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Expression of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration
experimental studies in the rabbit eye. Am J Ophthalmol 1998;125:627– 634. 5. de Juan E, Loewenstein A, Bressler NM, Alexander J. Translocation of the retina for management of subfoveal choroidal neovascularization, II: a preliminary report in humans. Am J Ophthalmol 1998;125:635– 646.
Expression of Matrix Metalloproteinase-7 in Choroidal Neovascular Membranes in Age-related Macular Degeneration Kazuaki Kadonosono, MD, Futoshi Yazama, PhD, Norihiko Itoh, DVM, Hajime Sawada, MD, and Shigeaki Ohno, MD Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes associated with neovascularization. We evaluated the expression and localization of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration. METHODS: Immunofluorescence and transmission electron microscopic examinations were performed on subfoveal neovascular membranes that had been surgically removed from seven eyes of seven patients with agerelated macular degeneration. RESULTS: Matrix metalloproteinase-7 was expressed in all specimens and distinctly expressed in the thickened layer of Bruch membrane and basement membrane–like structure around retinal pigment epithelial cells. CONCLUSIONS: Matrix metalloproteinase-7 was expressed in Bruch membrane of choroidal neovascular membranes in age-related macular degeneration. Matrix metalloproteinase-7 may be an important factor for the development of the submacular neovascular membrane in age-related macular degeneration. (Am J Ophthalmol 1999;128: 382–384. © 1999 by Elsevier Science Inc. All rights reserved.) PURPOSE:
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FIGURE 1. (Top) Immunolocalization of matrix metalloproteinase-7 in surgical specimens of subretinal neovascular membranes from an eye with age-related macular degeneration. Semi-thin frozen sections were labeled with mouse anti– human matrix metalloproteinase-7 monoclonal antibody, and fluorescein isothiocyanate– coupled goat anti–mouse IgG antibody was used for detection. Retinal pigment epithelial cells show yellow autofluorescence. Anti-matrix metalloproteinase-7 antibody is labeled intensely in the layer below retinal pigment epithelial cells (arrows), showing specific apple-green fluorescence. A weak signal is also seen around the retinal pigment epithelial cells (arrowheads). (Bottom) Nomarski views of the same fields as in the top panel. The pigment of retinal pigment epithelial cells is recognized as orange particles (arrows). Arrowheads point to the margin of the thickened Bruch membranes.
HOROIDAL NEOVASCULAR MEMBRANES ARE RESPON-
sible for severe vision loss in age-related macular degeneration, which is becoming a major cause of legal blindness in Japan, as in the United States and European countries. Precursors to choroidal neovascular membranes are soft drusen or pigment epithelial detachment observed by biomicroscopy. Visual acuity is not markedly compromised at this stage. However, in the developed stage of
choroidal neovascular membranes, vision loss occurs. Previous pathologic examinations have shown that the choroidal neovascular membrane extends from the choroid, perforates Bruch membrane, and then proliferates in the sub–pigment epithelium space.1 The matrix metalloproteinases are a group of enzymes involved in physiologic and pathologic processes associated with extracellular matrix remodeling. Matrix metalloproteinases function to degrade and remodel the extracellular matrix and play an important role in various diseases associated with neovascularization. A few studies have demonstrated matrix metalloproteinase-2,9 expression in the choroidal neovascular membranes, but little is known about the distribution and
Accepted for publication April 1, 1999. From the Departments of Ophthalmology (K.K., N.I., S.O.) and Anatomy (F.Y., H.S.), Yokohama City University School of Medicine, Yokohama, Japan. Inquiries to Kazuaki Kadonosono, MD, Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fukuura Kanazawaku, Yokohama 236-0004, Japan; fax: 81-45-781-9755.
382
AMERICAN JOURNAL
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OPHTHALMOLOGY
SEPTEMBER 1999
FIGURE 2. Transmission electron micrograph of subretinal neovascular membranes surgically removed from an eye with age-related macular degeneration. Retinal pigment epithelial (RPE) cells varying in size and shape can be seen. Asterisks indicate basement membrane–like structures around the retinal pigment epithelial cells. A thickened layer of homogeneous, globular deposits of moderate electron density can be seen beneath the retinal pigment epithelial cells. L ⴝ lumen of capillary. The inset shows a higher magnification around the retinal pigment epithelial cells. Note the basement membrane–like structures (arrows) and the long-spacing banded collagen material (arrowheads) in the thickened layer under the retinal pigment epithelial cells.
localization of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration.2,3 Matrix metalloproteinase-7 has been identified as a putative metalloproteinase and is thought to be a useful marker of biologic aggressiveness in human carcinomas.4 We used immunofluorescence and transmission electron microscopic examination of the subfoveal fibrovascular membranes surgically removed from seven eyes of seven patients with age-related macular degeneration (three women and four men, aged 71 to 79 years) to examine the expression of matrix metalloproteinase-7. Four eyes had laser treatment, and the other three eyes had no therapy. The specimens were prepared for semi-thin frozen sectioning,5 and 1-m frozen sections were rehydrated with phosphate-buffered saline and blocked with 50% calf serum in phosphate-buffered saline. Then, anti-matrix metalloproteinase-7 antibody or antilaminin antibody diVOL. 128, NO. 3
luted in 50% calf serum was applied to the section, which was then incubated for 2 hours at room temperature. The sections were washed three times with phosphatebuffered saline, 10 minutes each time, at room temperature and mixed with fluorescein isothiocyanate– conjugated goat anti–mouse immunoglobulin at a dilution of 1:100 in 50% calf serum for 2 hours. In control specimens, primary antibodies were omitted. These specimens were observed and photographed with a fluorescence microscope. In all the surgically removed membranes, anti-matrix metalloproteinase-7 antibody was labeled intensely in the layer below retinal pigment epithelial cells, and a weak signal of matrix metalloproteinase-7 was also recognized around the retinal pigment epithelial cells (Figure 1). These areas positive for matrix metalloproteinase-7 corresponded to laminin localization (not shown). Transmission electron microscopic observation disclosed that the
BRIEF REPORTS
383
layer beneath the retinal pigment epithelial cells was the basal laminar deposit, which was characterized by scattered long-spacing banded collagen material, and positive areas around retinal pigment epithelial cells corresponded to basement membrane–like structures (Figure 2). These results show that matrix metalloproteinase-7 may be present in Bruch membrane in choroidal neovascular membranes and strongly implicate matrix metalloproteinase-7 in degradation of basement membranes and neovascularization in the choroid. Matrix metalloproteinase-7 may degrade the extracellular matrix in Bruch membrane and be associated with the perforation of new vessels into Bruch membrane. These results suggest that matrix metalloproteinase-7 and matrix metalloproteinase-2,9 are potentially important factors in the development of choroidal neovascular membranes.
RESULTS:
Postoperatively, at 6 and 5 months, respectively, both patients reported new visual sensation in the visual field corresponding to the transplant. In both patients, the visual sensation continued to be present after transplantation, at 12 and 8 months, respectively. In Patient A, a transient multifocal electroretinography (mfERG) response was observed in the transplant area 4 months postoperatively but was not detectable in Patient A at 6.0 and 9.5 months post–retinal transplantation. In Patient B, no positive mfERG responses were seen up to 5 months postoperatively. No rejection (presenting as cystoid macular edema, macular pucker, and extensive intraretinal edema with disrupted retinal pigment epithelium) to the transplanted tissue was seen up to 13 months in Patient A and 9 months in Patient B by fluorescein angiography. CONCLUSION: Transplantation of intact sheets of fetal human retina in two patients with retinitis pigmentosa was not associated with evidence of transplant rejection. Subjective improvement and an indication of objective improvement 4 months postoperatively were seen in Patient A, and subjective improvement only was seen in Patient B. (Am J Ophthalmol 1999;128:384 –387. © 1999 by Elsevier Science Inc. All rights reserved.)
REFERENCES
1. Das A, Puklin JE, Frank RN, Zhang NL. Ultrastructural immunocytochemistry of subretinal neovascular membranes in age-related macular degeneration. Ophthalmology 1992;99: 1368 –1376. 2. Steen B, Sejersen S, Berglin L, Seregard S, Kvanta A. Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1998;39:2194 –2199. 3. De La Paz MA, Itoh Y, Toth CA, Nagase H. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci 1998;39:1256 –1260. 4. Mori M, Barnard GF, Mimori K, et al. Overexpression of matrix metalloproteinases-7 mRNA in human colon carcinomas. Cancer 1995;75:1516 –1519. 5. Yazama F, Esaki M, Sawada H. Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: electron microscopic localization with improved methodology. Anat Rec 1997;248:51– 62.
R
diseases that may result in legal or total blindness. Rod photoreceptors degenerate first, and cone photoreceptors and retinal pigment epithelium degenerate in later stages. Sheets of adult human photoreceptor cells harvested from human cadaveric eyes have been safely transplanted to the subretinal space of two retinitis pigmentosa patients without systemic immunosuppression. However, no improved function has been reported.1 The motivation for this human trial was our success in transplanting intact sheets of fetal retina to restore damaged rat retinas.2 Those transplants developed an organized photoreceptor layer with outer segments in contact with the host retinal pigment epithelium2 and showed a phototransduction protein dark-light shift like normal photoreceptors.3 We have developed a unique instrument and method to
Preliminary Report: Indications of Improved Visual Function After Retinal Sheet Transplantation in Retinitis Pigmentosa Patients
Accepted for publication June 28, 1999. From the Department of Ophthalmology and Visual Sciences (N.D.R., R.B.A., M.S., H.M.P.), the Department of Anatomical Sciences and Neurobiology (R.B.A., M.S.), and the Department of Psychological and Brain Sciences (H.M.P.), University of Louisville School of Medicine, Louisville, Kentucky. This work was supported by the Vitreoretinal Research Foundation, Louisville, Kentucky (N.D.R.), the Jewish Hospital Foundation, Louisville, Kentucky (R.B.A., M.S.), National Institutes of Health grant R01EY08519, Bethesda, Maryland (R.B.A., M.S.), the Murray Foundation, Inc, New York, New York (R.B.A., M.S.), Foundation Fighting Blindness, Hunt Valley, Maryland (R.B.A., M.S.), an unrestricted grant from Research to Prevent Blindness, Inc, New York, New York (Department of Ophthalmology, University of Louisville), and an anonymous donor (R.B.A., M.S., N.D.R.). Norman D. Radtke, MD, Robert B. Aramant, PhD, and Magdalene Seiler, PhD, have a proprietary interest in the instrument and method (patent pending) discussed. Reprint requests to Norman D. Radtke, MD, FACS, Retina Vitreous Resource Center, 240 Audubon Medical Plaza, Louisville, KY 40217; Fax: (502) 634-1646; e-mail: [email protected].
Norman D. Radtke, MD, FACS, Robert B. Aramant, PhD, Magdalene Seiler, PhD, and Heywood M. Petry, PhD PURPOSE:
To report indications of new visual function after retinal transplantation in two blind patients with retinitis pigmentosa. METHODS: Intact sheets of fetal retina (15 and 17 weeks gestational age) were transplanted subretinally (between the neurosensory retina and the retinal pigment epithelium) near the fovea in the left eye of a 23-year-old white man (Patient A) and in the left eye of a 72-year-old white woman (Patient B), both with autosomal-recessive retinitis pigmentosa. 384
AMERICAN JOURNAL
ETINITIS PIGMENTOSA IS A GROUP OF HEREDITARY
OF
OPHTHALMOLOGY
SEPTEMBER 1999
Expression of Matrix Metalloproteinase-7 in Choroidal Neovascular Membranes in Age-related Macular Degeneration Kazuaki Kadonosono, MD, Futoshi Yazama, PhD, Norihiko Itoh, DVM, Hajime Sawada, MD, and Shigeaki Ohno, MD Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes associated with neovascularization. We evaluated the expression and localization of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration. METHODS: Immunofluorescence and transmission electron microscopic examinations were performed on subfoveal neovascular membranes that had been surgically removed from seven eyes of seven patients with agerelated macular degeneration. RESULTS: Matrix metalloproteinase-7 was expressed in all specimens and distinctly expressed in the thickened layer of Bruch membrane and basement membrane–like structure around retinal pigment epithelial cells. CONCLUSIONS: Matrix metalloproteinase-7 was expressed in Bruch membrane of choroidal neovascular membranes in age-related macular degeneration. Matrix metalloproteinase-7 may be an important factor for the development of the submacular neovascular membrane in age-related macular degeneration. (Am J Ophthalmol 1999;128: 382–384. © 1999 by Elsevier Science Inc. All rights reserved.) PURPOSE:
C
FIGURE 1. (Top) Immunolocalization of matrix metalloproteinase-7 in surgical specimens of subretinal neovascular membranes from an eye with age-related macular degeneration. Semi-thin frozen sections were labeled with mouse anti– human matrix metalloproteinase-7 monoclonal antibody, and fluorescein isothiocyanate– coupled goat anti–mouse IgG antibody was used for detection. Retinal pigment epithelial cells show yellow autofluorescence. Anti-matrix metalloproteinase-7 antibody is labeled intensely in the layer below retinal pigment epithelial cells (arrows), showing specific apple-green fluorescence. A weak signal is also seen around the retinal pigment epithelial cells (arrowheads). (Bottom) Nomarski views of the same fields as in the top panel. The pigment of retinal pigment epithelial cells is recognized as orange particles (arrows). Arrowheads point to the margin of the thickened Bruch membranes.
HOROIDAL NEOVASCULAR MEMBRANES ARE RESPON-
sible for severe vision loss in age-related macular degeneration, which is becoming a major cause of legal blindness in Japan, as in the United States and European countries. Precursors to choroidal neovascular membranes are soft drusen or pigment epithelial detachment observed by biomicroscopy. Visual acuity is not markedly compromised at this stage. However, in the developed stage of
choroidal neovascular membranes, vision loss occurs. Previous pathologic examinations have shown that the choroidal neovascular membrane extends from the choroid, perforates Bruch membrane, and then proliferates in the sub–pigment epithelium space.1 The matrix metalloproteinases are a group of enzymes involved in physiologic and pathologic processes associated with extracellular matrix remodeling. Matrix metalloproteinases function to degrade and remodel the extracellular matrix and play an important role in various diseases associated with neovascularization. A few studies have demonstrated matrix metalloproteinase-2,9 expression in the choroidal neovascular membranes, but little is known about the distribution and
Accepted for publication April 1, 1999. From the Departments of Ophthalmology (K.K., N.I., S.O.) and Anatomy (F.Y., H.S.), Yokohama City University School of Medicine, Yokohama, Japan. Inquiries to Kazuaki Kadonosono, MD, Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fukuura Kanazawaku, Yokohama 236-0004, Japan; fax: 81-45-781-9755.
382
AMERICAN JOURNAL
OF
OPHTHALMOLOGY
SEPTEMBER 1999
FIGURE 2. Transmission electron micrograph of subretinal neovascular membranes surgically removed from an eye with age-related macular degeneration. Retinal pigment epithelial (RPE) cells varying in size and shape can be seen. Asterisks indicate basement membrane–like structures around the retinal pigment epithelial cells. A thickened layer of homogeneous, globular deposits of moderate electron density can be seen beneath the retinal pigment epithelial cells. L ⴝ lumen of capillary. The inset shows a higher magnification around the retinal pigment epithelial cells. Note the basement membrane–like structures (arrows) and the long-spacing banded collagen material (arrowheads) in the thickened layer under the retinal pigment epithelial cells.
localization of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration.2,3 Matrix metalloproteinase-7 has been identified as a putative metalloproteinase and is thought to be a useful marker of biologic aggressiveness in human carcinomas.4 We used immunofluorescence and transmission electron microscopic examination of the subfoveal fibrovascular membranes surgically removed from seven eyes of seven patients with age-related macular degeneration (three women and four men, aged 71 to 79 years) to examine the expression of matrix metalloproteinase-7. Four eyes had laser treatment, and the other three eyes had no therapy. The specimens were prepared for semi-thin frozen sectioning,5 and 1-m frozen sections were rehydrated with phosphate-buffered saline and blocked with 50% calf serum in phosphate-buffered saline. Then, anti-matrix metalloproteinase-7 antibody or antilaminin antibody diVOL. 128, NO. 3
luted in 50% calf serum was applied to the section, which was then incubated for 2 hours at room temperature. The sections were washed three times with phosphatebuffered saline, 10 minutes each time, at room temperature and mixed with fluorescein isothiocyanate– conjugated goat anti–mouse immunoglobulin at a dilution of 1:100 in 50% calf serum for 2 hours. In control specimens, primary antibodies were omitted. These specimens were observed and photographed with a fluorescence microscope. In all the surgically removed membranes, anti-matrix metalloproteinase-7 antibody was labeled intensely in the layer below retinal pigment epithelial cells, and a weak signal of matrix metalloproteinase-7 was also recognized around the retinal pigment epithelial cells (Figure 1). These areas positive for matrix metalloproteinase-7 corresponded to laminin localization (not shown). Transmission electron microscopic observation disclosed that the
BRIEF REPORTS
383
layer beneath the retinal pigment epithelial cells was the basal laminar deposit, which was characterized by scattered long-spacing banded collagen material, and positive areas around retinal pigment epithelial cells corresponded to basement membrane–like structures (Figure 2). These results show that matrix metalloproteinase-7 may be present in Bruch membrane in choroidal neovascular membranes and strongly implicate matrix metalloproteinase-7 in degradation of basement membranes and neovascularization in the choroid. Matrix metalloproteinase-7 may degrade the extracellular matrix in Bruch membrane and be associated with the perforation of new vessels into Bruch membrane. These results suggest that matrix metalloproteinase-7 and matrix metalloproteinase-2,9 are potentially important factors in the development of choroidal neovascular membranes.
RESULTS:
Postoperatively, at 6 and 5 months, respectively, both patients reported new visual sensation in the visual field corresponding to the transplant. In both patients, the visual sensation continued to be present after transplantation, at 12 and 8 months, respectively. In Patient A, a transient multifocal electroretinography (mfERG) response was observed in the transplant area 4 months postoperatively but was not detectable in Patient A at 6.0 and 9.5 months post–retinal transplantation. In Patient B, no positive mfERG responses were seen up to 5 months postoperatively. No rejection (presenting as cystoid macular edema, macular pucker, and extensive intraretinal edema with disrupted retinal pigment epithelium) to the transplanted tissue was seen up to 13 months in Patient A and 9 months in Patient B by fluorescein angiography. CONCLUSION: Transplantation of intact sheets of fetal human retina in two patients with retinitis pigmentosa was not associated with evidence of transplant rejection. Subjective improvement and an indication of objective improvement 4 months postoperatively were seen in Patient A, and subjective improvement only was seen in Patient B. (Am J Ophthalmol 1999;128:384 –387. © 1999 by Elsevier Science Inc. All rights reserved.)
REFERENCES
1. Das A, Puklin JE, Frank RN, Zhang NL. Ultrastructural immunocytochemistry of subretinal neovascular membranes in age-related macular degeneration. Ophthalmology 1992;99: 1368 –1376. 2. Steen B, Sejersen S, Berglin L, Seregard S, Kvanta A. Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1998;39:2194 –2199. 3. De La Paz MA, Itoh Y, Toth CA, Nagase H. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci 1998;39:1256 –1260. 4. Mori M, Barnard GF, Mimori K, et al. Overexpression of matrix metalloproteinases-7 mRNA in human colon carcinomas. Cancer 1995;75:1516 –1519. 5. Yazama F, Esaki M, Sawada H. Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: electron microscopic localization with improved methodology. Anat Rec 1997;248:51– 62.
R
diseases that may result in legal or total blindness. Rod photoreceptors degenerate first, and cone photoreceptors and retinal pigment epithelium degenerate in later stages. Sheets of adult human photoreceptor cells harvested from human cadaveric eyes have been safely transplanted to the subretinal space of two retinitis pigmentosa patients without systemic immunosuppression. However, no improved function has been reported.1 The motivation for this human trial was our success in transplanting intact sheets of fetal retina to restore damaged rat retinas.2 Those transplants developed an organized photoreceptor layer with outer segments in contact with the host retinal pigment epithelium2 and showed a phototransduction protein dark-light shift like normal photoreceptors.3 We have developed a unique instrument and method to
Preliminary Report: Indications of Improved Visual Function After Retinal Sheet Transplantation in Retinitis Pigmentosa Patients
Accepted for publication June 28, 1999. From the Department of Ophthalmology and Visual Sciences (N.D.R., R.B.A., M.S., H.M.P.), the Department of Anatomical Sciences and Neurobiology (R.B.A., M.S.), and the Department of Psychological and Brain Sciences (H.M.P.), University of Louisville School of Medicine, Louisville, Kentucky. This work was supported by the Vitreoretinal Research Foundation, Louisville, Kentucky (N.D.R.), the Jewish Hospital Foundation, Louisville, Kentucky (R.B.A., M.S.), National Institutes of Health grant R01EY08519, Bethesda, Maryland (R.B.A., M.S.), the Murray Foundation, Inc, New York, New York (R.B.A., M.S.), Foundation Fighting Blindness, Hunt Valley, Maryland (R.B.A., M.S.), an unrestricted grant from Research to Prevent Blindness, Inc, New York, New York (Department of Ophthalmology, University of Louisville), and an anonymous donor (R.B.A., M.S., N.D.R.). Norman D. Radtke, MD, Robert B. Aramant, PhD, and Magdalene Seiler, PhD, have a proprietary interest in the instrument and method (patent pending) discussed. Reprint requests to Norman D. Radtke, MD, FACS, Retina Vitreous Resource Center, 240 Audubon Medical Plaza, Louisville, KY 40217; Fax: (502) 634-1646; e-mail: [email protected].
Norman D. Radtke, MD, FACS, Robert B. Aramant, PhD, Magdalene Seiler, PhD, and Heywood M. Petry, PhD PURPOSE:
To report indications of new visual function after retinal transplantation in two blind patients with retinitis pigmentosa. METHODS: Intact sheets of fetal retina (15 and 17 weeks gestational age) were transplanted subretinally (between the neurosensory retina and the retinal pigment epithelium) near the fovea in the left eye of a 23-year-old white man (Patient A) and in the left eye of a 72-year-old white woman (Patient B), both with autosomal-recessive retinitis pigmentosa. 384
AMERICAN JOURNAL
ETINITIS PIGMENTOSA IS A GROUP OF HEREDITARY
OF
OPHTHALMOLOGY
SEPTEMBER 1999