The Role Of Bone Marrow-derived Cells In The Pathogenesis And Tissue Repair Of Aortic Aneurysm And Dissection

Objective The aortic wall is constantly exposed to biologic insults and hemodynamic stress, which can cause injury. Insufficient vascular repair may promote aortic aneurysm and dissection (AAD). The reparative process in AAD is poorly understood; however, it is well known that bone marrow-derived cells (BMDCs) can be recruited to the injury to repair damaged tissue. AKTsignaling pathway is an important pathway which is involved in cell metabolism, cell survival, cell proliferation, cell migration, etc. It was reported that AKT2signaling is an important protective pathway in the aorta. Impaired AKT2signaling may contribute to increased susceptibility to the development of AAD. In this study, we tested the BMDC recruitment, potential differentiation, and growth factors production in a mouse model of sporadic AAD, so as to explore the the role of BMDC in the pathogenesis and tissue repair of AAD. To investigate the underlying mechanism, we chose AKT2as a potential contributing molecule, and discussed the role of AKT2signaling pathway in the BMDC-mediated pathogenesis and tissue repair of AAD through animal and cell experiments.Methods Wild-type C57BL/6mice were fed a high-fat (challenged; n=19) or chow (control; n=7) diet for4weeks and then were lethally irradiated and transplanted with BMDCs from transgenic mice expressing green fluorescent protein;4weeks later, we infused angiotensin Ⅱ or saline into challenged and control mice, respectively. The aortas were harvested4weeks after pump implantation, and followed with immunofluorescence studies to understand the BMDC recruitment, differentiation potential, and growth factor production. To investigate the role of AKT2signaling pathway in the BMDC-mediated pathogenesis and tissue repair of AAD, we created a hybrid of Akt2-/-homozygote and GFP transgenic mouse, mated the subsequent generations, and finally got a homozygote of Akt2-/-GFP transgenic mouse. The genotypes were confirmed by polymerase chain reaction (PCR). Use the same method as previously discribed to establish the AAD model and us bone marrow trasplatation to label all the BMDCs. All the recipients were wild type C57BL/6mice, and donors were GFP transgenic mice (WW; n=14) or Akt2-/-GFP transgenic mice (WA; n=14). We measured the diameters of the aorts and cut slices to observe the cross sections to compare the incidence of AAD between the groups of WW and WA, so as to determine whether or not AKT2has participated in regulating the BMDC-mediated pathogenesis and tissue repair of AAD. We used histochemical staining to compare the histologicl difference between WW and WA. Immunofluorescence double staining was used to detect the co-expression of GFP and a series of cell surface markers, and then the cell types and amount of specific cell types were compared between WW and WA, so as to decide whether AKT2in BMDC can affect the cell types in the aortic wall and contribute to the pathogenesis of AAD. We then isolated the aortic vascular smooth muscle cells (VSMCs) from wild type mice and bone marrow mesenchymal stem cells (MSCs) from GFP transgenic mice (WT MSCs) and Akt2-/-GFP transgenic mice (Akt2-/-MSCs), and did the primary culture of those three kinds of cells. We cocultured the VSMCs with WT MSCs or Akt2-/-MSCs, and set up a cell model of oxidative stress, then the expression of Wnt signaling related molecules, Wnt5and β-catenin in those two kinds of MSCs or VSMCs cocultured with them respectively were assessed using western blotting (WB), so as to discover the molecular mechanism of AKT2regulating the BMDC-mediated pathogenesis and tissue repair of AAD. By means of immunofluorescence double staining, WB and real-time quantitative PCR, we studied the expression levels of Notch signaling-related molecules, so as to understand if the Notch signaling pathway plays a role in the pathogenesis and tissue repair of AAD.Results Of challenged mice,37%(11/19) developed AAD, and the number of BMDCs recruited to the aortic wall was significantly increased, particularly in AAD areas. Although we did not detect differentiation of BMDCs into smooth muscle cells, we did observe an abundance of bone marrow-derived Sca-1+stem/progenitor cells, NG2+pericytes, FSP-1+fibroblasts, and CD68+macrophages in the aortas of challenged mice. Furthermore, the expression of NG2, FSP-1, and CD68in Sca-1+BMDCs in the aortas of challenged mice suggested that Sca-1+BMDCs differentiated into pericytes, fibroblasts, and macrophages, respectively. Moreover, BMDCs, including Sca-1+cells, produced insulin-like growth factor, platelet-derived growth factor, and vascular endothelial growth factor. To further investigate the mechanism of BMDC-mediated pathogenesis and tissue repair of AAD, we successfully obtained the Akt2-/-GFP transgenic mice by hybridizing Akt2-/-homozygote and GFP transgenic mice, and mating the subsequent generations. This kind of mice was used as the donor of bone marrow cells for WA group. WA group has a significantly higher incidence of AAD than WW group, which assepted bone marrow cells from normal GFP transgenic mice (64%vs.0%). Thinner aortic media compared with WW group was observed in the aortas without AAD formation of the mice in WA group, while the thickness of the aortic media of mice in WA group which developed AAD was uneven even on the same cross section. The elastic fiber layers were fragmented and disordered, and turned flat at some point in WA group. The immunofluorescent staining showed that the BMDCs migrated to the aortic wall in the WA group was significantly fewer than in the WW group, and the composition of different cell types was different between groups. In the WA group, there were less NG2+pericytes, FSP-1+fibroblasts, and more CD68+macrophages than in WW group. There were also less NG2+BMDCs, and resident FSP-1+fibroblasts in WA group, and higher CD68+macrophage ratio in the BMDCs that migrated to the aortas of mice in WA group. Moreover, we observed higher apoptosis ratio of medial cells in the aortas of WA group compared with WW group. In the cell experiment, we discovered that there were more apoptotic cells in the VSMCs co-cultured with Akt2-/-MSCs than those co-cultured with WT MSCs. Akt2-/-MSC produced less Wnt5than WT MSC with or without oxidative stress, and the VSMCs oc-cultured with Akt2-/-MSCs contained less β-catenin than those co-cultured with WT MSCs. These findings suggest that AKT2in MSCs protected VSMC from apoptosis throught Wnt-β-catenin singaling pathway. After studying the aortic samples of AAD patients, we found that in the VSMCs of aortic media from AAD patients, the Notch signaling was down regulated, while it was up regulated in the CD34+or Stro-1+stem/progenitor cells, fibroblasts and macrophages.Conclusions Our results indicate that aortic injury induces BMDCs to migrate to the aortic wall, differentiate into macrophages, pericytes, and fibroblasts, and participate in aortic inflammation, repair, and remodeling in AAD. Meanwhile, BMDCs produce several growth factors, which provide a suitable microenviroment for tissue repair. The AKT2in BMDCs can affect the composition of cell types in the aortic wall. The defect of AKT2in BMDCs can reduce the ratio of pericytes and firbroblasts in the aortic wall, and raise the ratio of macrophages. On the other hand, AKT2in BMDCs can protect VSMCs from apoptosis through Wnt-β-catenin signaling pathway. In conclusion, AKT2plays an important role in the BMDC-mediated pathogenesis and tissue repair of AAD, and is a potential new molecular target for the treatment of AAD. Notch signaling pathway was changed in the aortas of AAD patients, indicating that Notch signaling may play a role in the aortic inflammation and post-injury aortic repair and tissue remodeling of AAD.