The Role And Mechanism Of BMP4 In Corneal Injury Repair

BackgroundIn general,the cornea is avascular,transparent,and an essential component of the refractive system.Corneal neovascularization(CNV)is a common ocular pathology,the fourth leading cause of vision impairment worldwide,and one of the most difficult to treat clinically.CNV is caused by a variety of reasons,including infection,inflammation,and chemical injury.Treatments for CNV include topical steroids,non-steroidal anti-inflammatory agents,laser cautery,fine-needle diathermy,amniotic membrane transplantation,anti-vascular endothelial growth factor(VEGF)agents,and even gene therapy.However,all of the above methods have limited effectiveness and have associated side effects.Therefore,the study of the pathogenesis of CNV and the exploration of effective therapeutic measures,as well as the search for alternative drugs with fewer side effects,will be of great practical significance for the prevention and treatment of CNV,as well as for the prevention and treatment of blindness.Inflammation is a key pathological process in the formation of CNV and the main mechanism is the imbalance between pro-and anti-angiogenic factors.A gradient of VEGF-A triggers the selection of individual endothelial cells as tip cells that guide sprouting.A dynamic balance of tip and stalk cells that always maintains a certain ratio collaborates to promote angiogenesis.However,the mechanism of tip and stalk cell selection is still unclear.CNV formation is often accompanied by neutrophil over infiltration.Neutrophils induce inflammatory response by generating neutrophil extracellular traps(NETs)that lead to the expansion of the inflammatory response and the formation of CNV.NETs also play a pro-angiogenic role in various disease processes.Currently,the effect of NETs on CNV in the corneal suture model has not been reported.The apical junctional complexes(AJCs)of the corneal epithelium are key structures in maintaining the corneal barrier function.It consists of adhesive junctions beneath the corneal epithelial cells and tight junctions at the top.Various etiological factors that lead to CNV cause varying degrees of damage to corneal epithelial AJCs.Damage to AJCs can further accelerate the development of CNV.Therefore,maintaining the normal structure and function of the cornea depends on the integrity of the AJCs,and repairing damaged epithelial AJCs can help prevent the formation of CNV.As a cytokine involved in cell differentiation,proliferation,metastasis and apoptosis,bone morphogenetic proteins(BMP4)plays a special regulatory role in tissue healing and angiogenesis.The group previously found that BMP4 could significantly reduce CNV formation,downregulate VEGF-A expression,and promote corneal epithelial healing.However,the specific mechanism by which BMP4 inhibits CNV formation is unknown.PurposeThis study started from the establishment of a corneal suture-induced CNV model,explored the regulatory mechanism of BMP4 on NETs during CNV and its effect on tip cells,investigated whether BMP4 inhibits CNV by repairing corneal epithelial AJCs,and loaded BMP4 into F127-OSA hydrogel to prolong the retention time of the drug on the ocular surface and to improve the efficiency of its use.Methods1.Human umbilical vein endothelial cells(HUVECs)were cultured,and VEGF-A was applied to induce angiogenesis.The proliferation ability of the cells was detected by CCK-8 assay,and the optimal concentration of VEGF-A was determined.Cell proliferation,migration and lumen formation were observed by tube forming experiment.The tube formation assay was used to observe cell proliferation,migration,and to study the effect of BMP4 on angiogenesis.In addition,immunofluorescence co-staining of CD34 and phalloidine was applied to analyse the effect of BMP4 on CD34-stained tip cell.2.To establish a suture-induced CNV model,three interrupted 10-0 sutures were placed 1.5 mm from the limbus to induce CNV in wistar rats.The experiment was divided into a control group(Untreated),a suture control group(Suture),a solvent control group(BMP4 solvent),and a BMP4 group(BMP4).After modelling,20μg/ml BMP4 was dropped into the left eye as the BMP4 group,and 5μl of buffer(0.1%bovine serum albumin,4 m M HCl)was dropped into the right eye as the solvent control group,3 times a day for 3,5 and 7 days.There was also a suture group(no drug dropped after modelling)and a control group(no modelling,no drug dropping).Photographs were taken by slit lamp biomicroscopy for corneal analysis on days 3,5 and 7 after modelling respectively.The degree of CNV in neovascularisation was determined according to the length,area and number of bifurcation points;HE stained sections were applied to observe the morphological changes and inflammatory reactions in corneal epithelium;Western Blot experiments were performed to detect the expression of VEGF-A and VEGFR2;and the mode of neovascularisation in CNV was observed by electron microscopy.Immunofluorescence staining was used to detect the expression of MPO and Histone H3.3.A suture-induced rat CNV model was established and the experiment was divided into a control group(Untreated),a suture control group(Suture),a solvent control group(DMSO solvent)and a DPI group(DPI).To study the role of NOX in suture-induced CNV,the NOX inhibitor DPI was used in eye drops.DPI eye drops were applied immediately after modelling,four times a day for 7 days.Saline with an equal concentration of DMSO was used as a control.The expression of MPO and Histone H3in each group was detected by immunofluorescence staining;the expression of NOX-2was detected by Western Blot.4.A suture-induced rat CNV model was established,and the experiment was divided into the untreated group,the suture group,the BMP4 solvent group and the BMP4 group.Western Blot was applied to detect the expression of NOX-2 protein.5.A suture-induced rat CNV model was established,and the experiment was divided into the untreated group,the suture group,the BMP4 solvent group and the BMP4 group.The drug spotting was started 7 days after modelling,3 times a day,and the rat corneal tissues were taken after 3 days for circ RNA microarray to select the target genes for further validation.6.A suture-induced rat CNV model was established,and the experiment was divided into the untreated group,the suture group,the BMP4 solvent group and the BMP4 group.The expression of tight junction protein ZO-1 and adhesion junction proteins E-cadherin andβ-catenin were detected by immunofluorescence staining and Western Blot after 7 days of modelling.7.A suture-induced rat CNV model was established,and the experiment was divided into a control group(Untreated),a suture control group(Suture),a gel group(Gel),a BMP4 group(BMP4),and a BMP4+gel group(BMP4+Gel).Corneal analysis was performed by slit-lamp biomicroscopy on days 3,5 and 7 after modelling;the thickness of corneal epithelium and the morphology of epithelial cells were observed in HE-stained sections.And the damage and repair of corneal epithelium in each group were observed by transmission electron microscopy on days 7 after modelling.Results1.CCK-8 showed that different concentrations of VEGF-A had significant effects on the proliferation of HUVECs.20 ng/ml VEGF-A was the optimal concentration to promote the proliferation of HUVECs.In vitro tube formation experiments showed that HUVECs in the VEGF-A control group formed multiple densely arranged tube structures in each image,whereas those in the 20μg/ml BMP4group were fewer and more sparsely arranged.2.Immunofluorescence staining showed significantly lower expression of CD34and filamentous morphology of CD34~+tip cells in the BMP4 group compared with the control group.3.In vivo,the length of CNV was shortened and the area of CNV was significantly reduced in the BMP4,and the corneal inflammation was reduced.BMP4 mainly decreased the protein expression of VEGF-A and vascular endothelial growth factor receptor 2(VEGFR2)in cornea after suture injury.By observing the ultrastructure of the cornea,BMP4 inhibited the sprouting of tip cells and brought forward the appearance of intussusception.4.Immunofluorescence staining of the corneas showed that mesh NETs labeled with DNA,MPO and Histone H3 were obviously accumulated in the corneal stroma of the suture group over time.In the BMP4 group,there was less extracellular colocalization of DNA with MPO and Histone H3.5.Immunofluorescence staining of cornea showed that DPI reduced the formation of NETs.Western Blot experimental assay showed that NOX-2 expression was up-regulated in rat cornea after suture-induced CNV,and DPI inhibited NOX-2 expression.6.Western Blot assay showed that the expression of NOX-2 was also decreased in the BMP4 group compared with the suture group and the solvent control group.7.CircRNA microarray analysis.The first step showed the differentially expressed circ RNA in the BMP4 group compared to the suture group,and filtered out the down-regulated circ RNA in the suture group compared to the normal group by the t-test,and up-regulated circ RNA in the BMP4 group after excluding the effect of solvent group.The second step was verified by RT-q PCR test,and selected circ＿009413 for our study.According to the ce RNA regulation machine,rno＿circ RNA＿009413 can target micro RNAs by sponge adsorption,which in turn regulates the expression of the downstream target gene Raf1.Western Blot experiments were used to detect the expression of Raf1 in the untreated group,the suture group,the BMP4 solvent group and the BMP4 group.The results showed that suture caused an increase in Raf1expression,while Raf1 expression was significantly reduced in the BMP4 group.8.The corneal epithelial cells in the suture group and the solvent control group showed loose inter-cellular connections,vacuoles,and a significant decrease in the number of inter-cellular connections,while the BMP4 group showed neat cell organisation and tight inter-cellular connections.The expression levels of ZO-1,E-cadherin,andβ-catenin in the suture group and solvent control group were also significantly lower than those in the control group,whereas BMP4 group significantly increased the expression levels of the above proteins.9.On day 5,the length and area of CNV in the BMP4+Gel group decreased significantly compared to the BMP4 group.By HE section staining,corneal epithelial cell growth was found to be abnormal and disorganised in the suture and gel groups.BMP4 eye drops and BMP4+Gel inhibited the abnormal proliferation and irregular arrangement of corneal epithelial cells.In addition,the thickness of the corneal epithelium increased significantly after suturing and continued to grow with the duration of stimulation.At all time intervals,BMP4 eye drops and BMP4+Gel significantly reduced corneal epithelial thickness,attenuated corneal oedema,and restored the corneal thickness to the normal level.BMP4+Gel outperformed BMP4 on day 5.10.The corneal epithelial microvilli in the untreated group were numerous and neatly organised,whereas the corneal epithelial microvilli in the suture group and the gel control group were flattened,with obvious detachment and fusion.The BMP4 group and the BMP4+Gel group improved the morphology and arrangement of the epithelial cells and repaired the damaged microvilli.The control group had tight intercellular junctions,intact nuclei and nuclear membranes,dense chromatin,and typical cell structures.In the suture group,the cell gap was significantly enlarged,the intercellular junctions were damaged,the chromatin was abnormal,the nuclear membrane was widened,the cytoplasm was locally lysed,the rough endoplasmic reticulum was enlarged,and the mitochondria were swollen.BMP4 group and BMP4+Gel group can be used to repair these injuries,with the thermal hydrogel being superior to the eye drops.Conclusions1.BMP4 inhibits corneal neovascularization.2.BMP4 can significantly inhibit the expression of VEGF-A in suture-induced CNV model,significantly inhibit the degree of corneal CNV,shorten the length of neovascularization,reduce the intervascular connections,and reduce the density of vascular reticular structure.3.BMP4 inhibits the formation of tip cells,destroys the angiogenesis sprouting of tip cells,breaks the dynamic balance of tip and stalk cells,and prevents the orderly process of angiogenesis,thus inhibiting CNV.4.BMP4 can reduce corneal inflammation and inhibit NOX-2-mediated NETs formation by down-regulating Raf1,thus inhibiting CNV.5.BMP4 has a restorative effect on corneal epithelium,which is achieved by regulating connexin.6.The temperature-sensitive gel wrapped BMP4 can slow down the BMP4efficacy,prolong the residence time of BMP4 in the ocular surface,and promote the faster repair of the cornea.