The Role Of Human Endometrial Perivascular Cells With Elevated CYR61 Expression In The Repair Of A Full-thickness Uterine Injury In Rat

Objective: To observe the effects of the transplantation of human endometrial perivascular cells(En-PSCs)overexpressing CYR61 on structural and functional regeneration in rat models of partial full thickness uterine excision.Methods: We first sorted human En-PSCs from an endometrial single cell suspension by flow cytometry.Human En-PSCs expressing low or high levels of CYR61(si-CYR61 En-PSCs or ov-CYR61 En-PSCs)were then generated via transfection with a CYR61-specific si RNA construct or overexpression plasmid.Transfected En-PSCs were co-cultured with human umbilical vein endothelial cells(HUVECs)for tube formation assays and migration assays.To establish a rat model of uterine injury,a subset,1.5cm × 0.5cm,opposite to the mesometium in the middle part of uterine wall was then resected from each uterine horn in experimental animals.Female rats were randomly assigned to five groups,including a shamoperated group(sham group,uterine horns were intact without excision)and four repair groups.Repair groups included a Collagen/PBS group,a Collagen/En-PSCs group,a Collagen/si-CYR61 En-PSCs group and a Collagen/ov-CYR61 En-PSCs group.On days 30 and 90 after transplantation,a subset of rats in each group were sacrificed,and uterine tissue was recovered and serially sectioned in paraffin.Hematoxylin and eosin staining and immunohistochemistry were then performed.Finally,the remain rats of each group were mated with male rats on day 90 for a two-week period.Results: Sorted En-PSCs expressed all recognized markers of MSCs,including CD10,CD13,CD44,CD73,CD90,and CD105,and didn’t express hematopoietic markers(CD34,CD45,CD56,CD144 and HLA-DR).En-PSCs had differentiation potential toward adipocytes,osteoblasts,and neuron-like cells,indicating that En-PSCs are a subset of MSCs.ov-CYR61 En-PSCs increased the tube forming and migration ability of HUVECs showed by tube formation assays and migration assays.After 30 days of repair,endometrial thickness in the Collagen/ov-CYR61 En-PSCs group was greater than that in the Collagen/En-PSCs group,and the Collagen/En-PSCs group was significantly greater than the Collagen/PBS group;the percentage of a-SMA positive area in the collagen/ov-CYR61 En-PSCs group was higher than that in the collagen/PBS group,and there was no significant difference between the Collagen/En-PSCs group and the Collagen/PBS group;the density of blood vessels in the Collagen/ov-CYR61 En-PSCs group was greater than that in the Collagen/PBS group,Collagen/En-PSCs group was significantly greater than the Collagen/PBS group,and there was no significant difference between the Collagen/ov-CYR61 En-PSCs group and the Collagen/En-PSCs group.At days 90 after transplantation,endometrial thickness in the Collagen/ov-CYR61 En-PSCs group was greater than that in the Collagen/En-PSCs group or the Collagen/PBS group,and there was no significant difference between the Collagen/En-PSCs group and the Collagen/PBS group;the percentage of α-SMA positive area in the Collagen/ov-CYR61 En-PSCs group was higher than that of the Collagen/PBS group,Collagen/En-PSCs group was significantly greater than the Collagen/PBS group,and there was no statistical difference between the Collagen/ov-CYR61 En-PSCs group and the Collagen/En-PSCs group;blood vessel density in the collagen/ov-CYR61 En-PSCs group was greater than that in the collagen/ En-PSCsgroup and the Collagen/PBS group,and there was no significant difference between the Collagen/En-PSCs group and the Collagen/PBS group.Pregnancy rates differed among five groups: sham group,100%;Collagen/PBS group,40%;Collagen/En-PSCs,80%;Collagen/si-CYR61 En-PSCs group,12.5%;Collagen/ovCYR61 En-PSCs group,80%.In addition,the pregnancy rates in rat uterine regeneration areas were: Collagen /PBS group,0;Collagen/En-PSCs group,20%;Collagen/si-CYR61 En-PSCs group,12.5%;Collagen/ov-CYR61 En-PSCs group,40%.Conclusion: The present results showed that CYR61 plays an important role in angiogenesis.ovCYR61 En-PSCs improved endometrial,myometrial and vascular regeneration in the injured uterus of rat.Transplantation of En-PSCs or ov-CYR61 En-PSCs can increase the pregnancy rate of injured uterus in rats.ov-CYR61 En-PSCs increased the pregnancy rate of damaged parts of the uterus in rats compared with En-PSCs.