Characterization of hucMSCs
In this study, P0 hucMSCs samples were obtained from Hui Yisen Cell Gene Engineering Company (Changsha, Hunan, China), and it exhibited a flattened and spindle shape as shown in (Fig. 1A). By using in vitro cell differentiation strategy, we have successfully differentiated hucMSCs into adipocytes or osteoblasts under the induction of a special induction medium (Fig. 1B, C). Cell surface markers were identified by flow cytometry. The results showed that CD29, CD44, CD90, CD105, and CD73 were positive, while CD31, CD34, CD45, and HLA-DR were negative (Fig. 1D). Flow cytometry results and stem cell differentiation ability determination results confirmed that the cells used in the experiment were hucMSCs.
Pretreatment with Ang II promotes the migration of hucMSCs
To determine the optimal treatment concentration of Ang II for hucMSCs treatment,hucMSCs were pretreated with different concentrations of Ang II (0 M, 10− 8 M, 10− 7 M and 10− 6 M) and pretreated- hucMSCs were used for scratch healing assay. Cell scratch experiments showed that hucMSCs had the strongest migration ability at a concentration of 10− 7 M (Fig. 2A, B).
Besides, wound healing could result from enhancement of cell migration or the increase in the number of hucMSCs or likely from both factors. Surprisingly, the CCK-8 proliferation experiment of hucMSCs showed that there was no significant difference in proliferation ability of hucMSCs treated with different concentrations of Ang II (Fig. 2C). Therefore, pretreatment with different concentrations of Ang II can enhance the migration ability of hucMSCs and has little effect on the proliferation of hucMSCs.
Ang II mediates homing of transplanted hucMSCs at the injury site through binding of its receptor target AT2R
Ang II acts mainly by binding to receptors such as AT1R and AT2R(31). Our RT-PCR results showed that AT1R and AT2R were significantly overexpressed at 10-7M of Ang II with a 2.5 amplification index of AT1R (Fig. 3A) and 60 amplification index of AT2R (Fig. 3B), which is significantly higher than that of AT1R. RT-PCR results showed that Ang II pretreatment could enhance the expression of AT2R in hucMSCs. Thus, Ang II mainly binds with the AT2R receptor at the optimal concentration to promote the migration of hucMSCs.
To determine the role of Ang II mediating homing of engrafted hucMSCs, on day 15, after BLM modeling, SD rats in different groups were injected with PBS, DiR Iodide (DiLC18 (7)) tagged-hucMSCs, or DiR Iodide (DiLC18 (7)) tagged-hucMSCs-Ang II cells, respectively (Fig. 3C). Tracking through a live imaging system revealed that a significant number of Ang-Ⅱ-treated hucMSCs were navigated through the tail vein and observed more numerous occupying surfaces at the injury site and the highest fluorescence intensity was observed in BLM + hucMSCs-Ang II treated SD rat group (Fig. 3D, E).
Transplantation of hucMSCs-Ang II reduces the inflammatory response in SD rat lung
Bleomycin in the airway burns the lungs and this leads to a loss of water and food intake by the rodents. Therefore, the weight of SD rats would be reduced after BLM injection. After treatment with hucMSCs, the body weight of BLM + hucMSCS-Ang II group increased faster than that of BLM + PBS group (Fig. 4A). To determine the role of hucMSCs-Ang II in reducing the inflammatory response in SD following injury, we performed pathological assays such as hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stainings. H&E stained tissue sections revealed a significantly reduced inflammation in animals treated with hucMSCs or hucMSCs-Ang II compared with control-treated counterparts (Fig. 4B). Similarly, IHC determination of infiltrating neutrophils at fibrotic lung tissue revealed that a reduced number of cells were observed in SD rats treated with hucMSCs-Ang II group compared with control PBS-treated SD rats (Fig. 4C, D). In addition, the expression of anti-inflammatory factor IL-10 in the hucMSCs-Ang II group was up-regulated in BLM + PBS and hucMSCs groups compared with control-treated groups (Fig. 4E). Taken together, these results suggest that the transplantation of hucMSCs-Ang II reduced inflammatory responses in SD rat lung by decreasing infiltrating neutrophils at injury sites and up-regulating anti-inflammatory IL-10.
Transplantation of hucMSCs-Ang II reduced the expression of α-SMA and HYP
Hydroxyproline (HYP) is low in elastin and absent in other proteins. Therefore, the amount of HYP reflects the collagen metabolism of connective tissue diseases(32). Based on this notion, we next sought to determine the role of hucMSCs-Ang II in α-SMA and HYP expression in SD rats following transplantation. The results showed that the BLM + PBS treated group had more HYP than the Sham group, and less HYP content in the hucMSCs-Ang II group, which indicated that the collagen content in the hucMSCs-Ang II group was reduced (Fig. 5A). We next performed Western blot analysis to determine the accumulation of α-SMA protein level, a marker of pathological remodeling of pulmonary fibrosis. The result showed that the expression of α-SMA in the BLM + PBS group was significantly increased compared with control groups (Fig. 5B, C). After treatment with hucMSCs, reduced accumulation of α-SMA was detected in SD rats. More importantly, the transplantation of hucMSCs-Ang II has significantly improved the accumulation of α-SMA at the injury locus. Similarly, the results from IHC analysis revealed that the number of α-SMA positive cells reduced at injury sites after hucMSCs treatment compared with the PBS-treated group (Fig. 5D). Likewise, treatment of SD rats with hucMSCs-Ang II reduced significantly the accumulation of α-SMA positive cells at injury sites (Fig. 5E). Therefore, the transplantation of hucMSCs and hucMSCs-Ang II cells into PF SD rats could reduce the expression of α-SMA and HYP.
Studies highlighted that the ACE-Ang II-AT1R axis can promote pulmonary fibrosis(33–35). Conversely, ACE2-Ang-(1–7)-Mas axis inhibits fibrosis(35–37). We next detected the expression of AT1R in SD rats after hucMSCs transplantation. Western blot analysis demonstrated that the expression of AT1R was up-regulated in the BLM + PBS group (Fig. 5F), and down-regulated after hucMSCs treatment (Fig. 5G). The results showed that the degree of pulmonary fibrosis in SD rats was alleviated after hucMSCs-Ang II treatment.
Transplantation of hucMSCs-Ang II promotes the synthesis of MMP-9 and degradation of collagen fibers
Next, we determined the deposition of collagen (the main ECM component of tissue fibrogenesis) and MMP-9 after hucMSCs-Ang II transplantation. Western blotting results showed that the expression of MMP-9 (the main collagen degrading enzyme) in the BLM + hucMSCs-Ang II group was significantly increased (Fig. 6A, B). In addition, IHC staining demonstrated that the expression of MMP-9 was significantly increased in the BLM + hucMSCs-Ang II group, compared with a single treatment of hucMSCs (Fig. 6C, D). It is believed that the synthesis of MMP-9 can promote the degradation of collagen and collagen fibers(38). Therefore, we next determined the deposition of collagen in PF SD rats after injury induction. For this purpose, we induced PF in SD rats by injection of BLM. The results from Massion and Sirius red staining revealed that the BLM + PBS group exhibited severe fibrosis, while Ang Ⅱ-hucMSCs treatment significantly reduced PF compared with sole hucMSCs application (Fig. 6E-G). Therefore, transplantation of hucMSCs-Ang II in BLM-induced pulmonary fibrotic SD rats could reduce collagen deposition at injury locus through increased synthesis of MMP-9.