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To characterize the involvement of Semaphorin 7A (Sema7a) in corneal neovascularization (NV).
We generated anti-Sema7A antibodies to detect protein expression. Corneal fibroblast cells were cultured, stimulated with bFGF, immunostained with anti-Sema7A antibodies, and visualized by confocal microscopy. bFGF pellets were implanted in mouse corneal micropockets for 3–10 days, and corneal sections were immunostained with anti-Sema7A antibodies. Mouse corneas were injected with naked Sema7A DNA and vector control for 3, 7, and 10 days. Mouse corneas were imaged by slit lamp microscopy, and areas of corneal NV were calculated using the ImageJ program. Mouse corneal sections were also immunostained with anti-macrophage marker (F4/80) and anti-vascular endothelial growth factor (VEGF)-A antibodies.
Our data showed enhanced Sema7A expression levels in bFGF-stimulated cultured corneal fibroblasts. bFGF corneal implantation also demonstrated enhanced Sema7A expression. Corneas injected with a Sema7A expression vector showed evidence of significant corneal NV compared to controls on day 10 (1.8 mm2 vs. 0.11 mm2; p<0.02). Additionally, immunolocalization of Sema7A DNA-injected corneas (at day 7) revealed macrophage recruitment and enhanced VEGF-A levels.
We demonstrated that Sema7A was expressed in vascularized corneas and showed pro-angiogenic properties in our corneal model. Understanding the mechanism of Sema7A in angiogenesis may provide a therapeutic target for the treatment of corneal angiogenesis-related disorders.
The semaphorin family of transmembrane and secreted proteins was initially described as composed of axon guidance factors involved in the development of the central nervous system 1–3. Recent studies have reported that semaphorins, including Semaphorin 3B, 3F, 3E, 4D, and 6A, can either promote or inhibit tumor angiogenesis and progression 4–7. We previously detected Semaphorin 7A (Sema7a) expression in mouse ocular tissues during the course of ocular angiogenesis analysis 8, 9.
Sema7A has been implicated in the regulation of the immune and nervous systems 1, 10, 11. Sema7A is associated with the cell surface through a glycophosphatidylinositol linkage 12 and exerts pronounced effects on axon outgrowth and nerve sprouting through its integrin-binding motif (Arg-Gly-Asp) and effects on the mitogen-activated protein kinase pathway 13. Emerging evidence suggests that axon growth cones and capillary tip cells use common signaling cues to regulate their guidance 14.
The normally avascular cornea is a good model for evaluating pro- and anti-angiogenic factors 8, 15. The cornea requires low levels of angiogenic factors and high levels of anti-angiogenic factors under basal conditions; shifting the balance towards higher levels of angiogenic factors is associated with pathological processes. Corneal neovascularization (NV) is induced by the upregulation of angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and chemokines 15. Under corneal hypoxia and inflammatory conditions, VEGF is secreted by macrophages, T cells, astrocytes, and keratocytes, and binds to endothelial cell-specific receptor tyrosine kinases (i.e., VEGFR-1 and VEGFR-2) to mediate angiogenic responses.
In the present study, we demonstrate that Sema7A, previously known for its immunomodulatory and neuronal effects, mediates vascular growth. We observed an increase in the expression levels of corneal Sema7A after bFGF pellet implantation. Sema7A expressed in bFGF-stimulated corneal fibroblasts was visualized by confocal immunostaining. In addition, macrophage recruitment, VEGF-A expression, and NV were evident in corneas injected with Sema7A cDNA. These data suggest a pro-angiogenic role for Sema7A in corneal NV.
Animal studies were conducted in accordance with the Animal Care and Use Committee Guidelines of the University of Illinois at Chicago and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 wild-type mice were used in this research.
A Sema7A peptide (REDNPDKNPEAPLNVSRVAQLC) was synthesized based on the mouse Sema7A amino acid sequence (242–264) and injected into a rabbit. Rabbit serum was collected, and anti-Sema7A antibodies were purified using a Sema7A peptide affinity column. The specificity of the anti-Sema7A antibody was confirmed by peptide competition assay.
Pellets were made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate), which contains sucralfate (45 ng/pellet; Sigma-Aldrich, St. Louis, MO) and human bFGF (120 ng/pellet; R&D Systems, Minneapolis, MN) as previously described 16. A suspension of recombinant bFGF and sucralfate was prepared in sterile saline and concentrated for 10 minutes, and then 12% Hydron in ethanol (10 μl) was added. The suspension was then deposited onto a sterilized nylon mesh (LAB Pak, Sefar America, Depew, NY) and embedded between the fibers. The resulting grid (10×10-mm squares) was allowed to dry on a sterile Petri dish for 1 hour. The fibers of the mesh were separated under a microscope, and among the approximately 100 pellets produced, 30–40 pellets of uniform size (0.4×0.4×0.2 mm) were selected for implantation.
Corneal micropockets were created with a modified von Graefe knife in adult mice, and the pellets were implanted into corneal pockets. On days 1, 3, and 10 after implantation, the eyes were harvested, embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN), and frozen in liquid nitrogen prior to immunolocalization assay.
Cryostat sections (8 μm) of the OCT-embedded corneas were fixed in 4% paraformaldehyde for 15 minutes. After blocking with 1% bovine serum albumin (Sigma-Aldrich) for 30 minutes, the sections were incubated for 1 hour with rabbit anti-mouse Sema7A polyclonal antibody, goat anti-mouse VEGF-A antibody (R&D Systems), and rat anti-mouse F4/80 antibody (ebiosciences, San Diego, CA), followed by Incubation with fluorescein-conjugated donkey anti-rabbit IgG, FITC-donkey anti-goat IgG, and Cy5 donkey anti-rat IgG secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Negative controls were prepared in the same manner, but without the primary antibody. Propidium iodide (PI; Vector Laboratories, Burlingame, CA) was used for nuclear staining. Immunostained sections were viewed by confocal microscopy (TCS 4D; Leica, Heidelberg, Germany).
The expression vector containing mouse Sema7A cDNA (pCMV-Sport6-Sema7A) was purchased from ATCC (Manassas, VA). Mice received a 5-μl injection of pCMV-Sport6-Sema7A (400 ng/μl; n=9) into the central corneal stroma, or a 5-μl injection of empty vector (400 ng/μl pCMV-Sport6; n=9). An antibiotic ophthalmic ointment was used after injection.
Mouse corneas were evaluated on days 3, 7, and 10 after DNA injection. Corneal NV was digitally photographed using a slit lamp microscope (Nikon FS-2, Tokyo, Japan). The neovascular area was measured and analyzed with NIH ImageJ software (NIH, Bethesda, MD). Statistical analysis was performed with SPSS 14.0 software. p<0.05 was considered significant.
A 22-mer peptide (REDNPDKNPEAPLNVSRVAQLC) based on the mouse Sema7A sequence was used to generate an anti-Sema7A antibody (Fig. 1a). To determine whether Sema7A is expressed in mouse fibroblasts, cultured corneal fibroblasts were immunostained for Sema7A (Fig. 1b). Only low levels of Sema7A were detected in unstimulated fibroblasts (Fig. 1b, A–C); however, an increase in Sema7A expression was found in bFGF-stimulated fibroblasts (Fig. 1b, D–F). The specificity of the anti-Sema7A antibody was determined by the positive staining of Sema7A in bFGF-stimulated corneal fibroblasts (Fig. 1c, A–C). Negative immunofluorescence staining of Sema7A in bFGF-stimulated corneal fibroblasts after preincubation of the anti-Sema7A antibodies with cognate Sema7A peptides further confirmed the specificity of the anti-Sema7A antibodies (Fig. 1c, D–F).
Mouse eyes implanted with a bFGF pellet were immunostained for Sema7A on days 1, 3, and 10 post-implantation. On day 1 after implantation, little to no Sema7A was detected in the cornea (Fig. 2A–C). However, on days 3 and 10, increased expression levels of Sema7A were observed in the stroma between the implanted pellet and the corneal limbus (Fig. 2D–I).
To determine whether Sema7A could induce NV, mouse corneas were injected with Sema7A cDNA or empty vector pCMV-Sport6 (Fig. 3). No corneal NV was detected in the control group (empty vector) at days 3, 7, and 10 (Fig. 3A–C). In contrast, corneal NV was evident on day 7 in corneas injected with Sema7A cDNA (Fig. 3E), and was considerable on day 10 (Fig. 3F). New vessels developed through the superficial cornea from the limbus towards the central area, where the Sema7A cDNA was injected. Compared with controls, the neovascular area appeared to increase in Sema7A cDNA-injected corneas on days 3 (0.1 mm2 vs. 0.05 mm2; p=0.39) and 7 (0.49 mm2 vs. 0.06 mm2; p=0.12), but the amount of corneal NV was not significantly higher until day 10 (1.8 mm2 vs. 0.11 mm2; p<0.02; Fig. 3G).
Sema7A has been shown to stimulate human monocytes during T-cell responses. To assay the effect of naked Sema7A DNA in injection-induced corneal NV, recruitment of inflammatory cells and VEGF-A expression levels in Sema7A cDNA- and empty vector-injected mouse corneas were determined. Both Sema7A cDNA- and empty vector-injected corneas were immunostained with antibodies against both the macrophage-specific marker F4/80 and VEGF-A. Sections from the Sema7A cDNA- and vector-injected corneas on day 1 showed neither F4/80 (Fig. 4a) nor VEGF-A (Fig. 4b) immunostaining. By day 7, vector-injected corneas showed low levels of F4/80 (Fig. 4a M) and VEGF-A (Fig. 4b A) immunostaining; furthermore, by day 7, Sema7A-injected corneas showed increases in F4/80 (Fig. 4a P) and VEGF-A (Fig. 4b D) immunostaining.
In the present study, we demonstrated that Sema7A is not constitutively expressed in mouse corneas; however, Sema7A expression was observed after bFGF stimulation. Furthermore, mouse corneas injected with Sema7A cDNA developed significant NV by day 10 compared to corneas injected with empty vector. These findings suggest a previously unknown pro-angiogenic role for Sema7A in corneal NV.
Semaphorins have been described as inhibitory signals because of their ability to prevent cell migration and axon outgrowth 17. However, semaphorins can, at times, promote cell chemotaxis and axon/dendrite outgrowth and attraction 13, 18, 19. Several reports have shown that Sema7A induces chemotaxis, inflammatory cytokine production [e.g., tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8], and superoxide release in monocytes 20. In this report, the increase in corneal NV in Sema7A-injected corneas coincided with the presence of F4/80, a macrophage marker, suggesting that Sema7A may mediate an inflammatory response and recruit infiltrating cells, including macrophages. Additionally, the presence of VEGF-A in Sema7A cDNA-injected vascularized corneas supports the hypothesis that Sema7A-induced NV is mediated through VEGF-A production.
Although the roles of semaphorins have not been characterized as extensively in the vascular system as in the nervous and immune systems, members of the semaphorin family appear to be important in vascular remodeling. Sema3A has been reported to play a crucial role in regulating endothelial cell migration and angiogenesis 21–23, and appears to mediate the topographic congruence of nerves and blood vessels 24. Sema3F inhibits angiogenesis in vitro and in vivo and is a potent inhibitor of tumor angiogenesis 25. The soluble extracellular domain of Sema6A-1 blocks VEGF-mediated endothelial cell migration in vitro, and its intraperitoneal administration inhibited both bFGF- and VEGF-induced NV in vivo 26. Other semaphorins may exert opposing effects. Sema4D was reported to potentially induce chemotaxis and tubulogenesis in endothelial cells in vitro and promote blood vessel formation in an in vivo mouse model; these functions are believed to be mediated by the plexin B1 receptor 27–29.
The characterization of Sema7A receptors remains controversial. Plexin C1 was identified first as a Sema7A receptor 30; further studies demonstrated a β1 subunit-containing integrin receptor as a Sema7A receptor 13. Further research is needed to characterize the mechanisms and receptors involved in the actions of Sema7A in corneal NV.
In the present report, we demonstrated that Sema7A was expressed in vascularized corneas and showed pro-angiogenic properties in our corneal model. These findings indicate a previously unknown role for Sema7A in corneal NV. Understanding the mechanism of Sema7A in angiogenesis may provide a therapeutic target for the treatment of corneal NV and other angiogenesis-related disorders, such as diabetic retinopathy, macular degeneration, and cancer.
Grant support: An unrestricted departmental grant from Research to Prevent Blindness New York (NY) and National Institutes of Health Grants EY10101 (DTA) and EY001792 (DTA).
Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.