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Angiogenic activity of the corneal epithelium
Exp.
Eye Res. (1985),
41, 721-732
Angiogenic
Activity
of the Cornea1
JOSEPH
Division
ELIASON
Stanford University Medical CA 94305, U.S.A.
of Ophthalmology, Stanford,
(Received 19 April
A.
Epithelium
1985 and accepted 5 September
Center,
1985, New York)
A homogenate of cornea1 epithelial cells was tested to determine if it could induce vascularization in the cornea. Both fresh and cultured cells were used &s sources of the homogenate which was evaluated in vivo using a self-contained system to perfuse it constantly into the cornea1 stroma. A vigorous growth of vessels resulted when the concentration of the homogenate exceeded a threshold of 20 yg per ml total protein. This capacity was not destroyed when the homogenate was heated. As a preliminary refinement of this response white blood cells were eliminated from the model with whole body X-irradiation. Vaacularization occurred in leukopenic animals but was less than that observed in normal animals. It is concluded that the epithelial homogenate is able to provoke cornea1 vascularization in the absence of leukocytes. Key war&: angiogenesis; cornea1 epithelium: vasostimulat,ing factor: leucopenia; cell culture.
1. Introduction The cornea, as a consequenceof its ready availability to observation is an excellent site to study the process of new blood vessel growth. Early investigators have presented convincing evidence that, in addition to edema, which separates the compact cornea1 lamella to permit vessel ingrowth mechanically (Cogan, 1949), a diffusible substance must be involved both to initiate and direct the growth of blood vessels in the cornea (Ashton and Cook, 1953; Campbell and Michaelson, 1948). Maurice, Zauberman and Michaelson (1966) concluded that, such a material must have a continuous cellular source in order to maintain a gradient throughout the duration of active vessel growth. In previously reported work a model wasdescribed in which a discrete cautery burn produced cornea1vascularization in animals depleted of white blood cells (Eliason, 1978). Others have reported similar results (Sholley, Gimbrone and Cotran, 1978). When the area of the injury was examined histologically in this model, at a time prior to the first appearance of vessels,the only cellular population that appeared viable was the cornea1epithelium. This was a regenerating layer. The leukopenia eliminated all leukocytes from the stroma and the cornea1endothelial and keratocyte populations had been destroyed by the cautery burn. It was concluded that the epithelium might be the source of a vasostimulating substance in this model. As an extension of this observation, this paper reports that the cornea1 epithelium is capable of inciting vascularization. 2. Methods Preparation
of howmgenate
A homogenateof rabbit cornea1epithelium waspreparedsimilarly from two sources.Fresh cells were obtained the cornea directly Please University
from with
the eyes of New a scalpel blade.
Zealand albino rabbits of varying age by scraping Care was taken to exclude conjunctival cells or
send correspondence to Joseph A. Eliason, Medical Center, Stanford, CA 94305, U.S.A.
0014-4835/85/120721+
12 $03.00/O
M.D..
Division
@ 1985 Academic
of Ophthalmology,
Press Inc. (London)
Stanford
Limited
.J. :I. E:I,IASOK
,“” WI
stroma. A second source of cells was a primary outgrowth in tissue culture obtained in a slightly modified manner from that described by Stocker, Eiring, Georgiade and Georgiade (1958). Corneas were excised from enucleated eyes within the limbus and the endothelial cell layer was removed with a cotton-tipped applicator stick (Gospodarowicz. Greenburg and Alvarado, 1979). This cornea1 button was then cut into four pie-shaped wedges and these were placed in a 60 mm tissue culture dish. It was found that a denuded Descemrt’s membrane both facilitated adhesion of the tissue pieces to the bottom of the dish and eliminated any contamination of the culture with cornea1 endothelial cells. Incubation was at 37 ‘C in a 5 y0 CO,-air atmosphere. The culture medium was TC 199 supplemented with 20’4 newborn calf serum (Microbiological Associates). At 4 days the tissue wedges were removed from the dishes leaving behind a monolayer of migrating epithelium which continued to spread and cover the surface in 7-9 days. Contamination by keratocytes was avoided since these ce!ls require about 7 days for their slower migration to bring them onto the surface of a culture dish from an explant (Newsome, Tagasugi, Kenyon, Stark and Opelz, 1974). Epithelial cells and keratocytes present distinctly different morphology in cultures. Phase-contrast microscopy was used to check for contamination by other cell types. Cells were harvested by first removing the medium, washing three times with cold saline and then scraping the dish surface with a rubber policeman. The suspension of cells obtained from culture or fresh from the eye was homogenized in a Potter-Elvejheim tissue grinder, centrifuged for 20 min at 1000 g, the sediment discarded and the supernatant then passed through a 922 pm filter to remove particulate material. The solvent used was saline prepared as a 99 “6 solution in double-distilled and de-ionized water. When indicated, the homogenate was heated at 80 “C for 10 min followed by repeat centrifugation and filtration. A total protein level in the homogenate was determined using the Lowry assay with a standard of human albumin-globulin (Sigma Chemical Co.) (Lowry, Rosebrough, Farr and Randall, 1951). Prepared homogenate was stored in glass at 4 “C and used within 7 days. In vivo
assay
system
The perfusion system has been previously described (Eliason and Maurice, 1980). In brief, an osmotic pump (Alzet, Alza Pharmaceuticals) was employed to perfuse continuously a solution into the cornea1 stroma through an implanted cannula. The pump was located subcutaneously in the scalp. A length of polyethylene tubing, heat-drawn to a fine tip (3G-50 pm o.d.), extended from the pump along a subcutaneous then subconjunctival path to a point at the superior limbus. It then entered the cornea1 stroma and continued to a point 3 mm from the inferior limbal vascular arcade. The pump, with its reservoir, will maintain a constant output of 1 yl hr-’ over a 7-8 day period. In order to monitor this function, sodium fluorescein was added to all solutions used (final concentration of 905 %). The presence of fluorescence as well as localized edema surrounding the tip of the cannula confirmed the operation of the system. New Zealand albino rabbits of 2-3 kg were used. Systems were implanted bilaterally, in most but not all cases, using a control solution in one eye and a test solution in the other. Leukopenia was produced as previously described (Eliason, 1978). In brief, X-irradiation was delivered with a Phillips 250 kV unit with the eyes shielded by 92 cm of lead and half of the dose given to each side of the animal. It was measured as a midline-absorbed dose. A total of 1500 rad was given in two doses separated by 48 hr. Daily white blood cell counts were measured in a hemocytometer chamber using venous blood obtained from the ear. A maximum level for the peripheral white count was set at 200. This selection was based upon previous work with this leukopenic model (Eliason, 1978). Serial sections on the animals in that study confirmed the absence of any infiltrating leukocytes in the stroma at 24 and 48 hr following the onset of the stimulus. Generally, animals reached a white count of 200 or less 3 days following the last dose of radiation at which time the implantation procedure was performed. Animals that failed to drop to this level were not used in the study. Tissue prepara!ion Animals were killed with a barbiturate overdose. Eyes were enucleated and fixed in 10 y0 buffered formaldehyde. After fixation, the cornea, with a surrounding rim of sclera, was
EPITHELTAL
ANGIOGEKIC
ACTIVITY
carefully excised. Cuts were made immediately outside the to produce a rectangular section of cornea which included central cornea. The tissue was imbedded in paraffin and long axis of the specimen (i.e. radially with respect to the were cut in a serial fashion from each specimen. Sections and eosin, dehydrated and mounted in permount. Measurements
and
723
region of limbal vascular ingrowth the limbus at each end and the 8,um sections were out along the cornea). A total of 40-M) sections were then stained in hematoxylin
observations
Changes in the cornea were observed with a Haag-Streit model 300 slit-lamp and documented with photographs taken through the slit-lamp. Vasoularization was deemed present if any new blood vessels, regardless of size, were noted under high magnification. Two methods were used for quantitative evaluation. Measurements were taken from the photographs and from the histologic sections. Photographs were projected and on the image three points were marked: two at the circumferential extent of vascular invtlsion at the limbus and one at the most central extent of invasion. In control corneas lacking vascular invasion, an area of vascular engorgement was usually present. Points were positioned at the extent of this area (Fig. 1). Using these three points a triangle was constructed and an area calculated
FIG. I. Slit-lamp photograph of cornea in a leukopenic animal receiving epithelial homogenate after 5 days. Arrow indicates tip of tube in the stroma and dots demonstrate placement of points to measure area and extent of vescularization.
to include the triangle plus the section of the cornea1 circle marked by its base. Measurements were standardized by using a photographed and projected millimeter scale. On the histologic sections the distance of vascular invasion into the cornea was determined by measuring from the most central vascular structure on each section to the junction of cornea1 and conjunctival epithelium. An eyepiece graticule was used with 100x magnification. The junction was determined both by the appearance of subconjunctival connective tissue rend by the onset of a variable thickness and irregular basal layer in the conjunctival epithelium. The measurements from the individual sections were averaged to arrive at a mean value for each eye.
724
.l. A. ELIASOK
3. Results After testing several potential control solutions in the perfusion system, 0.9 y0 saline was settled upon. Several trials with TC 199 with and without serum added and Eagle’s minimal essential medium without added serum invariably produced a vigorous inflammatory reaction in the cornea with a dense white cell infiltrate and vascularization. Even when autologous serum was used, a modest vascular growth resulted. Phosphate buffered saline (Xliason and Maurice, 1980) led to a modest infiltrate and vessel growth only in some cases. Saline (0*9O/“) with or without potassium chloride rarely excited any vascularization. Mean values of soluble protein in the homogenate were 280 ,ug per dish of cultured cells and 490 pg per cornea for fresh epithelium. For these experiments, non-heated homogenate solutions were adjusted to approximately 300 ,ug cm-” total protein. Heat-precipitating the homogenate resulted in a loss of approximately 90% of the original soluble protein. Heated homogenate solutions were adjusted to approximately 60 ,ug crnp3 unless otherwise indicated. At first, 42 eyes of 21 normal animals were used. Saline was perfused in 21 control eyes, seven eyes were perfused with homogenate from fresh epithelium and 14 eyes received homogenate from cultured cells. In one eye of the latter group the perfusion system failed to function and it was eliminated from the study. The results are indicated in Table I based upon slit-lamp observations over the &day period that the TABLE
1
Presence and absence of vessel growth in corneas perfused with saline control or a homogenate of cornea1 epithelium indicated as number of eyes for each group. Vascularization
Controls Fresh epithelium Cultured epithelium Totals Chi-squared
value
as a 2 x 2 table
is 237,
Yes
No
Totals
3 6 13 22
18 1 0 19
21 7 13
hence P < OGOl,
perfusion system operated. As indicated above, the appearance of any new vessels regardless of size was sufficient for vascularization to be judged present. Vasrularization occurred in three of 21 control eyes, six of seven eyes perfused with a homogenate from fresh cells and all eyes perfused with a homogenate from cultured cells. When the results of both of the homogenate groups are combined, chi-squared analysis employing the Yates correction factor indicates that the observed differences are statistically significant at a level of P < O*OOi. It is important to note that in the three control eyes showing vascularization, vessel invasion was estimated to extend less than 1 mm into the cornea. All of the eyes perfused with homogenate and showing vascularization, save one, demonstrated more extensive growth than this minimal amount. The histology of control corneas (Figs. 2, 3) showed edematous disruption of the collagen layers surrounding the tip of the tube. The overlying epithelium was slightly irregular and thinned but not disorganized. Mononuclear cells were present along the
EPITHELIAL
Fm. animal
ANGIOGENIC
ACTIVITY
2. Epithelium and anterior stroma immediately above the tip of the perfusion tube receiving normal saline after 7 days. Arrows indicate region of tube tip. x 63.
725
in a control
FIG. 3. (a) Stroma anterior to tube tip in a control animal receiving saline after 7 days. Note infiltration of inflammatory cells (arrows). The level of the tube is at the bottom of the photomicrograph. x 100. (b) Stroma posterior to the tube tip bearing few infiltrating cells. The tube level is near the top of the photomicrograph. x 100.
FIG. 4. (a) Epithelium and anterior stroma above the tip of the perfusion tube in an animal receiving an epithelial homogenate after 7 days. Arrows indicate region of tube tip. x 50. (b) Stroma anterior to the tube showing vascular invasion and inflammatory infiltration. Arrows indicate vessels. x 100. of the perfusion tube with some inflammatory cells in the surrounding stroma at the level of the tube and anterior to it. Infiltrating cells were rarely found in the stroma posterior to the level of the tube. In corneas perfused with homogenate (Fig. 4) there was a dense infiltrate in the region of the tube with scattered mononuclear and polymorphonuclear cells present throughout the stroma from the tube tip to the limbus. This infiltrate was. as in the controls, most prominent in the stroma anterior tract
EPITHELIAL
ANGIOGEKI(’
ACTIVITY
727
to the level of the tube. New vessels were present in the anterior stroma accompanied by collections of inflammatory cells and hemorrhage. The epithelium was again irregular and thinned as in the controls. Non-heated homogenate (nine eyes) was compared with heated homogenate (12 eyes) in 11 normal animals (a perforation of the anterior chamber occurred in one eye that was excluded from the study). The results are presented in Table II and expressed as mean areas calculated from slit-lamp photographs on successive days following implantation. The values for heated homogenate are greater than those for non-heated material. However, the observed differences are not statistically significant. Thus heated material retains its ability to incite vascularization. TABLE
Comparison
of the vascular
II
response to heated and non-heated Days following 3
Heated homogenate Non-heated homogenate
0.53 * 054 1.03kO.67
Area of vaacularization as calculated from photographs Mean values+s.~. are given. Measured area from control on given days are not statistically significant.
2.ol3+0.93 1.69+@6
corneas
homogenate,
implantation 5
on days
epithelial
8 351f1.32 304 f 1.62
following implantation as indicated. was O-65+028. Observed differences
Heated homogenate from cultured cells was lyophilyzed and then reconstituted in varying concentrations in saline: Solutions were perfused in seven eyes with protein concentrations from 50-1500 pg ml-l. Vascularization occurred in all eyes without any observable difference in the intensity of the vesselgrowth. Minor variations in the density of the vascular responsesappeared more to be related to individual variability in responseamong the test animals than the concentration of the solutions used. A threshold was encountered at 20 pugml- ‘. Two out of three eyes perfused with a solution at this concentration failed to show any vessel growth. In six animals, leukopenia was induced with radiation as noted above. In each animal, one eye was perfused with non-heated homogenate and the fellow eye with saline. Here, as in the normal animals, the first visible growth of new vessels commenced at about 72 hr after implantation of the perfusion system. One animal died on the fourth day and four others either died or were killed on the fifth day after implantation. A sixth animal died on the third day. It was excluded from vessel measurementsbut sections of the cornea are included here to demonstrate the lack of an inflammatory response.One pump system in a control eye failed to operate and was also excluded. Fig. 1 is a slit-lamp photograph of a leukopenic animal with vascular growth. Mean values for the most central vascular structure measuredfrom serial sectionsin the control and homogenate perfused corneasare presented in Table III. Using Student’s t test, the difference between these means is statistically significant with P c O@Ol. Measurementsof the area vascularized are presented in the same table. In order to make some comparison between leukopenic and normal animals, measurements from photographs were made for four of the leukopenic animals (photographs were not obtained for one of the leukopenic animals) and for
J. A. ELIASCIV
728
TABLE
III
Vascular invasion in leukopenic animals Vascular Distance Control Epithelial Distance photographs.
(mm)
038fO99 970 + @OS
homogenate
invasion Area
(mm-*)
065 + 028 1.28+019
of vascular invasion measured from serial sections of the cornea, and area calculated Mean values +s.D. are given. (P < 0901 for distance; P < 001 for area). TABLE
Area of vascuhrization
from
IV
in normal and leukopenic animals Area of vascularization (mm-*)
Normal WBC Leukopenic
2.08+_093 1.28&@19
count
Measurements were made from photographs an epithelial homogenate. Values are expressed
of normal and leukopenic as mean + S.D.
animals,
both
perfused
with
11 normal animals at 5 days after implantation. These values are indicated in Table IV; the difference of the means is not statistically significant. Histological examination revealed slight irregularity and thinning of the epithelium as noted in normal animals in both controls and homogenate perfused corneas (Fig. 5). The stroma was devoid of inflammatory cells, even immediately surrounding the perfusion tube. The keratocytes appeared normal. Invading vessel sprouts were unaccompanied by inflammatory cells and were present in both the mid-stromal and subepithelial levels. 4. Discussion It is evident from these results that the perfusion of a homogenate of cornea1 epithelium into the stroma will produce vascularization. Although, this response does not require leukocytes, these cells do seem to play an amplifying role, as was noted in previous reports (Eliason, 1978; Sholley, et al., 1978). Problems have been encountered with in vivo testing systems for vascularization. Principal among these in rabbit models is the ease with which its cornea will vascularize. Almost any noxious insult will result in new vessel growth (Fromer and Klintworth, 1975; Cogan, 1962). It is likely that many of these causes act in a non-specific and indirect fashion by inciting an inflammatory reaction in the cornea which in turn produces vascularization (Fromer and Klintworth, 1975, 1976). The difficulty encountered in finding a non-reactive control solution points out this sensitivity of the system. Consequently, a positive response lends limited weight to the specificity of an agent. Nevertheless, it is critical for any potential vasostimulating substance to evoke vascularization in such a basic model before it can come under further scrutiny. In vitro assay systems using vascular endothelial cells can give
EPITHELIAL
ANGIOGENIC
ACTIVITY
F ‘IQ. 5. (a) Epithelium end anterior stroma above the tip of the perfusion tube in B leukopenic animal reef 4ving epitheliel homogenate after 72 hr. Arrow indicates tip of tube. x 125. (b) Invading vessel sprout animal receiving epithelial homogenate after 5 days. x 125. in a leukopenic
730
J. A. ELIASOS
valuable complementary data. However, the proliferative response elicited by a substance in culture may not necessarily translate into organized vascular growth. Thus, in vivo studies provide useful information. In this report, the minimal response found in three control corneas is statistically insignificant compared with the uniformly positive response to the homogenate group. The vascularization that occurred in the leukopenic animals argues against the homogenate producing an inflammatory response which then incites the vessel growth. The perfusion system simulates the continuous production of a vasostimulating factor. If it were to act indirectly, the only available mediators would be native epithelium, endothelium or keratocytes; the more likely leukocyte source having been eliminated. This model does not exclude a role for the keratocytes or endothelium which may be recruited to contribute to the angiogenic signal. However, in the previous report (Eliason, 1978), neither of these cell types were necessary as both were absent during vascularization. The perfusion system supplies a known and constant output and thus permits reliable distinctions between homogenate concentrations. A threshold was found in the range of 5&20 yg ml-’ protein in the homogenate. This was attended by a greater than 50 y0 reduction in the incidence of vascularization. Cultured and freshly harvested cells produced a similar response. There is insufficient information from this study to draw any distinctions between the two sources of homogenate. The response to fresh epithelium suggests either a rapid synthesis or preformed active material. The histologic specimens demonstrated some thinning and irregularity of the epithelium in both control and homogenate corneas in the region of the perfusion tube. This effect was perhaps caused by the elevation produced in the cornea1 surface by the fluid input from the perfusion system and the mechanical action of the lids. The presence of the tube in the stroma was not significant since these epithelial changes were not observed in central areas of the cornea where proximal lengths of the tube were present but there was no perfusion. However, a direct effect of the homogenate on the epithelium cannot be ruled out. A very limited inflammatory reaction, insufficient to excite vascular growth, was present in control corneas. The stability of the angiogenic activity to heating suggests that it might be caused by a small-molecular-weight compound. While a large molecule can be heat stable, this is more commonly a property of small species, A small compound will give a greater concentration gradient when introduced from a point source in the stroma, this being determined by the rate of loss across the permeability barrier of the endothelium. If a gradient is required for vascularization as has been implicated by others (Maurice et al., 1966), then this, as well as its stability, would suggest that a smal1 rather than large molecular species is involved. The process of vascularization in the cornea is a complex one, perhaps with many components both intrinsic and extrinsic to the cornea participating. Recent work has pointed out the presence of endogenous agents produced by the cornea (Raju, Alessandri and Gullino, 1984). It is possible that the epithelium may be the source of one of these ingredients. It may act early in the process providing initial signals for vessel activation and growth. It is probable that the epithelium is not the sole source of a vasogenic factor in the cornea. Many such sources have been reported in other tissues. These range from neoplastic tissues, (Folkman, 1971; Folkman, Merier, Abernathy and Williams, 1971; Weiss, Brown, Kumar and Phillips, 1979; Fenseiau, Watt and Mello, 1981), retina (D’Amore, Glaser, Brunson and Fenselau, 1981; Kissun, etal., 1982),adipocytes, (Castellot, KarnovskyandSpiegelman, 1980)aorticendothelial
EPITHELIAL
ANGIOGENIC
ACTIVITY
731
cells, (Harris-Hacker, Gajdusek, Wight and Schwartz, 1983) macrophages (Knighton et al., 1983), lymphocytes (Kaminski, Nowack, Skopinska-Rozewska, Kaminska and Bern, 1981), polymorphonuclear cells (Fromer and Klintworth, 1976) to platelets (Knighton, Hunt, Thakral and Goodson, 1982). Wolf and Harrison (Wolf and Harrison, 1973) extracted a heat-labile, non-dialyzable and apparently polar substance from skin epidermis which incited the growth of vessels in the hamster cheek pouch. With such a diversity of tissues it seems possible that the production of an angiogenic factor may be a property shared by many tissues. ACKNOWLEDGMENTS This work was supported in part by NIH grants EYOO421 and EYO6051, and by The Pemberton Fund. I express my gratitude to Dr David M. Maurice for his invaluable criticism and timely encouragement. REFERENCES Ashton, N. and Cook, C. (1953). Mechanism of cornea1 vascularization. Br. J. Ophthdmol, 37, 193. Campbell, F. N. and Michaelson, I. C. (1948). Blood vessel formation in the cornea. Br. J. OphthalmoE. 33, 248. Castellot, J. J., Karnovsky, M. J. and Spiegelman, B. M. (1980). Potent stimulation of vascular endothelial cell growth by differentiated 3T3 adipocytes. Proc. Nut. Ad. Sci. U.S.A. 77, 6007. Cogan, D. G. (1949). Vascularization of the cornea. Arch. Ophthdmol. 92, 158. Cogan, D. G. (1962). Cornea1 vascularization. Invest. Ophthalmol. 1, 253. D’Amore, P. A., Glaser, B. M., Brunson, S. K. and Feneelau, A. H. (1981). Angiogenic activity from bovine retina: partial purification and characterization. Proc. Nat. Acad. Sci. U.S.A. 78, 3068. Ellason, J. A. (1978). Leukocytes and experimental cornea1 vascularization. Invest. Ophthdmol. 17, 1087. Eliason, J. A. and Maurice, D. M. (1980). An ocular perfusion system. Invest. Ophthdmd. Vis. sci. 19, 102. Fenselau, A., Watt, S. and Mello, R. J. (1981). Tumor angiogenic factor. Purification from Walker rat tumor. J. Biol.. Chem. 256, 9605. Folkman, J. (1971). Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285,1182. Folkman, J., Merier, E., Abernathy, C. and Williams, G. (1971). Isolation of a tumor factor responsible for angiogenesis. J. Exp. Med. 133, 275. Fromer, C. H. and Klintworth, G. K. (1975). An evaluation of the role of leukocytes in the pathogenesis of experimentally induced cornea1 vasoularization. I. Comparison of experimental models of cornea1 vascularization. Am. J. Path&. 79, 537. Fromer, C. H. and Klintworth, G. K. (1976). A n evaluation of the role of leukocytes in the pathogenesis of experimentally induced cornea1 vascularization. III. Studies related to the vasoproliferative capability of polymorphonuclear leukocytes and lymphocytes. Am. J. Pathol. 82, 157. Gospodarowicz, D., Greenburg, G. and Alvarado, J. (1979). Transplantation of cultured bovine cornea1 endothelial cells to rabbit cornea: Clinical implications for human studies. Proc. Nat. Ad. Sci. U.S.A. 76, 464. Harris-Hooker, S. A., Gajdusek, C. M., Wight, T. N. and Schwartz, S. M. (1983). Neovascular response induced by cultured aortic endothelial cells. J. Cell Physiol. 114, 302. Kaminski, M. J., Nowack, M., Skopinska-Rozewska, E., Kaminska, G. and Bern, W. (1981). Human peripheral blood T lymphocyte subpopulation isolated on the basis of their affinity for sheep blood cells differ in angiogenesis-inducing capability. C&n. Exp. Immurwl. 46, 327. Kissun, R. D., Hill, C. R., Garner, A., Phillips, P., Kumar, S. and Weiss, J. B. (1982). A low-molecular-weight angiogenic factor in cat retina. Br. J. Ophthdmol. 66, 165.
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Knighton, D. R., Hunt, T. K., Scheuensthul, H., Halliday, B. J., Werb, Z. and Banda, M. J. (1983). Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 221, 1283. Knighton, D. R., Hunt, T. K., Thakral, K. K. and Goodson, W. H. (1982). Role of platelets and fibrin in the healing sequence. An in vivo study of angiogenesis and collagen synthesis. Ann. Surg. 196, 379. Lowry, 0. H., Rosebrough, N. J., Parr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin Phenol reagent, J. Biol. Chem. 193, 265. Maurice, D. M., Zauberman, H. and Michaelson, I. C. (1966). The stimulus to neovascularization in the cornea. Exp. Eye Res. 5, 168. Newsome, D. A., Takasugi, M., Kenyon, K. R., Stark, W. F. and Opelz, G. (1974). Human cornea1 cells in vitro: morphology and histocompatibility (HL-A) antigens of pure cell populations. Invest. Ophdudmol. 13, 23. Raju, K. S., Alessandri, G. and Gullino, P. M. (1984). Characterization of a chemoattractant for endothelium induced by angiogenesis effecters. Cancer Res. 44, 1579. Sholley, M. M., Gimbrone, M. A. and Cotran, R. S. (1978). The effects of leukocyte depletion on cornea1 neovascularization. Lab. Invzvest.38, 32. Stocker, F. W., Eiring, A., Georgiade, M. A. and Georgiade, N. (1958). A tissue culture technique for growing cornea1 epithelial, stromal, and endothelial tissues separately. Am. J. Ophthalmol. 46, 294. Weiss, J. B., Brown, R. A., Kumar, S. and Phillips, P. (1979). An angiogenic factor isolated from tumors: A potent low-molecular-weight compound. Br. J. Cancer 40, 493. Wolf, J. E. and Harrison, R. G. (1973). Demonstration and characterization of an epidermal angiogenic factor. J. Invest. Dermatol. 61, 130.
Eye Res. (1985),
41, 721-732
Angiogenic
Activity
of the Cornea1
JOSEPH
Division
ELIASON
Stanford University Medical CA 94305, U.S.A.
of Ophthalmology, Stanford,
(Received 19 April
A.
Epithelium
1985 and accepted 5 September
Center,
1985, New York)
A homogenate of cornea1 epithelial cells was tested to determine if it could induce vascularization in the cornea. Both fresh and cultured cells were used &s sources of the homogenate which was evaluated in vivo using a self-contained system to perfuse it constantly into the cornea1 stroma. A vigorous growth of vessels resulted when the concentration of the homogenate exceeded a threshold of 20 yg per ml total protein. This capacity was not destroyed when the homogenate was heated. As a preliminary refinement of this response white blood cells were eliminated from the model with whole body X-irradiation. Vaacularization occurred in leukopenic animals but was less than that observed in normal animals. It is concluded that the epithelial homogenate is able to provoke cornea1 vascularization in the absence of leukocytes. Key war&: angiogenesis; cornea1 epithelium: vasostimulat,ing factor: leucopenia; cell culture.
1. Introduction The cornea, as a consequenceof its ready availability to observation is an excellent site to study the process of new blood vessel growth. Early investigators have presented convincing evidence that, in addition to edema, which separates the compact cornea1 lamella to permit vessel ingrowth mechanically (Cogan, 1949), a diffusible substance must be involved both to initiate and direct the growth of blood vessels in the cornea (Ashton and Cook, 1953; Campbell and Michaelson, 1948). Maurice, Zauberman and Michaelson (1966) concluded that, such a material must have a continuous cellular source in order to maintain a gradient throughout the duration of active vessel growth. In previously reported work a model wasdescribed in which a discrete cautery burn produced cornea1vascularization in animals depleted of white blood cells (Eliason, 1978). Others have reported similar results (Sholley, Gimbrone and Cotran, 1978). When the area of the injury was examined histologically in this model, at a time prior to the first appearance of vessels,the only cellular population that appeared viable was the cornea1epithelium. This was a regenerating layer. The leukopenia eliminated all leukocytes from the stroma and the cornea1endothelial and keratocyte populations had been destroyed by the cautery burn. It was concluded that the epithelium might be the source of a vasostimulating substance in this model. As an extension of this observation, this paper reports that the cornea1 epithelium is capable of inciting vascularization. 2. Methods Preparation
of howmgenate
A homogenateof rabbit cornea1epithelium waspreparedsimilarly from two sources.Fresh cells were obtained the cornea directly Please University
from with
the eyes of New a scalpel blade.
Zealand albino rabbits of varying age by scraping Care was taken to exclude conjunctival cells or
send correspondence to Joseph A. Eliason, Medical Center, Stanford, CA 94305, U.S.A.
0014-4835/85/120721+
12 $03.00/O
M.D..
Division
@ 1985 Academic
of Ophthalmology,
Press Inc. (London)
Stanford
Limited
.J. :I. E:I,IASOK
,“” WI
stroma. A second source of cells was a primary outgrowth in tissue culture obtained in a slightly modified manner from that described by Stocker, Eiring, Georgiade and Georgiade (1958). Corneas were excised from enucleated eyes within the limbus and the endothelial cell layer was removed with a cotton-tipped applicator stick (Gospodarowicz. Greenburg and Alvarado, 1979). This cornea1 button was then cut into four pie-shaped wedges and these were placed in a 60 mm tissue culture dish. It was found that a denuded Descemrt’s membrane both facilitated adhesion of the tissue pieces to the bottom of the dish and eliminated any contamination of the culture with cornea1 endothelial cells. Incubation was at 37 ‘C in a 5 y0 CO,-air atmosphere. The culture medium was TC 199 supplemented with 20’4 newborn calf serum (Microbiological Associates). At 4 days the tissue wedges were removed from the dishes leaving behind a monolayer of migrating epithelium which continued to spread and cover the surface in 7-9 days. Contamination by keratocytes was avoided since these ce!ls require about 7 days for their slower migration to bring them onto the surface of a culture dish from an explant (Newsome, Tagasugi, Kenyon, Stark and Opelz, 1974). Epithelial cells and keratocytes present distinctly different morphology in cultures. Phase-contrast microscopy was used to check for contamination by other cell types. Cells were harvested by first removing the medium, washing three times with cold saline and then scraping the dish surface with a rubber policeman. The suspension of cells obtained from culture or fresh from the eye was homogenized in a Potter-Elvejheim tissue grinder, centrifuged for 20 min at 1000 g, the sediment discarded and the supernatant then passed through a 922 pm filter to remove particulate material. The solvent used was saline prepared as a 99 “6 solution in double-distilled and de-ionized water. When indicated, the homogenate was heated at 80 “C for 10 min followed by repeat centrifugation and filtration. A total protein level in the homogenate was determined using the Lowry assay with a standard of human albumin-globulin (Sigma Chemical Co.) (Lowry, Rosebrough, Farr and Randall, 1951). Prepared homogenate was stored in glass at 4 “C and used within 7 days. In vivo
assay
system
The perfusion system has been previously described (Eliason and Maurice, 1980). In brief, an osmotic pump (Alzet, Alza Pharmaceuticals) was employed to perfuse continuously a solution into the cornea1 stroma through an implanted cannula. The pump was located subcutaneously in the scalp. A length of polyethylene tubing, heat-drawn to a fine tip (3G-50 pm o.d.), extended from the pump along a subcutaneous then subconjunctival path to a point at the superior limbus. It then entered the cornea1 stroma and continued to a point 3 mm from the inferior limbal vascular arcade. The pump, with its reservoir, will maintain a constant output of 1 yl hr-’ over a 7-8 day period. In order to monitor this function, sodium fluorescein was added to all solutions used (final concentration of 905 %). The presence of fluorescence as well as localized edema surrounding the tip of the cannula confirmed the operation of the system. New Zealand albino rabbits of 2-3 kg were used. Systems were implanted bilaterally, in most but not all cases, using a control solution in one eye and a test solution in the other. Leukopenia was produced as previously described (Eliason, 1978). In brief, X-irradiation was delivered with a Phillips 250 kV unit with the eyes shielded by 92 cm of lead and half of the dose given to each side of the animal. It was measured as a midline-absorbed dose. A total of 1500 rad was given in two doses separated by 48 hr. Daily white blood cell counts were measured in a hemocytometer chamber using venous blood obtained from the ear. A maximum level for the peripheral white count was set at 200. This selection was based upon previous work with this leukopenic model (Eliason, 1978). Serial sections on the animals in that study confirmed the absence of any infiltrating leukocytes in the stroma at 24 and 48 hr following the onset of the stimulus. Generally, animals reached a white count of 200 or less 3 days following the last dose of radiation at which time the implantation procedure was performed. Animals that failed to drop to this level were not used in the study. Tissue prepara!ion Animals were killed with a barbiturate overdose. Eyes were enucleated and fixed in 10 y0 buffered formaldehyde. After fixation, the cornea, with a surrounding rim of sclera, was
EPITHELTAL
ANGIOGEKIC
ACTIVITY
carefully excised. Cuts were made immediately outside the to produce a rectangular section of cornea which included central cornea. The tissue was imbedded in paraffin and long axis of the specimen (i.e. radially with respect to the were cut in a serial fashion from each specimen. Sections and eosin, dehydrated and mounted in permount. Measurements
and
723
region of limbal vascular ingrowth the limbus at each end and the 8,um sections were out along the cornea). A total of 40-M) sections were then stained in hematoxylin
observations
Changes in the cornea were observed with a Haag-Streit model 300 slit-lamp and documented with photographs taken through the slit-lamp. Vasoularization was deemed present if any new blood vessels, regardless of size, were noted under high magnification. Two methods were used for quantitative evaluation. Measurements were taken from the photographs and from the histologic sections. Photographs were projected and on the image three points were marked: two at the circumferential extent of vascular invtlsion at the limbus and one at the most central extent of invasion. In control corneas lacking vascular invasion, an area of vascular engorgement was usually present. Points were positioned at the extent of this area (Fig. 1). Using these three points a triangle was constructed and an area calculated
FIG. I. Slit-lamp photograph of cornea in a leukopenic animal receiving epithelial homogenate after 5 days. Arrow indicates tip of tube in the stroma and dots demonstrate placement of points to measure area and extent of vescularization.
to include the triangle plus the section of the cornea1 circle marked by its base. Measurements were standardized by using a photographed and projected millimeter scale. On the histologic sections the distance of vascular invasion into the cornea was determined by measuring from the most central vascular structure on each section to the junction of cornea1 and conjunctival epithelium. An eyepiece graticule was used with 100x magnification. The junction was determined both by the appearance of subconjunctival connective tissue rend by the onset of a variable thickness and irregular basal layer in the conjunctival epithelium. The measurements from the individual sections were averaged to arrive at a mean value for each eye.
724
.l. A. ELIASOK
3. Results After testing several potential control solutions in the perfusion system, 0.9 y0 saline was settled upon. Several trials with TC 199 with and without serum added and Eagle’s minimal essential medium without added serum invariably produced a vigorous inflammatory reaction in the cornea with a dense white cell infiltrate and vascularization. Even when autologous serum was used, a modest vascular growth resulted. Phosphate buffered saline (Xliason and Maurice, 1980) led to a modest infiltrate and vessel growth only in some cases. Saline (0*9O/“) with or without potassium chloride rarely excited any vascularization. Mean values of soluble protein in the homogenate were 280 ,ug per dish of cultured cells and 490 pg per cornea for fresh epithelium. For these experiments, non-heated homogenate solutions were adjusted to approximately 300 ,ug cm-” total protein. Heat-precipitating the homogenate resulted in a loss of approximately 90% of the original soluble protein. Heated homogenate solutions were adjusted to approximately 60 ,ug crnp3 unless otherwise indicated. At first, 42 eyes of 21 normal animals were used. Saline was perfused in 21 control eyes, seven eyes were perfused with homogenate from fresh epithelium and 14 eyes received homogenate from cultured cells. In one eye of the latter group the perfusion system failed to function and it was eliminated from the study. The results are indicated in Table I based upon slit-lamp observations over the &day period that the TABLE
1
Presence and absence of vessel growth in corneas perfused with saline control or a homogenate of cornea1 epithelium indicated as number of eyes for each group. Vascularization
Controls Fresh epithelium Cultured epithelium Totals Chi-squared
value
as a 2 x 2 table
is 237,
Yes
No
Totals
3 6 13 22
18 1 0 19
21 7 13
hence P < OGOl,
perfusion system operated. As indicated above, the appearance of any new vessels regardless of size was sufficient for vascularization to be judged present. Vasrularization occurred in three of 21 control eyes, six of seven eyes perfused with a homogenate from fresh cells and all eyes perfused with a homogenate from cultured cells. When the results of both of the homogenate groups are combined, chi-squared analysis employing the Yates correction factor indicates that the observed differences are statistically significant at a level of P < O*OOi. It is important to note that in the three control eyes showing vascularization, vessel invasion was estimated to extend less than 1 mm into the cornea. All of the eyes perfused with homogenate and showing vascularization, save one, demonstrated more extensive growth than this minimal amount. The histology of control corneas (Figs. 2, 3) showed edematous disruption of the collagen layers surrounding the tip of the tube. The overlying epithelium was slightly irregular and thinned but not disorganized. Mononuclear cells were present along the
EPITHELIAL
Fm. animal
ANGIOGENIC
ACTIVITY
2. Epithelium and anterior stroma immediately above the tip of the perfusion tube receiving normal saline after 7 days. Arrows indicate region of tube tip. x 63.
725
in a control
FIG. 3. (a) Stroma anterior to tube tip in a control animal receiving saline after 7 days. Note infiltration of inflammatory cells (arrows). The level of the tube is at the bottom of the photomicrograph. x 100. (b) Stroma posterior to the tube tip bearing few infiltrating cells. The tube level is near the top of the photomicrograph. x 100.
FIG. 4. (a) Epithelium and anterior stroma above the tip of the perfusion tube in an animal receiving an epithelial homogenate after 7 days. Arrows indicate region of tube tip. x 50. (b) Stroma anterior to the tube showing vascular invasion and inflammatory infiltration. Arrows indicate vessels. x 100. of the perfusion tube with some inflammatory cells in the surrounding stroma at the level of the tube and anterior to it. Infiltrating cells were rarely found in the stroma posterior to the level of the tube. In corneas perfused with homogenate (Fig. 4) there was a dense infiltrate in the region of the tube with scattered mononuclear and polymorphonuclear cells present throughout the stroma from the tube tip to the limbus. This infiltrate was. as in the controls, most prominent in the stroma anterior tract
EPITHELIAL
ANGIOGEKI(’
ACTIVITY
727
to the level of the tube. New vessels were present in the anterior stroma accompanied by collections of inflammatory cells and hemorrhage. The epithelium was again irregular and thinned as in the controls. Non-heated homogenate (nine eyes) was compared with heated homogenate (12 eyes) in 11 normal animals (a perforation of the anterior chamber occurred in one eye that was excluded from the study). The results are presented in Table II and expressed as mean areas calculated from slit-lamp photographs on successive days following implantation. The values for heated homogenate are greater than those for non-heated material. However, the observed differences are not statistically significant. Thus heated material retains its ability to incite vascularization. TABLE
Comparison
of the vascular
II
response to heated and non-heated Days following 3
Heated homogenate Non-heated homogenate
0.53 * 054 1.03kO.67
Area of vaacularization as calculated from photographs Mean values+s.~. are given. Measured area from control on given days are not statistically significant.
2.ol3+0.93 1.69+@6
corneas
homogenate,
implantation 5
on days
epithelial
8 351f1.32 304 f 1.62
following implantation as indicated. was O-65+028. Observed differences
Heated homogenate from cultured cells was lyophilyzed and then reconstituted in varying concentrations in saline: Solutions were perfused in seven eyes with protein concentrations from 50-1500 pg ml-l. Vascularization occurred in all eyes without any observable difference in the intensity of the vesselgrowth. Minor variations in the density of the vascular responsesappeared more to be related to individual variability in responseamong the test animals than the concentration of the solutions used. A threshold was encountered at 20 pugml- ‘. Two out of three eyes perfused with a solution at this concentration failed to show any vessel growth. In six animals, leukopenia was induced with radiation as noted above. In each animal, one eye was perfused with non-heated homogenate and the fellow eye with saline. Here, as in the normal animals, the first visible growth of new vessels commenced at about 72 hr after implantation of the perfusion system. One animal died on the fourth day and four others either died or were killed on the fifth day after implantation. A sixth animal died on the third day. It was excluded from vessel measurementsbut sections of the cornea are included here to demonstrate the lack of an inflammatory response.One pump system in a control eye failed to operate and was also excluded. Fig. 1 is a slit-lamp photograph of a leukopenic animal with vascular growth. Mean values for the most central vascular structure measuredfrom serial sectionsin the control and homogenate perfused corneasare presented in Table III. Using Student’s t test, the difference between these means is statistically significant with P c O@Ol. Measurementsof the area vascularized are presented in the same table. In order to make some comparison between leukopenic and normal animals, measurements from photographs were made for four of the leukopenic animals (photographs were not obtained for one of the leukopenic animals) and for
J. A. ELIASCIV
728
TABLE
III
Vascular invasion in leukopenic animals Vascular Distance Control Epithelial Distance photographs.
(mm)
038fO99 970 + @OS
homogenate
invasion Area
(mm-*)
065 + 028 1.28+019
of vascular invasion measured from serial sections of the cornea, and area calculated Mean values +s.D. are given. (P < 0901 for distance; P < 001 for area). TABLE
Area of vascuhrization
from
IV
in normal and leukopenic animals Area of vascularization (mm-*)
Normal WBC Leukopenic
2.08+_093 1.28&@19
count
Measurements were made from photographs an epithelial homogenate. Values are expressed
of normal and leukopenic as mean + S.D.
animals,
both
perfused
with
11 normal animals at 5 days after implantation. These values are indicated in Table IV; the difference of the means is not statistically significant. Histological examination revealed slight irregularity and thinning of the epithelium as noted in normal animals in both controls and homogenate perfused corneas (Fig. 5). The stroma was devoid of inflammatory cells, even immediately surrounding the perfusion tube. The keratocytes appeared normal. Invading vessel sprouts were unaccompanied by inflammatory cells and were present in both the mid-stromal and subepithelial levels. 4. Discussion It is evident from these results that the perfusion of a homogenate of cornea1 epithelium into the stroma will produce vascularization. Although, this response does not require leukocytes, these cells do seem to play an amplifying role, as was noted in previous reports (Eliason, 1978; Sholley, et al., 1978). Problems have been encountered with in vivo testing systems for vascularization. Principal among these in rabbit models is the ease with which its cornea will vascularize. Almost any noxious insult will result in new vessel growth (Fromer and Klintworth, 1975; Cogan, 1962). It is likely that many of these causes act in a non-specific and indirect fashion by inciting an inflammatory reaction in the cornea which in turn produces vascularization (Fromer and Klintworth, 1975, 1976). The difficulty encountered in finding a non-reactive control solution points out this sensitivity of the system. Consequently, a positive response lends limited weight to the specificity of an agent. Nevertheless, it is critical for any potential vasostimulating substance to evoke vascularization in such a basic model before it can come under further scrutiny. In vitro assay systems using vascular endothelial cells can give
EPITHELIAL
ANGIOGENIC
ACTIVITY
F ‘IQ. 5. (a) Epithelium end anterior stroma above the tip of the perfusion tube in B leukopenic animal reef 4ving epitheliel homogenate after 72 hr. Arrow indicates tip of tube. x 125. (b) Invading vessel sprout animal receiving epithelial homogenate after 5 days. x 125. in a leukopenic
730
J. A. ELIASOS
valuable complementary data. However, the proliferative response elicited by a substance in culture may not necessarily translate into organized vascular growth. Thus, in vivo studies provide useful information. In this report, the minimal response found in three control corneas is statistically insignificant compared with the uniformly positive response to the homogenate group. The vascularization that occurred in the leukopenic animals argues against the homogenate producing an inflammatory response which then incites the vessel growth. The perfusion system simulates the continuous production of a vasostimulating factor. If it were to act indirectly, the only available mediators would be native epithelium, endothelium or keratocytes; the more likely leukocyte source having been eliminated. This model does not exclude a role for the keratocytes or endothelium which may be recruited to contribute to the angiogenic signal. However, in the previous report (Eliason, 1978), neither of these cell types were necessary as both were absent during vascularization. The perfusion system supplies a known and constant output and thus permits reliable distinctions between homogenate concentrations. A threshold was found in the range of 5&20 yg ml-’ protein in the homogenate. This was attended by a greater than 50 y0 reduction in the incidence of vascularization. Cultured and freshly harvested cells produced a similar response. There is insufficient information from this study to draw any distinctions between the two sources of homogenate. The response to fresh epithelium suggests either a rapid synthesis or preformed active material. The histologic specimens demonstrated some thinning and irregularity of the epithelium in both control and homogenate corneas in the region of the perfusion tube. This effect was perhaps caused by the elevation produced in the cornea1 surface by the fluid input from the perfusion system and the mechanical action of the lids. The presence of the tube in the stroma was not significant since these epithelial changes were not observed in central areas of the cornea where proximal lengths of the tube were present but there was no perfusion. However, a direct effect of the homogenate on the epithelium cannot be ruled out. A very limited inflammatory reaction, insufficient to excite vascular growth, was present in control corneas. The stability of the angiogenic activity to heating suggests that it might be caused by a small-molecular-weight compound. While a large molecule can be heat stable, this is more commonly a property of small species, A small compound will give a greater concentration gradient when introduced from a point source in the stroma, this being determined by the rate of loss across the permeability barrier of the endothelium. If a gradient is required for vascularization as has been implicated by others (Maurice et al., 1966), then this, as well as its stability, would suggest that a smal1 rather than large molecular species is involved. The process of vascularization in the cornea is a complex one, perhaps with many components both intrinsic and extrinsic to the cornea participating. Recent work has pointed out the presence of endogenous agents produced by the cornea (Raju, Alessandri and Gullino, 1984). It is possible that the epithelium may be the source of one of these ingredients. It may act early in the process providing initial signals for vessel activation and growth. It is probable that the epithelium is not the sole source of a vasogenic factor in the cornea. Many such sources have been reported in other tissues. These range from neoplastic tissues, (Folkman, 1971; Folkman, Merier, Abernathy and Williams, 1971; Weiss, Brown, Kumar and Phillips, 1979; Fenseiau, Watt and Mello, 1981), retina (D’Amore, Glaser, Brunson and Fenselau, 1981; Kissun, etal., 1982),adipocytes, (Castellot, KarnovskyandSpiegelman, 1980)aorticendothelial
EPITHELIAL
ANGIOGENIC
ACTIVITY
731
cells, (Harris-Hacker, Gajdusek, Wight and Schwartz, 1983) macrophages (Knighton et al., 1983), lymphocytes (Kaminski, Nowack, Skopinska-Rozewska, Kaminska and Bern, 1981), polymorphonuclear cells (Fromer and Klintworth, 1976) to platelets (Knighton, Hunt, Thakral and Goodson, 1982). Wolf and Harrison (Wolf and Harrison, 1973) extracted a heat-labile, non-dialyzable and apparently polar substance from skin epidermis which incited the growth of vessels in the hamster cheek pouch. With such a diversity of tissues it seems possible that the production of an angiogenic factor may be a property shared by many tissues. ACKNOWLEDGMENTS This work was supported in part by NIH grants EYOO421 and EYO6051, and by The Pemberton Fund. I express my gratitude to Dr David M. Maurice for his invaluable criticism and timely encouragement. REFERENCES Ashton, N. and Cook, C. (1953). Mechanism of cornea1 vascularization. Br. J. Ophthdmol, 37, 193. Campbell, F. N. and Michaelson, I. C. (1948). Blood vessel formation in the cornea. Br. J. OphthalmoE. 33, 248. Castellot, J. J., Karnovsky, M. J. and Spiegelman, B. M. (1980). Potent stimulation of vascular endothelial cell growth by differentiated 3T3 adipocytes. Proc. Nut. Ad. Sci. U.S.A. 77, 6007. Cogan, D. G. (1949). Vascularization of the cornea. Arch. Ophthdmol. 92, 158. Cogan, D. G. (1962). Cornea1 vascularization. Invest. Ophthalmol. 1, 253. D’Amore, P. A., Glaser, B. M., Brunson, S. K. and Feneelau, A. H. (1981). Angiogenic activity from bovine retina: partial purification and characterization. Proc. Nat. Acad. Sci. U.S.A. 78, 3068. Ellason, J. A. (1978). Leukocytes and experimental cornea1 vascularization. Invest. Ophthdmol. 17, 1087. Eliason, J. A. and Maurice, D. M. (1980). An ocular perfusion system. Invest. Ophthdmd. Vis. sci. 19, 102. Fenselau, A., Watt, S. and Mello, R. J. (1981). Tumor angiogenic factor. Purification from Walker rat tumor. J. Biol.. Chem. 256, 9605. Folkman, J. (1971). Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285,1182. Folkman, J., Merier, E., Abernathy, C. and Williams, G. (1971). Isolation of a tumor factor responsible for angiogenesis. J. Exp. Med. 133, 275. Fromer, C. H. and Klintworth, G. K. (1975). An evaluation of the role of leukocytes in the pathogenesis of experimentally induced cornea1 vasoularization. I. Comparison of experimental models of cornea1 vascularization. Am. J. Path&. 79, 537. Fromer, C. H. and Klintworth, G. K. (1976). A n evaluation of the role of leukocytes in the pathogenesis of experimentally induced cornea1 vascularization. III. Studies related to the vasoproliferative capability of polymorphonuclear leukocytes and lymphocytes. Am. J. Pathol. 82, 157. Gospodarowicz, D., Greenburg, G. and Alvarado, J. (1979). Transplantation of cultured bovine cornea1 endothelial cells to rabbit cornea: Clinical implications for human studies. Proc. Nat. Ad. Sci. U.S.A. 76, 464. Harris-Hooker, S. A., Gajdusek, C. M., Wight, T. N. and Schwartz, S. M. (1983). Neovascular response induced by cultured aortic endothelial cells. J. Cell Physiol. 114, 302. Kaminski, M. J., Nowack, M., Skopinska-Rozewska, E., Kaminska, G. and Bern, W. (1981). Human peripheral blood T lymphocyte subpopulation isolated on the basis of their affinity for sheep blood cells differ in angiogenesis-inducing capability. C&n. Exp. Immurwl. 46, 327. Kissun, R. D., Hill, C. R., Garner, A., Phillips, P., Kumar, S. and Weiss, J. B. (1982). A low-molecular-weight angiogenic factor in cat retina. Br. J. Ophthdmol. 66, 165.
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Knighton, D. R., Hunt, T. K., Scheuensthul, H., Halliday, B. J., Werb, Z. and Banda, M. J. (1983). Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 221, 1283. Knighton, D. R., Hunt, T. K., Thakral, K. K. and Goodson, W. H. (1982). Role of platelets and fibrin in the healing sequence. An in vivo study of angiogenesis and collagen synthesis. Ann. Surg. 196, 379. Lowry, 0. H., Rosebrough, N. J., Parr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin Phenol reagent, J. Biol. Chem. 193, 265. Maurice, D. M., Zauberman, H. and Michaelson, I. C. (1966). The stimulus to neovascularization in the cornea. Exp. Eye Res. 5, 168. Newsome, D. A., Takasugi, M., Kenyon, K. R., Stark, W. F. and Opelz, G. (1974). Human cornea1 cells in vitro: morphology and histocompatibility (HL-A) antigens of pure cell populations. Invest. Ophdudmol. 13, 23. Raju, K. S., Alessandri, G. and Gullino, P. M. (1984). Characterization of a chemoattractant for endothelium induced by angiogenesis effecters. Cancer Res. 44, 1579. Sholley, M. M., Gimbrone, M. A. and Cotran, R. S. (1978). The effects of leukocyte depletion on cornea1 neovascularization. Lab. Invzvest.38, 32. Stocker, F. W., Eiring, A., Georgiade, M. A. and Georgiade, N. (1958). A tissue culture technique for growing cornea1 epithelial, stromal, and endothelial tissues separately. Am. J. Ophthalmol. 46, 294. Weiss, J. B., Brown, R. A., Kumar, S. and Phillips, P. (1979). An angiogenic factor isolated from tumors: A potent low-molecular-weight compound. Br. J. Cancer 40, 493. Wolf, J. E. and Harrison, R. G. (1973). Demonstration and characterization of an epidermal angiogenic factor. J. Invest. Dermatol. 61, 130.