Structural characterization of the circulating soluble FGF receptors reveals multiple isoforms generated by secretion and ectodomain shedding.

Soluble fibroblast growth factor receptors (FGFRs) have been identified in multiple biological fluids, including blood. Efforts to examine the biological properties of these proteins have been hampered by the incomplete chemical characterization of the receptors within the second half of the third immunoglobulin (Ig)-like domain, where alternative splicing leads to receptor variants with different ligand binding properties. Using mass spectrometry techniques, we have mapped the soluble FGFRs to the secreted receptor, FGFR1(IIIa), the two and three Ig-like domain isoforms of FGFR1(IIIc) and a carboxyl-terminal cleavage peptide from the two and three Ig-like domain isoforms of FGFR1(IIIb). The secreted FGFR is produced by the translation of an alternatively spliced transcript and the cleaved receptors are released by ectodomain shedding of the transmembrane receptors.

Silicone oil tamponade to seal macular holes without position restrictions.

OBJECTIVE: The authors performed a study to determine the effectiveness and safety of silicone oil as a substitute for gas to fill the vitreous cavity to treat macular holes. DESIGN: Multicenter, nonrandomized, interventional trial. PARTICIPANTS: Thirty-seven consecutive patients chose vitrectomy with silicone tamponade instead of gas to treat 40 eyes with stage-2 to stage-4 idiopathic age-related macular holes. Stage-2 holes constituted 40% of the holes, and stage-3 and stage-4 holes made up 60%. INTERVENTION: All eyes were treated with vitrectomy, manual detachment of the posterior vitreous face (not done for stage-4 holes), autologous serum instillation, and silicone fill of the vitreous cavity. After insertion of the oil, the patients resumed normal activity with no restriction of head or eye position except to avoid faceup position. The oil was removed after approximately 6 weeks. MAIN OUTCOME MEASURES: The authors considered the seal of the macular hole and the preoperative and postoperative logarithm of the minimum angle of resolution (logMAR) visions the most significant measures for comparison to other studies. RESULTS: Eighty percent of all holes and 86% of holes not treated previously were sealed with a single silicone tamponade of the vitreous cavity. The logMAR value of visual acuity improved an average of 0.26 (2.6 lines) to 0.61 (20/81) for all eyes and 0.34 (3.4 lines) to 0.52 (20/66) when the macular hole sealed. Completeness of fill of the vitreous cavity with silicone affected seal of the macular hole. Three of eight eyes in which open holes developed after oil removal had less than 90% fill of the vitreous cavity by silicone. Sixty-nine percent of lenses increased opacity one grade or were removed after silicone tamponade. There were no significant adverse effects arising from silicone tamponade. CONCLUSIONS: Silicone oil tamponade of macular holes is effective and safe. Silicone may be optimal for the treatment of macular holes in persons who must travel, who cannot maintain facedown positioning, or who have monocular vision. The most important factor in the successful closure of the macular hole was the completeness of fill of the vitreous cavity with silicone oil.

FGF receptor-1 (flg) expression is correlated with fibre differentiation during rat lens morphogenesis and growth.

Our previous studies indicate an important role for fibroblast growth factor (FGF) in lens development. Here we study the expression of the flg variant of FGF receptor 1 (FGFR1) during lens development by immunohistochemistry and in situ hybridisation. FGFR1 was expressed throughout lens development. Prominent FGFR1 immunoreactivity was associated with cell nuclei, particularly in differentiating lens fibres, suggesting internalisation and nuclear translocation of the receptor. FGFR1 immunoreactivity was also associated with basolateral membranes of cells in the equatorial region and at lens sutures. FGFR1 mRNA was only weakly expressed during early lens morphogenesis but expression increased with the onset of lens fibre differentiation. Once the lens acquired its distinct polarity, an anteroposterior gradient in both protein reactivity and mRNA signal was evident. Anteriorly, central epithelial cells showed weak expression for FGFR1, whereas more posteriorly, in the germinative and transitional zones of the lens where cells maximally proliferate and undergo early stages of fibre differentiation, respectively, expression was significantly stronger. The anteroposterior gradient of increased expression of FGFR1 in the lens coincides with the previously documented anteroposterior gradient of FGF stimulation. In lens epithelial explants, FGF stimulation was found to upregulate FGFR1 expression. Such upregulation may be an important mechanism for generating a high level of FGF stimulation and ensuring a fibre differentiation response. In postnatal rat lenses, there was a significant age-related decline in FGFR1 expression; this correlates with the reduced rate of lens fibre differentiation with age. Overall, these studies support the hypothesis that FGF and FGFR1 are important for regulation of lens fibre differentiation throughout lens development.

Soluble forms of the high-affinity fibroblast growth factor receptor in human vitreous fluid.

PURPOSE: Fibroblast growth factor-binding proteins (FGF-BPs) have been identified recently in blood and other biologic fluids and have been shown to be identical to a truncated form of the high-affinity cell surface FGF receptor. The authors examined the hypothesis that FGF-BPs also are present in human vitreous fluid. METHODS: Vitreous fluid obtained from 12 patients was incubated overnight with heparin-Sepharose, FGF-2 heparin-Sepharose, and wheat germ agglutinin (WGA)-Sepharose, a lectin known to bind to high-affinity FGF receptors. The precipitated proteins were characterized by immunoblotting using two FGF receptor antibodies raised to either the extracellular domain of FGFR-1 or the intracellular domain of FGFR-1. RESULTS: A 70- to 85-kd FGF-BP was detected in the vitreous in each of the 12 eyes examined. A 55-kd FGF-BP was detected in six of the samples. Both the 70-to 85-kDa and the 55-kDa proteins were precipitated by FGF-2 heparin-Sepharose but not by heparin-Sepharose alone, suggesting that the interaction was dependent on the presence of FGF-2. The proteins bound avidly to WGA-Sepharose. Western blot analysis revealed that the proteins were recognized by an antibody raised to the extracellular domain of the high-affinity FGF receptor but not by an antibody raised to the intracellular domain of the FGF receptor, indicating they are likely to be truncated portions of the extracellular domain of the high-affinity FGF receptor. CONCLUSIONS: Vitreous fluid contains 70- to 85-kd and 55-kd FGF-BPs that have biochemical and immunologic characteristics similar to the extracellular domain of the high-affinity FGF receptor. These naturally occurring FGF-BPs may sequester free FGF in the vitreous cavity and may modulate the biologic activity of FGF in vitreoretinal diseases.

A fibroblast growth factor binding protein in human cerebral spinal fluid.

A soluble protein which binds basic fibroblast growth factor (FGF-2) has been detected in cerebral spinal fluid from normal individuals and patients with Alzheimer's disease and Parkinson's disease. This 70-85 kDa protein is recognized by an antibody to the extracellular domain of the high affinity FGF receptor and is not detected by an antibody to the intracellular domain of the FGF receptor, suggesting that it consists of a truncated portion of the extracellular domain of the high affinity FGF receptor. This FGF-BP appears to be identical to the two IgG-like loop form of FGFR-1 identified and purified from human and bovine blood. The possibility that this FGF-BP may play a role in transporting, sequestering, or even delivering the FGFs to target cells in the CNS is discussed.

High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.

Identification of soluble forms of the fibroblast growth factor receptor in blood.

We have purified three acidic (FGF-1) and basic (FGF-2) fibroblast growth factor binding proteins (FGF-BP1, FGF-BP2, and FGF-BP3) from human plasma and calf serum and demonstrate the presence of these circulating FGF-BPs in blood. Each are truncated forms of the high-affinity FGF receptor (FGFR-1). FGF-BP1 and FGF-BP2 have estimated molecular masses of 70-85 kDa and 55-60 kDa, respectively, and are detected by using 125I-labeled FGF-2 ligand blotting. Immunoblotting with four distinct antibodies to FGFR-1 reveals that FGF-BP1 and FGF-BP2 are immunologically and biochemically related to the extracellular domain of FGFR-1. Reverse-phase HPLC chromatography resolves FGF-BP2 into two proteins with estimated molecular masses of 55 kDa and 60 kDa. Protein sequencing of the amino terminus of FGF-BP2 and FGF-BP3 reveals identity with the extracellular domain of the two-IgG-loop form of human FGFR-1. The FGF-BPs do not require heparin to bind FGF-2 on affinity columns, but heparin does enhance their recovery from blood. These FGF-BPs may play an important physiological role in regulating the biological activity of FGF and the other members of the FGF family of growth factors.

Altered distribution of basic fibroblast growth factor in diabetic retinopathy.

Basic fibroblast growth factor (FGF) is a potent endothelial cell mitogen that has been proposed to play a role in proliferative diabetic retinopathy and other neovascular processes. Our understanding of the in vivo role of basic FGF in the pathogenesis of these disorders is limited. We studied the immunolocalization of basic FGF in 16 clinical cases of diabetic retinopathy to determine whether the normal retinal distribution of basic FGF changed during the development of diabetic retinopathy and correlated with the onset of retinal neovascularization. By using monoclonal and affinity-purified polyclonal antibodies against basic FGF and heparan sulfate proteoglycan (HSPG), we found that basic FGF colocalized with HSPG to vascular basement membranes. As the basement membranes thickened during the progression of diabetic retinopathy, the intraretinal stores of immunoreactive basic FGF and HSPG expanded. With the development of neovascularization, the colocalization of basic FGF and HSPG changed; HSPG localized to basement membranes, while basic FGF localized intracellularly, with only minimal basement membrane immunoreactivity. Incubations of the neovascular fronds with exogenous basic FGF demonstrated multiple HSPG glycosaminoglycan-binding sites for basic FGF, indicating that basic FGF had not been released from the matrix of neovascular fronds by heparitanase digestion.

The management of retinal detachments associated with choroidal colobomas by vitreous surgery.

We used vitreous surgery to treat seven patients (eight eyes) with complicated retinal detachments associated with choroidal colobomas. All eyes had large choroidal colobomas and no evidence of peripheral retinal breaks. Small, atrophic breaks were detected in five of the eyes and were located in the base of the coloboma in four of the five eyes. Adjunctive surgical techniques were necessary and included cyanoacrylate retinopexy in four eyes, silicone oil tamponade in five eyes, and retinectomy in two eyes. Retinal reattachment was ultimately attained in seven of the eight eyes. The number of surgical procedures ranged from one to five, with an average of three. Postoperative visual acuity of the eyes that underwent anatomically successful procedures ranged from 20/100 to light perception. Proliferative vitreoretinopathy was the most frequent cause of redetachment, occurring in six of the eight eyes.

Treatment of carotid-cavernous sinus fistulas using a detachable balloon catheter through the superior ophthalmic vein.

Four consecutive patients with carotid-cavernous sinus fistulas that could not be treated by the standard techniques of endoarterial balloon occlusion or embolization were successfully treated by advancement of a detachable balloon catheter through the ipsilateral superior ophthalmic vein. Under angiographic monitoring, the balloon was passed into the cavernous sinus, inflated to close the fistula, and detached. Three of the patients had a spontaneous fistula, and one had a traumatic fistula that had previously been trapped unsuccessfully. All patients had complete resolution of symptoms and signs after occlusion of the fistula. There were no intraoperative or postoperative complications. The transvenous approach to the cavernous sinus through the superior ophthalmic vein is a safe, effective treatment of carotid-cavernous sinus fistulas, whether they are direct or dural in nature.

Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina.

The basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15-amino-acid sequence from the amino terminus of basic FGF in order to avoid cross-reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.

Vitrectomy and scleral buckling methods for proliferative vitreoretinopathy.

The authors operated on 95 eyes with retinal detachment (RD) complicated by proliferative vitreoretinopathy (PVR) using vitrectomy combined with scleral buckling. The retina was successfully reattached in 10 (91%) of 11 eyes with mild PVR (B, C1), 14 (93%) of 15 eyes with moderate PVR (C2-C3), and 52 (75%) of 69 eyes with severe PVR (D1-D3). Thirteen (88%) of 15 eyes in which silicone oil was injected were successfully reattached. Eighty-eight percent of the 76 successfully reattached cases had a postoperative visual acuity of 5/200 or better; the median postoperative visual acuity was 20/200. Severe PVR was the cause of failure in 17 (89%) of 19 eyes in which the retina was not reattached.

Choroidopathy in systemic lupus erythematosus.

Choroidopathy in association with systemic lupus erythematosus (SLE) is a clinically unusual manifestation, previously described in only six patients, to our knowledge. We have followed up six patients with SLE and choroidopathy manifested by multifocal, serous elevations of the retinal pigment epithelium and sensory retina. In four patients, macular involvement was present, and they suffered visual loss. Two eyes of two patients progressed to large, bullous, exudative retinal detachments. In the three patients in whom control of the systemic disease was achieved, the serous detachments resolved. The pathogenesis is most likely related to choroidal vascular disease with resultant pigment epithelial damage and serous fluid leakage beneath the retina.

Uremic optic neuropathy.

Vision loss progressing over several days, reduced pupil reactions to light, and swollen optic nerves were the clinical features in six patients with severe renal disease manifested by uremia, anemia, and (in four patients) moderately or severely elevated blood pressure. In two patients pale edema of the optic nerve head extended into the macula. One patient with renal transplant rejection was in the early phases of cryptococcal meningitis that went undiagnosed for two weeks. Medical management with hemodialysis was followed by improvement of vision in four patients. In one patient, resumption of oral corticosteroid therapy was followed by improvement in vision. The patient whose vision improved the most rapidly was managed by prompt use of both dialysis and oral corticosteroid therapy. The patient with cryptococcal meningitis did not recover vision.

Osmotic tolerance of rabbit and human corneal endothelium.

Rabbit and human corneas were mounted in a specular microscope and perfused with a balanced salt solution of varying osmolality (200 to 500 mOsm). Measurements of corneal thickness were made throughout the perfusion period, and at selected times the corneas were fixed and prepared for scanning and transmission electron microscopy. A hypo-osmotic perfusion medium caused an increase in corneal thickness; by comparison, a hyperosmotic perfusion medium decreased corneal thickness in both rabbit and human corneas. Despite the marked changes in corneal thickness and the water movement that occurred across the endothelium, the cellular ultrastructure remained intact. In reversal studies (return to 300-mOsm perfusion medium), corneal thickness returned to control values with no marked changes in endothelial cell structure. These data indicate that the corneal endotheium can tolerate a wide range of solution osmolalities (200 to 400 mOsm) without marked endotheial cell breakdown if the essential ions are present.