Anti-Inflammatory and Anti-Fibrotic Effects of Human Amniotic Membrane Mesenchymal Stem Cells and Their Potential in Corneal Repair

Abstract

Acute ocular chemical burns are ophthalmic emergencies requiring immediate diagnosis and treatment as they may lead to permanent impairment of vision. The clinical manifestations of such burns are produced by exacerbated innate immune response via the infiltration of inflammatory cells and activation of stromal fibroblasts. New therapies are emerging that are dedicated to repair mechanisms that improve the ocular surface after damage; for example, transplantation of stem cells (SC) has been successfully reported for this purpose. The pursuit of easily accessible, noninvasive procedures to obtain SC has led researchers to focus on human tissues such as amniotic membrane. Human amniotic mesenchymal SC (hAM-MSC) inhibits proinflammatory and fibrotic processes in different diseases. hAM-MSC expresses low levels of classical MHC-I and they do not express MHC-II, making them suitable for regenerative medicine. The aim of this study was to evaluate the effect of intracameral injection of hAM-MSC on the clinical manifestations, the infiltration of inflammatory cells, and the activation of stromal fibroblasts in a corneal alkali-burn model. We also determined the in vitro effect of hAM-MSC conditioned medium (CM) on α-SMA⁺ human limbal myofibroblast (HLM) frequency and on release of neutrophil extracellular traps (NETs). Our results show that intracameral hAM-MSC injection reduces neovascularization, opacity, stromal inflammatory cell infiltrate, and stromal α-SMA⁺ cells in our model. Moreover, in in vitro assays, CM from hAM-MSC decreased the quantity of α-SMA⁺ HLM and the release of NETs. These results suggest that intracameral hAM-MSC injection induces an anti-inflammatory and anti-fibrotic environment that promotes corneal wound healing. Stem Cells Translational Medicine 2018;7:906-917.

Keywords: Corneal repair; Inflammation; NETs; hAM-MSC; α-SMA myofibroblasts.

Figure 1

Intracameral hAM‐MSC injection ameliorates neovascularization and corneal opacity on corneal burn. Clinical photographs of non‐injured cornea of control group (top and bottom‐left plots); alkali‐injured cornea of NaOH group and treated with intracameral BSS (top and bottom‐middle plots); and alkali‐injured cornea of NaOH‐hAM‐MSC group (top and bottom‐right plots) at day 1 (top plots) and 12 days after burn (bottom plots). Notice that hAM‐MSC injection ameliorates clinical features of the alkali‐corneal burn injury 12 days after the intracameral injection of hAM‐MSC. These are representative images of six independent assays (A). Graphical comparison of corneal opacity (B) and corneal neovascularization (C) scores, among NaOH and NaOH‐hAM‐MSC treated groups. Intracameral injection of hAM‐MSC (grey bars) reduces significantly corneal opacity (**p < .01; n = 6); and corneal neovascularization (***p < .001; n = 6) in comparison with alkali‐burned corneas (black bars). These results are obtained from the clinical evaluation at day 12 postinjury. Data are expressed as the mean value of clinical score ± SE. Abbreviation: CNV, corneal neovascularization.

Figure 2

hAM‐MSC locate into the anterior chamber after 12 days of intracameral injection. Fluorescence microscopy of the anterior chamber of an NaOH‐hAM‐MSC treated cornea. The hAM‐MSC was QD‐labeled and injected intracamerally at the same time of corneal injury. After 12 days, immunofluorescence microscopy was performed to visualize HNA marker. Interestingly, hAM‐MSC were found in the iris even after 12 days of injection and were not located in the cornea or adjacent tissues such as corneal endothelium or ciliary processes; arrows indicate the autofluorescence of erythrocytes (left plot, scale bar represents 100 μm); the magnification of inner square is presented in right plot (scale bar represents 5 μm). In both plots cell nuclei are shown with DAPI (blue), QD‐labeled‐hAM‐MSC in red and HNA marker in green. These are representative images from six‐independent assays. Abbreviations: AC, anterior chamber; DAPI, 4′,6‐diamidino‐2‐phenylindole; hAM‐MSC, human amniotic mesenchymal stem cell; HNA, human nuclear antigen; QD, quantum dot.

Figure 3

Intracameral hAM‐MSC injection diminishes stromal cell proliferation. Immunofluorescence micrographs from the corneal stroma stained with the proliferating cell marker Ki‐67 in the control group (top‐left plot), NaOH‐group (top‐right plot) and NaOH‐hAM‐MSC group (bottom‐left plot). Ki‐67 is preferentially expressed into the nucleus of the stromal proliferating cells as shown in the middle right plots. These are representative images from six‐independent experiments; scale bar represents 20 μm (A). The chemical burn induces a significant augmentation of the Ki‐67⁺ cells in the corneal stroma in comparison with the control group; interestingly, the NaOH‐hAM‐MSC group presented a significant reduction in comparison with the NaOH‐group as observed in the bars graphic (bottom‐right plot) (B). Three random fields were analyzed for each animal (n = 6). Data are expressed as mean ± SE (*p < .05).

Figure 4

Intracameral hAM‐MSC injection reduces corneal myofibroblasts differentiation. Immunofluorescence micrographs from the corneal stroma stained with anti‐α‐SMA in the control group (upper‐left panel), NaOH group (upper‐right panel) and NaOH‐hAM‐MSC (lower‐left panel). These are representative images from six independent experiments, scale bar represents 20 μm (A). Interestingly, the group of the alkali‐injured rabbits treated with hAM‐MSC (grey bar) presented a significant reduction of the α‐SMA positive cells in comparison with the alkali‐burned group (black bar) as observed in the bars graphic (lower‐right panel) (B). Three random fields were analysed for each animal (n = 6). Data are expressed as mean ± SE (*p < .05).

Figure 5

CM from hAM‐MSC significantly reduces the frequency of α‐SMA⁺ HLM. Histograms from human limbal myofibroblasts (HLM) cultured in the presence (gray line) or in the absence (black line) of CM from hAMSC during 12 hours; dashed line represents the staining negative control (A). CM from hAM‐MSC significantly reduce the frequency of α‐SMA⁺ HLM (gray bar) compared with the α‐SMA⁺ HLM incubated only with medium (black bar) (B). Each bar represents the mean of α‐SMA⁺ HLM frequency ± SE (*p < .05; n = 3).

Figure 6

CM from hAM‐MSC reduced NETs releasing cells. Human isolated neutrophils were stimulated or not with PMA and NETs release was identified with extracellular DNA and elastase. Nonstimulated neutrophils present their characteristic lobulated nuclei and the elastase were located in the cytoplasm (top‐left plot); in contrast, PMA‐stimulated neutrophils liberated NETs identified by the colocalization of both DNA and elastase (top‐right plot); meanwhile, PMA‐stimulated neutrophils cultured in the presence of CM from hAM‐MSC, presented nonlobulated nuclei and the release of NETs by these cells was reduced (bottom‐left plot). The images are representative of three independent assays; scale bar represents 20 μm (A). PMA increased NETs release up to fourfold change in comparison with the nonstimulated neutrophils; in contrast, and interestingly, CM from hAM‐MSC was able to significantly reduce NETs release in PMA‐stimulated neutrophils (bottom‐right plot) (B). Data are expressed as mean ± SE (***p < .001).