The role of bone morphogenetic protein 4 in corneal injury repair.

PURPOSE: Corneal injury may cause neovascularization and lymphangiogenesis in cornea which have a detrimental effect to vision and even lead to blindness. Bone morphogenetic protein 4 (BMP4) regulates a variety of biological processes, which is closely relevant to the regulation of corneal epithelium and angiogenesis. Herein, we aimed to evaluate the effect of BMP4 on corneal neovascularization (CNV), corneal lymphangiogenesis (CL), corneal epithelial repair, and the role of BMP4/Smad pathway in these processes. METHODS: We used MTT assay to determine the optimal concentration of BMP4. The suture method was performed to induce rat CNV and CL. We used ink perfusion and HE staining to visualize the morphological change of CNV, and utilized RT-qPCR and ELISA to investigate the expression of angiogenic factors and lymphangiogenic factors. The effects of BMP4 and anti-VEGF antibody on migration, proliferation and adhesion of corneal epithelium were determined by scratch test, MTT assay and cell adhesion test. RESULTS: BMP4 significantly inhibited CNV and possibly CL. Topical BMP4 resulted in increased expression of endogenous BMP4, and decreased expression of angiogenic factors and lymphangiogenic factors. Compared with anti-VEGF antibody, BMP4 enhanced corneal epithelium migration, proliferation and adhesion, which facilitated corneal epithelial injury repair. Simultaneously, these processes could be regulated by BMP4/Smad pathway. CONCLUSIONS: Our results demonstrated unreported effects of BMP4 on CNV, CL, and corneal epithelial repair, suggesting that BMP4 may represent a potential therapeutic target in corneal injury repair.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas.

Background: Corneal neovascularization (CNV) caused by alkali burn injury is tightly associated with an inflammatory reaction and can lead to vision loss. Melatonin is involved in anti-inflammation and anti-angiogenesis, but its role in CNV has not yet been investigated. Methods: We induced CNV using sodium hydroxide (NaOH) and compared the reactions of vehicle control and melatonin-treated male C57BL/6 mice at 7 and 14 days following the corneal burn. The infiltration of inflammatory cells and the expression of proangiogenic factors, chemokines, and inflammation-related molecules were quantified via immunohistochemical (IHC) analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Murine peritoneal macrophages were used in vitro to further verify the effect of melatonin in inflammatory CNV. Results: Compared with the vehicle control mice, the melatonin-treated mice showed significant inhibition of angiogenesis and reduction of corneal epithelial defects in alkali-burned corneas. Concomitantly, the infiltration of inflammatory cells and F4/80+ cells were dramatically reduced after melatonin treatment. The messenger RNA (mRNA) expression of proangiogenic factors [vascular endothelial growth factors (VEGF) and matrix metalloproteinase-9 (MMP-9)], monocyte chemotactic protein-1 (MCP-1), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) and IL-6] were down-regulated in the melatonin-treated mice. Moreover, melatonin inhibited the expression of these factors in murine peritoneal macrophages. Conclusions: Melatonin inhibits the neovascular and inflammatory responses in corneal alkali burn injury, suggesting that it may be a potential therapy for CNV.

Effect of Nintedanib Nanothermoreversible Hydrogel on Neovascularization in an Ocular Alkali Burn Rat Model.

PURPOSE: To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. METHODS: Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. RESULTS: NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 µM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. CONCLUSIONS: Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.

Combined delivery of small molecule and protein drugs as synergistic therapeutics for treating corneal neovascularization by a one-pot coassembly strategy.

Combined drug administration is a potential strategy to increase efficacy through therapeutic synergy. Small molecule drugs and protein drugs are the two most popular kinds of drugs in medicine. However, efficiently encapsulating these two drugs still have key challenges due to their distinct properties (molecular weight, hydrophilicity, chemical groups, etc.), weak ability to penetrate through various biobarriers (cell membrane, endosome escape, tissue barriers dependent on the method of administration, etc.) and the easy deactivation of protein drugs during the construction of carrier and delivery process. Here, we utilize the hexahistidine-metal assembly (HmA), which can encapsulate a wide spectrum of drugs with high loading efficiency, to coencapsulate Dexp (a small molecule drug) and BVZ (protein drug) by a one-pot coassembly strategy. Our data demonstrated that Dexp and BVZ were coloaded into Dexp&BVZ@HmA with high efficiency, while the bioactivity of BVZ was well-maintained. Most importantly, when evaluating the therapeutic outcomes of drugs@HmA in a corneal neovascularization (CNV) model in vitro and in vivo, the combination group presented overwhelming efficacy compared to the monotherapy group. This strategy offers a platform to codeliver protein and small drugs and has the potential for treating anterior segment diseases as well as other diseases that need combination therapy.

Loss of miR-673-5p expression in the cornea promotes rat corneal allograft rejection by promoting Th17 cell differentiation mediated by JAK2/STAT3.

BACKGROUND: Cluster of differentiation 4 positive (CD4+) T cells play an important role in corneal graft rejection, especially the dynamic balance between regulatory T cells and helper T cells. This study aims to explore the upstream and downstream regulatory mechanisms of Th17 cell differentiation-mediated corneal allograft rejection. METHODS: By establishing rat corneal allograft transplantation model, transcriptome analysis was carried out to screen the differentially expressed genes related to T helper 17 (Th17) cell differentiation, and then cell experiments were used to verify the effect of miR-673-5p/Janus Kinase 2 (JAK2) signal on naïve CD4+ T cell differentiation and the proliferation, migration, and tube formation ability of human umbilical vein endothelial cells (HUVECs). Finally, the role of miR-673-5p/JAK2 signal in corneal allograft rejection was verified by animal model in vivo. RESULTS: The results showed that JAK2/STAT3 signaling activation-mediated Th17 cell differentiation was significantly up-regulated during corneal allograft rejection, and miR-673-5p expression was down-regulated after corneal allograft rejection. Low expression of miR-673-5p promoted Th17 cell differentiation by up-regulating JAK2, and then promoted placental growth factor (PLGF)mediated corneal neovascularization (CNV). CONCLUSIONS: The results of this study suggested that low expression of miR-673-5p is a promoter of corneal allograft rejection. Overexpression of miR-673-5p can improve the survival rate of corneal allografts by inhibiting the differentiation and maturation of Th17 cells mediated by JAK2/STAT3 signaling.

Tetramethylpyrazine (TMP) ameliorates corneal neovascularization via regulating cell infiltration into cornea after alkali burn.

In the present study, we investigated the underlying mechanism of tetramethylpyrazine (TMP)-medicated inhibition of corneal neovascularization (CNV). Our data showed that TMP could effectively downregulate the expression levels of CXCR4 mRNA and protein, as well as inhibit HUVECs, endothelial cells, tubule formation in vitro. In vivo, alkali burn (1 M NaOH) could remarkably upregulate CXCR4 expression and increase the migration of TNF-α-positive cells to corneal stroma. TMP drops could significantly downregulate CXCR4 expression in cornea, compared to the control. However, there was no difference in the downregulation of CXCR4 between TMP and FK506, an immunosuppressive drug. Moreover, the immunofluorescent staining of CD45 showed TMP and FK506 could significantly restrain the bone marrow (BM)-derived infiltration while the F4/80 staining reflects the suppression of macrophage aggregation. Meanwhile TMP could regulate the Interleukin 10 (IL-10) and FK506 could restrain the Interleukin 2 (IL-2). Furthermore, TMP and FK506 significantly ameliorate corneal opacity and neovascularization. Clinical assessment detected an obvious improvement in TMP and FK506 treatment groups, compared to controls in vivo. Thus, TMP had similar effects in inhibition of immune response and CNV by suppressing BM-infiltrating cells into cornea as FK506. TMP could be a potential agent in eye-drop therapy for cornea damaged by Alkali Burn.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1ß, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.