Baoqi Yu

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal–Regulated Kinase/β-Catenin Signaling Pathway

Objective—Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. Approach and Results—Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22&agr; and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal–regulated kinase 1/2 activation that lead to &bgr;-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal–regulated kinase 1/2, or &bgr;-catenin attenuated sirolimus-induced SM-22&agr; promoter activation and SMC differentiation. Conclusions—These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal–regulated kinase/&bgr;-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.

Vascular Stem/Progenitor Cell Migration Induced by Smooth Muscle Cell-Derived Chemokine (C-C Motif) Ligand 2 and Chemokine (C-X-C motif) Ligand 1 Contributes to Neointima Formation

Recent studies have shown that Sca‐1+ (stem cell antigen‐1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca‐1+ progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC‐derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C‐C motif) ligand 2) and CXCL1 (chemokine (C‐X‐C motif) ligand 1), and their corresponding receptors on Sca‐1+ progenitors, CCR2 (chemokine (C‐C motif) receptor 2) and CXCR2 (chemokine (C‐X‐C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca‐1+ progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca‐1+ progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire‐injured mouse femoral arteries, a large proportion of GFP‐Sca‐1+‐cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post‐operation. Interestingly, Sca‐1+ progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2−/− mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016;34:2368–2380

Vascular Stem/Progenitor Cell Migration Induced by SMC‐derived CCL2 and CXCL1 Contributes to Neointima Formation

Recent studies have shown that Sca‐1+ (stem cell antigen‐1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca‐1+ progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC‐derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C‐C motif) ligand 2) and CXCL1 (chemokine (C‐X‐C motif) ligand 1), and their corresponding receptors on Sca‐1+ progenitors, CCR2 (chemokine (C‐C motif) receptor 2) and CXCR2 (chemokine (C‐X‐C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca‐1+ progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca‐1+ progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire‐injured mouse femoral arteries, a large proportion of GFP‐Sca‐1+‐cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post‐operation. Interestingly, Sca‐1+ progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2−/− mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016;34:2368–2380

Adventitial SCA-1+ Progenitor Cell Gene Sequencing Reveals the Mechanisms of Cell Migration in Response to Hyperlipidemia

Summary Adventitial progenitor cells, including SCA-1+ and mesenchymal stem cells, are believed to be important in vascular remodeling. It has been shown that SCA-1+ progenitor cells are involved in neointimal hyperplasia of vein grafts, but little is known concerning their involvement in hyperlipidemia-induced atherosclerosis. We employed single-cell sequencing technology on primary adventitial mouse SCA-1+ cells from wild-type and atherosclerotic-prone (ApoE-deficient) mice and found that a group of genes controlling cell migration and matrix protein degradation was highly altered. Adventitial progenitors from ApoE-deficient mice displayed an augmented migratory potential both in vitro and in vivo. This increased migratory ability was mimicked by lipid loading to SCA-1+ cells. Furthermore, we show that lipid loading increased miRNA-29b expression and induced sirtuin-1 and matrix metalloproteinase-9 levels to promote cell migration. These results provide direct evidence that blood cholesterol levels influence vascular progenitor cell function, which could be a potential target cell for treatment of vascular disease.

A cytokine-like protein dickkopf-related protein 3 is atheroprotective

Background: Dickkopf-related protein 3 (DKK3) is a secreted protein that is involved in the regulation of cardiac remodeling and vascular smooth muscle cell differentiation, but little is known about its role in atherosclerosis. Methods: We tested the hypothesis that DKK3 is atheroprotective using both epidemiological and experimental approaches. Blood DKK3 levels were measured in the Bruneck Study in 2000 (n=684) and then in 2005 (n=574). DKK3-deficient mice were crossed with apolipoprotein E-/- mice to evaluate atherosclerosis development and vessel injury-induced neointimal formation. Endothelial cell migration and the underlying mechanisms were studied using in vitro cell culture models. Results: In the prospective population-based Bruneck Study, the level of plasma DKK3 was inversely related to carotid artery intima-media thickness and 5-year progression of carotid atherosclerosis independently from standard risk factors for atherosclerosis. Experimentally, we analyzed the area of atherosclerotic lesions, femoral artery injury-induced reendothelialization, and neointima formation in both DKK3-/-/apolipoprotein E-/- and DKK3+/+/apolipoprotein E-/- mice. It was demonstrated that DKK3 deficiency accelerated atherosclerosis and delayed reendothelialization with consequently exacerbated neointima formation. To explore the underlying mechanisms, we performed transwell and scratch migration assays using cultured human endothelial cells, which exhibited a significant induction in cell migration in response to DKK3 stimulation. This DKK3-induced migration activated ROR2 and DVL1, activated Rac1 GTPases, and upregulated JNK and c-jun phosphorylation in endothelial cells. Knockdown of the ROR2 receptor using specific siRNA or transfection of a dominant-negative form of Rac1 in endothelial cells markedly inhibited cell migration and downstream JNK and c-jun phosphorylation. Conclusions: This study provides the evidence for a role of DKK3 in the protection against atherosclerosis involving endothelial migration and repair, with great therapeutic potential implications against atherosclerosis.

Adventitial Sca1+ Cells Transduced With ETV2 Are Committed to the Endothelial Fate and Improve Vascular Remodeling After Injury

Objective— Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. Approach and Results— We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control. Conclusions— These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.

DKK3 (Dickkopf 3) Alters Atherosclerotic Plaque Phenotype Involving Vascular Progenitor and Fibroblast Differentiation Into Smooth Muscle Cells

Objective— DKK3 (dickkopf 3), a 36-kD secreted glycoprotein, has been shown to be involved in the differentiation of partially reprogrammed cells and embryonic stem cells to smooth muscle cells (SMCs), but little is known about its involvement in vascular disease. This study aims to assess the effects of DKK3 on atherosclerotic plaque composition. Approach and Results— In the present study, we used a murine model of atherosclerosis (ApoE−/−) in conjunction with DKK3−/− and performed tandem stenosis of the carotid artery to evaluate atherosclerotic plaque development. We found that the absence of DKK3 leads to vulnerable atherosclerotic plaques, because of a reduced number of SMCs and reduced matrix protein deposition, as well as increased hemorrhage and macrophage infiltration. Further in vitro studies revealed that DKK3 can induce differentiation of Sca1+ (stem cells antigen 1) vascular progenitors and fibroblasts into SMCs via activation of the TGF-&bgr; (transforming growth factor-&bgr;)/ATF6 (activating transcription factor 6) and Wnt signaling pathways. Finally, we assessed the therapeutic potential of DKK3 in mouse and rabbit models and found that DKK3 altered the atherosclerotic plaque content via increasing SMC numbers and reducing vascular inflammation. Conclusions— Cumulatively, we provide the first evidence that DKK3 is a potent SMC differentiation factor, which might have a therapeutic effect in reducing intraplaque hemorrhage related to atherosclerotic plaque phenotype.

Over-expression of HSP47 augments mouse embryonic stem cell smooth muscle differentiation and chemotaxis.

In the recent decade, embryonic stem cells (ESC) have emerged as an attractive cell source of smooth muscle cells (SMC) for vascular tissue engineering owing to their unlimited self-renewal and differentiation capacities. Despite their promise in therapy, their efficacy is still hampered by the lack of definitive SMC differentiation mechanisms and difficulties in successful trafficking of the ESC towards a site of injury or target tissue. Heat shock protein 47 (HSP47) is a 47-kDa molecular chaperone that is required for the maturation of various types of collagen and has been shown to be a critical modulator of different pathological and physiological processes. To date, the role of HSP47 on ESC to SMC differentiation or ESC chemotaxis is not known and may represent a potential molecular approach by which ESC can be manipulated to increase their efficacy in clinic. We provide evidence that HSP47 is highly expressed during ESC differentiation into the SMC lineage and that HSP47 reduction results in an attenuation of the differentiation. Our experiments using a HSP47 plasmid transfection system show that gene over-expression is sufficient to induce ESC-SMC differentiation, even in the absence of exogenous stimuli. Furthermore, HSP47 over-expression in ESC also increases their chemotaxis and migratory responses towards a panel of chemokines, likely via the upregulation of chemokine receptors. Our findings provide direct evidence of induced ESC migration and differentiation into SMC via the over-expression of HSP47, thus identifying a novel approach of molecular manipulation that can potentially be exploited to improve stem cell therapy for vascular repair and regeneration.

B DKK3 Stabilises Atherosclerotic Plaque via Promoting Vascular Progenitor and Fibroblast Differentiation to Smooth Muscle Cells

Atherosclerosis, a chronic condition that can be converted into an acute clinical event caused by plaque rupture and thrombosis, has been the primary cause of mortality and morbidity worldwide. Dickkopf 3 (DKK3), a 36-kD secreted glycoprotein, has a role in cell differentiation, but little is known about its involvement in vascular disease. In the present study, we utilised a murine model of atherosclerosis (ApoE-/-) in conjunction with DKK3-/- to assess the effects of DKK3 on plaque stability. We found that the absence of DKK3 leads to vulnerable unstable atherosclerotic plaques, due to a reduced number of smooth muscle cells (SMCs) and reduced matrix protein deposition, as well as increased haemorrhage and macrophage infiltration. Using a cell linear tracing method, vascular progenitors and fibroblasts from SM22-LacZ transgenic mice were isolated and applied to the adventitial side of injured femoral arteries resulting in migration of both types of cells to the intima. Upon migration the cells displayed beta-gal positivity, indicating SMC differentiation. Further in vitro studies revealed that DKK3 can induce differentiation of Sca1+ vascular progenitors and fibroblasts into SMCs via activation of the TGFβ/ATF6 and Wnt signalling pathways. Finally, we assessed the therapeutic potential of DKK3 in mouse and rabbit models and found that DKK3 increases atherosclerotic plaque stability via an increase in SMC numbers and reduced vascular inflammation. Cumulatively, we provide the first evidence that DKK3 is a potent SMC differentiation factor, which may have a therapeutic effect in reducing acute haemorrhagic conditions through promotion of atherosclerotic plaque stability.