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The Inhibition Effect of Propranolol on the Corneal Neovascularization in Analkali-induced Injury Mouse Model

闫丽梦

南方医科大学

Abstract: Background Since1960s, p-adrenergic receptor blockers （β-blocker） are widely used in different area in clinic, especially in cardiologic diseases. In ophthalmology, the members in this family, e.g. timolol, are common in the treatment of glaucoma. Recently, the non-selective β-blocker propranolol is clearly known to inhibit the vessel endothelial cells, and down-regulate the vessel growth factors like VEGF, bFGF, which are mostly related to the hemangiomas growth. Therefore, it gets quite good recommendation in the treatment of hemangiomas, and in the hope of being one of the1st class medicine instead of the others. After that, some reports showed that propranolol also has certain inhibition effect on the neovascularizatio in a hyperoxic-induced retinopathy of premature （ROP） mouse model. There is no blood and lymph vessel in the normal cornea. It’s necessary for the cornea to maintain transparency and immuno privilege. When the stable, non-vessel state is broken by some pathologic factors as infection, injury or immunologic response, the neovascularization will invade into the cornea, destroy the micro-environment of it, and then lead to the injury of normal structure and function, even blindness. As one of the most commen factors of vision loss, corneal neovascularization （CNV） is also the high risk of the immunologic rejection after the cornea transplantation. That’s why it’s always amnong the popular topics in ophthalmology. The studies above all possess the similar mechanism with CNV. There is no any report about the inhibition effect of pronpranolol on the CNV yet. The animal model of CNV is an important way to study the mechanism and evaluate the therapic effect. Alkali-induced injury mouse model is one of the classic models. It can re-present the pathologic process of alkali burning in the corneas, and very useful in the study of inflammatory neovascularization. This model is widely applied due to the advantages like low cost, easy management and nice repeatability. The growth of CNV depends on the complicate effect of numerous vessel growth stimulators or inhibitors in the regulatory network, including inflammatory cytokines, growth factors and enzymes. The lots of relative studies provide reliable evidence for targets in CNV therapy. VEGF is the major blood-vessel growth factor. It’s involved in both vasculogenesis and angiogenesis. VEGF can improve the proliferation and migration of endothelial cells during angiogenesis, with degradation of base membrane and increase of permeability of blood vessels. bFGF is from the fibroblast growth factor family. It can stimulate the mitosis and induce the formation of vessels. It’s essential to the vasculogenesis, angiogenesis, development of nervous system and wound healing. IGF-1is a kind of polypetitde growth factor and exists in almost all the tissues. It’s a primary mediator of the effects of growth hormone, addition to the insulin-like effects. Both VEGF and bFGF are very closed to the neovascularization in hemangiomas. The down-regulated expression of them presents the obvious inhibition effect of propranolol in the hemangiomas. While in the studies of mouse hyperoxic-induced ROP model, the attenuation of neovascularization are performed by decrease of both VEGF and IGF-1. Part One The Effect of Propranolol on the Mouse Corneal Epithelial Cells, Stromal Fibroblast Cells and Human Cord-vein Endothelial Cells in Vitro Objective: To confirm the cytotoxicity effect of propranolol on the3main cells in vitro, which are involved into the CNV process--mouse corneal epithelial cells（MCEC）,mouse stromal fibroblast cells（MCSFC） and human umbilical-vein endothelial cells （HUVEC）--and get the evidence for later experiments and clinic research in theory. Material and Method: 1. Experiments were performed on6healthy, adult and germ-free Balb/c mice （9-11week-old）. The fresh corneas were harvested for in vitro culture of mouse corneal epithelial cells, mouse stromal fibroblast cells; and the fresh human cord was for in vitro culture of human cord-vein endothelial cells. The immunofluorescence of CD31was conducted to identify the P3of HCVEC. 2. The IC50of propranolol was measured using P1-MCEC, Pl-MSCFC and P3-HUVEC by MTT assay. The fresh concentration gradient of propranolol （10-1-10-8M） was made in cell culture medium.100μl/well was added in triplicate for each group. After24-Hr culture, MTT solution （5mg/ml） was added for20μl/well. The OD value was measured by enzyme-labelled meter at the wavelength of570nm. Then the Mean of death rate was calculated and the IC50（M） was confirmed by4PL Curve Analysis. 3. The cell viability of propranolol was measured using P1-MCEC, P1-MSCFC and P3-HUVEC by flowcytometry with7AAD-staining.The fresh propranolol （concentration of10-4M） was made in cell culture medium, and added into each group as130μg/5ml. After24-Hr,106cells+100μl7AAD （200μg/ml）+1ml PBS was mixed well to be sorted by flowcytometry, and analysed by GraphPad Prism. Result: 1. The adhesion of P1-MCEC was monolayer, with classic large, polygonal, paved-stone-like cells; the adhesion of P1-MCSFC was monolayer, with clear edge, rich cytoplasma, spindle-like cells; the adhesion of P3-HUVEC was monolayer, with clear edge, polygonal, round or oval, rich cytoplasma, nice-alignment cells. 2. The positive ratio of CD31-GFP immunofluorescence-staining was more than95%by fluorescence microscope, which is demonstrated as HUVEC. 3. The IC50in each group was:10-3,165M for MCEC,10-3,955M for MCSFC,10-4.866M for HUVEC. It showed the death-induced capability of propranolol was higher in HUVEC group than in the other2groups. 4. The viability of MCEC and MCSFC in the medium withpropranolol （concentration of10-4M） was stable, while it was much lower in HUVEC group. Conclusion: The IC50value was increasing as HUVEC＜MCSFC＜MCEC in the culture medium with propranolol. Under the same concentration of propranolol as10-4M, the three main cell components involved in CNV showed different response. The cytotoxicity of propranolol for HUVEC was lower than for MCEC and MCSFC. It meant that the death of blood-vessel endothelial cells would reduce the incidence of CNV, or promote the regression of it, without any effect on MCEC or MCSFC during the wound healing. The finding above was the in vitro experiment evidence for decreasing the side effect from CNV in the process of wound healing. Part Two The Inhibition Effect of Propranolol on the Corneal Neovascularization in an Alkali-induced Injury Mouse Model Objective: The reliable and efficient mouse model of alkali-induced injury was established for in vivo experiment. The proliferation of inflammatory CNV was observed, with histological variation of cornea samples in different time spots. Meanwhile, the inhibition effect of propranolol on the CNV by two drug delivery methods （per oral Vs intraperitoneal injection） was compared with the control group. The control group showed the pathologic process after alkali-induced injury without any other interfering factors. The experiment can provide the further evidence of the inhibition effect of propranolol on CNV treatment. Material and Method: 1. The model of alkali-induced injury was established on the left corneas in200Balb/c mice （the diameter of filter paper disc was2mm, o.5mol/L NaOH solution for20s）.The mice were randomly divided into three groups after3-day injury:0.5ml N.S./per mouse was injected intraperitoneally in the Control group; o.o4%propranolol solution was injected intraperitoneally for0.5ml/per mouse in the IP group; o.o4%propranolol solution was administrated intragastically for0.5ml/per mouse in the PO group. The delivery was performed twice everyday from the4th to6th day after establishment. 2. The observation of mice ocular anterior segment was recorded and analysed at the3rd D,7th D,10th D and14th D after establishment by slit lamp microscope, with the calculation of corneal inflammation points （including CNV score, corneal edema score and intraocular exudation or hyphema）. 3. The corneas from5mice/group were harvested randomly at the3rd D,7th D,10th D and14th D after establishment, for histological examination by H＆E staining. 4. The corneas from5mice/group were harvested randomly at the3rd D,7th D,10th D and14th D after establishment, for immuno-histological examination by CD31staining. The photos taken by confocal microscope were analyesd for CNV growth index by Image J. Result: 1. The observation record by the slit lamp demonstrated that the situation about CNV length, cornea edema and intraocular exudation or hyphema from the3rdD,7th D,10th D and14th D after the establishment of the models was mildest extent and fastest regression in IP group; severest extent and slowest regression in Control group; with PO group in the middle. 2. The corneal inflammation points showed:the inflammation state in Control group was increasing to the top with the time prolongation at the3rd D to10th D （2.92±0.51to4.33±0.49）, and attenuated at the14th D （2.17±0.72）; in IP group it was higher at the3rd D than at the7th D （2.92±0.51to2.25±0.45）, and stable at the10th D （2.42±0.51）, then obviously decreased at the14th D （1.08±0.29）; in PO group it was increasing to the top with the time prolongation at the3rd D to7th D （2.92±0.51to3.25±0.75）, and stable at the10th D （3.17±0.58）, then attenuated at the14th D （1.33±0.49）. The data above indicated the quantification of the inflammation state was mildest extent and shortest existence in IP group; severest extent and longest existence in Control group; with PO group in the middle. 3. The tendency of histological examination by H＆E staining was almost the same as the observation record above. The state about CNV growth, stroma edema, infiltration of inflammatory cells and epithelium healing was all mildest extent and shortest existence in IP group; severest extent and longest existence in Control group; with PO group in the middle. 4. According to the comparison of CNV growth area （%）, the area was increasing in Control group from the3rd D to10th D （11.81±1.210to35.47±1.780）, which matched the variation of inflammation state, and keep stable at the14th D （34.22±2.430）; in IP group, it kept stable between the3rd D and7th D （11.81±1.210to12.76±2.110）, started to decrease since the10th D （10.19±2.395）, and much less at the14th D （5.840±1.320） than it was at the3rd D; in PO group, it was increasing to the top with the time prolongation at the3rd D to10th D （11.81±1.210to27.69±1.930）, while decreased obviously at the14th D （20.33±1.050）. The variation was almost the same as the results above. The CNV growth extent was mildest in IP group with fastest regression, severest and longest in Control group; with PO group in the middle. Conclusion: The optimal method to establish the mouse model of alkali-induced injury was to burn the central area of cornea （diameter=2mm） using0.5mol/L NaOH-immersed filter paper disc for20s. It can achieve the inflammatory CNV with limited inflammation and avoidance of multiple interfering factors involved in. When evaluating the inhibition effect of propranolol on CNV, the method can improve the utilization of the models and clearly present the different therapeutic effect of propranolol in each group. After CNV came out, using0.04%propranolol as0.5ml/per mouse, twice a day for consecutive3days, can inhibit the growth of CNV markedly, with reduction of CNV area and limition of inflammation. It either exerted the positive effect from CNV by improving the metabolism in the microenvironment during the cornea healing, or induced the negative effect from the stimulation between inflammation and CNV, and finally accelerated the healing process. Considering that the bioavailability by oral administration would be affected by the fat-soluble characterization of propranolol, the inhibition effect on CNV was high in IP group＞PO group＞Control group. Part Three The Effect of Propranolol on the Expression of VEGF, IGF-1and bFGF in an Alkali-induced Injury Mouse Model Objective: The examination for the expression variation of three provascular growth factors-VEGF, IGF-1and bFGF, which were closed to the regulation of neovascularization by propranolol, would confirm the inhibition effect of propranolol on CNV in an alkali-induced injury mouse model in the level of gene and protein. It also can provide more credible evidence for the anti-CNV effect of propranolol, and give rise to the potential application of anti-CNV medicine. Material and Method: 1. The model of alkali-induced injury was established on the left corneas in200Balb/cmice （the diameter of filter paper disc was2mm, o.5mol/L NaOH solution for20s）.The mice were randomly divided into three groups after3-day injury:0.5ml N.S./per mouse was injected intraperitoneally in the Control group; o.o4%propranolol solution was injected intraperitoneally for0.5ml/per mouse in the IP group; o.o4%propranolol solution was administrated intragastically for0.5ml/per mouse in the PO group. The delivery was performed twice everyday from the4th to6th day after establishment. 2. The corneas from5mice/group were harvested randomly at the3D,7D,10D and14D after establishment, for VEGF, bFGF and IGF-1examination by RT-PCR. 3. The corneas from5mice/group were harvested randomly at the3D,7D,10D and14D after establishment, for VEGF, bFGF and IGF-1examination by ELISA. Result: 1. From the resulte of RT-PCR, we can know:the expression （%） of VEGF gene in Control group was increasing to the top with the time prolongation from the3rd D to10th D （41.00±2.95to83.42±5.62）, while decreased obviously at the14th D （32.92±3.58）; in IP group, it kept stable between the3rd D and7th D （41.00±2.95to41.42±3.94）, then decreased along with the time prolongation, especially at the14th D（4.50±2.28）; in PO group, it was much higher at the7th D （55.50±4.78） than it was at the3rd D（41.00±2.95）, while kept stable to the10th D （57.58±5.60）, then decreased obviously at the14th D （21.33±2.84）. The expression （%） of VEGF gene was lowest and decreased most quickly in IP group; highest and decreased most showly in Control group; while with PO group in the middle. 2. From the resulte of RT-PCR, we can know:the expression （%） of IGF-1gene in Control group kept stable between the3rd D and7th D （61.00±3.86to61.33±3.85）, increased at the10th D （72.33±3.55）, while decreased since the14th D （59.75±2.93）; in IP group, it was decreasing markedly with the time prolongation from the3rd D to14th D （61.00±3.86to37.17±3.04）; in PO group, it kept stable between the3rd D and10th D （61.00±3.86to59.75±3.96）, while decreased obviously at the14th D （47.42±2.99）. The expression （%） of IGF-1gene was around the same level between Control group and PO group at the7th D, while it was much lower in IP group; at the10th D and14th D, it was high in IP group＞PO group＞Control group. 3. From the resulte of RT-PCR, we can know:the expression （%） of bFGF gene in Control group, it was higher at the7th D （60.08±3.03） than it was at the3rd D（55.83±2.72）, then decreased at the10th D （50.00±3.36）, while kept stable to the14th D （49.17±2.37）; in IP group, it was decreasing markedly with the time prolongation from the3rd D to10th D （55.83±2.72至45.17±3.38）, and kept stable to the14th D （42.75±2.93）; in PO group, it kept stable between the3rd D and7th D （55.83±2.72to56.83±3.30）, then decreased obviously at the10th D （48.00±3.30）, while kept stable again to the14th D （46.58±4.68）. The expression （%） of bFGF gene was around the same level between Control group and PO group during the process, while it was much lower in IP group. 4. From the resulte of Elisa, we can know:the expression （pg/ml） of VEGF protein in Control group was increasing to the top with the time prolongation from the3rd D to10th D （105.56±8.21to150.24±4.70）, while decreased obviously at the14th D （74.06±4.39）; in IP group, it was decreasing markedly with the time prolongation from the3rd D to10th D （105.56±8.21to19.88±1.43）; in PO group, it kept stable between the3rd D and7th D （105.56±8.21to112.82±7.92）, while decreased obviously to the14th D （42.01±4.94）. The variation almost matched the results in gene expression above. It was lowest and decreased most quickly in IP group; highest and slowest in Control group, while PO group was in the middle. 5. From the resulte of Elisa, we can know:the expression （pg/ml） of IGF-1protein in Control group was increasing from the3rd D to7th D （173.33±10.44to254.69±13.53）, and decreased since the10th D to14th D （227.59±14.11to167.92±3.94）; in IP group, it was decreasing markedly with the time prolongation from the3rd D to14th D（173.33±10.44to82.90±4.45）; in PO group, it kept stable between the3rd D and7th D （173.33±10.44to187.34±10.70）, then decreased obviously to the14th D （132.31±2.98）. The variation almost matched the results in gene expression above. 6. From the resulte of Elisa, we can know:the expression （pg/ml） of bFGF protein in Control group was increasing to the top with the time prolongation from the3rd D to7th D （140.67±3.69to177.78±3.29）, while decreased since the10th D （162.61±6.48）; in IP group, it kept stable between the3rd D and7th D （140.67±3.69to140.48±7.04）, then decreased obviously to the14th D （71.06±3.02）; in IP group, it was higher at the7th D （153.36±3.90） than it was at the3rd D（140.67±3.69）, then decreased at the10th D （131.99±5.38）, while kept stable to the14th D （127.68±4.0）. The variation almost matched the results in gene expression above. It was high in Control group＞ PO group＞ IP group from the7th D to10th D; and almost the same between Control and IP group at the14D, while much lower in IP group. Conclusion: The inhibition effect of propranolol on CNV was based on the multiple regulations of provascular growth factors. It down-regulated the cytokines to the different levels during the process of inflammatory CNV to suppress the formation of CNV and also promote the regression, which proved the conclusion above in the mouse model in the micro-level. We verified the inhibition effect of propranolol on CNV in an alkali-induced injury mouse model for the first time. It would play a role as the foundation for the later relative studies. Simultaneously, with the advantages of safety, low-price and visible therapeutic effect, the classic medicine propranolol might be the potential medicine for diversity of the CNV treatment.
Keywords：

Propranolol; Alkali-induced Injury mouse model; Neovascularization; VEGF; IGF-1; bFGF;

  • Series：

    (E) Medicine ＆ Public Health

  • Subject：

    Ophthalmology and Otolaryngology

  • Classification Code：

    R779.1

Tutor：

陆晓和;

Retraction:

眼科学

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