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Neovasculogenesis. Triggering factors and possible mechanisms
SURVEY OF OPHTHALMOLOGY
CURRENT
VOLUME 24
l
NUMBER 3
l
NOVEMBER-DECEMBER
1979
RESEARCH
EDWARD COTLIER, EDITOR
Neovasculogenesis.
Triggering
Factors
and Possible Mechanisms
DAVID BEN EZRA, M.D., PH.D. Clinical Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, and The Department of Ophthalmology, Hadassah Hebrew University Hospital, Jerusalem, Israel Abstract. The possible regulating mechanism(s)
of neovascularization are discussed. Experimental data supporting the view that ocular neovascularization might be triggered by a fundamental metabolic mechanism are presented. Based on his own data and scattered evidence from the literature, the author suggests a model for a possible regulating mechanism of the neovascular process in which the prostaglandins fulfill the role of neovascular mediating substances. Preliminary results demonstrating that indomethacin limits the extent of the neovascular process support this concept and may have a therapeutic implication. Further studies are in progress in order to better understand the pivotal role fulfilled by the prostaglandins in neovasculogenesis.
(Sun Ophthalmol 24:167-176, 1979) . chemoattractants . growth factors . indomethacin Key words. keratocytes . leukocytes . mitogens . neovascular attracting factor . neovasculogenesis . prostaglandins
T
proliferation of he pathological blood vessels in the eye is one of the most detrimental processes for the visual function. Although intensively investigated for more than half a century, the growth of ocular blood vessels in health and disease remains one of the most enigmatic ocular phenomena. Recent advances in photocoagulation therapy’l and vitreous surgerys8 may have some beneficial effects and reduce the morbidity due to proliferative retinopathy. 167
However, these effects are very limited and, at best, of short duration. A better understanding of the fundamental processes leading to the triggering of neovasculogenesis would eventually provide the clinical ophthalmologist with a possible mode of therapy. therapy. As with any other biological tissue, the modulation of blood vessel growth (or involution) is governed by the net effect of inhibiting versus stimulating factor(s)’ accumulating
168
SurvOphthalmol
24
(3)
November-December
locally. The inhibiting factor(s) may be due to intrinsic characteristics of the involved tissue. However, a possible systemic origin is conceivable. The stimulating factor(s), on the other hand, should be considered to be of strictly local origin; they are produced following changes in the metabolism of the local cells. This concept, which in my opinion is the essence of the neovasculogenic mechanism, has been forwarded by Professor I. C. Michaelson in his treatise of the retinal circulation in man and animalszB This review provides an overview of the neovascular activating (or attracting) factor(s) [NAF], with particular emphasis on possible regulating mechanisms.
Background The various experimental designs of cornea1 vascularization have been reviewed recently by Klintworth,21 while a thoughtful discussion on ocular neovascularization has been presented by Henkind in his Krill Memorial Lecture.18 Therefore, I will limit the present discussion to the three major “schools of thought” regarding the characteristics of the neovascular stimulus. COMPACTNESS
OF THE CORNEAL
AS A FACTOR
FOR NEOVASCULARIZATION
STROMA
(COGAN)
Studying the sequence of events following the induction of small cornea1 lesions in approximately 200 rabbit eyes, Cogan” observed the following: a) Swelling of the cornea was the most constant phenomenon closely associated with or preceeding vascular changes. b) Hemorrhagic extravasates had no influence on neovasculogenesis as blood injected intracorneally did not induce any constant vascularization of the cornea. c) The role of “toxins” appeared unlikely since extracts of vascularized corneas had effects similar to those obtained from nonvascularized corneas. X-FACTOR
8EN EZRA
1979
OF NEOVASCULARIZATION
(MICHAELSON)
The proliferation of blood vessels is closely associated with the metabolism of the tissue. In certain conditions due to local environmental changes, basic cell metabolites accumulate and trigger the vessel growth.lE This possibility has been reinforced by
clinical and experimental observations.10~16~a’-2s These can be summarized as follows: a) New blood vessels are attracted toward the site of highest potential concentration of the X-factor. These form a triangular shaped tuft of new vessels with a large base at the origin. b) The sprouting of new blood vessels is always from the side of the pre-existing vessel that faces the origin of the X-factor. c) The degree of vessel proliferation is closely associated with the X-factor concentration. d) On cessation of the factor production, there is an involution of the blood vessels. OXYGEN
THEORY
(ASHTON,
PATZ)
OF RELATIVE
HYPOXIA
An early phase of vasoconstriction is followed by vessel proliferation under conditions of lower oxygen tension.‘*‘*” This theory provided an adequate explanation for the clinical and experimental phenomena observed in retrolental fibroplasia. Considering the activation of blood vessel growth, one cannot stop wondering about the avascularity of some tissues (cartilage, nail, vitreous, lens, cornea). The easiest explanation for this situation is to postulate on the presence of local inhibitor(s). Indeed, an inhibitor of neovascularization has been demonstrated in cartilageg*zs,a7 and in vitreous.** During histologic observation of new vessel growth into the cornea after the insertion of implants sequestering various mitogens,*’ I was struck by the fact that the leukocyte and new vessel invasion of the cornea was, at least initially, always confined to the upper half or two-thirds of the cornea (Fig, 1). The lower third to half of the cornea nearer to the anterior chamber remained free of cellular invasion unless the reaction was so intense that a uveitis was induced. This pattern of invasion was attributed at first to the mid-stromal position of the implant and to the possible greater edema in the upper half of the cornea facilitating the infiltration. Therefore, experiments were performed in which the implant was inserted and placed “Mitogens: These are plant extracts (Con A, PHA, PWM) or products from bacterial origin (LPS, staphylococcal filtrate) that have a nonspecific mitogenic effect on lymphocytes in vitro.
CURRENT
RESEARCH
near the Descemet’s membrane (Fig. 2). Under these conditions, the same pattern in which the lower part of the cornea, although edematous, remains free of cellular or vessel invasion is observed. The most appealing explanation for these observations is the postulation of a circulating inhibitor present
169
in relatively high concentrations in the aqueous humor. This could be in line with other observations concerning the presence of an inhibitor of lymphocyte blast transformation in primary aqueous humor of rabbit eyes8 From these data, it is conceivable to assume that, once neovascularization is
FIG 1. Implants at the mid-stromal level. a and b: The leukocyte and new vessel infiltrations are limited to the upper two-thirds of the cornea. c and e show the reaction in the upper third of the cornea; d and f illustrate the histology of the lower third of the cornea nearer to the anterior chamber. Note that although edematous, the lower third remains free of neovascularization. Ep = epithellum; AC = anterior chamber; Imp = implant, V = new vessel; Mn = monuclear cell; Plm = polymorphonuclear cells; K = keratocytes; Ds = Descemet’s membrane. (Hematoxylin and eosin. a and b, X 20; c-f, X 60)
170
Surv Ophtholmol
24 (3) November-December
1979
triggered, its extent is regulated via the continuous local production of attracting factor(s) on one hand and the presence of local and/or systemic inhibitor(s) on the other hand.
Possible Activating Mechanism(s) The general consensus, at present, among researchers in the field is that Michaelson’s
BEN EZRA
postulation concerning the release of an Xfactor best explains the neovascular phenomena. However, the characteristics of that factor(s) remain an enigma. In an attempt to correlate the relative hypoxia with the blood vessel proliferation, experiments testing the potential vasoproliferative ability of lactic acid were carried out.‘* A more sophisticated approach to the problem was reported by Zauberman et al.‘” Using a plastic tube in-
FIG. 2. Implants positioned near the Descemet’s membrane. The leukocyte and new vessel infiltration is slightly deeper than in cases shown in Fig. 1. However, the pattern of invasion is similar. The lower third of the cornea nearer to the anterior chamber remains free of new vessels (a and b). Few leukocytes are observed near the Descemet’s membrane in these cases (d and f). (Hematoxylin and eosin. a and b, X 20; c-f, x 60)
171
serted into the rabbit cornea as a slow release device, these investigators found that biogenic amines (acetylcholine, histamine, serotonin and bradykinin) induced a neovascularization in approximately 35% of the cases. More recently, it was shown that tumors produce and release an angiogenic factor (TAF).*’ Finally, the cornea1 vasculogenic capacity of a “non-sedimentable” fraction obtained from a sonicate of peritoneal cell exudates has been reported.” In an attempt to elucidate the role of lymphoid cell products in neovasculogenesis, an autologous system was used.3,7 Leukocytes obtained from rabbit peripheral blood were stimulated in vitro by various mitogens. The culture supernatants and the activated leukocytes were injected back to autologous* and allogeneic** corneas. Supernatants and leukocytes activated by concanavalin A (Con A) or lipopolysaccharide (LPS) showed a strong NAF activity in both the autologous and the allogeneic systems. Cultures stimulated by phytohemagglutinin (PHA) or pokeweed mitogen (PWM) were seldom weakly active, while nonactivated cultures had no detectable NAF. It is interesting that supernatants from rabbit keratocyte cultures had no neovasculogenic activity alone or when supplemented with mitogens.s*7 It is not improbable, however, that keratocytes under various metabolic conditions would demonstrate some NAF activity. From these data, it was postulated that the various experimental means for the triggering of new blood vessel proliferation might act through the release of neovascular mediating substances (NMS). The NMS would influence local cells in the target organ or trigger some cells involved in the defense mechanism (lymphoid) to produce NAF, or else NMS might directly affect the vessel endothelial cells (Fig. 3). These postulations were reinforced by the recent finding of Polverini et aLsa that activated macrophages were able to release a neovasculogenic substance. The degree of positive neovasculogenesis *Autologous system: Activated cells and supernatants are tested on corneas of same animal that donated the cells. **Allogeneic system: Activated cells and supernatants are tested on corneas of another animal of the same species, e.g. cells obtained from rabbit (a) are tested on corneas of rabbit (b), (c), etc.
TABLE 1 Schematic Representation of Active Neovasculogenic Substances
System
Neovasculogenesis*
Activated leukocytes LPS Con A PHA
++++ ++ _**
Prostaglandins PGE, PGE, PGF,(r PGF,a
+++ + + -
Growth factors FGF EGF NGF
f + -
*Number of + denotes intensity of neovasculogenesis under optimal conditions. **Denotes no neovasculogenesis.
observed under various conditions is schematically illustrated in Table 1. Cyclic AMP and cyclic GMP* and dibutyryl cyclic AMP did not induce any noticeable NAF activity. All implants sequestering these compounds remained inactive, as did all cont’tils of “empty” implants of Elvax-40. Prostaglandin El** was the strongest and most consistent inducer of neovasculogenesis. PGE, and to some extent PGF,, showed a moderate NAF activity. The formylated peptides, which are very potent in vitro chemoattractantsas,as and induce only a slight infiltration of polymorphonuclears in viva,* had no NAF activity. The purified growth factors (GF)***
*Cyclic nucleotides: Cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP). **Prostaglandins (PG): Series of hormones that are designated as A, D, E, F, etc. In each series there are few compounds that are labeled numerically, e.g. PGE,, PGb, etc. ***Growth Factors (GF): Polypeptides that demonstrate a growth promoting potential similar to or exceeding that observed with the addition of serum (or plasma) in cell cultures. The biological role of these peptides in vivo is still unclear. They are extracted from various organs.
172
Surv Ophthalmol
24 (3) November-December
\ NORMAL
A
BEN EZRA
1979
TISSUE
(
tttt
Hypoxia, Cautery, Chemical, Mechanical
// / / _--L-
-.-__. \ \ - _.... - --)
Neovascular
Mediating
Endothel-Mural
Substances
* - --
---1,
%
Cells
FIG. 3. Possible mechanism(s) for the induction of neovasculogenesis.
[epidermal growth factor (EGF), tibroblast growth factor (FGF) and nerve growth factor (NGF) ] varied in their capacity to induce neovasculogenesis. EGF induced a mild NAF activity in concentration of 10 pg per implant and a stronger neovasculogenesis at 20 pg per implant. FGF had similar effects, but was a milder stimulant than EGF. In connection with the neovasculogenesis induced by 10 or 20 pg EGF, two histological observations are noteworthy: The blood vessel proliferation is accompanied by a very mild leukocytic infiltration and a marked epithelial proliferation (Fig. 4). NGF in concentrations of one to 10 units per implant had no NAF activity.
Discussion From the presented data and a gathering of reported facts concerning the role of prostaglandins as intercellular messengers, it is very tempting to speculate that Michaelson’s X-factor, TAF, NAF or any
neovasculogenic activity is mediated through local release of prostaglandins. Moreover, various prostaglandins may have antagonistic effects and thus, may provide a possible regulatory influence on the neovascular process. In some preliminary studies, we have observed that PGF1, may have an inhibitory effect on the NAF activity of PGE,. Therefore, the possibility that under various different prostaglandins circumstances, produced locally may regulate the NAF activity is very attractive. In this regard, it is interesting that growing tumor cells as well as activated leukocytes and macrophages synthesize and release prostaglandins which are used as intercellular messengers.16J8~20~22 Also, platelets from patients with diabetes synthesize and release more prostaglandins than platelets from nondiabetics in the platelet aggregation reaction.” The influence of PGE, on the sickling of erythrocytes in a hypoxic state has also been reported.s’ From the scattered evidence, it is very tempting to con-
FIG. 4. Effect of epidermal growth factor (EGF). a: Implant sequestering 10 pg EGF. Note the mild vessel proliferation (V), the little leukocyte infiltration and the marked epithelial hyperplasia. b: Higher magnification of the site of vessel infiltration (V). c: Higher magnification of epithelium above the implant. d-f; Similar sites from cornea with empty implant of Elvax-40 (control). (Hematoxylin and eosin. a and d, X 20; b and e, X 60; c and f, X 80)
ceive that the ocular neovascularization phenomena are all activated through a common pathway. The vascular proliferation, as observed in retrolental fibroplasia, diabetic retinopathy, sickle cell retinopathy, central retinal vein occlusion and chronic inflammatory or immune reactions, can be
rationalized on the basis of the local production of prostaglandins induced by the various stimuli (Fig. 5). The above postulation is also attractive in view of its possible therapeutic implications. In ongoing experiments, we are studying the effect of indomethacin on the neovascular
174
Surv Ophthalmol
1’ ,-------)
24 (3) November-December
BEN EZRA
1979
NeovascuC Mediating Substances+-----_t, (NW
InflamZiG3n
Met&
ibeases
-m-.-.-m
+ 1 Endothel-Mural Cells 1
FIG. 5. Schematic mechanism(s) of neovasculoaenesis neovascular mediating substances ‘(NMS). process. Preliminary results show that indomethacin, although it does not completely abolish the NAF activity of PGE, in any of the concentrations used, markedly limits the extent of the neovascular process (as represented best by the reduction of the neovascular surface). These data may be interpreted as an indication of the perpetuation of the neovascular process by further local synthesis of prostaglandins or other sensitive metabolites. Indomethacin probably has no effect on the prostaglandin sequestered in the implant, but limits the extent of neovascularization by inhibiting the local synthesis and release of newly formed mediators. At the present stage of our investigation, it is very difficult to totally exclude the possible involvement of metabolites other than the stable prostaglandins in the neovascular process. Also, the role of precursors of prostaglandins and other unstable intermediate compounds must be considered. Nevertheless, a carefully monitored treatment with inhibitors of prostaglandin syn-
postulating the role of prostaglandins
as main
and/or the prevention of ocular neovasculogenesis is warranted even at these early stages of our knowledge of the phenomenon. At least in proliferative retinopathies, the use of adequate doses of salicylates or indomethacin might be a logical and, if carefully monitored, a “harmless” mode of treatment. thesis for the limitation
Acknowledgments I am thankful to Ms. Sammy Bornstein for her technical assistance and her skill in the preparation of the histology slides, to Ms. Joan Lee and Ms. Iris Kivitz for their persevering typing and retyping, and to Dr. Elmer Ballintine for his discussions and encouragement. I am also indebted to Mr. James Ingram for his patient and skillful assistance with the experimental animals and to Ms. Louise Smith for her pre- and post-operative care of the rabbits.
1.
References Ashton N: Oxygen and the growth and development of retinal vessels. Am J Ophthalmol 62:412-435, 1966
CURRENT
RESEARCH
2. Ashton N: Retinal vascularization in health and disease. Am J Ophthalmol 44:7-17, 1957 3. BenEzra D: Mediators of immunological reactions and neovascularization. The Jerusalem Conference on Impaired Vision in Childhood, May, 1977 4. BenEzra D: Mediators of immunological reactions. Function as inducers of neovascularization. Metabolic Ophthalmol (in press) 5. BenEzra D: A microculture technique for the evaluation of cornea1 cell metabolism in vitro. Invest Ophthalmol Vis Sci 16:893, 1977 6. BenEzra D: Neovasculogenic ability of prostaglandins, growth factors and synthetic chemoattractants. Am J Ophthalmol (in press) 7. BenEzra D: Possible mediators of vasculogenesis by products of immune reactions. The Second International Symposium on Ocular Immunology and Immunopathology. Paris, Masson and Company (in press) 8. BenEzra D, Sachs U: Growth factors in aqueous humor of normal and inflamed eyes of rabbits. Invest Ophthalmol 13:868-870, 1974 9. Brem H, Folkman J: Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 141: 427-439, 1975 10. Campbell FW, Michaelson IC: Blood vessel formation in the cornea. Br J Ophthalmol 33~248-255, 1949 11. Cogan DG: Vascularization of the cornea: Its experimental induction by small lesions and a new theory of its pathogenesis. Arch Ophthalmol 41:406-416, 1949 12. The Diabetic Retinopathy Study Research Group: Preliminary report on effects of photocoagulation therapy. Am J Ophthalmol 81:383-396, 1976 13. Folkman J, Merler E, Abernathy C, Williams G: Isolation of a tumor factor responsible for angiogenesis. J Exp Med 133:275-288, 1971 14. Fromer CH, Klintworth GK: An evaluation of the role of leukocytes in the pathogenesis of experimentally induced cornea1 vascularization. Am J Pathol 82:157-168, 1976 15. Goodwin JS, Bankhurst AD, Messner RP: Suppression of human T-cell mitogenesis by prostaglandins. Existence of a prostaglandinproducing suppressor cell. J Exp Med 146:1719-1734, 1977 16. Henkind P: Ocular neovascularization. Am J Ophthalmol 85:287-301, 1978 17. Halushka PV, Weiser G, Chambers A, Colwell J: Synthesis of prostaglandin “E like” in diabetic and normal platelets. Adv ProstagI Thromb Res 2:853, 1976 18. Humes JL, Bonney RJ, Pelus L, et al; synthesise and release Macrophages prostaglandins in response to inflammatory stimuli. Nature 269:149-151, 1977 19. Imre G: Studies on the mechanism of retinal
175
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
neovascularization. Role of lactic acid. Br J Ophthalmol 48:75-82, 1964 Jaffee BM: Prostaglandins and cancer. An update. Prostaglandins 6:453-461, 1974 Klintworth GK: The cornea - structure and macromolecules in health and disease. Am J Pathol 89:718-808, 1977 Kurland JI, Bockman R: Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages. J Exp Med 147:952-957, 1978 Langer R, Brem H, Falterman K, et al: Isolation of a cartilage factor that inhibits tumor neovascularization. Science 193:70-9 1, 1976 Langer R, Folkman J: Polymers for the sustained release of proteins and other macromolecules. Nature 263:797-800, 1976 Maurice DM, Zauberman H, Michaelson IC: The stimulus to neovascularization in the cornea. Exp Eye Res 5:168-184, 1966 Michaelson IC: The mode of development of the retinal vessels and some observations of its significance in certain retinal diseases. Tram Ophthalmol Sot UK 68:137-180, 1948 Michaelson IC: Proliferation of limbal melanoblasts into the cornea in response to a cornea1 lesion. Br J Ophthalmol 36:657-665, 1952 Michaelson IC: Retinal Circulation in Man and Animals. Springfield, Charles C Thomas, 1954, pp 118-131 Michaelson IC: Unilateral budding in new vessel formation in the eye. Br J OphthaImol 40:36-39, 1956 Oppenheim JJ, Rosenstrich DL: Signals regulating in vitro activation of lymphocytes. Prog Allergy 20:65-194, 1976 Patz A: Oxygen studies in retrolental fibroplasia. IV. Clinical and experimental observations. Am J Ophthalmol 38:291-308, 1954 Preis I, Langer R, Brem H, et al: Inhibition of neovascularization by an extract derived from vitreous. Am J Ophthalmol 84:323-328, 1977 Polverini PJ, Cotran RS, Gimbrone MA, Unanue ER: Activated macrophages induce vascular proliferation. Nature 269:804-806, 1977 Rabinowitz I, Wolf PL, Berman S, et al: Prostaglandin E, and calcium in the sickling cycle of sickle cell erythrocytes. Adv Prostagl Thromb Res 2:883, 1976 Schiffman E, Corcoran B, Wahl SM: NFormylmethionyl peptides as chemoattractants for leucocytes. Proc Nat1 Acad Sci USA 72:1059-1062, 1975 Schiffman E, Showell HV, Corcoran BA, et al: The isolation and partial characterisation of neutrophil chemotactic factors from Esherichia Coli. J Immunol 114:1831-1837, 1975
176
37.
SW Ophthalmol 24 (3) November-December 1979
Sorgente N, Kuettner KE, Soble LW, Eisenstein R: The resistance of certain tissues to invasion. II. Evidence for extractable factors in cartilage which inhibit invasion by vascularized mesenchyme. Lab Invest 32:217-222, 1975 38. Triester G, Machemer R: Results of vitrectomy for rare proliferative and hemorrhagic diseases. Am J Ophthlmol 84:394-412, 1977 39. Waksman BH, Namba Y: On soluble mediators of immunologic regulation. Cell Immunol 21:161-176, 1976
MN lZRA
40. Zauberman H, Michaelson IC, Bergmann F, Maurice DM: Stimulation of neovascularization of the cornea by biogenic amines. Exp Eye Res 8:77-83, 1969
Reprint requests should be addressed to David BenEzra, M.D., Ph.D., Department of Ophthalmology, Hadassah Hebrew University Hospital, Kytiat Hadassah, Jerusalem, Israel.
CURRENT
VOLUME 24
l
NUMBER 3
l
NOVEMBER-DECEMBER
1979
RESEARCH
EDWARD COTLIER, EDITOR
Neovasculogenesis.
Triggering
Factors
and Possible Mechanisms
DAVID BEN EZRA, M.D., PH.D. Clinical Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, and The Department of Ophthalmology, Hadassah Hebrew University Hospital, Jerusalem, Israel Abstract. The possible regulating mechanism(s)
of neovascularization are discussed. Experimental data supporting the view that ocular neovascularization might be triggered by a fundamental metabolic mechanism are presented. Based on his own data and scattered evidence from the literature, the author suggests a model for a possible regulating mechanism of the neovascular process in which the prostaglandins fulfill the role of neovascular mediating substances. Preliminary results demonstrating that indomethacin limits the extent of the neovascular process support this concept and may have a therapeutic implication. Further studies are in progress in order to better understand the pivotal role fulfilled by the prostaglandins in neovasculogenesis.
(Sun Ophthalmol 24:167-176, 1979) . chemoattractants . growth factors . indomethacin Key words. keratocytes . leukocytes . mitogens . neovascular attracting factor . neovasculogenesis . prostaglandins
T
proliferation of he pathological blood vessels in the eye is one of the most detrimental processes for the visual function. Although intensively investigated for more than half a century, the growth of ocular blood vessels in health and disease remains one of the most enigmatic ocular phenomena. Recent advances in photocoagulation therapy’l and vitreous surgerys8 may have some beneficial effects and reduce the morbidity due to proliferative retinopathy. 167
However, these effects are very limited and, at best, of short duration. A better understanding of the fundamental processes leading to the triggering of neovasculogenesis would eventually provide the clinical ophthalmologist with a possible mode of therapy. therapy. As with any other biological tissue, the modulation of blood vessel growth (or involution) is governed by the net effect of inhibiting versus stimulating factor(s)’ accumulating
168
SurvOphthalmol
24
(3)
November-December
locally. The inhibiting factor(s) may be due to intrinsic characteristics of the involved tissue. However, a possible systemic origin is conceivable. The stimulating factor(s), on the other hand, should be considered to be of strictly local origin; they are produced following changes in the metabolism of the local cells. This concept, which in my opinion is the essence of the neovasculogenic mechanism, has been forwarded by Professor I. C. Michaelson in his treatise of the retinal circulation in man and animalszB This review provides an overview of the neovascular activating (or attracting) factor(s) [NAF], with particular emphasis on possible regulating mechanisms.
Background The various experimental designs of cornea1 vascularization have been reviewed recently by Klintworth,21 while a thoughtful discussion on ocular neovascularization has been presented by Henkind in his Krill Memorial Lecture.18 Therefore, I will limit the present discussion to the three major “schools of thought” regarding the characteristics of the neovascular stimulus. COMPACTNESS
OF THE CORNEAL
AS A FACTOR
FOR NEOVASCULARIZATION
STROMA
(COGAN)
Studying the sequence of events following the induction of small cornea1 lesions in approximately 200 rabbit eyes, Cogan” observed the following: a) Swelling of the cornea was the most constant phenomenon closely associated with or preceeding vascular changes. b) Hemorrhagic extravasates had no influence on neovasculogenesis as blood injected intracorneally did not induce any constant vascularization of the cornea. c) The role of “toxins” appeared unlikely since extracts of vascularized corneas had effects similar to those obtained from nonvascularized corneas. X-FACTOR
8EN EZRA
1979
OF NEOVASCULARIZATION
(MICHAELSON)
The proliferation of blood vessels is closely associated with the metabolism of the tissue. In certain conditions due to local environmental changes, basic cell metabolites accumulate and trigger the vessel growth.lE This possibility has been reinforced by
clinical and experimental observations.10~16~a’-2s These can be summarized as follows: a) New blood vessels are attracted toward the site of highest potential concentration of the X-factor. These form a triangular shaped tuft of new vessels with a large base at the origin. b) The sprouting of new blood vessels is always from the side of the pre-existing vessel that faces the origin of the X-factor. c) The degree of vessel proliferation is closely associated with the X-factor concentration. d) On cessation of the factor production, there is an involution of the blood vessels. OXYGEN
THEORY
(ASHTON,
PATZ)
OF RELATIVE
HYPOXIA
An early phase of vasoconstriction is followed by vessel proliferation under conditions of lower oxygen tension.‘*‘*” This theory provided an adequate explanation for the clinical and experimental phenomena observed in retrolental fibroplasia. Considering the activation of blood vessel growth, one cannot stop wondering about the avascularity of some tissues (cartilage, nail, vitreous, lens, cornea). The easiest explanation for this situation is to postulate on the presence of local inhibitor(s). Indeed, an inhibitor of neovascularization has been demonstrated in cartilageg*zs,a7 and in vitreous.** During histologic observation of new vessel growth into the cornea after the insertion of implants sequestering various mitogens,*’ I was struck by the fact that the leukocyte and new vessel invasion of the cornea was, at least initially, always confined to the upper half or two-thirds of the cornea (Fig, 1). The lower third to half of the cornea nearer to the anterior chamber remained free of cellular invasion unless the reaction was so intense that a uveitis was induced. This pattern of invasion was attributed at first to the mid-stromal position of the implant and to the possible greater edema in the upper half of the cornea facilitating the infiltration. Therefore, experiments were performed in which the implant was inserted and placed “Mitogens: These are plant extracts (Con A, PHA, PWM) or products from bacterial origin (LPS, staphylococcal filtrate) that have a nonspecific mitogenic effect on lymphocytes in vitro.
CURRENT
RESEARCH
near the Descemet’s membrane (Fig. 2). Under these conditions, the same pattern in which the lower part of the cornea, although edematous, remains free of cellular or vessel invasion is observed. The most appealing explanation for these observations is the postulation of a circulating inhibitor present
169
in relatively high concentrations in the aqueous humor. This could be in line with other observations concerning the presence of an inhibitor of lymphocyte blast transformation in primary aqueous humor of rabbit eyes8 From these data, it is conceivable to assume that, once neovascularization is
FIG 1. Implants at the mid-stromal level. a and b: The leukocyte and new vessel infiltrations are limited to the upper two-thirds of the cornea. c and e show the reaction in the upper third of the cornea; d and f illustrate the histology of the lower third of the cornea nearer to the anterior chamber. Note that although edematous, the lower third remains free of neovascularization. Ep = epithellum; AC = anterior chamber; Imp = implant, V = new vessel; Mn = monuclear cell; Plm = polymorphonuclear cells; K = keratocytes; Ds = Descemet’s membrane. (Hematoxylin and eosin. a and b, X 20; c-f, X 60)
170
Surv Ophtholmol
24 (3) November-December
1979
triggered, its extent is regulated via the continuous local production of attracting factor(s) on one hand and the presence of local and/or systemic inhibitor(s) on the other hand.
Possible Activating Mechanism(s) The general consensus, at present, among researchers in the field is that Michaelson’s
BEN EZRA
postulation concerning the release of an Xfactor best explains the neovascular phenomena. However, the characteristics of that factor(s) remain an enigma. In an attempt to correlate the relative hypoxia with the blood vessel proliferation, experiments testing the potential vasoproliferative ability of lactic acid were carried out.‘* A more sophisticated approach to the problem was reported by Zauberman et al.‘” Using a plastic tube in-
FIG. 2. Implants positioned near the Descemet’s membrane. The leukocyte and new vessel infiltration is slightly deeper than in cases shown in Fig. 1. However, the pattern of invasion is similar. The lower third of the cornea nearer to the anterior chamber remains free of new vessels (a and b). Few leukocytes are observed near the Descemet’s membrane in these cases (d and f). (Hematoxylin and eosin. a and b, X 20; c-f, x 60)
171
serted into the rabbit cornea as a slow release device, these investigators found that biogenic amines (acetylcholine, histamine, serotonin and bradykinin) induced a neovascularization in approximately 35% of the cases. More recently, it was shown that tumors produce and release an angiogenic factor (TAF).*’ Finally, the cornea1 vasculogenic capacity of a “non-sedimentable” fraction obtained from a sonicate of peritoneal cell exudates has been reported.” In an attempt to elucidate the role of lymphoid cell products in neovasculogenesis, an autologous system was used.3,7 Leukocytes obtained from rabbit peripheral blood were stimulated in vitro by various mitogens. The culture supernatants and the activated leukocytes were injected back to autologous* and allogeneic** corneas. Supernatants and leukocytes activated by concanavalin A (Con A) or lipopolysaccharide (LPS) showed a strong NAF activity in both the autologous and the allogeneic systems. Cultures stimulated by phytohemagglutinin (PHA) or pokeweed mitogen (PWM) were seldom weakly active, while nonactivated cultures had no detectable NAF. It is interesting that supernatants from rabbit keratocyte cultures had no neovasculogenic activity alone or when supplemented with mitogens.s*7 It is not improbable, however, that keratocytes under various metabolic conditions would demonstrate some NAF activity. From these data, it was postulated that the various experimental means for the triggering of new blood vessel proliferation might act through the release of neovascular mediating substances (NMS). The NMS would influence local cells in the target organ or trigger some cells involved in the defense mechanism (lymphoid) to produce NAF, or else NMS might directly affect the vessel endothelial cells (Fig. 3). These postulations were reinforced by the recent finding of Polverini et aLsa that activated macrophages were able to release a neovasculogenic substance. The degree of positive neovasculogenesis *Autologous system: Activated cells and supernatants are tested on corneas of same animal that donated the cells. **Allogeneic system: Activated cells and supernatants are tested on corneas of another animal of the same species, e.g. cells obtained from rabbit (a) are tested on corneas of rabbit (b), (c), etc.
TABLE 1 Schematic Representation of Active Neovasculogenic Substances
System
Neovasculogenesis*
Activated leukocytes LPS Con A PHA
++++ ++ _**
Prostaglandins PGE, PGE, PGF,(r PGF,a
+++ + + -
Growth factors FGF EGF NGF
f + -
*Number of + denotes intensity of neovasculogenesis under optimal conditions. **Denotes no neovasculogenesis.
observed under various conditions is schematically illustrated in Table 1. Cyclic AMP and cyclic GMP* and dibutyryl cyclic AMP did not induce any noticeable NAF activity. All implants sequestering these compounds remained inactive, as did all cont’tils of “empty” implants of Elvax-40. Prostaglandin El** was the strongest and most consistent inducer of neovasculogenesis. PGE, and to some extent PGF,, showed a moderate NAF activity. The formylated peptides, which are very potent in vitro chemoattractantsas,as and induce only a slight infiltration of polymorphonuclears in viva,* had no NAF activity. The purified growth factors (GF)***
*Cyclic nucleotides: Cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP). **Prostaglandins (PG): Series of hormones that are designated as A, D, E, F, etc. In each series there are few compounds that are labeled numerically, e.g. PGE,, PGb, etc. ***Growth Factors (GF): Polypeptides that demonstrate a growth promoting potential similar to or exceeding that observed with the addition of serum (or plasma) in cell cultures. The biological role of these peptides in vivo is still unclear. They are extracted from various organs.
172
Surv Ophthalmol
24 (3) November-December
\ NORMAL
A
BEN EZRA
1979
TISSUE
(
tttt
Hypoxia, Cautery, Chemical, Mechanical
// / / _--L-
-.-__. \ \ - _.... - --)
Neovascular
Mediating
Endothel-Mural
Substances
* - --
---1,
%
Cells
FIG. 3. Possible mechanism(s) for the induction of neovasculogenesis.
[epidermal growth factor (EGF), tibroblast growth factor (FGF) and nerve growth factor (NGF) ] varied in their capacity to induce neovasculogenesis. EGF induced a mild NAF activity in concentration of 10 pg per implant and a stronger neovasculogenesis at 20 pg per implant. FGF had similar effects, but was a milder stimulant than EGF. In connection with the neovasculogenesis induced by 10 or 20 pg EGF, two histological observations are noteworthy: The blood vessel proliferation is accompanied by a very mild leukocytic infiltration and a marked epithelial proliferation (Fig. 4). NGF in concentrations of one to 10 units per implant had no NAF activity.
Discussion From the presented data and a gathering of reported facts concerning the role of prostaglandins as intercellular messengers, it is very tempting to speculate that Michaelson’s X-factor, TAF, NAF or any
neovasculogenic activity is mediated through local release of prostaglandins. Moreover, various prostaglandins may have antagonistic effects and thus, may provide a possible regulatory influence on the neovascular process. In some preliminary studies, we have observed that PGF1, may have an inhibitory effect on the NAF activity of PGE,. Therefore, the possibility that under various different prostaglandins circumstances, produced locally may regulate the NAF activity is very attractive. In this regard, it is interesting that growing tumor cells as well as activated leukocytes and macrophages synthesize and release prostaglandins which are used as intercellular messengers.16J8~20~22 Also, platelets from patients with diabetes synthesize and release more prostaglandins than platelets from nondiabetics in the platelet aggregation reaction.” The influence of PGE, on the sickling of erythrocytes in a hypoxic state has also been reported.s’ From the scattered evidence, it is very tempting to con-
FIG. 4. Effect of epidermal growth factor (EGF). a: Implant sequestering 10 pg EGF. Note the mild vessel proliferation (V), the little leukocyte infiltration and the marked epithelial hyperplasia. b: Higher magnification of the site of vessel infiltration (V). c: Higher magnification of epithelium above the implant. d-f; Similar sites from cornea with empty implant of Elvax-40 (control). (Hematoxylin and eosin. a and d, X 20; b and e, X 60; c and f, X 80)
ceive that the ocular neovascularization phenomena are all activated through a common pathway. The vascular proliferation, as observed in retrolental fibroplasia, diabetic retinopathy, sickle cell retinopathy, central retinal vein occlusion and chronic inflammatory or immune reactions, can be
rationalized on the basis of the local production of prostaglandins induced by the various stimuli (Fig. 5). The above postulation is also attractive in view of its possible therapeutic implications. In ongoing experiments, we are studying the effect of indomethacin on the neovascular
174
Surv Ophthalmol
1’ ,-------)
24 (3) November-December
BEN EZRA
1979
NeovascuC Mediating Substances+-----_t, (NW
InflamZiG3n
Met&
ibeases
-m-.-.-m
+ 1 Endothel-Mural Cells 1
FIG. 5. Schematic mechanism(s) of neovasculoaenesis neovascular mediating substances ‘(NMS). process. Preliminary results show that indomethacin, although it does not completely abolish the NAF activity of PGE, in any of the concentrations used, markedly limits the extent of the neovascular process (as represented best by the reduction of the neovascular surface). These data may be interpreted as an indication of the perpetuation of the neovascular process by further local synthesis of prostaglandins or other sensitive metabolites. Indomethacin probably has no effect on the prostaglandin sequestered in the implant, but limits the extent of neovascularization by inhibiting the local synthesis and release of newly formed mediators. At the present stage of our investigation, it is very difficult to totally exclude the possible involvement of metabolites other than the stable prostaglandins in the neovascular process. Also, the role of precursors of prostaglandins and other unstable intermediate compounds must be considered. Nevertheless, a carefully monitored treatment with inhibitors of prostaglandin syn-
postulating the role of prostaglandins
as main
and/or the prevention of ocular neovasculogenesis is warranted even at these early stages of our knowledge of the phenomenon. At least in proliferative retinopathies, the use of adequate doses of salicylates or indomethacin might be a logical and, if carefully monitored, a “harmless” mode of treatment. thesis for the limitation
Acknowledgments I am thankful to Ms. Sammy Bornstein for her technical assistance and her skill in the preparation of the histology slides, to Ms. Joan Lee and Ms. Iris Kivitz for their persevering typing and retyping, and to Dr. Elmer Ballintine for his discussions and encouragement. I am also indebted to Mr. James Ingram for his patient and skillful assistance with the experimental animals and to Ms. Louise Smith for her pre- and post-operative care of the rabbits.
1.
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CURRENT
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Reprint requests should be addressed to David BenEzra, M.D., Ph.D., Department of Ophthalmology, Hadassah Hebrew University Hospital, Kytiat Hadassah, Jerusalem, Israel.