By David N. Leff

In rocky open country of sub-Saharan Africa, an unwary traveler may encounter a 14-foot-long black mamba (Dendroaspis polylepsis). This predatory snake makes its living by hunting small tree-dwelling animals. Cornered on the ground, the black mamba rears up and bites a man on the head or trunk. Without antivenin serum, the death rate is close to 100 percent.

This virulent viper¿s current claim to fame derives from its genomic resemblance to a newly discovered molecular relative of VEGF ¿ the vascular endothelial growth factor. VEGF¿s new cousin has been named EG-VEGF ¿ endocrine-gland VEGF.

¿The EG-VEGF gene resides on human chromosome 1,¿ observed its discoverer, reproductive endocrinologist Napoleone Ferrara, at Genentech Inc. in South San Francisco. ¿It expresses a small protein with a molecular mass of 8,600,¿ he continued. ¿The intriguing thing is it¿s an orthologue [genomic look-alike] of a snake. That protein was identified long ago in the venom of a black mamba. Snake venom has been the source of many important factors, like nerve growth factor, which is expressed at very high levels in snake venom.

¿There are no obvious implications in this 80 percent homology,¿ Ferrara pointed out, ¿because in humans EG-VEGF is very selectively expressed in adrenal glands, and a very few other endocrine glands. It probably has a completely different biology from the role it might play in the snake. With 58 percent identity, EG-VEGF also belongs to the same gene family as the yellow-bellied toad (Bombina variegata) and Xenopus laevis, the African clawed frog.¿

The Genentech staff scientist is senior author of a paper in today¿s Nature dated Aug. 30, 2001, which reports EG-VEGF¿s discovery. Ferrara¿s paper bears the title: ¿Identification of an angiogenic mitogen selective for endocrine gland endothelium.¿

¿We made a lot of progress in angiogenesis in the last 10 years,¿ Ferrara told BioWorld Today. ¿Especially, the discovery of vascular endothelial growth factor ¿ a cell-specific mitogen ¿ explained many phenomena. I think this discovery of EG-VEGF has put our understanding on a much higher level of science. We have for the first time a molecule that is specific for a particular class of endothelial cells ¿ those that reside in endocrine glands. This new molecule is active only, or predominantly, in endothelial cells that are expressed in human endocrine glands that secrete steroids ¿ mainly ovary, testis, adrenal glands and placenta. Those are probably the organs where it¿s likely to play a special role.¿

First Of A Highly Choosy Class?

¿The future implications could be,¿ he added, ¿that with similar special-tissue growth factors, you could select mitogens for the heart, the lung, the brain epithelia. Such selectivity would make for a much safer kind of therapy ¿ because it means using angiogenesis only for delivering important proteins with minimal impact on outside target organs. Until now, all the growth factors, like VEGF, seem to be acting across the board on all endothelial cells.¿

To isolate EG-VEGF, Ferrara and his co-authors ¿ all at Genentech ¿ expressed and purified a broad collection of secreted human proteins. Testing for organ selectivity, they assayed bovine adrenal cortex endothelial cells. ¿And we found this EG-VEGF molecule to be a very potent mitogen for those cells,¿ he recounted. ¿When we tested other endothelial cell types, we got little or no effect. This applied to human umbilical vein, bovine brain capillary and adult bovine aorta endothelial cells.¿

Their verification of endocrine selectivity also held up in in vivo experiments.

¿We tested VEGF and EG-VEGF side by side in a number of animal models,¿ Ferrara related, ¿such as in the corneal pocket assay. This consists of putting mitogenic pellets into the corneas of rats. With VEGF there is already an extensive literature, which shows a strong angiogenic effect. EG-VEGF had little or no effect. And the same was true with skeletal muscle, which is another organ where VEGF has been shown to be strongly angiogenic. Again, EG-VEGF was completely inactive.¿

The team then delivered its EG-VEGF gene to rat ovaries and other local sites, in experiments using recombinant adenovirus as a delivery vector. ¿We expected angiogenesis and increased endothelial cell permeability,¿ Ferrara recalled, ¿which is exactly what we got. There was a very large increase in ovarian mass and lots of angiogenesis, linked to formation of ovarian cysts. The study of EG-VEGF,¿ he suggested, ¿may offer insights into pathophysiology and therapy of endocrine disorders characterized by excessive angiogenesis, such as in the human polycystic ovary syndrome.¿

Heimlicher For Blood-Thirsty Tissues

Permeability is the key ploy of EG-VEGF endothelial cells. The keyholes are tiny apertures in the otherwise blood-tight cell wall. They are called fenestra ¿ ¿windows¿ in Latin. The leakiness they create allows the blood to off-load its cargo of hormones and other blood-borne substances for which client tissues are urgently thirsty. Both EG and VEGF independently unlock those fenestra.

¿The fenestra have some very nice discontinuities,¿ Ferrara described, ¿like little round holes within the cell-wall membrane, about 60 nanometers in diameter. They facilitate the permeability. Fenestra cluster in microvessels ¿ capillary endothelia. In the endocrine glands, the secretory cells are in very close proximity to the endothelial cells, so you can have this class of hormones crossing over.¿

Hypoxia ¿ shortage of oxygen ¿ gives the heads-up signal to the body¿s blood supply. Perhaps its best-known operative is the hormone erythropoietin, which tells red blood cells it¿s high time to replenish their oxygen-freighting hemoglobin. Ferrara related how the expression of EG-VEGF is goosed up by hypoxia, explaining that expanding endocrine glands add on numerous and specialized blood vessels. And because arteries and smooth muscle cells derive from precursors other than veins, the co-authors suspect that their tissue-specific angiogenic molecules orchestrate these processes as well. ¿Embryonic development, reproductive functions, tumorigenesis and many other proliferative processes,¿ Ferrara pointed out, ¿are critically dependent on the development of a blood supply.¿

An editorial titled ¿Creating unique blood vessels,¿ commenting on the Nature paper, cautioned: ¿The downside of the long-term use of such general molecules [as EG-VEGF] is that they might also stimulate angiogenesis elsewhere ¿ in hidden tumors, for example.¿ But it concluded, ¿This study has raised the hope that we might be able to achieve [therapeutic goals] more rapidly, and more safely, than we thought.¿