| Introduction：Good visual acuity is essencial for human being. Vision involves perception ofthe visual light, its transformation into electrical impulse and transfer to the corticalcenters for final processing. Only normal structure and functional ability of thecomponents of visual system can provide good visual acuity.Cornea is the outermost layer of the eye. It accounts for approximately2/3th ofoptical power of the eye. Cornea is transparent, convex and has lamellar structure. It iscomposed of five distinct layers: three cellular layers and two acellular membranes.Transparency is due to the special distinct arrangement of collagen fibrils of thestroma and avascularity. The latter one is also responsible for corneal immuneprivilege. The avascularity is maintained by the balanced level of pro-andanti-angiogenic factors of the cornea and adjacent tissues. Several factors can triggerthis equilibration with increase of pro-angiogenic and/or decrease of anti-angiogenicfactors and result in new vessel formation in the cornea. Causes of cornealneovascularization (CNV) can be congenital, for example aniridia, or acquired, suchas trauma, infection, inflammation, degeneration, etc. These factors induceneovascularization by different mechanisms, but most of them include hypoxia,inflammation and limbal stem cell deficiency.Around ten million people world-wide are bilaterally blind from cornealinvolvement. It is the second common cause of the reversible blindness after cataract.The corneal blindness is more frequent in developing countries, due to the poor livingconditions, hygiene and lack of the modern and expensive treatment possibilities.However, it is also an actual problem in developed countries. Unlike several blindingconditions, including retinal degenerations and glaucoma, corneal blindness ispreventable in most cases and reversible in almost every case. Improvement ofalready established treatment methods and investigation of new treatment modalitiesare very important in order to decrease the level of corneal blindness. Also, education of the people, improvement of the living conditions world-wide will decrease the levelof blindness due to the corneal involvement.Angiogenesis and neovascularization are very important also in tumor formation,growth and metastasis. Even though many researchers have worked on this topic, upto date precise mechanism of neovascularization are not fully defined. Nevertheless,the role of several inflammatory mediators and growth factors has been investigatedin this pathology. One of the key factors of angiogenesis and neovascularization is thefamily of vascular endothelial growth factors. VEGF include several subtypes offactors, from which VEGF-A is the most important and powerful one in angiogenesis,at least recognized today. It binds to VEGF receptor (VEGFR)-1or2, stimulatesautophosphorylation of specific receptor tyrosine residues and promotes growth,mobilization and division of vascular endothelial cells. Another two factors of thisfamily VEGF-C and VEGF-D bind to VEGFR-3and regulate lymphangiogenesis,which is especially important in corneal transplantation and graft rejection, becausethis route leads antigen presenting cells to proximal lymph nodes. Some other factors,also responsible for angiogenesis, are platelet-derived growth factor (PDGF), basicfibroblast growth factor (bFGF), certain interleukins and cytokines. Their roles arestill ill-defined. Matrix metalloproteinases (MMPs) can destroy extracellular matrixand facilitate ingrowth of new vessels. MMP-2and MMP-9play an important role incorneal neovascularization and can be targets of treatment.Extended wear of contact lenses induces hypoxia and can be a reason of cornealneovascularization. Different types of lenses, especially soft contact lenses, imposegreater risk of CNV formation. The mechanism by which soft contact lenses induceangiogenesis is induction of hypoxia-inducible factor1(HIF-1) formation by hypoxia,which then binds to the hypoxia response element (HRE) in the promoter of VEGFgenes, and the cascade continues as it was described previously. Abstention fromextended contact lens wearing, good hygiene and care for lenses can significantlydecrease the level of complications and neovascularization.The treatment modalities, which are acceptable used in clinical practicenowadays, can be classified into conservative and surgical treatment ways.Conservative or non-surgical treatment includes application of steroidal ornon-steroidal anti-inflammatory drugs. Lately, anti-VEGF factors were used in thetreatment of several neovascular conditions of the eye, including corneal neovascularization. Although, the beneficial effect was obvious and was presented inmany reports; these agents are still used as off-label worldwide. Several chemicalsubstances and gene-modification therapies have been used in research for treatmentof corneal neovascularization. However, none of them are used in clinical practice.Surgical treatment includes argon laser photocoagulation, photodynamic therapy,electrocoagulation and transplantations. Transplantation can involve whole orseveral layers of the cornea, amniotic membrane, conjunctiva, cultivated oral mucosaepithelial sheets or limbal stem cells. Limbal stem cells reside in stem cell niche,which are suggested to be palisades of Vogt. Limbal stem cell transplantation is atreatment of choice for corneal limbal stem deficiency and corneal neovascularizationin many ophthalmic centers. It has very good success rate. First time limbal stem celltransplantation included incision of the small limbal piece from healthy donor eye andtransplantation on the contralateral injured eye. Several modifications of thistechnique have been implemented, including amplification of limbal stem cells andsuccessful transplantation on the eye with amniotic membranes, carrier bio-scaffoldsor contact lenses. Several clinical trials have been carried out to investigate the safety,feasibility and effectiveness of these techniques.Limbal stem cell transplantation is a truly effective treatment option for cornealneovascularization, but it has several drawbacks: firstly, removal of the limbalsegment requires invasive surgery, which can be complicated by inflammation andinfection. Secondly, many ocular conditions with neovascularization are bilateral, so itis impossible to take even small graft from the limbus. Use of allograft imposes highrisk of immune rejection, especially with already compromised corneal immuneprivilege. For those reasons medical specialists are seeking for new treatment options,which can be safe and effective for many patients. Transplantation of stem cells is oneof those preferable treatment possibilities.Stem cells are undifferentiated cells with the self-renewal ability anddifferentiation into more specialized cells. There are three main types of stem cells:embryonic stem cells (ESC), adult stem cells, induced pluripotent stem cells (iPS).IPS and ESC grow faster, are being pluripotent and can differentiate into any kind ofcell upon appropriate stimulation, but they have practical problems, halting theirclinical use. They raise ethical issues and have high risks of immune rejection andtumorogenesis. In contrast to ESC and iPS, MSCs are more suitable for stem cell therapy, because of facile isolation and in-vitro culture, prolonged self-renewableability, auto-transplantation, low risk of tumor formation and a lack of ethical issues.There have been many studies showing that these cells are safe and have goodplasticity, without producing tumors. As such, they are preferred for transplantationtherapy. Mesenchymal stem cells can be isolated from different organs and tissues:bone-marrow, adipose tissue, teeth pulp and also amniotic fluid, umbilical cord,placenta, etc. The most researched and used cell type is bone-marrow derivedmesenchymal stem cell (BMSC). They are described with several features: isolatedfrom bone-marrow, adherent to the bottom of plastic culture dishes, obtain spindleshape like fibroblasts and express a combination of cell surface markers (CD105,CD90, CD29, etc. and negative for CD31, CD45, CD11b, etc). The beneficial actionof transplanted MSCs is suggested to be due to the production of trophic factors,however, the ability of integration and differentiation is still questionable. MSCs arepreferred for auto-transplantation. Recently researches showed that BMSCs don’texpress MHC class II antigens, co-stimulatory molecules CD80, CD84or CD40andonly express low quantity of MHC class I antigens, so they can be invisible forrecipient immune system. This makes MSCs universal also for allo-and evenxeno-transplantations. It was also shown that MSC itself suppresses immune systemby suppressing T-cell, B-cell and NK activity.MSC derived from adipose tissue, umbilical cord, amniotic fluid andbone-marrow showed generally similar characteristics, so cells for transplantationscan be obtained from medical wastes (liposuction, placenta, umbilical cord (UC) andamniotic fluid after delivery) and used safely for allotransplantations.The reports on MSCs use in treatment of corneal neovascularization appeared in2005-2006. After that, many groups have investigated the role of mesenchymal stemcells in corneal wound healing and corneal angiogenesis. There are at least35published reports available in Pub Med database. There are several in-vitro, in-vivoand combined experiments. All published papers reported beneficial action ofmesenchymal stem cell in corneal wound healing and decrease of the time requiredfor the surface re-epithelization. Whether this is due to the trophic factor productionand regulation of the inflammatory response or transdifferentiation ability ofmesenchymal stem cells into corneal cells is not clear. Several researchers reported positive transdifferentiation effect, but most of them hadn’t used correct and precisedetection methods.Most of the researchers cultivated stem cells on the amniotic membranes andtransplanted it on the corneal surface with different success rates. We could retrieveonly one paper from the Pub Med database that used subconjunctival injection ofBMSCs for treatment of corneal chemical injury. They have reported positive results.No reports have been made to compare these two modalities.Purpose:Our study is to further investigate the role of bone marrow derived mesenchymalstem cells used in corneal neovascularization, wound healing and compare thefeasibility and effectiveness of different application routes.Materials and Methods:Bone marrow derived mesenchymal stem cells were isolated from8days oldWistar rats and characterized by flow cytometry for several expressed surface marker.Cells from second or third passages were transduced by Lentivirus for GreenFluorescent Protein (GFP) expression. Human amniotic membranes were obtainedfrom healthy pregnant women during Cesarean section after their informed consent.Epithelial layers were removed by chemical and mechanical approaches, and denudedhuman amniotic membranes (d-HAM) were seeded with BMSCs. Totally36femaleWistar rats were used in this study to make the animal models of cornealneovascularization. Animals were anesthetized by peritoneal injection of chloralhydrate. Chemical injury was induced by application of3mm diameter filter discsoaked in1mol/L sodium hydroxide solution (NaOH) on the center of the cornea ofthe right eye for30seconds followed by thorough washing of the cornea with60mlsterile saline for one minute. After seven days rats were divided into three groups:first group received subconjunctival injection of bone marrow derived mesenchymalstem cells suspended in PBS, second group received transplantation of BMSCspreviously cultured on denuded human amniotic membrane (d-HAM), and third groupdidn’t receive treatment and served as control group. Special charts were designed andused for evaluation of corneal injury, including area of epithelial defect, significanceof the stromal edema and haze, area of neovascularization. Four point grading systemwas used for each characteristic. The eyes were examined daily by pen light andweekly by slit-lamp. Five weeks after injury and consequently four weeks after treatment administration animals were sacrificed with overdose of anesthetic, andcorneas were harvested. Five corneas from each group were used to make flat mountsafter ink perfusion for better visualization of new vessels, pictures were obtained bythe digital camera attached to the light microscope and were processed with specialsoftware (Image-Pro Plus6) to calculate the total length of the vessels for furtherstatistical analyses. Four corneas from each group were used for RNA extraction anddetection of VEGF-A, MMP9, Toll-like receptor2(TLR-2) and TLR-4in quantitativereal time polymerase chain reaction (q-PCR). The quantity of VEGF was detected byEnzyme linked immunosorbent analyses (ELISA), and corneal cryosections weremade for further histopathology and immunostaining for VEGF and MMP9.Statistical analyzes were performed with SPSS. One-Way ANOVA with post-hoc testwas used for comparison of3groups.All animal experiments were handled according to the animal protocols approvedby Jilin University.Results:Forty eight hours after isolation and culture in cell culture flasks small cellsadherent to the bottom of the flasks were visible. Cells from the passage two andpassage three showed typical characteristics of mesenchymal stem cells: they wereattached to the bottom of the culture dish, had spindle-like shape, and flow cytometryshowed positive expression of CD90and CD44, and negative expression of CD45andCD11b surface markers. Thus, it was confirmed, that cells were real mesenchymalstem cells. They were further transduced with gene of green fluorescent protein bylentiviral vectors. The positive transduction was confirmed by detection of green lightemission from the cells under inverted fluorescent microscope. Denudation of theamniotic membranes was confirmed by staining with DAPI and examination withconfocal laser microscope. Membranes didn’t contain any cell with stained nuclei.The same technique was used to examine the BMSCs culture and growth on denudedmembranes. This time many cells were obvious on the membranes: they had irregulararrangement, nuclei were stained with DAPI, and cells had green color emissionunder special filters.Examination of the rats seven days after the injury showed visible cornealopacities, growth of new vessels and epithelial defects stained with fluorescent dye.After division into groups, statistical analyze was performed to measure the level of the injury in each group. One-Way ANOVA showed no difference between groups(P=103, CI95%). Four weeks later results were obtained and compared by ANOVAagain. This analyze showed statistically significant difference between groups(P=0.002, CI=95%). It was obvious that the difference was the result of differenttreatment strategies. Post-hoc test revealed that the difference was between group1and group2, and group1and group3. There was no significant difference betweengroup2and group3. Thus, single subconjunctival injection of BMSCs had greaterimpact on corneal wound healing and neovascularization, than amniotic membranetransplantation with BMSCs.Preparation of flat mounts after ink injection also showed that the area andexpansion of new vessels were bigger in group2and group3compare to group1(P=0.01, CI95%).Sections of the corneas stained with Hematoxylin and Eosin showed that thecorneas from the group1were thinner, stromas were compact with less infiltration ofinflammatory cells, and the epithelial layers were fully recovered. Immunostainingwith DAPI, Anti-VEGF and Anti-MMP9antibodies didn’t reveal any usefulinformation with the sections having inadequate low level of staining in all groups.The quantity of VEGF was under the detectable threshold of the kit for ELISA.Q-PCR results for VEGF, MMP9and TLR2and TLR4were obtained, andrelated gene expression to b-Actin was calculated for each factor. Statistical analyzesshowed difference between the groups for each factor, but only for VEGF expressionthere was a statistically significant difference (P=0.03, CI95%).Discussion:Corneal blindness, being the second common cause of reversible blindness, hasgross economical and psychological impact on the society world-wide. Millions ofpeople are bilaterally blind with corneal involvement, with higher rate in developingcountries. Inappropriate living conditions, poor hygiene, bad environmental factors,lacking of education and absence of modern treatment modalities are the importantfactors responsible for this unequal distribution. However, corneal blindness isactual problem even in developed countries. Strategies to improve this situation are inthe center of interest of many international organizations, including World HealthOrganization (WHO). Medical specialists are seeking for possibilities to improve current treatmentmodalities and research is going on to find new treatment methods. One of thistreatment options is application of mesenchymal stem cell in the treatment of cornealneovascularization. Many groups have worked on this topic and reported satisfactoryresults. The aim of our study was to further investigate the role of bone marrowderived mesenchymal stem cells in corneal neovascularization and to compare theefficacy of several application routes.We have treated chemically injured corneas of animals with singlesubconjunctival injection of BMSCs or with transplantation of denuded amnioticmembranes previously seeded with BMSCs. As long as we know, there isn’t anyprevious report to compare simple subconjunctival injection of BMSCs withtransplantation on the amniotic membrane. Our main interest was the assessment ofthe clinical effect of cell transplantations and their impact on neovascularization.Our work showed that stem cells enhance corneal wound healing and decrease thelevel of neovascularization. Surprisingly, it revealed that simple subconjunctivalinjection of BMSCs is more effective, than transplantation of amniotic membraneseeded with the stem cells. We assume that the difference in the effectiveness wasconnected with the short existence of amniotic membranes on the corneal surface(after4days most of the membranes have been melted). However, the reasons of thisdifference should be further investigated for more precise and correct explanation.The differentiation ability of stem cells into corneal cells was out of the interestof our study. Nevertheless, for tracking the movements of transplanted cells, theywere previously transduced for GFP expression by lentivirus. Four weeks aftertransplantation we were not able to detect any transplanted cells in the cornea. Weassume that the main mechanism of action was trophic factor production and also theeffect of the BMSCs on inflammation. The level of VEGF and MMP9geneexpression also showed significant decrease after subconjunctival injection. ForTLR-2and TLR-4the difference was insignificant. However, with the lack of thereports on this matter in the previous published papers similar to ours, we can onlyassume that this finding was connected with the prolonged period of observation. Aswe know, TLRs are responsible for innate immune response, and their level isincreased in the beginning of the inflammatory process. After four weeks the inflammation has decreased significantly in all groups with the levels of TLRsdecreased to normal rates.Conclusion:Corneal blindness imposes great impact on the societies of developing and alsodeveloped countries. The inexpensive, feasible treatment modalities are preferableworld-wide, especially in the developing part of the world.We have compared subconjunctival injection of stem cells with alreadyestablished transplantations with amniotic membranes. Surprisingly, the resultsshowed that simple injection can be more effective, than transplantation of the cellson amniotic membranes. We do understand that this idea should be investigated andall pros and cons should be evaluated before proceeding to the wide use in clinicalpractice. However, we hope this technique will pass all the trials and will beimplemented in clinical practice in the near future. |
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