Current Eye Research
Volume 4 number 10 1990 ~~~
Transforming growth factor-@in human aqueous humor ~
~~~
Henry D.Jarnpel, Nan Roche', Walter J.Stark2 and Anita B.Roberts'
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Glaucoma and Tornea Services, Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD and 'Laboratory of Chernoprevention, National Cancer Institute, National lnstitutcs of Health, Bethesda, MD, USA
ABSTRACT The transforming growth factor-8s are peptide growth factors known to play a central role in wound healing. Using a specific, in vitro assay of cell growth inhibition, we have detected transforming growth factor-a (TGF-8) in 24/24 aqueous humor specimens from eyes undergoing cataract extraction with intraocular lens implantation. The amount of TGF-a ranged from 2.3 to 8.1 ng/ml (mean r SD = 4.5 f 1.7 ng/ml), with 61% present in the active form. Subtyping of TGF-8 was performed by addition of antibodies specific for the 81 and 132 isoforms to the growth inhibition assay, and confirmed with a sandwich enzyme-linked immunosorbent assay. None of the TGF-13 detected was of the 81 isoform; in contrast, the a2 isoform was present in every sample, implying that it might have originated from ocular tissues. The presence of this potent modulator of tissue repair in aqueous humor suggests a role in the healing processes following intraocular surgery, including glaucoma filtration surgery.
aqueous humor, does not inhibit lymphocyte proliferation. TGF-a is another peptide growth factor thought to play an important role in wound healing. It is found in high concentrations in the a granules of platelets (6), and is secreted by activated T lymphocytes (7), macrophages (8),and many types of cultured cells. TGF-8 is present in vitreous aspirates from eyes with retinal detachments, and is found in particularly high concentrations in eyes with proliferative vitreoretinopathy (9). TGF-I3 shares several of the activities of aqueous humor, including being chemotactic for monocytes (10) and fibroblasts (1l ) , and inhibiting the proliferation of T (7) and B (12) lymphocytes. When injected subcutaneously in mice, it causes rapid fibrosis and angiogenesis (13), and increases the breaking
INTRODUCTION The degree and nature of the wound healing response following glaucoma filtration surgery in large
strength of surgical wounds (14). TGF-8 exists in at least 5 genetically distinct
part determines its success or failure. Because the
isoforms, although only 3 isoforms, 61, 82, and 133, have been found in humans and other mammalian
aqueous humor bathes the surgical wound after
species (15). The isoform of TGF-a found in
filtration surgery, its composition may influence
mammalian platelets and serum is predominantly R1.
wound healing after surgery.
Many tissues express all 3 isoforms of TGF-A; an
Aqueous humor exerts effects in vitrQthat suggest a potential role in wound healing in vivq. Dilute
exception is neural tissue where the principal isoforms expressed are 82 and I33 (K. Flanders and K.
aqueous humor stimulates the proliferation of cultured
Unsicker, personal communication). The TGF-0 in the
fibroblasts (l), but inhibits lymphocyte proliferation
vitreous is predominantly of the 62 subtype (9).
(2,3). It is chemodttractive for conjunctival fibroblasts (4). Although aqueous humor has been shown to
Because aqueous humor appears to play a role in ocular wound healing, and because TGF-B and aqueous humor share some biological activities, we
contain basic fibroblast growth factor (3,to the best of our knowledge, this peptide growth factor, unlike
have investigated whether human aqueous humor
~~
Receivcd on June 1 , 1990; accepted on September 26, 1990
963
Current Eye Research normally contains TGF-a. If so, it might have an
for hemostasis; and the limbus was cleared of all
important effect on wound healing following glaucoma
visible blood and dried with a Weck Cel.
filtration surgery and other ocular surgery. We now report the identification, quantification, and subtyping
Aqueous humor sampling Because the technique used for aqueous humor
of TGF-A in human aqueous humor.
collection can affect its composition (16), all sampling was performed by one surgeon (WJS) using a
MATERIALS AND METHODS
standardized technique. The globe was stabilized with
Patients
a forceps near the sampling site. A 5/8 inch long, 27
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Twenty-four consecutive patients undergoing
gauge needle was placed through the superior limbus,
cataract extraction with intraocular lens implantation
bevel up, and parallel to the plane of the iris. When
were studied. Informed written consent for aqueous
the entire bevel was within the anterior chamber, 50-
humor sampling was obtained in all cases. Patients
150 uls of aqueous humor, depending upon the size
were excluded if they were taking topical medications,
of the anterior chamber, were aspirated. Extreme care
or had ocular conditions, such as glaucoma, that were
was taken not to touch the iris or lens with the needle.
ever treated with topical or systemic medications.
Aspiration of aqueous humor was halted before the
Patients were taking a variety of systemic medications. The characteristics of the patients are presented in the
anterior chamber became so shallow that the needle tip might contact the corneal endothelium. After the
Table.
needle was removed from the anterior chamber, a
Interventions precedina aqueous humor samDlinq
sharp blade was used to enlarge the entrance site,
All eyes had received the following topical
sodium hyaluronate was placed in the anterior
medications preoperatively: Gentamycin sulfate
chamber, and the operation continued in routine
solution, twice on the day preceding surgery, and 2 to
fashion.
4 times on the day of surgery; flurbiprofen sodium
All specimens were rapidly transferred to 500
0.03% solution and prednisolone acetate 1%, both 4
microliter Eppendorf centrifuge tubes and immediately
times in tho 2 hours before surgery; and
placed on dry ice for transport to a -70 O Cfreezer for
phenylephrine hydrochloride 2.5% and cyclopentolate
storage.
hydrochloride 1% solutions, both 6 times in the 2
Heat activation of the aaueous humor samoles
hours before surgery. All surgery was performed under local anesthesia
Because most of the TGF-a activity found in wound fluid and in vitreous aspirates is latent (9,17),
with intravenous sedation. Medications used for
most samples were assayed both with and without
sedation included fentanyl citrate, midazolam
prior heat activation to determine the relative
hydrochloride, and thiamylal sodium. Local anesthesia
proportion of active and latent forms. Briefly, 20 ul of
consisted of 1% lidocaine hydrochloride and 0.375%
a 50 ug/ml solution of bovine serum albumin was
bupivicaine hydrochloride administered by retrobulbar
added to 10 ul of human aqueous humor in a
and facial injection. A Honan balloon was employed
siliconized tube, and the sample was either left
to lower the intraocular pressure after the retrobulbar injection.
untreated or heated at 80 OCfor 8 minutes, a
The following ocular manipulations occurred prior to obtaining the aqueous humor sample: A superior rectus stay suture was placed; a fornix based conjunctival flap was made; wetfield cautery was used
964
procedure known to activate irreversibly latent TGF-13 (18).
Detection and auantification of TGF-13 The amount of TGF-a activity present in the
aqueous humor samples was determined in an assay
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Current Eye Research based on inhibition of the growth of CCL-64 mink lung
activity of purified porcine TGF-I31 (R & D Systems,
epithelial cells (19). The sensitivity of the assay is 10-
Minneapolis, MN). Duplicate or triplicate
20 pg/mL TGF-13, ED.,
measurements of most samples were made. TGFs 13's
These cells have been shown
to be sensitive to inhibition by TGF-13 (20). Briefly,
1, 2, and 3 have equivalent activity in this assay.
serially diluted samples of aqueous humor were treated as described above and added to the cells;
Determination of the relative amounts of TGF-Bl and
I32
was determined incorporation of ['2sl]iodode~xyuridine
The relative amounts of TGF-I31 and 02 were
as a measure of cell proliferation. The concentration of TGF-A in the aqueous humor samples was
determined by 2 methods. First, heat-activated aqueous humor specimens were preincubated with
determined by comparison with a standard curve of
either turkey anti-TGF-81 (19) or rabbit anti-TGF-I32
TABLE TRANSFORMING GROWTH FACTOR-BETA IN HUMAN AQUEOUS HUMOR PATIENT # AGE/SEX/RACE TRANSFORMING GROWTH % PRESENT IN % TYPE 2
FACTOH BETA (NG/ML)* 7.8
t
2.6
5.6
t
1.9
15
3
5.7
t
2.4
36
4
6.6 t 3.7
23
78
5
5.6
t
1.8
39
76
6
3.3
* 1.4
60
65
74
7
3.6 t 1.0
48
8
5.8 (5.0, 6.61 (2)
35
9
5.1 (4.6, 5.61 (2)
76
10
3.9
t
0.9
100
11
3.7
t
2.1
48 100
12
4.1 t 0.9
13
2.8
1.7
53
14
3.6 i2.3
100
15
5.1
94
16
2.8 [2.3, 3.31 (2)
17
4.5
t
1.8
88
18
3.9
t
0.8
76
19
2.4 (2.3, 2.51 (2)
20
6.2
t
*
28
1 2
i
% PlPE 1
ACTIVE FORMt lSOFORM* ISOFORM
2.2
(11
21
8.1
(1)
54
22
2.3
(11
65
23
2.5
(11
76
24
3.2
(11
95 64
0 0
94
0
94
0
*Mean * SD of 3 separate determinations, except where number Is indicated in parentheses. Where only two determinations were made, the actual values are given within brackets.
'Single determination of the % of the total assayable TGF-beta that Is actlve. *Values represent a single determination (see Figure 2). -
965
Current Eye Research (21) before addition to the CCL-64 growth inhibition
The ability of aqueous humor samples to inhibit the
assay. These polyclonal antibodies, whose specificlty
CCL-64 growth assay was unaltered by preincubation
has been rigorously tested (20), specifically block the
with anti-TGF-A1 (n= 8 ) , nor was any TGF-I31 detected in the sandwich ELISA assay (n = 10). In contrast,
activity of TGF-A1 and 02, respectively. Secondly, the relative amounts of these 2 isoforms of TGF-A were determined in a sandwich ELSA assay (21).
between 64 and 95% (mean ? SD = 80 t 13%) of the ability of the aqueous humor samples (n=8) to inhibit CCL-64 growth was blocked by preincubation with
RESULTS
antibodies against TGF-fi2 (Figure 2). Furthermore,
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The patients ranged in age from 45 to 87 years
TGF 02 was detected in 10/10 samples tested in the
(mean t SD; 67 t 14). There were 12 men and 12
sandwich ELlSA assay (data not shown); TGF-A1 was
women. 23 patients were white, one was black
not detected in any of these samples.
(Table). Each aqueous humor sample contained
Comparison of the activity of the aqueous humor
detectable levels of TGF-B ranging from 2.3 to 8.1
samples in the CCL-64 growth inhibition assay before
ng/ml (mean ? S.D. = 4.5 t 1.7 ng/ml; Table). The
and after heat activation (n =20) revealed that between
dose-response curves for inhibition of the CCL-64
15 and 100% (mean ? SD= 61 k 27%) of the TGF-13
growth assay by aqueous humor were similar in shape
was present in active form in aqueous humor.
to the curve generated by the addition of purified porcine TGF-A1 (Figure 1).
40
! Human Aqueous (pl/mL)
TGF-I31 (pglmL) or Aqueous (pllmL)
Figure 1. Growth inhibitory activrty of human aqueous humor for mink lung epithelial CCL-64 cells. Serial dilutions of heat-activated human aqueous humor (patients 1,4; 4p; and 5,o)were tested for their ability to inhibit uptake of '251-iodo-deoxyuridine. Concentrations of transforming growth factor-8 in the aqueous humor were estimated by comparison with a standard curve of porcine transforming growth factor4 ( 0 ) . Shown is a representative experiment of 3 such assays for each sample. Maximum incorporation of untreated cells (control) was 2670 cpm.
966
Figure 2. Anti-transforming growth factor-oZ antibodies, but not anti-transforming growth factor-81 antibodies, block the growth inhibitory activity of human aqueous humor. To aliquots of human aqueous humor (patient #4) were added either turkey control serum (1 pl/mL;o); turkey anti-transforming growth factor-A1 #366 (1 pl/mL;m); rabbit IgG (45 pg/mL;o), or rabbit anti-transforming growth factor-02 SAM (50pg/mL;o). Samples were then assayed for their growth inhibitory activity. The proportion of transforming growth factor42 was calculated by comparing the level of assayable transforming growth factor4 in the presence and absence of the antibodies. Only anti-transforminggrowth factor-I32 antibodies reduced the activity. Maximum incorporation was 7840 cpm.
Current Eye Research No correlation was detected between the age (r=
is unknown at the present time. source of the TGF-A~
0.08, p= 0.72, linear regression analysis) or gender
However, retinal pigment epithelial cells in culture have
(males, 4.3 5 1.5; females 4.8 5 1.9; p =0.49, unpaired
been shown to secrete predominantly TGF-132 (26).
t test) of the patient and the concentration of TGF-8.
Moreover, the neural tissue of the eye including the photoreceptor cells, ganglion cells, and bipolar cells all
DISCUSSION
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The TGF-As are a set of multifunctional proteins
stain intensely for TGF-13’s 2 and 3; no staining for TGF-A1 is detected in these cells (K. Flanders and K.
with a variety of effects (15). A growing body of
Unsicker, personal communication).
evidence suggests that they play a pivotal role in the process of wound repair (22). For this reason they
The significance of this preponderance of the 82 isoform in aqueous humor is unclear. In most
may also be important in wound healing processes
bioassays, including the CCL-64 growth inhibition
within the eye and on the ocular surface.
assay that we employed, the isoforms of TGF-A are
TGF-D has been detected in the rat cornea after
functionally indistinguishable. One notable exception
epithelial wounding (23), in the retina and choroid of
is the almost 100-fold greater ability of TGF-A1 to
monkeys following argon laser photocoagulation (24),
inhibit growth of endothelial cells in culture (27). This
in the embryonic mouse eye (25), and in the vitreous
suggests differential activities of these 2 isoforms in
of eyes with retinal detachments (9). Granstein et al.
control of angiogenesis which could result in an
(2) have reported the presence of TGF-A in murine
altered healing response pattern. In some aqueous humor samples, a portion of the
and human aqueous humor at levels of 200 to lo00 pg/ml after acid activation. We now report the
TGF-A activity was not neutralized by antisera against
presence of significantly higher concentrations of TGF-
either the a1 or 132 isoforms. Two possible
D in a series of carefully obtained aqueous humor
explanations are that some of the inhibitory activity in
specimens from human eyes with well-defined clinical characteristics. Furthermore, we have characterized it
the assay was not due to TGF-A or that a portion of the TGF-A in the aqueous humor represents another
as the 82 isoform and have demonstrated that the
isoform, ~3 (15). The latter possibility is supported by
majority is present in the activated form. The composition of our aqueous humor samples
the observation that both messenger RNA for A3 (by
may have been influenced by the presence of cataract
imrnunohistochemistry) are detectable in the mouse
or by the pharmacologic and mechanical interventions
retina and many other tissues of neural origin (B.
that preceded the sampling. These samples,
Marascalco and K. Unsicker, personal
however, represent the closest to unaltered aqueous
communication). Therefore, it is possible that TGF-83
humor that we could obtain. The presence of TGF-13
may be present in aqueous humor. Unfortunately, no
in the samples is unlikely to be due solely to
antibodies are currently available for selective detection of this isoform.
preoperative interventions because we and others have detected TGF-A in other species that have
in situ hybridization) and the peptide itself (by
In most biological fluids, including vitreous (9),
undergone only minimal manipulation before sampling
most of the TGF-A is present in a biologically inactive
(2).
form (28), and requires either heat or acid treatment to We detected only the A2 isoform of TGF-R. Since
express its activity (18). Therefore, the large
TGF-A2 is not found in human serum or platelets, the
proportion of TGF-A present in active form in the
TGF-8 detected in the aqueous humor does not reflect
aqueous humor (15 to loo%,with an mean value of 61 27%), is striking. Although the significance of
contamination from the serum during sampling, The
967
Current Eye Research this observation is not clear, the presence of so much active form of TGF-I3 in aqueous humor suggests a possible role in normal ocular physiology. Joseph et al. (4) have determined that aqueous
CORRESPONDING AUTHOR Henry D. Jampel, M.D., Maumenee 8-110, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.
humor is chemotactic for ocular surface fibroblasts and have reported that this activity resides in substances with molecular weights greater than 30 kD and is deactivated by low pH (29). TGF-I3 has been shown to have potent chemotactic activity for human Curr Eye Res Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
fibroblasts (11); however, it has a molecular weight of 25 kD and is activated by acid treatment (15). These
data suggest that the chemoattractant activity of aqueous humor might be due to factors other than TGF-A. Our identification of TGF-13 in aqueous humor and the availability of specific blocking antibodies to TGF-B’s 1 and 2 now permit direct testing of a possible role for this peptide in chemotaxis of ocular fibroblasts. The detection of TGF-A in the aqueous humor raises the question of its origin. Preliminary results suggest that cells fcom either the iris and/or ciliary body may secrete TGF-13 in vitro (30). Immunohistochemical techniques and in situ hybridization should permit localization of the protein in tissues of the anterior segment, and the determination of which cells are actually synthesizing TGF-8. Glaucoma filtration surgery generally fails due to scarring in the subconjunctival space (31). The TGF-s present in the aqueous humor may play a role in the wound healing process following filtration surgery.
ACKNOWLEDGMENTS Supported in part by a grant from National Glaucoma Research, American Health Assistance Foundation, Rockville, MD (Dr. Jampel).
The authors would like to thank David Danielpour, PhD. for his help with the immunoassays and with the sandwich ELISA.
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Current Eye Research Kang, A.H. (1987) Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor-B. J. Exp. Med. 165,251 1-2556. 12. Kehrl, J.H., Roberts, A.B., Wakefield, L.M., Jakowlew, S.B., Sporn, M.B., and Fauci, A.S. (1986) Transforming growth factor beta is an important immunomodulatory protein for human Blymphocytes. J. Immunol. l;lz,3855-3860. 13. Roberts, A.B., Sporn, M.B., Assoian, R.K., Smith, J.M., Roche, N.S., Wakefield, L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H., and Fauci, A S . (1986) Transforming growth factor typebeta:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA, 83, 4167-4171. 14. Mustoe, T.A., Pierce, G.F., Thomason, A., Gramates, P., Sporn, M.B., and Deuel, T.F. (1987) Transforming growth factor type beta induces accelerated healing of incisional wounds in rats. Science, 1333-1336. 15. Roberts, A.B., and Sporn, M.B. (1990) The transforming growth factor-8s. In "Growth Factors and their Receptors." Handbook of Experimental Pharmacology, Vol 95. (Eds. Sporn, M.B., and Roberts, A.B.) Pp. 419-472. Springer-Verlag, Heidelberg. 16. Tripathi, R.C., Millard, C.B., and Tripathi B.J. (1989) Protein composition of human aqueous humor: SDS-PAGE analysis of surgical and postmortem samples. Exp. Eye Res. %, 117-130. 17. Cromack, D.T., Sporn, M.B., Roberts, A.B., Merino, M.J., Dart, L.L., and Norton, J.A. (1987) Transforming growth factor-beta levels in the rat wound chamber. J. Surg. Res. 42, 622-628. 18. Brown, P.D., Wakefield, L.M., Levinson, A.D., and Sporn, M.B. (1990) Physicochemical activation of recombinant latent transforming growth factorbeta's 1, 2, and 3. Growth Factors. 3, 35-43. 19. Danielpour, D., Dart, L.L., Flanders, K.C., Roberts, A.B., and Sporn, M.B. (1989) lmmunodetection and quantification of the two forms of transforming growth factor-beta (TGF-B1 and TGF-82) secreted by cells in culture. J. Cell Physiol. B, 79-86. 20. Tucker, R.F., Shipley, G.D., Moses, H.L., and Holley, R.W. (1984) Growth inhibitor form BSC-1 cells closely related to platelet type 8 transforming growth factor. Science, 226, 705-707. 21. Danielpour, D., Kim, K-Y, Dart, L.L., Watanabe, S., Roberts, A., and Sporn, M.B. (1989) Sandwich enzyme-linked immunosorbent assays (SELISAs) quantitate and distinguish two forms of transforming growth factor-beta (TGF-81 and TGFB2) in complex biological fluids. Growth Factors, 2, 61-71. 22. Sporn, M.B., and Roberts, A.B. (1989) Transforming growth factor-beta. Multiple actions and potential clinical applications. J.A.M.A. 938-941.
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23. Hayashi, K., Frangieh, G., Wolf, G.,and Kenyon, K.R. (1989) Expression of transforming growth factor-a in wound healing of vitamin A-deficient rat corneas. Invest. Ophthalmol. Vis. Sci. 3, 239-247. 24. Hayashi, H., Jerdan, J., Kato, H., and Glaser, B. (1988) Localization of transforming growth factorbeta within the retina and choroid following argon laser photocoagulation. Invest. Ophthalmol. Vis. Sci. 29 ( s u m , 221. 25. Heine, U.I., Munoz, E.F., Flanders, K.C., Ellingsworth, L.R., Lam, H.Y.P., Thompson, N.L., Roberts, A.B., and Sporn, M.B. (1987) Role of transforming growth factor-o in the development of the mouse embryo. J. Cell Bio 2861-2876. 26. Connor, T., Roberts, A., Sporn, M., Davis, J., and Glaser, B. (1988) RPE cells synthesize and release transforming growth factor-& a modulator of endothelial cell growth and wound healing. Invest. Ophthalmol. Vis. Sci. 29 f s u w , 307. 27. Jennings, J.C., Mohan, S., LinkhaC, T.A., Widstrom, R., and Baylink, B. (1988) Comparison of the biological activities of TGF-beta 1 and TGFbeta 2: differential activity in endothetial cells. J. Cell Physiol. 167-172. 28. Pircher, R., Jullien, P., and Lawrence, D.A. (1986) &transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem. Biophys. Res. Commun. 30-37. 29. Joseph, J.P., Grierson, I., and Hitchings, R.A. (1989) Partial characterization of the fibroblast chemotactic constituents of human aqueous humour. International Ophthalmology, U, 125130. 30. Knisely, T.L., and Granstein, R.D. (1990) Analysis of inhibitory factors produced by iris/ciliary body cells in vitro. Invest. Ophthalmol. Vis. Sci. 241. 31. Addicks, E.M., Quigley, H.A., Green, W.R., and Robin, A.L. (1983) Histologic characteristics of filtering blebs in glaucomatous eyes. Arch. Ophthalmol. 795-798.
m,
m,
m,
~
m,
m,
969
Volume 4 number 10 1990 ~~~
Transforming growth factor-@in human aqueous humor ~
~~~
Henry D.Jarnpel, Nan Roche', Walter J.Stark2 and Anita B.Roberts'
Curr Eye Res Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Glaucoma and Tornea Services, Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD and 'Laboratory of Chernoprevention, National Cancer Institute, National lnstitutcs of Health, Bethesda, MD, USA
ABSTRACT The transforming growth factor-8s are peptide growth factors known to play a central role in wound healing. Using a specific, in vitro assay of cell growth inhibition, we have detected transforming growth factor-a (TGF-8) in 24/24 aqueous humor specimens from eyes undergoing cataract extraction with intraocular lens implantation. The amount of TGF-a ranged from 2.3 to 8.1 ng/ml (mean r SD = 4.5 f 1.7 ng/ml), with 61% present in the active form. Subtyping of TGF-8 was performed by addition of antibodies specific for the 81 and 132 isoforms to the growth inhibition assay, and confirmed with a sandwich enzyme-linked immunosorbent assay. None of the TGF-13 detected was of the 81 isoform; in contrast, the a2 isoform was present in every sample, implying that it might have originated from ocular tissues. The presence of this potent modulator of tissue repair in aqueous humor suggests a role in the healing processes following intraocular surgery, including glaucoma filtration surgery.
aqueous humor, does not inhibit lymphocyte proliferation. TGF-a is another peptide growth factor thought to play an important role in wound healing. It is found in high concentrations in the a granules of platelets (6), and is secreted by activated T lymphocytes (7), macrophages (8),and many types of cultured cells. TGF-8 is present in vitreous aspirates from eyes with retinal detachments, and is found in particularly high concentrations in eyes with proliferative vitreoretinopathy (9). TGF-I3 shares several of the activities of aqueous humor, including being chemotactic for monocytes (10) and fibroblasts (1l ) , and inhibiting the proliferation of T (7) and B (12) lymphocytes. When injected subcutaneously in mice, it causes rapid fibrosis and angiogenesis (13), and increases the breaking
INTRODUCTION The degree and nature of the wound healing response following glaucoma filtration surgery in large
strength of surgical wounds (14). TGF-8 exists in at least 5 genetically distinct
part determines its success or failure. Because the
isoforms, although only 3 isoforms, 61, 82, and 133, have been found in humans and other mammalian
aqueous humor bathes the surgical wound after
species (15). The isoform of TGF-a found in
filtration surgery, its composition may influence
mammalian platelets and serum is predominantly R1.
wound healing after surgery.
Many tissues express all 3 isoforms of TGF-A; an
Aqueous humor exerts effects in vitrQthat suggest a potential role in wound healing in vivq. Dilute
exception is neural tissue where the principal isoforms expressed are 82 and I33 (K. Flanders and K.
aqueous humor stimulates the proliferation of cultured
Unsicker, personal communication). The TGF-0 in the
fibroblasts (l), but inhibits lymphocyte proliferation
vitreous is predominantly of the 62 subtype (9).
(2,3). It is chemodttractive for conjunctival fibroblasts (4). Although aqueous humor has been shown to
Because aqueous humor appears to play a role in ocular wound healing, and because TGF-B and aqueous humor share some biological activities, we
contain basic fibroblast growth factor (3,to the best of our knowledge, this peptide growth factor, unlike
have investigated whether human aqueous humor
~~
Receivcd on June 1 , 1990; accepted on September 26, 1990
963
Current Eye Research normally contains TGF-a. If so, it might have an
for hemostasis; and the limbus was cleared of all
important effect on wound healing following glaucoma
visible blood and dried with a Weck Cel.
filtration surgery and other ocular surgery. We now report the identification, quantification, and subtyping
Aqueous humor sampling Because the technique used for aqueous humor
of TGF-A in human aqueous humor.
collection can affect its composition (16), all sampling was performed by one surgeon (WJS) using a
MATERIALS AND METHODS
standardized technique. The globe was stabilized with
Patients
a forceps near the sampling site. A 5/8 inch long, 27
Curr Eye Res Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Twenty-four consecutive patients undergoing
gauge needle was placed through the superior limbus,
cataract extraction with intraocular lens implantation
bevel up, and parallel to the plane of the iris. When
were studied. Informed written consent for aqueous
the entire bevel was within the anterior chamber, 50-
humor sampling was obtained in all cases. Patients
150 uls of aqueous humor, depending upon the size
were excluded if they were taking topical medications,
of the anterior chamber, were aspirated. Extreme care
or had ocular conditions, such as glaucoma, that were
was taken not to touch the iris or lens with the needle.
ever treated with topical or systemic medications.
Aspiration of aqueous humor was halted before the
Patients were taking a variety of systemic medications. The characteristics of the patients are presented in the
anterior chamber became so shallow that the needle tip might contact the corneal endothelium. After the
Table.
needle was removed from the anterior chamber, a
Interventions precedina aqueous humor samDlinq
sharp blade was used to enlarge the entrance site,
All eyes had received the following topical
sodium hyaluronate was placed in the anterior
medications preoperatively: Gentamycin sulfate
chamber, and the operation continued in routine
solution, twice on the day preceding surgery, and 2 to
fashion.
4 times on the day of surgery; flurbiprofen sodium
All specimens were rapidly transferred to 500
0.03% solution and prednisolone acetate 1%, both 4
microliter Eppendorf centrifuge tubes and immediately
times in tho 2 hours before surgery; and
placed on dry ice for transport to a -70 O Cfreezer for
phenylephrine hydrochloride 2.5% and cyclopentolate
storage.
hydrochloride 1% solutions, both 6 times in the 2
Heat activation of the aaueous humor samoles
hours before surgery. All surgery was performed under local anesthesia
Because most of the TGF-a activity found in wound fluid and in vitreous aspirates is latent (9,17),
with intravenous sedation. Medications used for
most samples were assayed both with and without
sedation included fentanyl citrate, midazolam
prior heat activation to determine the relative
hydrochloride, and thiamylal sodium. Local anesthesia
proportion of active and latent forms. Briefly, 20 ul of
consisted of 1% lidocaine hydrochloride and 0.375%
a 50 ug/ml solution of bovine serum albumin was
bupivicaine hydrochloride administered by retrobulbar
added to 10 ul of human aqueous humor in a
and facial injection. A Honan balloon was employed
siliconized tube, and the sample was either left
to lower the intraocular pressure after the retrobulbar injection.
untreated or heated at 80 OCfor 8 minutes, a
The following ocular manipulations occurred prior to obtaining the aqueous humor sample: A superior rectus stay suture was placed; a fornix based conjunctival flap was made; wetfield cautery was used
964
procedure known to activate irreversibly latent TGF-13 (18).
Detection and auantification of TGF-13 The amount of TGF-a activity present in the
aqueous humor samples was determined in an assay
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Current Eye Research based on inhibition of the growth of CCL-64 mink lung
activity of purified porcine TGF-I31 (R & D Systems,
epithelial cells (19). The sensitivity of the assay is 10-
Minneapolis, MN). Duplicate or triplicate
20 pg/mL TGF-13, ED.,
measurements of most samples were made. TGFs 13's
These cells have been shown
to be sensitive to inhibition by TGF-13 (20). Briefly,
1, 2, and 3 have equivalent activity in this assay.
serially diluted samples of aqueous humor were treated as described above and added to the cells;
Determination of the relative amounts of TGF-Bl and
I32
was determined incorporation of ['2sl]iodode~xyuridine
The relative amounts of TGF-I31 and 02 were
as a measure of cell proliferation. The concentration of TGF-A in the aqueous humor samples was
determined by 2 methods. First, heat-activated aqueous humor specimens were preincubated with
determined by comparison with a standard curve of
either turkey anti-TGF-81 (19) or rabbit anti-TGF-I32
TABLE TRANSFORMING GROWTH FACTOR-BETA IN HUMAN AQUEOUS HUMOR PATIENT # AGE/SEX/RACE TRANSFORMING GROWTH % PRESENT IN % TYPE 2
FACTOH BETA (NG/ML)* 7.8
t
2.6
5.6
t
1.9
15
3
5.7
t
2.4
36
4
6.6 t 3.7
23
78
5
5.6
t
1.8
39
76
6
3.3
* 1.4
60
65
74
7
3.6 t 1.0
48
8
5.8 (5.0, 6.61 (2)
35
9
5.1 (4.6, 5.61 (2)
76
10
3.9
t
0.9
100
11
3.7
t
2.1
48 100
12
4.1 t 0.9
13
2.8
1.7
53
14
3.6 i2.3
100
15
5.1
94
16
2.8 [2.3, 3.31 (2)
17
4.5
t
1.8
88
18
3.9
t
0.8
76
19
2.4 (2.3, 2.51 (2)
20
6.2
t
*
28
1 2
i
% PlPE 1
ACTIVE FORMt lSOFORM* ISOFORM
2.2
(11
21
8.1
(1)
54
22
2.3
(11
65
23
2.5
(11
76
24
3.2
(11
95 64
0 0
94
0
94
0
*Mean * SD of 3 separate determinations, except where number Is indicated in parentheses. Where only two determinations were made, the actual values are given within brackets.
'Single determination of the % of the total assayable TGF-beta that Is actlve. *Values represent a single determination (see Figure 2). -
965
Current Eye Research (21) before addition to the CCL-64 growth inhibition
The ability of aqueous humor samples to inhibit the
assay. These polyclonal antibodies, whose specificlty
CCL-64 growth assay was unaltered by preincubation
has been rigorously tested (20), specifically block the
with anti-TGF-A1 (n= 8 ) , nor was any TGF-I31 detected in the sandwich ELISA assay (n = 10). In contrast,
activity of TGF-A1 and 02, respectively. Secondly, the relative amounts of these 2 isoforms of TGF-A were determined in a sandwich ELSA assay (21).
between 64 and 95% (mean ? SD = 80 t 13%) of the ability of the aqueous humor samples (n=8) to inhibit CCL-64 growth was blocked by preincubation with
RESULTS
antibodies against TGF-fi2 (Figure 2). Furthermore,
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The patients ranged in age from 45 to 87 years
TGF 02 was detected in 10/10 samples tested in the
(mean t SD; 67 t 14). There were 12 men and 12
sandwich ELlSA assay (data not shown); TGF-A1 was
women. 23 patients were white, one was black
not detected in any of these samples.
(Table). Each aqueous humor sample contained
Comparison of the activity of the aqueous humor
detectable levels of TGF-B ranging from 2.3 to 8.1
samples in the CCL-64 growth inhibition assay before
ng/ml (mean ? S.D. = 4.5 t 1.7 ng/ml; Table). The
and after heat activation (n =20) revealed that between
dose-response curves for inhibition of the CCL-64
15 and 100% (mean ? SD= 61 k 27%) of the TGF-13
growth assay by aqueous humor were similar in shape
was present in active form in aqueous humor.
to the curve generated by the addition of purified porcine TGF-A1 (Figure 1).
40
! Human Aqueous (pl/mL)
TGF-I31 (pglmL) or Aqueous (pllmL)
Figure 1. Growth inhibitory activrty of human aqueous humor for mink lung epithelial CCL-64 cells. Serial dilutions of heat-activated human aqueous humor (patients 1,4; 4p; and 5,o)were tested for their ability to inhibit uptake of '251-iodo-deoxyuridine. Concentrations of transforming growth factor-8 in the aqueous humor were estimated by comparison with a standard curve of porcine transforming growth factor4 ( 0 ) . Shown is a representative experiment of 3 such assays for each sample. Maximum incorporation of untreated cells (control) was 2670 cpm.
966
Figure 2. Anti-transforming growth factor-oZ antibodies, but not anti-transforming growth factor-81 antibodies, block the growth inhibitory activity of human aqueous humor. To aliquots of human aqueous humor (patient #4) were added either turkey control serum (1 pl/mL;o); turkey anti-transforming growth factor-A1 #366 (1 pl/mL;m); rabbit IgG (45 pg/mL;o), or rabbit anti-transforming growth factor-02 SAM (50pg/mL;o). Samples were then assayed for their growth inhibitory activity. The proportion of transforming growth factor42 was calculated by comparing the level of assayable transforming growth factor4 in the presence and absence of the antibodies. Only anti-transforminggrowth factor-I32 antibodies reduced the activity. Maximum incorporation was 7840 cpm.
Current Eye Research No correlation was detected between the age (r=
is unknown at the present time. source of the TGF-A~
0.08, p= 0.72, linear regression analysis) or gender
However, retinal pigment epithelial cells in culture have
(males, 4.3 5 1.5; females 4.8 5 1.9; p =0.49, unpaired
been shown to secrete predominantly TGF-132 (26).
t test) of the patient and the concentration of TGF-8.
Moreover, the neural tissue of the eye including the photoreceptor cells, ganglion cells, and bipolar cells all
DISCUSSION
Curr Eye Res Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
The TGF-As are a set of multifunctional proteins
stain intensely for TGF-13’s 2 and 3; no staining for TGF-A1 is detected in these cells (K. Flanders and K.
with a variety of effects (15). A growing body of
Unsicker, personal communication).
evidence suggests that they play a pivotal role in the process of wound repair (22). For this reason they
The significance of this preponderance of the 82 isoform in aqueous humor is unclear. In most
may also be important in wound healing processes
bioassays, including the CCL-64 growth inhibition
within the eye and on the ocular surface.
assay that we employed, the isoforms of TGF-A are
TGF-D has been detected in the rat cornea after
functionally indistinguishable. One notable exception
epithelial wounding (23), in the retina and choroid of
is the almost 100-fold greater ability of TGF-A1 to
monkeys following argon laser photocoagulation (24),
inhibit growth of endothelial cells in culture (27). This
in the embryonic mouse eye (25), and in the vitreous
suggests differential activities of these 2 isoforms in
of eyes with retinal detachments (9). Granstein et al.
control of angiogenesis which could result in an
(2) have reported the presence of TGF-A in murine
altered healing response pattern. In some aqueous humor samples, a portion of the
and human aqueous humor at levels of 200 to lo00 pg/ml after acid activation. We now report the
TGF-A activity was not neutralized by antisera against
presence of significantly higher concentrations of TGF-
either the a1 or 132 isoforms. Two possible
D in a series of carefully obtained aqueous humor
explanations are that some of the inhibitory activity in
specimens from human eyes with well-defined clinical characteristics. Furthermore, we have characterized it
the assay was not due to TGF-A or that a portion of the TGF-A in the aqueous humor represents another
as the 82 isoform and have demonstrated that the
isoform, ~3 (15). The latter possibility is supported by
majority is present in the activated form. The composition of our aqueous humor samples
the observation that both messenger RNA for A3 (by
may have been influenced by the presence of cataract
imrnunohistochemistry) are detectable in the mouse
or by the pharmacologic and mechanical interventions
retina and many other tissues of neural origin (B.
that preceded the sampling. These samples,
Marascalco and K. Unsicker, personal
however, represent the closest to unaltered aqueous
communication). Therefore, it is possible that TGF-83
humor that we could obtain. The presence of TGF-13
may be present in aqueous humor. Unfortunately, no
in the samples is unlikely to be due solely to
antibodies are currently available for selective detection of this isoform.
preoperative interventions because we and others have detected TGF-A in other species that have
in situ hybridization) and the peptide itself (by
In most biological fluids, including vitreous (9),
undergone only minimal manipulation before sampling
most of the TGF-A is present in a biologically inactive
(2).
form (28), and requires either heat or acid treatment to We detected only the A2 isoform of TGF-R. Since
express its activity (18). Therefore, the large
TGF-A2 is not found in human serum or platelets, the
proportion of TGF-A present in active form in the
TGF-8 detected in the aqueous humor does not reflect
aqueous humor (15 to loo%,with an mean value of 61 27%), is striking. Although the significance of
contamination from the serum during sampling, The
967
Current Eye Research this observation is not clear, the presence of so much active form of TGF-I3 in aqueous humor suggests a possible role in normal ocular physiology. Joseph et al. (4) have determined that aqueous
CORRESPONDING AUTHOR Henry D. Jampel, M.D., Maumenee 8-110, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.
humor is chemotactic for ocular surface fibroblasts and have reported that this activity resides in substances with molecular weights greater than 30 kD and is deactivated by low pH (29). TGF-I3 has been shown to have potent chemotactic activity for human Curr Eye Res Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
fibroblasts (11); however, it has a molecular weight of 25 kD and is activated by acid treatment (15). These
data suggest that the chemoattractant activity of aqueous humor might be due to factors other than TGF-A. Our identification of TGF-13 in aqueous humor and the availability of specific blocking antibodies to TGF-B’s 1 and 2 now permit direct testing of a possible role for this peptide in chemotaxis of ocular fibroblasts. The detection of TGF-A in the aqueous humor raises the question of its origin. Preliminary results suggest that cells fcom either the iris and/or ciliary body may secrete TGF-13 in vitro (30). Immunohistochemical techniques and in situ hybridization should permit localization of the protein in tissues of the anterior segment, and the determination of which cells are actually synthesizing TGF-8. Glaucoma filtration surgery generally fails due to scarring in the subconjunctival space (31). The TGF-s present in the aqueous humor may play a role in the wound healing process following filtration surgery.
ACKNOWLEDGMENTS Supported in part by a grant from National Glaucoma Research, American Health Assistance Foundation, Rockville, MD (Dr. Jampel).
The authors would like to thank David Danielpour, PhD. for his help with the immunoassays and with the sandwich ELISA.
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m,
m,
m,
~
m,
m,
969
