E X P E R I M E N T A L R E T I N A L N E O V A S C U L A R I Z A T I O N I N D U C E D B Y I N T R A V I T R E A L T U M O R S

D A N I E L F I N K E L S T E I N , M . D .

Baltimore, Maryland

S T E V E N B R E M , M . D .

Bethesda, Maryland

A R N A L L P A T Z , M . D . ,

Baltimore, Maryland

J U D A H F O L K M A N , M . D .

Boston, Massachusetts

A N D

S T E P H E N M I L L E R , B . A . , A N D C H U N G H O - C H E N , M . D . Baltimore, Maryland

Michaelson 1 and Ashton, Ward, and Serpell 2 proposed that a vasoformative substance might be produced by retina, causing growth of retinal vessels. Mi-chaelson suggested that the vasoforma-tive substance might be responsible for the normal process of embryologie vascu-larization, and Ashton, Ward, and Serpell suggested that an oversupply of this vaso-formative substance might be induced by the retinal ischemia of retrolental fibro-plasia.

The hypothesis that a vasoformative substance is liberated from ischemic or hypoxic tissue is still plausible and has received further support from a number of clinical observations. Firstly, fluores-cein angiography frequently demon-

From the Retinal Vascular Center, Wilmer Oph-thalmological Institute, Johns Hopkins Hospital (Drs. Finkelstein, Pätz, and Chen, and Mr. Miller), Baltimore, and the National Cancer Institute (Dr. Brem), Bethesda, Maryland; and the Department of Surgery, Children's Hospital Medical Center and Harvard Medical School (Dr. Folkman), Boston, Massachusetts. This study was supported by Na-tional Institutes of Health research grants EY-01368 and CA-14019 from the National Cancer Institute, and a career award (Dr. Pätz) from The Seeing Eye, Inc.

Reprint requests to Daniel Finkelstein, M.D..Wil-mer Institute, Woods Research Bldg., Johns Hop-kins Hospital, Baltimore, MD 21205.

strates areas of capillary nonperfusion before the development of retinal neovas-cularization. Secondly, the conditions as-sociated with retinal neovascularization, such as diabetic retinopathy, retrolental fibroplasia, sickle cell disorders, and reti-nal vein occlusion, always include areas of retinal capillary nonperfusion. Third-ly, retinal neovascularization occasional-ly occurs at a distance from the ischemic areas, such as when disk neovasculariza-tion occurs after branch vein occlusion. Fourthly, the rubeosis seen with diabetic retinopathy appears to respond to ablative photocoagulation of the retina. The mechanism for this effect may be the conversion of ischemic retina to dead retina, thereby decreasing the source of a vasoproliferative stimulus.

In the present study, we attempted to find substances that would produce reti-nal neovascularization. T h e demonstra-tion of such a substance would be helpful to elucidate the mechanism of diabetic and other proliferative retinopathies.

One substance that produces neovas-cularization, tumor angiogenesis factor (TAF) , has been isolated from many solid tumors. 3 We investigated the possible ef-fect of this substance on retinal vessels. T h e rabbit V-2 carcinoma has demon-strated angiogenic activity and can be

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transplanted to other adult rabbits with-out immune rejection. 4 Therefore, we se-lected the rabbit as the model in our study of retinal vessel proliferation.

M E T H O D S

To provide a source of T A F , a homolo-gous rabbit tumor, the V-2 carcinoma, was injected into the rabbit vitreous. T h e solid edge of a subcutaneous V-2 carcino-ma was passed through a cytosieve and the cells were suspended in 2 ml of cooled lactated Ringer 's solution. Tumor cell viability exceeded 9 0 % by trypan blue exclusion.

Tumor cells were transplanted to the vitreous bodies of 3- to 5-kg adult albino and Dutch belted rabbits. Under indirect ophthalmoscopy, 5 0 u,l ( 1 0 5 cells) were injected with a 27-gauge needle through the pars plana of 75 rabbit eyes. After the induction of intravenous pentobarbital anesthesia, each inoculation took less than one minute, was atraumatic to the lens and retina, and the vitreous remained transparent outside of the inoculum itself.

Tumor growth during the succeeding weeks was monitored by indirect oph-thalmoscopy, slit-lamp or fundus photog-raphy, or both, and fluorescein angi-ography. Tumor size was estimated by comparison with injected microspheres of known diameters (45-, 97-, and 320-u. aluminum microspheres).

At the completion of the experiments, some rabbits received an intracarotid in-fusion of colloidal carbon to outline the microvasculature. All eyes were enucle-ated, fixed in 1 0 % buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin for histologic ex-amination.

R E S U L T S

Of 7 5 adult albino rabbit eyes that were injected, growth of the rabbit V-2 carcino-ma was seen in 6 6 eyes. As long as the tumor's growth was confined to the vitre-

ous up to 100 days, it remained unvascu-larized. We saw no retinal vessel abnor-malities while the tumor was confined to the vitreous. Proliferation of retinal ves-sels only occurred when the tumors were contiguous with the retinal surface.

Initial growth of tumor in the vitreous occurred slowly over a period of weeks. As growth began, the eyes contained gen-erally one but occasionally as many as 12 nodules, and displayed two patterns of growth: spheroidal nodules or cylindrical stalks growing from the midvitreous to-ward the disk. Occasionally, nodules later developed a stalk pattern of growth.

Once the tumor stalks reached the reti-nal surface, they became vascularized by the ingrowth of proliferating retinal ves-sels. Figure 1 demonstrates neovasculari-zation by fluorescein angiography at the optic disk after infiltration by the tumor. After vascularization, tumors entered a new, explosive phase of growth (Fig . 2 ) . Within two weeks, a large exophytic mass, representing approximately a 19,000-fold increase in volume, grew along the vascularized portion of the reti-na and protruded into the vitreous (Fig . 3 ) . After local invasion into the retina and optic nerve, the tumors infiltrated the choroid and sclera.

Histologically, the vascularized tumors contained anaplastic cells with mitotic figures as well as capillary endothelial cell proliferation. In contrast, the intra-vitreal tumors showed an outer layer of ten to 2 0 viable cells with an inner necrot-ic center. T h e tumors were devoid of capillary endothelial cel ls .

Ocular changes were absent in 2 0 con-trol eyes injected with substances consist-ing of boiled V-2 carcinoma, fresh liver rabbit homogenate, india ink, normal sa-line, or aluminum microspheres.

T h e inoculation of tumor and placebo substances was atraumatic to the lens, retina, and vitreous as judged by slit-lamp and ophthalmoscopic observations. There

662 AMERICAN JOURNAL OF OPHTHALMOLOGY MAY, 1977

Fig. 1 (Finkelstein and associates). Top left, Fluo-rescein angiogram of the optic disk of a control rabbit in the early phase. Top right, Fluorescein angiogram of an eye after tumor infiltration demon-strates disk neovascularization in the early phase. Bottom, Fluorescein angiogram in same animal (top right) in the late phase demonstrates leakage from neovascularization. These rabbits underwent vitrec-tomy, and vitrectomy resulted in tumor dispersion that permitted better angiographic definition of un-derlying neovascularization.

was no sign of intraocular inflammation during intravitreal tumor growth by oph-thalmoscopic and histopathologic crite-ria. Intraocular inflammation was noted only after massive exponential growth of the tumor. T h e vitreous remained clear during the stages of intravitreal tumor growth; there were no signs of retinal vasculitis or retinal edema as monitored by indirect ophthalmoscopy, slit-lamp contact lens examination, fluorescein an-giography, and histopathologic examina-tion.

D I S C U S S I O N

Retinal neovascularization is elicited by intravitreal tumor implants. Experi-mental models of intravitreal neovascu-larization are important when studying the mechanisms of production of new vessels, and the mechanisms to prevent, treat, or eliminate neovascularization. Previous animal models of retinal neovas-cularization are limited to retrolental fi-broplasia and perhaps retinal vessel oc-clusion.

Previous studies have demonstrated

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Fig. 2 (Finkelstein and associates). Mean tumor volume after implantation into rabbit vitreous. Growth is slow during first nine weeks of avascular intravitreal growth. Arrow marks time of vascular-ization of tumor by retinal vessels, with rapid growth ensuing from the ninth to the 11th week.

that solid tumors produce a substance known as T A F that elicits neovasculariza-tion from the host vessels. T A F isolated from the tumor induced neovasculariza-tion without accompanying inflamma-t ion. 5 The ability of nontumor tissue to induce neovascularization has been ex-tensively investigated; chorioallantoic membrane, lung buds, l imb buds, skin,

Fig. 3 (Finkelstein and associates). Schematic representation of tumor growth. A, A spheroidal colony one week after implantation. B, Slow tumor growth for several weeks as a cylindrical stalk directed toward the retinal vessels. Neovasculariza-tion occurs only after the tumor is contiguous with the retina in C. Two weeks later, a large, vascular-ized mass protrudes from the retina (D).

heart, liver, spleen, pancreas, and intes-tine have never induced neovasculariza-t ion. 5 There may be an exception to the general rule that normal adult and embry-onic tissues do not induce neovasculari-zation; mouse trophoblast and human placenta stimulate mild neovascularizati-on. 5

T h e growth of tumor nodules in the vitreous for periods over three months provides a novel technique for the in vivo observation of tumor growth, free of con-tamination by host endothelial or inflam-matory cells. Characteristics of avascular tumor growth using homologous V-2 car-cinomas and heterologous tumors have been detailed. 6

In the cornea, implants of rabbit V-2 carcinoma, 2 .5 mm from the corneoscleral l imbus, attract new vessels within four days, and the tumors are vascularized after seven days. 4 In the vitreous, howev-er, these tumors remain unvascularized for up to 100 days. Even at a distance of 0.1 mm from the retinal vessels, blood vessels fail to proliferate toward the tu-mor. Vascularization occurs only when the growing edge of the tumor contacts the vascularized retinal surface. Unlike previous bioassays for T A F , retinal vessels of the rabbit do not respond to the tumor until contact occurs. Other models (the vascular rat back, the chick chorioallanto-ic membrane, the rabbit iris, and the rabbit cornea) demonstrate the effect of T A F at a distance from the tumor. 3 A number of factors, unique to the eye, that prevent T A F from inducing neovasculari-zation include physical and biochemical properties of the vitreous, physical prop-erties of the internal limiting membrane, and reactivity of the retinal vessels them-selves. These factors may be modified. For example, the vitreous may interfere with the transfer of a diffusible vasopro-liferative stimulus from the tumor to the host endothelial cells. Interference could be caused by a change in the activity of

664 AMERICAN JOURNAL OF OPHTHALMOLOGY MAY, 1977

T A F , the diffusion of T A F from the tu-

mor, or the result of a direct inhibition of

endothelial proliferation by the vitreous.

Such an inhibitor of tumor-induced neo-

vascularization has recently been isolated

from avascular ca r t i l age . 7 - 9

S U M M A R Y

Adult rabbit retinal vessels underwent

neovascularization in response to tumor

implantation within the vitreous body.

The neovascular response was presum-

ably elicited by the tumor angiogenesis

factor (TAF) . The response of adult reti-

nal vessels to an angiogenic stimulus rais-

es the possibility that a similar substance

may cause retinal neovascularization in

humans, and that in normal conditions

the vitreous may be able to suppress angi-

ogenic activity.

R E F E R E N C E S

1. Michaelson, I. C : The mode of development of the vascular system of the retina. With some obser-

vations on its significance for certain retinal diseas-es. Trans. Ophthalmol. Soc. U. K. 68:137, 1948.

2. Ashton, N., Ward, B., and Serpell,.G.: Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasia. Br. J . Ophthalmol. 38:397, 1954.

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

4. Gimbrone, M. A., Jr., Cotran, R. S., Leapman, S. B., and Folkman, J . : Tumor growth and neovascu-larization: An experimental model using the rabbit cornea. J . Natl. Cancer Inst. 52:413, 1974.

5. Folkman, J . : Tumor angiogenesis. In Klein, G., and Weinhous, S. (eds.): Advances in Cancer Re-search. New York, Academic Press, Inc., 1974, p. 331.

6. Brem, S., Brem, H., Folkman, J . , Finkelstein, D., and Pätz, A.: Prolonged tumor dormancy by prevention of vascularization in the vitreous. Cancer Res. 36:2807, 1976.

7. Brem, H., Arensman, R., and Folkman, J . : Inhibition of tumor angiogenesis by a diffusable factor from cartilage. In Slavkin, H. (ed.): Extracel-lular Matrix Influences on Gene Expression. New York, Academic Press, 1975, pp. 769-772.

8. Brem, H., and Folkman, J . : Inhibition of tumor angiogenesis mediated by cartilage. J . Exp. Med. 141:427, 1975.

9. Langer, R., Brem, H., Falterman, K., Klein, M., and Folkman, J . : Isolation of a cartilage factor that inhibits tumor neovascularization. Science 193:70, 1976.