The field of angiogenesis research, an exotic concept to most physicians a quarter century ago, now commands two journals devoted exclusively to answering such questions as how growth factors and other molecules regulate vessel growth and how this process is altered in early neoplasms and again during metastasis. Another intriguing question is how to control and possibly even reverse new vessel formation.

Efforts to curtail growth of the new vessels that nourish tumors and support tumor growth have taken three major directions:

- Countering the actions of the numerous factors (> 15 at last count) that maintain endothelial cell growth and proliferation. Two of the best known are vascular endothelial growth factor and what may be the most potent of all angiogenic substances, the misnamed basic fibroblast growth factor. It might prove possible genetically to alter growth factor receptors in tumor cells.

- Inhibiting matrix metalloproteases, enzymes that break down normal tissues to clear a path for new "tumor vessels."

- Using angiogenesis inhibitors, substances that paradoxically are produced by tumor cells but strongly suppress new vessel growth (neovascularization). This approach is the most interesting to clinicians because it seems to hold the promise of a biologically grounded and safe way to limit neoplastic growth.

What Happens in Tumors

Conventional thinking has long maintained that malignant tumors simply take over existing circulation and do not form their own blood vessels. Although some researchers still adhere to this view, persuasive arguments to the contrary have arisen-that is, that tumors are "angiogenesis-dependent" and must supply their own blood if they are to grow beyond a very limited size. This revolutionary view, which immediately suggests a way to discourage neoplastic growth, has evolved largely from nearly four decades of research by Judah Folkman, MD, and many colleagues. Folkman is professor of pediatric surgery and cell biology at Children's Hospital and Harvard Medical School in Boston.

Working with Frederick Becker, MD, in the early 1960s at the Naval Medical Research Institute to develop blood substitutes, Folkman perfused rabbit thyroid glands with hemoglobin solution and then inserted tiny melanoma implants (which are black and easily seen) to learn whether the blood substitute supported tumor growth. Unexpectedly, all the implants reached a few millimeters in size and then abruptly stopped growing. When replaced subcutaneously in their original mouse host, the implants developed new blood vessels and rapidly grew to a large size. The conclusion that the absence of blood vessels in the thyroid restricted tumor growth was irresistible (Surg Forum. 1962; 13:164-6). This work was replicated by a study showing that tumors implanted in the anterior chamber of the eye grow only as large as the head of a pin, but after being teased back into the vascularized iris they grow rapidly. This research is recounted in what many investigators consider the seminal paper in angiogenesis research, wherein Folkman proposed that "some diffusible message is released from tumor to nearby endothelial cells" that are then "switched" from their resting state to divide rapidly and form new capillary sprouts. These capillaries can grow as rapidly as 1 mm per day (N Engl J Med. 1971; 285:1182-6).

Just such an endothelial cell mitogen, named tumor-angiogenesis factor, was soon isolated from human and animal tumors (J Exp Med. 1971; 133:275-88). The first proof that numerous angiogenic proteins stimulate new vessel formation arose from an elegant feat of chemical engineering by Robert Langer, who devised a polymer bead. The bead, when placed in the avascular cornea, slowly and continuously released these proteins to stimulate the formation of new vessels (Nature. 1976; 263:797-800).

Balancing these stimulators of angiogenesis are such inhibitors as platelet factor 4, certain "angiostatic" steroids, and interferon. The presence of these inhibitors, which were first identified in the 1980s, may explain the difficulty of growing endothelial cells under normal conditions. These opposite-acting molecules conceivably constitute a system of "checks and balances" that regulates angiogenesis in much the same way that an elaborate network of antagonistic coagulation factors controls blood clotting. Later work showed that a two-step process involving upregulation of an angiogenic stimulator and down-regulation of an inhibitor turns on an angiogenic switch that rapidly induces neovascularization (Nature. 1989; 339:58-61).

In the Laboratory

The angiogenic switch is an essential part of any "successful" tumor. Now demonstrated in diverse animal and human cell types, the switch is a critical point in early tumor development that conceivably could be manipulated to tip the balance in favor of angiogenesis inhibitors (Cell. 1996; 86:353-64). Surgeons and oncologists have long known that removing a primary tumor, such as a sarcoma or melanoma, may quickly lead to aggressive metastasis. Although metastasis has traditionally been ascribed to "seeding" of tumor cells in the local tissues and later to immune suppression, it now seems clear that metastasis is stimulated by removal of the angiogenesis inhibitor along with the primary tumor. In the early 1990s, O'Reilly and colleagues found that a naturally occurring protein termed angiostatin, which resembles a fragment of human plasminogen, strongly suppressed metastasis of Lewis lung carcinoma in mice (Cell. 1994; 79:315-28). As shown in Figure 1, several types of human cancer implanted in mice stopped growing almost entirely after systemic treatment with angiostatin, which specifically inhibits endothelial cell proliferation. No toxicity or resistance was evident (Nat Med. 1996; 2:689-92). An analogous and even more potent inhibitor, endostatin, was found soon thereafter (Cell. 1997; 88:277-85).

View larger version (87K): [in this window] [in a new window]   Figure 1. Representative untreated and treated tumors. The mice on the left of all three panels were left untreated; the mice on the right were treated with angiostatin. Human prostate cancer (top), breast cancer (middle), and colon cancer (bottom) are shown. From Nat Med. 1996; 2:689-92, with permission.

There is evidence that human cancer cells express an enzyme that converts plasminogen to angiostatin (Cancer Res. 1996; 56:4887-90). Treating experimental cancer with endostatin does not produce a drug-resistant state (Nature. 1997; 390:404-7). Oncology aside, the downregulation of angiogenesis inhibitors may play a key pathogenetic role in disorders of excessive neovascularization, such as diabetic retinopathy, psoriasis, and even the formation of atherosclerotic plaque.

A big question: Why do tumors produce substances that prevent formation of blood vessels on which they depend so greatly? Folkman agrees that this is a good teleologic question: "There are so many hundreds of steps over millions of years that led to it, that many times you can't track it back. One answer is: ‘that's just the way we found it.’" Charles Erlichman, MD, director of phase I testing of new cancer treatments at the Mayo Clinic in Rochester, Minnesota, believes that the inhibitors may explain why some tumors just "sit in patients and do not grow."

In the Clinic

The great virtue of antiangiogenesis agents is that they stop new vessel growth without attacking the healthy vessels that perfuse normal tissues. Several potential angiogenesis inhibitors are under study in phase I and II (and a few in phase III) clinical trials (Table 1). The inhibitors themselves have not yet been produced in the amount needed for large-scale assessments, but phase I trials may be feasible in 1999. Gene therapy may provide one approach: Link the angiostatin gene with an adenovirus and implant it in muscle tissue, which may become a "factory" that produces the protein.

At least initially, these agents will probably be used as an adjunct to radiotherapy, chemotherapy, interferon, and other antitumor therapies. Erlichman has proposed that inhibitors be administered after removal of a primary tumor when there is a high risk for recurrence, or after a tumor has spread and other adjunctive measures are impractical. Rapidly growing tumors mechanically compress their new vessels, limiting the reach of systemic chemotherapy, and angiogenesis inhibitors hold hope of reversing this process (N Engl J Med. 1995; 333:1757-63).

To date, inhibitors have been tried in patients with advanced cancer-often of the lung, pancreas, or kidney. Folkman's general impression from early trials presented at meetings in the United States and Europe was that these inhibitors cause disease to stabilize or even regress slowly in some patients. It may soon be possible to amend his stated view that, "If you have cancer and you are a mouse, we can take good care of you." Angiogenesis inhibitors may also be useful for treating blinding eye diseases caused by neovascularization, skin conditions such as psoriasis, life-threatening hemangiomas, and refractory gastrointestinal ulcers.

The following myths about the clinical use of angiogenesis inhibitors can now be dispelled:

- Inhibitors seriously delay wound healing. The angiostatins (the most potent inhibitors) have no such effect. Some other types, however, may delay healing by up to 15%.

- Inhibitors must be avoided in postoperative patients. The tumor vessels that inhibitors affect obviously differ in subtle and unknown (but important) respects from normal vessels.

- Inhibitors are hazardous during pregnancy. Thalidomide certainly has teratogenic effects, and some other angiogenesis inhibitors are abortifacients. The angiostatins, however, do not seem to adversely affect pregnancy or the fetus.

Summing Up

Looking back to the 1960s at the inception of angiogenesis research and forward to the new century, Folkman has a broad vision of the potential uses of angiogenesis that extend beyond oncology: "I think it's a very exciting biomedical field. It has many principles in it that affect other aspects of medical practice." According to Erlichman, angiogenesis "is interesting and important work that has great potential in terms of clinical application to cancer therapy." That this potential has not been realized reflects the fact that, in the context of what is yet to come, this work is still at a preliminary stage.

- David A. Cramer, MD

Suggested Reading

Boehm T, Folkman J, Browder J, O'Reilly M. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature. 1997; 390:404-7.

Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971; 285:1182-6.

Folkman J. Clinical applications of research on angiogenesis. N Engl J Med. 1995; 333:1757-63.

Folkman J, Long DM, Becker FF. Tumor growth in organ culture. Surg Forum. 1962; 13:164-6.

Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible for angiogenesis. J Exp Med. 1971; 133:275-88.

Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature. 1989; 339:58-61.

Gately S, Twardowski P, Stack MS, Patrick M, Boggio L, Cundiff DL, et al. Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin. Cancer Res. 1996; 56:4887-90.

Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996; 86:353-64.

Langer R, Folkman J. Polymers for the sustained release of proteins and other macromolecules. Nature. 1976; 263:797-800.

O'Reilly M, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997; 88:277-85.

O'Reilly M, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med. 1996; 2:689-92.

O'Reilly M, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994; 79:315-28.