September 2016
Volume 57, Issue 11
Open Access
Retinal Cell Biology  |   September 2016
Is Angiostatin Involved in Physiological Foveal Avascularity?
Author Affiliations & Notes
  • Michael R. R. Böhm
    Department of Ophthalmology Clinic for Diseases of the Anterior Segments of the Eyes, Essen University Hospital, Essen, Germany
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Florian Hodes
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Katrin Brockhaus
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Stephanie Hummel
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Stefan Schlatt
    Centre for Reproductive Medicine and Andrology, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Harutyun Melkonyan
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Solon Thanos
    Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University of Münster, Münster, Germany
  • Correspondence: Michael R. R. Böhm, Department of Ophthalmology, Clinic for Diseases of the Anterior Segments of the Eyes, Essen University Hospital, 45147 Essen, Germany; michael.boehm@uk-essen.de
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4536-4552. doi:10.1167/iovs.16-19286
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      Michael R. R. Böhm, Florian Hodes, Katrin Brockhaus, Stephanie Hummel, Stefan Schlatt, Harutyun Melkonyan, Solon Thanos; Is Angiostatin Involved in Physiological Foveal Avascularity?. Invest. Ophthalmol. Vis. Sci. 2016;57(11):4536-4552. doi: 10.1167/iovs.16-19286.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The primate central retina is characterized by an avascular fovea and well-defined perifoveal capillary plexus. Neither blood vessels nor their accompanying astrocytes enter the fovea during any stage of retinal development; a balance of angiogenic and angiostatic factors probably maintains foveal avascularity throughout life. The aim of this study was to identify potentially angiorepulsive factors involved in the development of the avascular primate retinal fovea.

Methods: Retinas of newborn, juvenile, and adult Callithrix jacchus and Macaca fascicularis monkeys and control human retinas were studied to determine the localization of angiostatin relative to III β-tubulin, glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule-1 (PECAM), and the angiostatin receptor αvβ3-integrin in the foveal, macular, and peripheral retina. Expression studies were performed using immunohistochemistry (IHC) on retinal whole-mount and paraffin sections, and Western blotting on frozen material. The complex network of the main retinal cell types was identified by IHC of retinal whole mounts.

Results: In general, lifetime expression of angiostatin was found in all retinas. Colabeling with different markers revealed retinal ganglion cells as the main source of angiostatin expression in the primate retina, whereas PECAM-immunopositive blood capillaries expressed the angiostatin receptor αvβ3-integrin, and capillary-associated astrocytes expressed VEGF.

Conclusions: This study provides the first evidence of angiostatin expression in the primate retina; the expression of angiostatin in the avascular foveal region and the peripheral retina suggests that angiostatin may play a role in the regulation of retinal vascularization, providing a possible explanation for the development and persistence of an avascular fovea.

The macular region accounts for approximately 0.01% of the retina in macula-bearing primates. The central-most portion of the human macula, the fovea centralis, has a structural depression (i.e., the foveal groove) with a diameter of approximately 0.35 mm. The fovea is devoid of retinal vessels, and is known as the foveal avascular zone (FAZ).1 
According to previous investigations, vascular endothelial growth factor (VEGF) is the key factor that induces and maintains the ingrowth of retinal vessels during development.25 Retinal ganglion cells (RGCs) express VEGF strongly during retinal development and inhibit the migration of astrocytes and endothelial cells into the macula.6 Furthermore, it has been proposed that angiostatic factors are expressed in the macula during development, regulating vascular growth through repellent mechanisms.7 Although the nature of the inhibitory factor expression involved in the avascularity of the fovea throughout life has yet to be clarified,8 expression of several angiostatic proteins including ephrins A1 and A4 and their receptor EphA6, brain natriuretic peptide (BNP), and pigment epithelium–derived factor (PEDF)1,710 was associated with avascularity. In particular, the critical balance between the angiogenic VEGF and the angiostatic PEDF appears to be essential for preserving11 and stabilizing5,12,13 the avascular environment of the outer retina. There have been no reports to date on the differential expression of PEDF or other angiostatic factors involved in the development and persistence of the FAZ. 
Pathologic conditions such as ischemia and inflammation likely influence this physiological balance of angiogenic and angiostatic factors.12,1418 The angiorepellent factor angiostatin and its receptor integrin αvβ3 have been found in the retina.19 Angiostatin is a circulating inhibitor that is derived from plasminogen and was first described in a mouse model of Lewis lung carcinoma.20 It is generated from circulating plasminogen by proteolytic cleavage.2123 Several studies have described that angiostatin reduces retinal and choroidal neovascularization (CNV), prevents retinal leakage, and affects endothelial cells2427; and a clinical phase I study is currently being performed with RetinoStat (Oxford Biomedica [Oxford, UK], NCT01301443; clinicaltrials.gov). Angiostatin, which has been detected in several species, reduces the proliferation and migration of endothelial cells, inhibits vascular tube formation, increases endothelial cell apoptosis, and exhibits angiostatic properties in vivo.28 
The aim of this study was to describe the lifelong expression and retinal localization of angiostatin in the primate retina. The distribution profiles and cellular localizations of angiostatin are demonstrated herein, which assigns it a potential role in the maintenance of foveal avascularity. 
Materials and Methods
Retinal Tissues
All animal work was conducted under the guidelines of the Animal Welfare Act, in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the oversight and approval of the University and Governmental Institutional Animal Care and Use Committee (LANUV-NRW, permission nos. 39.32.7.2.1 and 84.02.005.20.12.0.18). Human retinas (n = 8 of five individuals) free of known retinal diseases as determined by the requirements for eye banking and transplantation were obtained with written consent from each cornea donor, in accordance with the Declaration of Helsinki and the permit issued by the Ethics Committee of the University for Anonymous Use of Histological Material for Research Purposes. Eyes from marmosets (Callithrix jacchus; 25 retinas of 14 animals) and cynomolgus monkeys (Macaca fascicularis; 16 retinas of 9 animals) were obtained from cadavers. No matching stages of age between the two strains of monkeys were available, therefore limiting direct comparison between the two strains at the same ages. Data regarding the experimental approach and additional information regarding age, sex, and the method used are provided in Table 1
Table 1
 
Experimental Approach
Table 1
 
Experimental Approach
Tissue Preparation
Retinas were obtained by removing eyes from monkey corpses at 10 to 20 minutes after death. The eye cups were removed and placed into ice-cold Hanks' balanced salt solution, in which all subsequent preparation steps were conducted. The retina was freed from other ocular tissues and isolated, flat-mounted on a nitrocellulose filter, and separated from the vitreous body with fine forceps. For whole-mount IHC staining, dissected retinas were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C for 30 minutes. After rinsing in PBS twice (5 minutes each), the retinas were transferred to 30% sucrose (Merck, Darmstadt, Germany) and stored at 4°C for 24 hours. The retinas were then frozen in a mixture of 2-methylbutan/fluid nitrogen (Merck) and stored in PBS at 4°C until further use.29 For Western blotting (WB) the isolated retinas were immediately frozen in liquid nitrogen and stored at −80°C until required for analysis. Paraffin-embedded retinas required for IHC were fixed in 4% PFA for 72 hours, transferred to 70% ethanol for 8 hours, and then soaked sequentially in the following for 15 minutes each: 70% ethanol, 96% ethanol, xylene (twice), and hot paraffin (twice). They were then infiltrated with low-melting-point paraffin wax (Sasol Wax, Hamburg, Germany) by immersion for 1 hour at 56°C three times, with each eye cup being embedded separately. The paraffin-embedded eye cups were kept dry at 4°C, cut at a thickness of 4 μm using a sliding microtome (CM 1500; Leica, Bremen, Germany), and mounted onto glass slides. Before IHC, the selected slides/sections were warmed (at 60°C for 30 minutes) and then deparaffinized by soaking them twice in xylene for 10 minutes each time (Panreac Appli-Chem, Darmstadt, Germany), followed by two 3-minute drenches in 99%, 96%, and 70% ethanol, followed by distilled water. After rinsing the slides with PBS (2 × 5 minutes), the sections were processed for IHC as described below. 
Immunohistochemistry and Western Blotting
For IHC analysis, the retinal whole mounts were incubated for 2 hours with blocking solution containing 10% goat serum (Sigma-Aldrich, Munich, Germany) and 0.5% Triton X-100 (Sigma-Aldrich) at room temperature, washed twice with PBS for 5 minutes each, and then incubated with primary antibodies for 72 hours at 4°C in darkness. The retinas were then washed with PBS and incubated with secondary antibodies (also for 72 hours at 4°C in darkness), washed again with PBS, and then coverslipped with antifade mounting medium (Mowiol; Hoechst, Frankfurt, Germany). The paraffin or cryo sections were processed according to protocols previously described.30 For WB, the tissues were processed according to previous protocols.30 Used antibodies for IHC and WB were provided in Table 2
Table 2
 
Antibodies Used for Immunohistochemistry and Western Blotting; Double Amount of Antibody Solution Was Used in the Case of Paraffin-Embedded Tissue
Table 2
 
Antibodies Used for Immunohistochemistry and Western Blotting; Double Amount of Antibody Solution Was Used in the Case of Paraffin-Embedded Tissue
Morphologic Study of the Fovea
The morphology of the macula and fovea was analyzed by IHC analysis of whole mounts (Table 1). Major subsets of retinal cells were stained with III β-tubulin for RGCs and VEGF for retinal vessels. ApoTome images by z-plane stacks were made to provide the three-dimensional context; the slides were viewed with the appropriate filter on a microscope equipped with epifluorescence (ApoTome 2; Carl Zeiss, Jena, Germany) and appropriate software (ZEN 2012; Carl Zeiss). The double labeling of angiostatin with selected retinal antigens was determined by superimposing a counting box with dimensions of 300 × 300 μm. The number of retinal cells obtained from bisbenzimide-merged images within the region of interest was counted with the cell-count function of ImageJ software (http://imagej.nih.gov/ij/; in the public domain). These data were used to calculate the rates of costaining (% = [number of cell bodies positive for selected protein/number of cortical cell type] × 100) in the retina of each region, as well as the means of the individual experimental groups. The data are presented as mean ± SD values. The method to determine counting and calculating the rates of double staining was modified after He et al.31 
Cocultivation of Primate Fovea and Human Umbilical Vein Endothelial Cells
The involvement of endothelial cells in the angiostatic properties of the fovea was determined by coculturing M. fascicularis foveal tissue with commercially obtained human umbilical vein endothelial cells (HUVEC) (Table 1). After preparing the retina as described above, either foveas or peripheral retinal regions were mechanically excised using a self-fabricated glass microtrephane with a diameter of 0.5 mm. The selected retinal tissue probes were cocultured with HUVEC in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) (Gibco, Karlsruhe, Germany) for 72 hours. Photodocumentation was performed at different time points to establish whether or not the HUVEC migrated into the retinal tissue. The HUVEC behavior in the cocultures was also time-lapse video recorded in selected cases. 
The expression of integrin αVβ3 (vitronectin receptor, which is a target for angiostatin), the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM), von Willebrand factor (vWF), and endothelin-1 (ET-1) by HUVEC was determined by IHC, as described above. 
Data Evaluation and Statistical Analysis
All data obtained for the specific costaining studies from IHC and optical density from WB analyses were analyzed with a test for two independent samples (MedCalc statistical software, version 15.2; Ostend, Belgium) to examine conformity with the Gaussian distribution, and processed using ANOVA (for a Gaussian distribution) or Friedman's test (for a non-Gaussian distribution). Local P values were corrected for multiple comparisons using the Holm-Bonferroni method as appropriate. The threshold for statistical significance was set at P < 0.05. The figures were prepared with the aid of image-processing software (Photoshop; Adobe Systems, San Jose, CA, USA), and the overall brightness and contrast were adjusted without retouching. 
Results
First, the morphology of different subsets of retinal cells and their relations were studied in the primate macular region using IHC on retinal whole mounts. The expression and localization of the angiostatin protein in subsets of cells in the retina were then explored using retinal sections, and finally, the presence of angiostatin expression in the human neuroretina was confirmed. 
RGCs (III β-Tubulin)
The central axons of the RGCs fasciculate and convert into circumferentially oriented bundles that traverse toward the optic nerve head (Fig. 1A). 
Figure 1
 
Ganglion cells in the retina of C. jacchus and M. fascicularis. Immunohistochemistry (IHC) staining of III β-tubulin (green) in the central primate retina to identify RGCs. (AH) Foveal morphology of RGCs (green) in (AD) newborn C. jacchus and (EH) adult M. fascicularis monkeys in retinal whole mounts. ApoTome images show an x-y en face view of the presented retinal whole mount obtained at (AD) 1.26-μm [22.75 μm] and (EH) 3.58-μm [39.4 μm] intervals [whole section]. (A, E) Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. (CD, FH) Selected z-plane stacks showing different sections of the foveal morphology. (AH) The top and side of each image is a cross section through the z-plane of multiple optical slides. An antibody raised against III β-tubulin was used to stain RGCs; a cyanine (Cy)-2–labeled secondary antibody (green) was used to reveal the antigen staining. The negative control was performed with the Cy-2–labeled secondary antibody alone (data not shown). Scale bars: 50 μm (AD); 100 μm (EH).
Figure 1
 
Ganglion cells in the retina of C. jacchus and M. fascicularis. Immunohistochemistry (IHC) staining of III β-tubulin (green) in the central primate retina to identify RGCs. (AH) Foveal morphology of RGCs (green) in (AD) newborn C. jacchus and (EH) adult M. fascicularis monkeys in retinal whole mounts. ApoTome images show an x-y en face view of the presented retinal whole mount obtained at (AD) 1.26-μm [22.75 μm] and (EH) 3.58-μm [39.4 μm] intervals [whole section]. (A, E) Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. (CD, FH) Selected z-plane stacks showing different sections of the foveal morphology. (AH) The top and side of each image is a cross section through the z-plane of multiple optical slides. An antibody raised against III β-tubulin was used to stain RGCs; a cyanine (Cy)-2–labeled secondary antibody (green) was used to reveal the antigen staining. The negative control was performed with the Cy-2–labeled secondary antibody alone (data not shown). Scale bars: 50 μm (AD); 100 μm (EH).
There was an interspecies difference in the cellular staining of III β-tubulin within the fovea. III β-tubulin–immunopositive cells were seen within the fovea of M. fascicularis retinas (Figs. 1E–H)32,33 but not in that of C. jacchus retinas (Figs. 1A–D). In terms of morphologic classification, foveal III β-tubulin–expressing cells did not appear to fit into the usual criteria applicable to either parasol or midget RGCs,34 raising the question as to their putative function within the fovea. 
Although not quantified, there was no visible immunohistochemical staining difference between the stages of age analyzed within M. fascicularis. There were no differences in the expression of GFAP and VEGF within the strains of monkeys examined. Astrocytes were labeled with glial fibrillary acidic protein (GFAP) (Figs. 2A–D; 4 years) and VEGF staining that was strongly associated with perivascular astrocytes wrapping retinal capillaries (Figs. 2E–H; 12 years). No astrocytes were seen within the fovea. Comparison of the morphometric features of the monkey retina with those in the human retina was achieved through IHC analyses in adult human tissue (Fig. 3). Low-magnification images revealed a comparable distribution of neurons and capillaries in the monkey and human retinas. III β-tubulin–positive cells were found in the human fovea. Double labeling of III β-tubulin and VEGF revealed a layer characterized by intense staining of RGCs and their axons, vessels, and capillaries in the inner retina. Those axons and vessels ran strictly parallel toward the fovea, with the following exceptions: Axons engulfing vessels with a larger diameter and axons crossing vessels with smaller diameter appeared in a disorganized arrangement (Figs. 3A–F). In peripheral regions there was no wrapping of vessels by axons, and there were more retinal vessels and capillaries that were crossed by axons (Figs. 3G–L). When retinal sections of either species were stained for PECAM and VEGF, PECAM was exclusively localized within capillary endothelial cells, whereas VEGF was expressed only by perivascular astrocytes (Figs. 4A–D). When retinal sections of either species were processed for PECAM and angiostatin, distinct cells were stained for either antigen with RGCs to become angiostatin positive and capillary endothelial cells to become PECAM positive (Figs. 4E, 4F). Further double staining revealed that III β-tubulin RGCs did not express PECAM (data not shown). 
Figure 2
 
Astrocytes and vessels in the retina of M. fascicularis. Glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) staining in the central retina of M. fascicularis. (AH) Foveal morphology of (AD) astrocytes (green) in adult (4 years) and (EH) vessels (red) in elderly M. fascicularis (12 years) revealed by IHC analysis of retinal whole mounts. (AH) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (A) 3.31-μm [26.5 μm] (magnification: ×10) and (BD, III β-tubulin green and GFAP red) 1.27-μm [21.6 μm] (magnification: ×20) intervals in adult M. fascicularis and at (E) 2.9-μm [57 μm] (magnification: ×20) and (FH, III β-tubulin green and VEGF red) 0.97-μm [33 μm] (magnification: ×40) intervals [whole section] in elderly M. fascicularis. Selected coherent z-planes ([A] 16.8 μm, [BD] 12.7 μm, [E] 22.8 μm, and [FH] 16.5 μm) show morphologic foveal features of (AD) astrocytes and (EH) silhouettes of vessels surrounded by labeled astrocytes. Antibodies detecting GFAP and VEGF were used to detect astrocytes and perivascular astrocytes, respectively, and III β-tubulin staining was used to identify RGCs. The antigens (GFAP, VEGF, and III β-tubulin) were revealed using secondary antibodies labeled with Cy-2 (green) and tetramethylrhodamine (TRITC; red), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A); 50 μm (BE); 50 μm (FH).
Figure 2
 
Astrocytes and vessels in the retina of M. fascicularis. Glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) staining in the central retina of M. fascicularis. (AH) Foveal morphology of (AD) astrocytes (green) in adult (4 years) and (EH) vessels (red) in elderly M. fascicularis (12 years) revealed by IHC analysis of retinal whole mounts. (AH) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (A) 3.31-μm [26.5 μm] (magnification: ×10) and (BD, III β-tubulin green and GFAP red) 1.27-μm [21.6 μm] (magnification: ×20) intervals in adult M. fascicularis and at (E) 2.9-μm [57 μm] (magnification: ×20) and (FH, III β-tubulin green and VEGF red) 0.97-μm [33 μm] (magnification: ×40) intervals [whole section] in elderly M. fascicularis. Selected coherent z-planes ([A] 16.8 μm, [BD] 12.7 μm, [E] 22.8 μm, and [FH] 16.5 μm) show morphologic foveal features of (AD) astrocytes and (EH) silhouettes of vessels surrounded by labeled astrocytes. Antibodies detecting GFAP and VEGF were used to detect astrocytes and perivascular astrocytes, respectively, and III β-tubulin staining was used to identify RGCs. The antigens (GFAP, VEGF, and III β-tubulin) were revealed using secondary antibodies labeled with Cy-2 (green) and tetramethylrhodamine (TRITC; red), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A); 50 μm (BE); 50 μm (FH).
Figure 3
 
The fovea in the elderly human retina. Foveal morphology of RGCs (A, D, G, I, green) and perivascular astrocytes (B, E, H, K, red) in the macula and the periphery in the elderly human retina, revealed by IHC analysis of retinal whole mounts. (AL) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (AC) 4.2-μm [37.8 μm], (DF) 0.52-μm [8.8 μm], (GI) 3.4-μm [30.2 μm], and (JL) 19.9-μm [9.4 μm] intervals [whole section]. Images are displayed in maximum-intensity projection through the z-axis to provide an overall overview through the retinal layers. The top and side of each image is a cross section through the z-plane of multiple optical slides. Antibodies detecting III β-tubulin (green) and VEGF (red) were used to stain RGCs and perivascular astrocytes, respectively. Secondary antibodies labeled with Alexa Fluor 488 (green; RGCs) and Alexa Fluor 594 (red; perivascular astrocytes) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC, GI); 50 μm (DF, JL).
Figure 3
 
The fovea in the elderly human retina. Foveal morphology of RGCs (A, D, G, I, green) and perivascular astrocytes (B, E, H, K, red) in the macula and the periphery in the elderly human retina, revealed by IHC analysis of retinal whole mounts. (AL) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (AC) 4.2-μm [37.8 μm], (DF) 0.52-μm [8.8 μm], (GI) 3.4-μm [30.2 μm], and (JL) 19.9-μm [9.4 μm] intervals [whole section]. Images are displayed in maximum-intensity projection through the z-axis to provide an overall overview through the retinal layers. The top and side of each image is a cross section through the z-plane of multiple optical slides. Antibodies detecting III β-tubulin (green) and VEGF (red) were used to stain RGCs and perivascular astrocytes, respectively. Secondary antibodies labeled with Alexa Fluor 488 (green; RGCs) and Alexa Fluor 594 (red; perivascular astrocytes) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC, GI); 50 μm (DF, JL).
Figure 4
 
Expression of PECAM, VEGF, and angiostatin in the human retina. Immunohistochemical examination of PECAM expression in relation to other retinal cell markers in human retinal sections. (AD) Colabeling of PECAM (green) with VEGF (red) revealed that PECAM is expressed in capillary endothelial cells whereas VEGF was exclusively found in the perivascular astrocytes as expected from the whole-mount images of Figure 3. (EF) Colabeling of PECAM (green) with angiostatin (red) reveals a distinct localization of PECAM in retinal capillaries and angiostatin in RGCs. Secondary antibodies labeled with Alexa Fluor 488 (green; PECAM) and Alexa Fluor 594 (red; [C, D] perivascular astrocytes; [E, F] angiostatin) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RGC, retinal ganglion cell; bv, blood vessel; ec, endothelial cells. Scale bars: 20 μm (AD, F); 50 μm (E).
Figure 4
 
Expression of PECAM, VEGF, and angiostatin in the human retina. Immunohistochemical examination of PECAM expression in relation to other retinal cell markers in human retinal sections. (AD) Colabeling of PECAM (green) with VEGF (red) revealed that PECAM is expressed in capillary endothelial cells whereas VEGF was exclusively found in the perivascular astrocytes as expected from the whole-mount images of Figure 3. (EF) Colabeling of PECAM (green) with angiostatin (red) reveals a distinct localization of PECAM in retinal capillaries and angiostatin in RGCs. Secondary antibodies labeled with Alexa Fluor 488 (green; PECAM) and Alexa Fluor 594 (red; [C, D] perivascular astrocytes; [E, F] angiostatin) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RGC, retinal ganglion cell; bv, blood vessel; ec, endothelial cells. Scale bars: 20 μm (AD, F); 50 μm (E).
Angiostatin Expression
The retinal localization and expression of angiostatin were determined initially in the retinas of C. jacchus at different ages. First, IHC and WB were used to verify the expression of angiostatin in the foveal and peripheral retina. The fluorescence intensity of immunohistochemically labeled angiostatin was then determined in the foveal, perifoveal, and peripheral retina. Immunolabeling was verified by the WB analysis, which revealed expression of angiostatin in both the macula and retinal periphery (Fig. 5A). There appeared to be a slight reduction in angiostatin expression in the retinal periphery (92.1 ± 6.4%) compared to the macula; however, the difference did not reach statistical significance (P = 0.1; Fig. 5B). These data demonstrate the presence of angiostatin throughout the retina and a tendency toward changes in the level of protein expression in different retinal regions, which may reflect the decreasing density of RGCs. Sections of all regions and depths of the C. jacchus retina at different ages were examined by IHC. The data revealed stronger angiostatin staining in the fovea than in the perifoveal and peripheral retinal regions (Figs. 5C–K). In terms of the retinal layers, the angiostatin labeling was most intense in the RGC layer (GCL) and nerve fiber layer (NFL), followed by the inner plexiform layer (IPL)/inner nuclear layer (INL) and outer plexiform layer (OPL)/outer nuclear layer (ONL) in all retinal regions. However, the qualitatively estimated fluorescence intensity within the retinal layers was not quantified. 
Figure 5
 
Expression of angiostatin in the retina of C. jacchus. Expression of retinal angiostatin (red) in the fovea, perifovea, and periphery of C. jacchus. (A) Western blotting (WB) analyses of the macula and retinal periphery, and (B) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. Expression of angiostatin in the (CE) fovea, (FH) perifovea, and (IK) periphery revealed by IHC staining of 4-μm-thick retinal sections. The antigen was revealed using a secondary antibody labeled with Alexa Fluor 594 (red). The negative control was performed with the fluorescently labeled secondary antibody alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. Scale bars: 50 μm (CK).
Figure 5
 
Expression of angiostatin in the retina of C. jacchus. Expression of retinal angiostatin (red) in the fovea, perifovea, and periphery of C. jacchus. (A) Western blotting (WB) analyses of the macula and retinal periphery, and (B) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. Expression of angiostatin in the (CE) fovea, (FH) perifovea, and (IK) periphery revealed by IHC staining of 4-μm-thick retinal sections. The antigen was revealed using a secondary antibody labeled with Alexa Fluor 594 (red). The negative control was performed with the fluorescently labeled secondary antibody alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. Scale bars: 50 μm (CK).
Cell Counts After Fluorescence Labeling
Colabeling of angiostatin and III β-tubulin within RGCs was found throughout the retina (Fig. 6). Comparable colabeling was found in the fovea (87.3 ± 2.2%) and perifovea (81.9 ± 7.1%; P = 0.2; Fig. 6I). The proportion of angiostatin-positive RGCs was significantly lower in the peripheral region (45.2 ± 16.8%; P < 0.05 each) than in the central region of the retina (Fig. 6I). 
Figure 6
 
Colabeling of angiostatin and III β-tubulin in the retina of C. jacchus. Expression of retinal angiostatin (red) and III β-tubulin detecting RGCs (green) in the perifovea and periphery of adult C. jacchus. Colabeling of angiostatin and III β-tubulin in the (AD) perifovea and (EH) periphery revealed by IHC staining of 4-μm-thick retinal sections. (I) The rate of costained cells [% = (no. of angiostatin+/RGC+)/( no. of RGC+)] (He et al.31) is shown with III β-tubulin–positive RGCs in the perifovea and periphery. The antigens (angiostatin and III β-tubulin) were revealed using secondary antibodies labeled with Alexa Fluor 594 (red) and Alexa Fluor 594 (green), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 20 μm (AH). *Significantly different at P < 0.05.
Figure 6
 
Colabeling of angiostatin and III β-tubulin in the retina of C. jacchus. Expression of retinal angiostatin (red) and III β-tubulin detecting RGCs (green) in the perifovea and periphery of adult C. jacchus. Colabeling of angiostatin and III β-tubulin in the (AD) perifovea and (EH) periphery revealed by IHC staining of 4-μm-thick retinal sections. (I) The rate of costained cells [% = (no. of angiostatin+/RGC+)/( no. of RGC+)] (He et al.31) is shown with III β-tubulin–positive RGCs in the perifovea and periphery. The antigens (angiostatin and III β-tubulin) were revealed using secondary antibodies labeled with Alexa Fluor 594 (red) and Alexa Fluor 594 (green), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 20 μm (AH). *Significantly different at P < 0.05.
Colabeling with angiostatin and Tau-1, which is expressed in the telodendrons of terminal photoreceptor axons,35,36 revealed a strong association between the proteins in the OPL. Weaker angiostatin/Tau-1 colabeling was found in the INL (Figs. 7A–D). Colabeling with angiostatin and synaptophysin, which is expressed in the synaptic vesicles within the dense neuronal network of the retina,37 was found in the IPL, while no such double labeling was found in the OPL (Figs. 7E–H). 
Figure 7
 
Colabeling of angiostatin, Tau-1, and synaptophysin in the retina of C. jacchus. Colabeling of retinal angiostatin (red) and Tau-1 (green) or synaptophysin (green) in the macular region of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AD) Tau-1–positive terminal axons of photoreceptors and (EH) synaptophysin-positive synaptic vesicles. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; Tau-1 and synaptophysin). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD), 20 μm (EH).
Figure 7
 
Colabeling of angiostatin, Tau-1, and synaptophysin in the retina of C. jacchus. Colabeling of retinal angiostatin (red) and Tau-1 (green) or synaptophysin (green) in the macular region of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AD) Tau-1–positive terminal axons of photoreceptors and (EH) synaptophysin-positive synaptic vesicles. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; Tau-1 and synaptophysin). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD), 20 μm (EH).
Glial Staining
Due to the physiological absence of glial cells in the FAZ, colabeling studies of GFAP and vimentin were focused on the perifoveal and peripheral regions of the retina of C. jacchus (Fig. 8). The colabeling of angiostatin and GFAP within glial cells did not differ significantly between the perifoveal (41.1 ± 8.4%) and peripheral (30.2 ± 4.9%; P = 0.06) retinal sections (Figs. 8A–H, 8Q). In contrast, a significantly large proportion of vimentin-positive cells was also immunostained with angiostatin in the perifoveal region (35.1 ± 4.4%) compared to the retinal periphery (24.9 ± 0.8%; P < 0.05; Figs. 8I–P, 8R). Thus, IHC revealed an association between angiostatin and RGCs, photoreceptor terminal axons in the OPL, and synaptic vesicles in the IPL. The weak angiostatin immunolabeling in glial and amacrine cells yielded a negligible correlation with these subsets of retinal cells. 
Figure 8
 
Colabeling of angiostatin, GFAP, and vimentin in the retina of C. jacchus. Expression of retinal angiostatin (red), GFAP (green), and vimentin (green) in the perifovea and periphery of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AH) GFAP-positive astrocytes and (IP) vimentin-positive Müller cells. The rate of costained cells [% = (no. of angiostatin+/retinal glial cell+)/( no. of retinal glial cell+] (He et al.31) is shown with (Q) GFAP-positive astrocytes and (R) vimentin-positive Müller cells in the perifovea and periphery. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; GFAP and vimentin). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD); 20 μm (EH); 50 μm (IP). *Statistically significantly different at P < 0.05.
Figure 8
 
Colabeling of angiostatin, GFAP, and vimentin in the retina of C. jacchus. Expression of retinal angiostatin (red), GFAP (green), and vimentin (green) in the perifovea and periphery of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AH) GFAP-positive astrocytes and (IP) vimentin-positive Müller cells. The rate of costained cells [% = (no. of angiostatin+/retinal glial cell+)/( no. of retinal glial cell+] (He et al.31) is shown with (Q) GFAP-positive astrocytes and (R) vimentin-positive Müller cells in the perifovea and periphery. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; GFAP and vimentin). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD); 20 μm (EH); 50 μm (IP). *Statistically significantly different at P < 0.05.
The expression of angiostatin in the human retina (macula and periphery) was confirmed by performing IHC analyses on whole-mounted tissue. Diffuse staining of angiostatin was found in the foveola (Figs. 9A–C). Angiostatin-positive RGCs were observed skirting the border of the foveal pit (Figs. 9D–F), and this protein was detected in the retinal periphery, though not in association with RGCs and astrocytes (Figs. 9G–I). Proteochemical methods confirmed the expression of angiostatin in both the macula and retinal periphery (Figs. 9J, 9K). Integrin αvβ3 (an angiostatin-related receptor) was detected in the retinal capillaries in both the macula and retinal periphery (Figs. 9L–N). Vascular endothelial growth factor expression was also observed in the retinal capillaries in the macular region and retinal periphery (Figs. 9O–Q). The expression pattern of angiostatin observed in the human retina was comparable to that described for the monkey retina. The findings of foveal RGCs expressing angiostatin and capillaries expressing integrin αvβ3 indicate that these cells may be involved in maintaining foveal avascularity. 
Figure 9
 
Expression of angiostatin, integrin αvβ3, and VEGF in the human retina. Expression of retinal angiostatin (B, E, H; red) and integrin αvβ3 (vibronectin receptor, target for angiostatin; [M] red) in the macula and retinal periphery in whole mounts of the human retina. Antibodies detecting III β-tubulin (A, D, L, O; green), GFAP (G; green), and VEGF (P; red) were used to stain RGCs, astrocytes, and perivascular astrocytes, respectively. (AI, LQ) ApoTome images showing an x-y en face view of the presented retinal whole mounts obtained at (AC) 3.6-μm [64.2 μm], (DF) 2.0-μm [49.0 μm], (GI) 0.7-μm [34 μm], (LN) 1.3-μm [23.5 μm], and (OQ) 3.4-μm [30.2 μm] intervals [whole section]. Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. The top and side of each image is a cross section through the z-plane of III β-tubulin (A, D) in the macular and foveal regions. (GI) No colabeling was detected with angiostatin (red) and GFAP (green) immunostaining in astrocytes. (J) WB analyses of the macula and retinal periphery, and (K) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. (LN) Integrin αvβ3 (vibronectin receptor, target for angiostatin) and (OQ) VEGF were expressed by perivascular astrocytes but not RGCs (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green), Cy-2 (green), Alexa Fluor 594 (red), and TRITC (red). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC); 50 μm (DQ).
Figure 9
 
Expression of angiostatin, integrin αvβ3, and VEGF in the human retina. Expression of retinal angiostatin (B, E, H; red) and integrin αvβ3 (vibronectin receptor, target for angiostatin; [M] red) in the macula and retinal periphery in whole mounts of the human retina. Antibodies detecting III β-tubulin (A, D, L, O; green), GFAP (G; green), and VEGF (P; red) were used to stain RGCs, astrocytes, and perivascular astrocytes, respectively. (AI, LQ) ApoTome images showing an x-y en face view of the presented retinal whole mounts obtained at (AC) 3.6-μm [64.2 μm], (DF) 2.0-μm [49.0 μm], (GI) 0.7-μm [34 μm], (LN) 1.3-μm [23.5 μm], and (OQ) 3.4-μm [30.2 μm] intervals [whole section]. Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. The top and side of each image is a cross section through the z-plane of III β-tubulin (A, D) in the macular and foveal regions. (GI) No colabeling was detected with angiostatin (red) and GFAP (green) immunostaining in astrocytes. (J) WB analyses of the macula and retinal periphery, and (K) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. (LN) Integrin αvβ3 (vibronectin receptor, target for angiostatin) and (OQ) VEGF were expressed by perivascular astrocytes but not RGCs (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green), Cy-2 (green), Alexa Fluor 594 (red), and TRITC (red). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC); 50 μm (DQ).
Cocultures of Foveal Tissue and HUVEC
The putative angiostatic properties of the fovea when confronted with endothelial cells were examined by monitoring whether HUVEC invade the fovea during coculture with mechanically excised foveas. While most of the HUVEC did avoid the foveal tissue, they were found to invade retinal pieces taken from the paracentral, equatorial, and peripheral areas (Figs. 10A, 10B). Immunohistochemistry analysis revealed expression of integrin αvβ3 within HUVEC (Fig. 10C). Furthermore, the endothelial markers vWF, ET-1, and PECAM were expressed abundantly by the cultured HUVEC (Figs. 10D–F). 
Figure 10
 
Coculturing of primate fovea and HUVEC cells. Coculture of primate (n = 5) and human umbilical vein endothelial cells (HUVEC) and identification of associated receptors. (A) Central (fovea) and (B) peripheral regions (periphery) of retinas (n = 5) were excised mechanically from the eyes of adult M. fascicularis monkeys and cocultured with HUVEC. IHC analysis revealed (C) expression of angiostatin (red), and integrin αVβ3 (green), (D) von Willebrand Factor (vWF) (red), (E) endothelin-1 (red), and (F) PECAM (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A, B); 50 μm (CE).
Figure 10
 
Coculturing of primate fovea and HUVEC cells. Coculture of primate (n = 5) and human umbilical vein endothelial cells (HUVEC) and identification of associated receptors. (A) Central (fovea) and (B) peripheral regions (periphery) of retinas (n = 5) were excised mechanically from the eyes of adult M. fascicularis monkeys and cocultured with HUVEC. IHC analysis revealed (C) expression of angiostatin (red), and integrin αVβ3 (green), (D) von Willebrand Factor (vWF) (red), (E) endothelin-1 (red), and (F) PECAM (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A, B); 50 μm (CE).
Discussion
Angiostatin appears to be strongly expressed in the RGCs throughout the primate retina. In C. jacchus, the lifelong expression of this angiorepellent assigns the molecule a role in interactions between RGCs and different retinal cell types, and in particular with capillary cells expressing the angiostatin receptor. Despite these unequivocal results, the work presented here must be considered in light of the limitation that tissues from all species at all stages of development were not tested. 
Development and Postnatal Maturation of the Macula
The fovea accounts for about 0.01% of the entire retina area. The macula is highly specialized and contains elevated densities of all subsets of neural cells.32-34,38-41 The novel finding of the present work is that III β-tubulin–immunostained parasol-like RGCs were found in the fovea of macaque monkeys and humans, but not in that of C. jacchus monkeys. The retinal vasculature in primates develops at a relatively late stage, starting at around 14 weeks after gestation in humans, fetal day (FD) 70 in macaques, and FD 100 in marmosets. Retinal vessels invade the developing human retina but grow more slowly toward the developing macula compared with the rest of the retina.5,9,42,43 Blood vessels and glial cells are absent from the fovea throughout life under physiological conditions,9 indicating an anatomic advantage serving to maximize central visual acuity.4447 
Angioattractant Factors During Retinal Development
Angioattractant factors such as netrin G11,48 and members of the Eph/ephrin family7,10,4951 guide developing RGCs during early fetal development and postnatal maturation.50,5254 Ligands for Eph-A6 and ephrin-A1 and -A4 on Pax-2–positive cells were found in astrocytes of the optic nerve head and NFL, which lead the developing vessels into the retina.7 Angiorepellent PEDF is expressed more strongly in RGCs in the incipient and emerging fovea than in other regions of the retina.8,5557 Proangiogenic factors such as VEGF,43 BNP, and c-Jun-N-terminal kinase regulate the proliferation of endothelial cells during vasculature development of the retina.6,5861 
The lifelong preservation of foveal avascularity is necessary for normal visual function. It is reasonable to assume that the same or similar factors associated with the developmental regulation of the avascular fovea operate to maintain the lifelong stability of the retinal vascular system.62 Under physiological conditions, inhibitory factors predominate and vessels remain quiescent. In a pathologic context, neovascularization occurs due to decreased production of inhibitors and/or increased production of angiogenic stimulators.6365 
Angiostatin and Endostatin
Endostatin prevents laser-induced CNV in mice66 and is reduced in the choroid of AMD patients compared to age-matched mates.67 Angiostatin has the potential to inhibit the proliferation, migration, differentiation, and tube formation of endothelial cells in vitro.68 Annexin, angiomotin, integrin ανβ3, and c-met are cell-surface proteins that bind angiostatin.69 The binding of angiostatin to tissue plasminogen activator results in a reduced cellular migration and invasion.70 Angiostatin binds to subunits of ATP synthase on the cell surface of endothelial cells,71 targets the Krebs cycle in mitochondria,72 and binds to integrin ανβ3, which is probably necessary for angiogenesis. Furthermore, angiostatin inhibits the response of stimulated endothelial cells and smooth muscle cells to hepatocyte growth factor by interrupting G2/M transition during the cell cycle,73 competitively antagonizes VEGF- and/or basic fibroblast growth factor–induced signaling (linked to AMD),74 and strongly blocks the neovascularization and growth of tumor metastases.20 The present findings showed that angiostatin is expressed in the GCL, IPL, and OPL throughout the neuroretina. To our knowledge, the present study is the first to demonstrate regional differences in angiostatin expression in the primate retina. Other studies have found retinal effects after intraocular angiostatin administration. The systemic administration of angiostatin completely prevented retinal neovascularization in a mouse model of oxygen-induced retinopathy.28 Recombinant angiostatin successfully suppressed experimental angiogenesis without signs of toxic effects to the retina.75 A clinical phase I dose escalation study is reported with endostatin and angiostatin subretinal injections in advanced neovascular CNV (reviewed in Ref. 76), however, with no clinical results available. 
The found comparable expression patterns of angiostatin in the macula and retinal periphery, indicating its potential role in regulating the vasculature homeostasis throughout the retina. The requirement in the macula to provide a physiological inhibitor of angiogenesis could be explained by elevated metabolic stress and increased levels of incoming light.77 The result that subsets of retinal cells express angiostatin reveals RGCs to be the main source of the protein. This concurs with the recent suggestion that a local lifelong-expressed factor that inhibits foveal vascularization could be expressed by RGCs.1 The finding of several known angiostatic factors maintained by the retinal pigment epithelium, Bruch's membrane, and choroid complex indicates that angiostatin may be a physiological determinant of vasculature homeostasis in the inner retina. 
Increased angiostatin staining was found within the OPL in the present study, whereby the terminal axons of photoreceptors connect to horizontal and bipolar cells as well as the IPL, where amacrine cells and RGCs are connected. It is known that synaptic plasticity occurs within the plexiform retinal layers throughout life7880; the involvement of angiostatin in these plastic events cannot be excluded. The findings of comparable expression patterns of angiostatin in the primate macula support the theory of a balance of proangiogenic and antiangiogenic factors maintaining the ocular vasculature. 
Acknowledgments
The authors thank Mechthild Wissing and Mechthild Langkamp-Flock for their skillful technical assistance, Magdalena Reis for typing the manuscript, and English Science Editing for native linguistic editing of the manuscript. We thank Hans Meyer-Rüsenberg for providing human material. 
Supported by an Innovative Medical Research (IMF) grant awarded by the School of Medicine, Westfalian Wilhelms-University of Münster (I-Bö221307 to MRRB), and a German Research Foundation (DFG) grant (BO 4556/1-1 to MRRB and Th 386/20-1 to ST). Novartis Company supported the participation of FH at the Symposium VEGF and Beyond with a poster presentation. 
Disclosure: M.R.R. Böhm, None; F. Hodes, None; K. Brockhaus, None; S. Hummel, None; S. Schlatt, None; H. Melkonyan, None; S. Thanos, None 
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Figure 1
 
Ganglion cells in the retina of C. jacchus and M. fascicularis. Immunohistochemistry (IHC) staining of III β-tubulin (green) in the central primate retina to identify RGCs. (AH) Foveal morphology of RGCs (green) in (AD) newborn C. jacchus and (EH) adult M. fascicularis monkeys in retinal whole mounts. ApoTome images show an x-y en face view of the presented retinal whole mount obtained at (AD) 1.26-μm [22.75 μm] and (EH) 3.58-μm [39.4 μm] intervals [whole section]. (A, E) Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. (CD, FH) Selected z-plane stacks showing different sections of the foveal morphology. (AH) The top and side of each image is a cross section through the z-plane of multiple optical slides. An antibody raised against III β-tubulin was used to stain RGCs; a cyanine (Cy)-2–labeled secondary antibody (green) was used to reveal the antigen staining. The negative control was performed with the Cy-2–labeled secondary antibody alone (data not shown). Scale bars: 50 μm (AD); 100 μm (EH).
Figure 1
 
Ganglion cells in the retina of C. jacchus and M. fascicularis. Immunohistochemistry (IHC) staining of III β-tubulin (green) in the central primate retina to identify RGCs. (AH) Foveal morphology of RGCs (green) in (AD) newborn C. jacchus and (EH) adult M. fascicularis monkeys in retinal whole mounts. ApoTome images show an x-y en face view of the presented retinal whole mount obtained at (AD) 1.26-μm [22.75 μm] and (EH) 3.58-μm [39.4 μm] intervals [whole section]. (A, E) Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. (CD, FH) Selected z-plane stacks showing different sections of the foveal morphology. (AH) The top and side of each image is a cross section through the z-plane of multiple optical slides. An antibody raised against III β-tubulin was used to stain RGCs; a cyanine (Cy)-2–labeled secondary antibody (green) was used to reveal the antigen staining. The negative control was performed with the Cy-2–labeled secondary antibody alone (data not shown). Scale bars: 50 μm (AD); 100 μm (EH).
Figure 2
 
Astrocytes and vessels in the retina of M. fascicularis. Glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) staining in the central retina of M. fascicularis. (AH) Foveal morphology of (AD) astrocytes (green) in adult (4 years) and (EH) vessels (red) in elderly M. fascicularis (12 years) revealed by IHC analysis of retinal whole mounts. (AH) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (A) 3.31-μm [26.5 μm] (magnification: ×10) and (BD, III β-tubulin green and GFAP red) 1.27-μm [21.6 μm] (magnification: ×20) intervals in adult M. fascicularis and at (E) 2.9-μm [57 μm] (magnification: ×20) and (FH, III β-tubulin green and VEGF red) 0.97-μm [33 μm] (magnification: ×40) intervals [whole section] in elderly M. fascicularis. Selected coherent z-planes ([A] 16.8 μm, [BD] 12.7 μm, [E] 22.8 μm, and [FH] 16.5 μm) show morphologic foveal features of (AD) astrocytes and (EH) silhouettes of vessels surrounded by labeled astrocytes. Antibodies detecting GFAP and VEGF were used to detect astrocytes and perivascular astrocytes, respectively, and III β-tubulin staining was used to identify RGCs. The antigens (GFAP, VEGF, and III β-tubulin) were revealed using secondary antibodies labeled with Cy-2 (green) and tetramethylrhodamine (TRITC; red), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A); 50 μm (BE); 50 μm (FH).
Figure 2
 
Astrocytes and vessels in the retina of M. fascicularis. Glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) staining in the central retina of M. fascicularis. (AH) Foveal morphology of (AD) astrocytes (green) in adult (4 years) and (EH) vessels (red) in elderly M. fascicularis (12 years) revealed by IHC analysis of retinal whole mounts. (AH) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (A) 3.31-μm [26.5 μm] (magnification: ×10) and (BD, III β-tubulin green and GFAP red) 1.27-μm [21.6 μm] (magnification: ×20) intervals in adult M. fascicularis and at (E) 2.9-μm [57 μm] (magnification: ×20) and (FH, III β-tubulin green and VEGF red) 0.97-μm [33 μm] (magnification: ×40) intervals [whole section] in elderly M. fascicularis. Selected coherent z-planes ([A] 16.8 μm, [BD] 12.7 μm, [E] 22.8 μm, and [FH] 16.5 μm) show morphologic foveal features of (AD) astrocytes and (EH) silhouettes of vessels surrounded by labeled astrocytes. Antibodies detecting GFAP and VEGF were used to detect astrocytes and perivascular astrocytes, respectively, and III β-tubulin staining was used to identify RGCs. The antigens (GFAP, VEGF, and III β-tubulin) were revealed using secondary antibodies labeled with Cy-2 (green) and tetramethylrhodamine (TRITC; red), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A); 50 μm (BE); 50 μm (FH).
Figure 3
 
The fovea in the elderly human retina. Foveal morphology of RGCs (A, D, G, I, green) and perivascular astrocytes (B, E, H, K, red) in the macula and the periphery in the elderly human retina, revealed by IHC analysis of retinal whole mounts. (AL) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (AC) 4.2-μm [37.8 μm], (DF) 0.52-μm [8.8 μm], (GI) 3.4-μm [30.2 μm], and (JL) 19.9-μm [9.4 μm] intervals [whole section]. Images are displayed in maximum-intensity projection through the z-axis to provide an overall overview through the retinal layers. The top and side of each image is a cross section through the z-plane of multiple optical slides. Antibodies detecting III β-tubulin (green) and VEGF (red) were used to stain RGCs and perivascular astrocytes, respectively. Secondary antibodies labeled with Alexa Fluor 488 (green; RGCs) and Alexa Fluor 594 (red; perivascular astrocytes) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC, GI); 50 μm (DF, JL).
Figure 3
 
The fovea in the elderly human retina. Foveal morphology of RGCs (A, D, G, I, green) and perivascular astrocytes (B, E, H, K, red) in the macula and the periphery in the elderly human retina, revealed by IHC analysis of retinal whole mounts. (AL) ApoTome images showing an x-y en face view of the presented retinal whole mount obtained at (AC) 4.2-μm [37.8 μm], (DF) 0.52-μm [8.8 μm], (GI) 3.4-μm [30.2 μm], and (JL) 19.9-μm [9.4 μm] intervals [whole section]. Images are displayed in maximum-intensity projection through the z-axis to provide an overall overview through the retinal layers. The top and side of each image is a cross section through the z-plane of multiple optical slides. Antibodies detecting III β-tubulin (green) and VEGF (red) were used to stain RGCs and perivascular astrocytes, respectively. Secondary antibodies labeled with Alexa Fluor 488 (green; RGCs) and Alexa Fluor 594 (red; perivascular astrocytes) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC, GI); 50 μm (DF, JL).
Figure 4
 
Expression of PECAM, VEGF, and angiostatin in the human retina. Immunohistochemical examination of PECAM expression in relation to other retinal cell markers in human retinal sections. (AD) Colabeling of PECAM (green) with VEGF (red) revealed that PECAM is expressed in capillary endothelial cells whereas VEGF was exclusively found in the perivascular astrocytes as expected from the whole-mount images of Figure 3. (EF) Colabeling of PECAM (green) with angiostatin (red) reveals a distinct localization of PECAM in retinal capillaries and angiostatin in RGCs. Secondary antibodies labeled with Alexa Fluor 488 (green; PECAM) and Alexa Fluor 594 (red; [C, D] perivascular astrocytes; [E, F] angiostatin) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RGC, retinal ganglion cell; bv, blood vessel; ec, endothelial cells. Scale bars: 20 μm (AD, F); 50 μm (E).
Figure 4
 
Expression of PECAM, VEGF, and angiostatin in the human retina. Immunohistochemical examination of PECAM expression in relation to other retinal cell markers in human retinal sections. (AD) Colabeling of PECAM (green) with VEGF (red) revealed that PECAM is expressed in capillary endothelial cells whereas VEGF was exclusively found in the perivascular astrocytes as expected from the whole-mount images of Figure 3. (EF) Colabeling of PECAM (green) with angiostatin (red) reveals a distinct localization of PECAM in retinal capillaries and angiostatin in RGCs. Secondary antibodies labeled with Alexa Fluor 488 (green; PECAM) and Alexa Fluor 594 (red; [C, D] perivascular astrocytes; [E, F] angiostatin) were used to reveal the antigens. The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RGC, retinal ganglion cell; bv, blood vessel; ec, endothelial cells. Scale bars: 20 μm (AD, F); 50 μm (E).
Figure 5
 
Expression of angiostatin in the retina of C. jacchus. Expression of retinal angiostatin (red) in the fovea, perifovea, and periphery of C. jacchus. (A) Western blotting (WB) analyses of the macula and retinal periphery, and (B) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. Expression of angiostatin in the (CE) fovea, (FH) perifovea, and (IK) periphery revealed by IHC staining of 4-μm-thick retinal sections. The antigen was revealed using a secondary antibody labeled with Alexa Fluor 594 (red). The negative control was performed with the fluorescently labeled secondary antibody alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. Scale bars: 50 μm (CK).
Figure 5
 
Expression of angiostatin in the retina of C. jacchus. Expression of retinal angiostatin (red) in the fovea, perifovea, and periphery of C. jacchus. (A) Western blotting (WB) analyses of the macula and retinal periphery, and (B) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. Expression of angiostatin in the (CE) fovea, (FH) perifovea, and (IK) periphery revealed by IHC staining of 4-μm-thick retinal sections. The antigen was revealed using a secondary antibody labeled with Alexa Fluor 594 (red). The negative control was performed with the fluorescently labeled secondary antibody alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. Scale bars: 50 μm (CK).
Figure 6
 
Colabeling of angiostatin and III β-tubulin in the retina of C. jacchus. Expression of retinal angiostatin (red) and III β-tubulin detecting RGCs (green) in the perifovea and periphery of adult C. jacchus. Colabeling of angiostatin and III β-tubulin in the (AD) perifovea and (EH) periphery revealed by IHC staining of 4-μm-thick retinal sections. (I) The rate of costained cells [% = (no. of angiostatin+/RGC+)/( no. of RGC+)] (He et al.31) is shown with III β-tubulin–positive RGCs in the perifovea and periphery. The antigens (angiostatin and III β-tubulin) were revealed using secondary antibodies labeled with Alexa Fluor 594 (red) and Alexa Fluor 594 (green), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 20 μm (AH). *Significantly different at P < 0.05.
Figure 6
 
Colabeling of angiostatin and III β-tubulin in the retina of C. jacchus. Expression of retinal angiostatin (red) and III β-tubulin detecting RGCs (green) in the perifovea and periphery of adult C. jacchus. Colabeling of angiostatin and III β-tubulin in the (AD) perifovea and (EH) periphery revealed by IHC staining of 4-μm-thick retinal sections. (I) The rate of costained cells [% = (no. of angiostatin+/RGC+)/( no. of RGC+)] (He et al.31) is shown with III β-tubulin–positive RGCs in the perifovea and periphery. The antigens (angiostatin and III β-tubulin) were revealed using secondary antibodies labeled with Alexa Fluor 594 (red) and Alexa Fluor 594 (green), respectively. The negative control was performed with the fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 20 μm (AH). *Significantly different at P < 0.05.
Figure 7
 
Colabeling of angiostatin, Tau-1, and synaptophysin in the retina of C. jacchus. Colabeling of retinal angiostatin (red) and Tau-1 (green) or synaptophysin (green) in the macular region of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AD) Tau-1–positive terminal axons of photoreceptors and (EH) synaptophysin-positive synaptic vesicles. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; Tau-1 and synaptophysin). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD), 20 μm (EH).
Figure 7
 
Colabeling of angiostatin, Tau-1, and synaptophysin in the retina of C. jacchus. Colabeling of retinal angiostatin (red) and Tau-1 (green) or synaptophysin (green) in the macular region of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AD) Tau-1–positive terminal axons of photoreceptors and (EH) synaptophysin-positive synaptic vesicles. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; Tau-1 and synaptophysin). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD), 20 μm (EH).
Figure 8
 
Colabeling of angiostatin, GFAP, and vimentin in the retina of C. jacchus. Expression of retinal angiostatin (red), GFAP (green), and vimentin (green) in the perifovea and periphery of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AH) GFAP-positive astrocytes and (IP) vimentin-positive Müller cells. The rate of costained cells [% = (no. of angiostatin+/retinal glial cell+)/( no. of retinal glial cell+] (He et al.31) is shown with (Q) GFAP-positive astrocytes and (R) vimentin-positive Müller cells in the perifovea and periphery. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; GFAP and vimentin). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD); 20 μm (EH); 50 μm (IP). *Statistically significantly different at P < 0.05.
Figure 8
 
Colabeling of angiostatin, GFAP, and vimentin in the retina of C. jacchus. Expression of retinal angiostatin (red), GFAP (green), and vimentin (green) in the perifovea and periphery of adult C. jacchus. IHC staining revealed colabeling of angiostatin and (AH) GFAP-positive astrocytes and (IP) vimentin-positive Müller cells. The rate of costained cells [% = (no. of angiostatin+/retinal glial cell+)/( no. of retinal glial cell+] (He et al.31) is shown with (Q) GFAP-positive astrocytes and (R) vimentin-positive Müller cells in the perifovea and periphery. The antigens were revealed using secondary antibodies labeled with Alexa Fluor 594 (red; angiostatin) and Alexa Fluor 594 (green; GFAP and vimentin). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Bisbenzimide (Hoechst 33258) was used to stain the cell nuclei. GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 50 μm (AD); 20 μm (EH); 50 μm (IP). *Statistically significantly different at P < 0.05.
Figure 9
 
Expression of angiostatin, integrin αvβ3, and VEGF in the human retina. Expression of retinal angiostatin (B, E, H; red) and integrin αvβ3 (vibronectin receptor, target for angiostatin; [M] red) in the macula and retinal periphery in whole mounts of the human retina. Antibodies detecting III β-tubulin (A, D, L, O; green), GFAP (G; green), and VEGF (P; red) were used to stain RGCs, astrocytes, and perivascular astrocytes, respectively. (AI, LQ) ApoTome images showing an x-y en face view of the presented retinal whole mounts obtained at (AC) 3.6-μm [64.2 μm], (DF) 2.0-μm [49.0 μm], (GI) 0.7-μm [34 μm], (LN) 1.3-μm [23.5 μm], and (OQ) 3.4-μm [30.2 μm] intervals [whole section]. Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. The top and side of each image is a cross section through the z-plane of III β-tubulin (A, D) in the macular and foveal regions. (GI) No colabeling was detected with angiostatin (red) and GFAP (green) immunostaining in astrocytes. (J) WB analyses of the macula and retinal periphery, and (K) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. (LN) Integrin αvβ3 (vibronectin receptor, target for angiostatin) and (OQ) VEGF were expressed by perivascular astrocytes but not RGCs (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green), Cy-2 (green), Alexa Fluor 594 (red), and TRITC (red). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC); 50 μm (DQ).
Figure 9
 
Expression of angiostatin, integrin αvβ3, and VEGF in the human retina. Expression of retinal angiostatin (B, E, H; red) and integrin αvβ3 (vibronectin receptor, target for angiostatin; [M] red) in the macula and retinal periphery in whole mounts of the human retina. Antibodies detecting III β-tubulin (A, D, L, O; green), GFAP (G; green), and VEGF (P; red) were used to stain RGCs, astrocytes, and perivascular astrocytes, respectively. (AI, LQ) ApoTome images showing an x-y en face view of the presented retinal whole mounts obtained at (AC) 3.6-μm [64.2 μm], (DF) 2.0-μm [49.0 μm], (GI) 0.7-μm [34 μm], (LN) 1.3-μm [23.5 μm], and (OQ) 3.4-μm [30.2 μm] intervals [whole section]. Maximum-intensity projection through the z-axis provides an overall overview of the fovea and surrounding macula. The top and side of each image is a cross section through the z-plane of III β-tubulin (A, D) in the macular and foveal regions. (GI) No colabeling was detected with angiostatin (red) and GFAP (green) immunostaining in astrocytes. (J) WB analyses of the macula and retinal periphery, and (K) corresponding densitometric analyses of the WB results relative to those measured in the macula (in %). Lysates of retinas treated as described in the text were prepared and tested for angiostatin (50 kDa) expression. Calnexin (90 kDa) expression verified the amount of protein loaded per lane. Protein bands are labeled in kDa. (LN) Integrin αvβ3 (vibronectin receptor, target for angiostatin) and (OQ) VEGF were expressed by perivascular astrocytes but not RGCs (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green), Cy-2 (green), Alexa Fluor 594 (red), and TRITC (red). The negative control was performed with fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (AC); 50 μm (DQ).
Figure 10
 
Coculturing of primate fovea and HUVEC cells. Coculture of primate (n = 5) and human umbilical vein endothelial cells (HUVEC) and identification of associated receptors. (A) Central (fovea) and (B) peripheral regions (periphery) of retinas (n = 5) were excised mechanically from the eyes of adult M. fascicularis monkeys and cocultured with HUVEC. IHC analysis revealed (C) expression of angiostatin (red), and integrin αVβ3 (green), (D) von Willebrand Factor (vWF) (red), (E) endothelin-1 (red), and (F) PECAM (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A, B); 50 μm (CE).
Figure 10
 
Coculturing of primate fovea and HUVEC cells. Coculture of primate (n = 5) and human umbilical vein endothelial cells (HUVEC) and identification of associated receptors. (A) Central (fovea) and (B) peripheral regions (periphery) of retinas (n = 5) were excised mechanically from the eyes of adult M. fascicularis monkeys and cocultured with HUVEC. IHC analysis revealed (C) expression of angiostatin (red), and integrin αVβ3 (green), (D) von Willebrand Factor (vWF) (red), (E) endothelin-1 (red), and (F) PECAM (green). The antigens were revealed using secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). The negative control was performed using fluorescently labeled secondary antibodies alone (data not shown). Scale bars: 100 μm (A, B); 50 μm (CE).
Table 1
 
Experimental Approach
Table 1
 
Experimental Approach
Table 2
 
Antibodies Used for Immunohistochemistry and Western Blotting; Double Amount of Antibody Solution Was Used in the Case of Paraffin-Embedded Tissue
Table 2
 
Antibodies Used for Immunohistochemistry and Western Blotting; Double Amount of Antibody Solution Was Used in the Case of Paraffin-Embedded Tissue
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