct. In particular, these data illustrate that VEGF120 is not able to substitute for the missing VEGF 188. Although the mechanistic explanation for the resulting developmental abnormalities has not been elucidated, it is appealing to speculate that expression of an isoform that is entirely soluble (i.e., has no capacity to bind to matrix) may be ineffective in establishing a gradient that is necessary to induce postnatal angiogenesis.
Pulmonary development was also markedly defective in VEGF 120/120 mice. These mice have delayed airspace development, and quantitation of air-blood barriers revealed that the 120/120 mice not only had as few as 30 percent of the air-blood barriers as their wild-type litter-mates, but that the architecture of barrier structures was abnormal. Whereas in the normal air-blood interface the pulmonary epithelium is closely apposed to the capillary endothelium, the pulmonary capillaries in the VEGF 120/ 120 mice were separated from the alveolar lumens by up to three cell layers. Interestingly, the heterozygous (120/+) mice display intermediate phenotypes.
Although these observations indicate distinct functions for the individual isoforms, they do not clarify the mechanisms whereby these different functions are mediated. Some insight into the possible means by which the isoforms act is provided by localization studies. VEGF isoform production in the lung appears to be developmentally regulated , with VEGF 188 levels relatively low until it dramatically increases at around embryonic day 16, the late canalicular and late saccular phases of lung development, when alveolar structures are forming. In addition to the temporal correlation between VEGF synthesis and pulmonary capillary formation, there is a clear spatial correlation. In situ hybridization has shown that VEGF is produced specifically by the type 2 pneumocytes, cells intimately associated with the forming vasculature. These findings strongly indicate that the intimate association between the source of the VEGF and the target cells directs the formation of the blood-air barrier. Furthermore, the fact that a majority of the VEGF produced by the pulmonary epithelium is VEGF 188, an isoform that is not freely diffusible but remains locally sequestered, adds further strength to this hypothesis of the paracrine regulation of alveolar formation.
Abnormalities in the formation of other tissues and organs have been described in the isoform-specific mice. Retinal vascularization in mice that express only VEGF120 is severely impaired; there is a dramatic reduction in the formation of the primitive vascular network, reduced remodeling, and a persistence of the hyaloid vasculature . Mice expressing only VEGF 188 also exhibit abnormal retinal vascularization. Although the primitive vessel plexus is normal at postnatal day 3 (P3), vessel number is reduced by half only two days later (P5), with ephrin staining indicating a loss of the arterial component. As for the VEGF120 mice, a persistent hyaloid vasculature appears to migrate into the retina by P9, perhaps compensating for the regression of the arteries. More recent investigations into the relative contribution of VEGF isoforms to pathologic retinal neovascular-ization have revealed that retinal vessel development is normal in VEGF120/188 (VEGF164-deficient) mice .
The relative contribution of the various VEGF isoforms to skeletogenesis has also been investigated. Analysis of mice expressing only VEGF120 leads to the suggestion that VEGF is involved in at least three aspects of bone formation, including vessel ingrowth into the perichondrium and primary ossification center, stimulation of vessel growth and osteoclast migration into hypertrophic cartilage, and induction of osteoblast activity in both intramembranous and endochondronal bones .
Thus, VEGF isoforms appear to play critical and rate-limiting roles in the formation of a wide range of tissues and organs. The continued, tissue-specific expression of VEGF isoforms in adult organs  suggests that these functions may persist in mature organs and tissues.
In Vivo Bioactivity: Pathologic Models
To date, there have been few comparative studies on the function of VEGF isoforms, but differences clearly exist. Overexpression of VEGF121 and VEGF165 in a brain tumor model led to rapid vessel growth and breakdown (hemorrhage) around tumors, whereas VEGF 189 overexpression led to tumors with a similar extent of vascularization as the other isoforms, but no hemorrhage .
Tumor cell lines expressing a single mouse VEGF isoform have been used to study the role of the isoforms during tumorigenesis . VEGF-null fibroblasts were immortalized by transfection with SV40 large T antigen and transformed by infection with H-ras. The fibroblasts were then infected with plasmids encoding individual VEGF iso-forms under control of the CMV promoter and used to generate fibrosarcomas in mice. Relative to VEGF-null cells that formed small, poorly vascularized tumors, VEGF164-expressing transformed fibroblasts completely rescued tumor growth, VEGF 188 failed to rescue tumor growth, and VEGF 120 had a partial effect. However, although VEGF188 did not rescue tumor growth, tumors were more vascularized compared to VEGF120 tumors . The authors proposed a model in which the isoforms act cooperatively during tumor vascularization with soluble forms acting at a distance to promote blood vessel recruitment, while ECM-bound forms act locally to expand the capillary bed within the tumor .
These results and others suggest that VEGF-A isoforms have different abilities to induce vascularization, tumor growth, and blood vessel leakiness in multiple models. However, all models to date have utilized overexpression systems with VEGF under the control of nonphysiological promoters such as the CMV promoter. Therefore, although these results strongly indicate differential functions for VEGF isoforms, the absence of endogeneous VEGF regulatory elements makes the data difficult to extrapolate. Development of models in which VEGF is under the control of its endogeneous regulatory elements would greatly enhance our understanding of the contribution of VEGF isoforms to vascularization during developmental and pathological processes.
Overexpression is not an issue in studies that have utilized mice that express single VEGF isoforms. A second set of pathologies in which VEGF isoform function has been studied is ocular neovascularization, including diabetic retinopathy and retinopathy of prematurity, two leading causes of blindness in which ischemia leads to aberrant retinal blood vessel proliferation. One study utilized mice that express single VEGF isoforms (see above). VEGF164 is derived from leukocytes, is more proinflammatory than VEGF120, and is preferentially induced during ocular neovascularization . Using a mouse model of retinopathy of prematurity, it was found that administration of a VEGF164-specific neutralizing reagent (aptamer) blocks leukocyte adhesion and pathologic neovascularization, with no effect on retinal revascularization or physiological (developmental) neovascularization . When all VEGF isoforms were blocked, revascularization and physiological neovascular-ization were also affected. In mice with only VEGF120 and VEGF188 (VEGF164-deficient), retinal development was normal, similar to the aptamer results, suggesting that VEGF164 has a primary role in pathologic but not developmental vessel growth . These data suggest that VEGF164 is essential for pathological retinal neovascularization, but not physiological revascularization, which can progress under the influence of only VEGF120 and VEGF188. Thus, in this ocular angiogenesis model, an inhibitor specific for VEGF164 has been developed, which eliminates the features of pathological neovascularization (i.e. leukocyte adhesion, vascular tufts) while allowing the ischemic retina to become revascularized normally.
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