Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

Abstract

Purpose: To evaluate the angiogenic properties of corneal derived mesenchymal stromal cells (Co-MSC).

Methods: Co-MSCs were extracted from human cadaver, and wild-type (C57BL/6J) and SERPINF1-/- mice corneas. The MSC secretome was collected in a serum-free medium. Human umbilical vein endothelial cell (HUVEC) tube formation and fibrin gel bead assay (FIBA) sprout formation were used to assess the angiogenic properties of Co-MSC secretome. Complete corneal epithelial debridement was used to induce corneal neovascularization in wild-type mice. Co-MSCs embedded in fibrin gel was applied over the debrided cornea to evaluate the angiogenic effects of Co-MSCs in vivo. Immunoprecipitation was used to remove soluble fms-like tyrosine kinase-1 (sFLT-1) and pigment epithelium-derived factor (PEDF, SERPINF1 gene) from the Co-MSC secretome.

Results: Co-MSC secretome significantly inhibited HUVECs tube and sprout formation. Co-MSCs from different donors consistently contained high levels of antiangiogenic factors including sFLT-1 and PEDF; and low levels of the angiogenic factor VEGF-A. In vivo, application of Co-MSCs to mouse corneas after injury prevented the development of corneal neovascularization. Removing PEDF or sFLT-1 from the secretome significantly diminished the antiangiogenic effects of Co-MSCs. Co-MSCs isolated from SERPINF1-/- mice had significantly reduced antiangiogenic effects compared to SERPINF1+/+ (wild-type) Co-MSCs.

Conclusions: These results illustrate the direct antiangiogenic properties of Co-MSCs, the importance of sFLT-1 and PEDF, and their potential clinical application for preventing pathologic corneal neovascularization.

Figures

Figure 1

Human and mouse corneal stromal cells demonstrate mesenchymal stromal cells (MSC) features. (A) Bright-field image of passage-4 human corneal MSCs. (B) Flow cytometry analysis demonstrated a homogenous MSC population. More than 95% of the cells were positive for cell surface markers CD73, CD90, CD105, and negative for CD19, CD45, HLA-DR, CD11b, and CD34 (n = 10). (C) Differentiation into the three mesenchymal lineages: I: Osteogenesis: calcium deposition stained with Alizarin Red; II: Adipogenesis: lipid formation stained with LipidTOX; III: Chondrogenesis: Glycosaminoglycans stained with Alcian Blue. (D) Bright-field microscopy image of passage-4 mouse corneal MSCs. (E) Flow cytometry analysis demonstrated a homogenous MSC population. More than 95% of the cells are positive for cell surface markers CD29, Sca-1, CD105, CD44 and CD106 and negative for CD11b, and CD45. (F) Differentiation into the three mesenchymal lineages: I: Calcium deposition stained with Alizarin Red; II: Lipid formation stained with LipidTOX; III: Glycosaminoglycans stained with Alcian Blue.

Figure 2

Corneal MSCs (Co-MSCs) are directly antiangiogenic in vitro. (A) Co-MSC secretome significantly inhibited mean sprout count per bead and mean sprout length in a fibrin-induced bead assay (n = 5). *P S, secretome.

Figure 3

Co-MSCs have an antiangiogenic profile by protein expression. (A) Human angiogenesis array was used to profile pro- and antiangiogenesis mediators in the Co-MSC secretome. It revealed it has high levels of antiangiogenic factors, whereas low levels of proangiogenic factors. (B) The secretome from Co-MSCs contains high levels of sFLT-1 (1875 ± 677 pg/mL); PEDF (4829 ± 2342 pg/mL); TSG-6 (643.3 ± 149.1 pg/mL); and low amount of VEGF-A (106.9 ±103.3 pg/mL; n = 5). The values shown are mean ± SD (error bars).

Figure 4

Co-MSCs inhibit pathologic corneal neovascularization in vivo. (A) Schematic illustration shows the steps for debriding the epithelium, MSC delivery using fibrin gel and temporarily closing the lids. (B) In vivo microscopy confirmed the presence of fluorescent-labeled MSCs in the cornea three days after the procedure. Scale bars: 200 μm. (C) The slit lamp photos and CD31 staining demonstrated both Co-MSCs significantly prevented corneal neovascularization compared to fibrin gel alone 14 days after injury. The values shown are mean ± SD (error bars). *P

Figure 5

Direct antiangiogenic properties of Co-MSCs depend on PEDF and sFLT-1. (A) Removing sFLT-1 and PEDF with neutralizing antibody capturing and immunoprecipitation abrogated the antiangiogenic property of Co-MSC secretome in a HUVEC assay (n = 5 1-way ANOVA and then 2-sided t-test were used to compare the groups). *P

Figure 6

Direct antiangiogenic properties of Co-MSCs are in part due to PEDF. Knocking down PEDF in Co-MSCs by siRNA obviates the antiangiogenic effects of their secretome and increases tubule formation in a HUVEC assay (n = 4, 1-way ANOVA, and then 2-sided t-test were used to compare the groups). *P

Figure 7

PEDF plays a key role on the antiangiogenic properties of Co-MSCs in vivo. (A) Western blot shows Co-MSCs isolated from SERPINF1−/− mice do not express PEDF. (B) Co-MSCs isolated from SERPINF1−/− mice had lower antiangiogenic effects, compared to wild-type (SERPINF1+/+) Co-MSCs, when placed in fibrin gels after corneal epithelial injury in wild-type mice (same model as in Fig. 4A; n = 5, 2-sided t-test: *P = 0.0001). Scale bars: 500 μm. The values shown are mean ± SD (error bars). Co-mMSCSF1, corneal mouse MSCSERPINF1; NV, neovascularization.

Figure 8

Schematic illustration summarizes the mechanisms by which Co-MSCs confer their antiangiogenic properties. (A) After corneal epithelial injury, DAMPS, chemokines, and cytokines released by the injured and resident cells directly induce angiogenesis while also recruiting inflammatory cells that further promote angiogenesis. The net effect is shifting the antiangiogenic balance of the cornea toward angiogenesis. (B) Co-MSCs applied to the cornea after injury modulate neovascularization directly through the secretion of antiangiogenic factors including PEDF and sFLT-1 and indirectly by suppressing inflammation via TSG6 and other anti-inflammatory factors. DAMP, damage-associated molecular pattern.