Study Of Gap Junction Protein Connexin43 In Stem Cell-based Trabecular Meshwork Regeneration

Glaucoma,the 2nd leading cause of blindness worldwide,is a degenerative retinal neuropathy caused by the apoptosis of retinal ganglion cells.High intraocular pressure（IOP）is an important pathogenic factor of glaucoma.Elevated IOP is the leading risk factor resulted from the imbalance of aqueous humor（AH）production and outflow.AH produced by the ciliary body enters the anterior chamber through the gap between iris and lens and drains from the eye through aqueous humor outflow tissues.Notably,80-90%of the outflow resistance is due to the obstacle of conventional outflow tissues comprising trabecular meshwork（TM）and Schlemm’s canal.Apoptosis or dysfunction of trabecular meshwork cells has been thought to be the primary cause of elevated IOP.Lowering the elevated IOP through increasing the outflow facility of the TM becomes an attractive and efficient way for glaucoma treatment.However,the current therapies to rebuild the damaged TM and restore the function in AH outflow are still limited.Our previous studies have found that TM cells derived from induced pluripotent stem cells,named iPSC-TM,could effectively regenerate the damaged trabecular meshwork tissue,restored and maintained the aqueous humor outflow in several models of glaucoma.However,we also found that the survival rate of the transplanted iPSC-TM in the TM is relatively low and affected the application process of stem cells.Thus,it is a challenge to maintain the therapeutic effect of iPSC-TM over a long-term period.We previously found that when we treated cells with CBX and FFA,inhibitors of gap junction proteins,the role of iPSC-TM in promoting cell proliferation is weakened.We thus hypothesized that gap junction,especially the robustly expressed gap junction protein Connexin43（Cx43）,playing critical roles in intercellular communication between the neighboring cells,might be essential for maintaining the function of iPSC-TM in stimulating endogenous TM cell division and could become a new therapeutic target influencing iPSC-TM-based TM regeneration.Objective:We aimed to investigate the role of gap junction,especially gap junction protein Cx43,in regulating iPSC-TM-based TM cell division in vitro,regenerating the damaged TM regeneration in vivo.Through this investigation,we would eventually find a way to enhance the effect of iPSCs on tissue regeneration,to provide new treatment ideas for glaucoma and explore new treatment methods.Methods:Mouse induced pluripotent stem cells were differentiated into a trabecular meshwork-like cell type using the medium conditioned by human TM cells.Differentiated cells were then characterized by q RT-PCR and immunohistochemical analysis of the expression of TM cell and stem cell biomarkers.Lentivirus carrying Cx43shRNA was used to down-regulate the expression of Cx43 in iPSC-TM cells.q RT-PCR and Western blot analysis were used to confirm the expression level of Cx43 in cells after infection.The effect of Cx43 on iPSC-TM-stimulated TM cell proliferation was evaluated in the co-culture system.To further confirm the Cx43’s function in iPSC-TM-based tissue regeneration,a mouse model with the elevated IOP was established by the intracameral injection of Ad5-MYOC^Y437H-EGFP virus in C57BL/6J mice.IOPs and the TM cellularities were detected to evaluate this model through the Tonometer and histological analysis.iPSC-TM with down-regulated Cx43 was injected into the anterior chamber of the above glaucomatous model.Compared to the regular iPSC-TM group,the therapeutic effect of iPSC-TM with down-regulated Cx43 was evaluated through IOP measurement and TM cellularity analysis.Results:1.Immunohistochemical analysis showed that TIMP3 and MMP3 proteins,TM biomarkers,were highly expressed in both TM cells of three human donors and mouse TM（m TM）cells extracted from the anterior segments of mice.Dexamethasone-induced up-regulation of myocilin protein,a common TM feature,could also be detected in human and mouse TM cells through IHC analysis.2.Immunohistochemical results showed that the differentiated iPSCs also highly expressed the TM biomarker proteins,TIMP3 and MMP3,whereas the expression levels of stem cell biomarkers,Nanog and SOX2,were decreased in these differentiated cells.q RT-PCR results showed that at the transcriptional level,the expression of TIMP3 was higher in iPSC-TM cells than that of iPSC,while the expression of Nanog in iPSC-TM was lower than that of iPSC.These results indicated that the differentiated cells showed TM-resembling features with diminished stem cell characterizations.3.iPSC-TM cells infected with lentivirus carrying Cx43 shRNA showed green fluorescence,indicating a successful infection of lentivirus in these cells.Western blot and q RT-PCR results indicated that the Cx43 expression was down-regulated by approximately 50%.Through the co-culture experiment,the effect of iPSC-TM after down-regulating Cx43 on stimulating m TM division was weakened.4.After injection of Ad5-MYOC^Y437H-EGFP into the anterior chamber,the IOPs of the mice were increased,and the TM cellularity was significantly decreased.The mouse model with elevated IOP and damaged trabecular meshwork could be established successfully using this approach.5.In Comparison with the regular iPSC-TM-treated group,the IOPs lowering effect of mice receiving iPSC-TM cells with down-regulated Cx43 could be weakened.Further,unlike the regeneration role of regular iPSC-TM,down-regulation of Cx43 in iPSC-TM could not increase the TM cellularity after transplantation.Conclusions:As we previously found,iPSC-TM cells could promote the proliferation of m TM cells in the co-culture model in vitro.Transplantation of iPSC-TM cells into a mouse model with elevated intraocular pressure could lower the IOP and restore the trabecular meshwork’s function via stimulating the endogenous TM cell division.However,down-regulation of Cx43 protein in iPSC-TM significantly weakens the therapeutic effects of iPSC-TM.Take together,Cx43,an essential protein in intercellular communication,plays a critical role in iPSC-TM-based trabecular meshwork regeneration,which could become a potential to improve the therapeutic effect of iPSC,promote the clinical application of iPSC.