OBJECTIVE: Vascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear.
METHODS: We used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development.
RESULTS: Here, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation.
CONCLUSIONS: Bves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, β-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R/ETAR antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long-term treatment with receptor antibodies. Imaging studies revealed that β2- but not β1-arrestin was recruited to ETAR in response to ET1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of β2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R- and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R/ETAR antibodies induce ERK1/2 and mTOR signaling involving β2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rational for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

Pathological vascular remodeling contributes to the development of restenosis following intraluminal interventions, transplant vasculopathy, and pulmonary arterial hypertension. Activation of the tumor suppressor p53 may counteract vascular remodeling by inhibiting aberrant proliferation of vascular smooth muscle cells and repressing vascular inflammation. In particular, the development of different lines of small-molecule p53 activators ignites the hope of treating remodeling-associated vascular diseases by targeting p53 pharmacologically. In this review, we discuss the relationships between p53 and pathological vascular remodeling, and summarize current experimental data suggesting that drugging the p53 pathway may represent a novel strategy to prevent the development of vascular remodeling.

Transplant vasculopathy (TV), a hallmark of chronic allograft rejection, is the primary cause of allograft loss after organ transplantation. Because multiple mechanisms are involved in TV pathogenesis, effective therapy for it remains elusive. Here, we identify the role of triptolide, which has a wide spectrum of immuno-suppressive activities, in inhibiting TV development. Murine aortic transplants models were constructed and divided into triptolide-treated and untreated groups. We found that triptolide significantly alleviated intima thickening of allografts by inhibiting multiple pathways. Triptolide significantly reduced infiltration of T lymphocytes and macrophages and inhibited the levels of pro-inflammatory (TNF-α, IL-2, and IL-6) and pro-fibrotic factors (TGF-β, α-SMA, and MMP-9) in the graft. Additionally, triptolide significantly decreased the numbers of IFN-γ-producing T lymphocytes, as well as the expression of IFN-γ and IFN-γ-inducing factor (CXCL9 and CXCL10) in recipient. Moreover, triptolide decreased the numbers of B lymphocytes and plasma cells, as well as the levels of donor specific antibodies (DSAs) in recipient. Furthermore, triptolide not only inhibited vascular smooth muscle cell (VSMC) viability and promoted VSMC apoptosis but also significantly inhibited VSMC migration in vitro. These results emphasize the efficacy of triptolide in inhibiting TV development and provide a basis for developing new treatments to prevent TV-related complications and improve the long-term survival of transplant recipients.

In this study, we tested the possibility that macrophages might contribute to neointima formation by stimulating microRNA expressions in mural cells. Thoracic aortas from F344 rats were transplanted into recipient Lewis rats. Clodronate liposome was used for in vivo macrophage depletion. Using miR-21 as a prototypic example of vascular enriched microRNA, we showed that macrophage depletion reduced the expression level of miR-21, which was upregulated in the allograft. This effect of macrophage depletion was accompanied by attenuations in neointimal hyperplasia and transplantation-induced vascular inflammation. Using in vitro assays, we identified that macrophages might stimulate miR-21 expression in smooth muscle cells and adventitial fibroblasts via the release of tumor necrosis factor-α. We also showed that silencing of miR-21 suppressed tumor necrosis factor-induced proliferation, migration, and inflammatory responses in mural cells. Our results suggest that macrophage may promote transplantation-induced neointima formation by stimulating miR-21 expression in vascular mural cells, which promotes mural cell proliferation, migration and/or inflammation. Moreover, we have established that tumor necrosis factor-α has a major role in mediating this paracrine process.

Increasing evidence has suggested that arterial adventitia may contribute to pathological vessel remodeling by producing reactive oxygen species and promoting neovascularization. However, these processes have not been studied yet in transplantation-induced vascular pathologies. We characterized the dynamic changes in NADPH oxidase expression and adventitial angiogenic response in a model of allograft aortic transplantation. The thoracic aorta from Fischer 344 rats were transplanted into the abdominal aorta of Lewis rats. Graft specimens were collected on days 0.5, 3, 7, and 14 for morphometry, immunohistochemistry, immunofluorescence staining, and quantitative PCR tests. Following transplantation, adventitial thickening was found as early as day 3, while neointima was observed from day 7. As compared to normal adventitial tissue, the expression levels of NADPH oxidase subunits gp91phox and p47phox in graft adventitia were elevated from day 3 and further increased up to day 14. Immunohistochemistry staining showed that infiltrating macrophages appeared to be a major source of NADPH oxidase expression. Increases in NADPH oxidase expression were also detected in fibroblasts isolated from the graft adventitia. Gene silencing of p47phox significantly suppressed proliferation and migration of the graft fibroblast cells. We also showed that adventitial thickening was accompanied by increased adventitial neovascularization; at day 14, there was a positive correlation between the density of adventitial microvessels and the neointimal thickness. Transplantation injury induces NADPH oxidase expression and neovascularization in the adventitia, raising the possibility that the activated adventitia may represent a target site for prevention of transplantation-induced transplant vasculopathy.