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Objective— To determine the role of a cytokine-like protein DKK3 (dikkopf-3) in directly transdifferentiating fibroblasts into endothelial cells (ECs) and the underlying mechanisms. Approach and Results— DKK3 overexpression in human fibroblasts under defined conditions for 4 days led to a notable change in cell morphology and progenitor gene expression. It was revealed that these cells went through mesenchymal-to-epithelial transition and subsequently expressed KDR (kinase insert domain receptor) at high levels. Further culture in EC defined media led to differentiation of these progenitors into functional ECs capable of angiogenesis both in vitro and in vivo, which was regulated by the VEGF (vascular endothelial growth factor)/miR (microRNA)-125a-5p/Stat3 (signal transducer and activator of transcription factor 3) axis. More importantly, fibroblast-derived ECs showed the ability to form a patent endothelium-like monolayer in tissue-engineered vascular grafts ex vivo. Conclusions— These data demonstrate that DKK3 is capable of directly differentiating human fibroblasts to functional ECs under defined media and provides a novel potential strategy for endothelial regeneration.

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-β (transforming growth factor-β)/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.

Supplemental Digital Content is available in the text., 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.

Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature.

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-22α 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 β-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal–regulated kinase 1/2, or β-catenin attenuated sirolimus-induced SM-22α 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/β-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3β (LC3-βII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1α (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3β expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt −537 to −755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1. Background: Apoptosis and autophagy are two closely related systems that induce cell death. Results: X-box-binding protein 1 (XBP1) mRNA splicing regulates BECLIN-1 transcriptional activation, a fundamental player in the initiation of autophagy. Conclusion: XBP1 splicing induces an autophagic response in endothelial cells. Significance: XBP1 could be used as an important pharmacological target that can regulate the autophagic machinery and endothelial cell death.

Rationale : Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function. Objective : This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism. Methods and Results : Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of β-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G 1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other β-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased β-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced β-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to β-catenin and forms a complex with 14-3-3 ε, ζ, and η proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLCγ-IP3K (phospholipase Cγ–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with β-catenin, disrupting the complex and releasing β-catenin to translocate into the nucleus. Conclusions : These findings demonstrate that HDAC7 interacts with β-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth.

Background: The role of DKK3 in vascular SMC differentiation has not been investigated. Results: DKK3 induces myocardin expression via ATF6 signaling. Conclusion: DKK3 is essential for the ESC-SMC differentiation. Significance: This study provides the molecular mechanisms underlying DKK3 regulated ESC-SMC differentiation., Smooth muscle cells (SMCs) are a key component of healthy and tissue engineered vessels and play a crucial role in vascular development and the pathogenic events of vascular remodeling i.e. restenosis. However, the cell source from which they can be isolated is limited. Embryonic stem (ES) cells that have the remarkable capability to differentiate into vascular SMCs in response to specific stimuli provide a useful model for studying SMC differentiation. Previous studies suggested that dickkopf homolog 3 (DKK3) has a role in human partially induced pluripotent stem cell to SMC differentiation. Here, we demonstrate that the expression of DKK3 is essential for the expression of SMC markers and myocardin at both the mRNA and protein levels during mouse ES cell differentiation into SMCs (ESC-SMC differentiation). Overexpression of DKK3 leads to further up-regulation of the aforementioned markers. Further investigation indicates that DKK3 added as a cytokine activates activating transcription factor 6 (ATF6), leading to the increased binding of ATF6 on the myocardin promoter and increased its expression. In addition, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) promotes the expression of ATF6 and leads to further increase of myocardin transcription. Our findings offer a novel mechanism by which DKK3 regulates ESC-SMC differentiation by activating ATF6 and promoting myocardin expression.

Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature.

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state. Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft. Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

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.

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

Background Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. The generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. Nevertheless, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. Methods and Results We hypothesised that a large number of SMCs could be obtained by direct reprogramming of fibroblasts to SMCs. Therefore, we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo and differentiated into SMCs when seeded on a collagen IV substrate and maintained in differentiation media. The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and calponin at mRNA and protein levels. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in this differentiation. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels. Additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by interaction of DKK3 with the transmembrane receptor Kremen 1, potentiation of Wnt signalling and ultimately β-catenin translocation. Finally, PiPS-SMCs repopulated decellularised vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with the previously established PiPS-endothelial cells, leading to increased survival of SCID mice after transplantation of the vessel as a vascular graft. Conclusion In the present study, we have developed a protocol to generate SMCs from PiPS cells through a DKK3 signalling pathway, which are useful for generating functional tissue-engineered vessels. These findings provide new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

The generation of induced pluripotent stem (iPS) cells is a fascinated tool for regenerative medicine. However, the main restriction for iPS cell application is a risk of tumour development. In this study we established a new method to generate a partially induced stem (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) to human fibroblasts. PiPS cells did not form tumours in vivo, and have abilities to specifically differentiate into different cell lineages, such as neurons, adipocytes, osteocytes, chondrocytes as well as endothelial cells in response to define media and culture conditions. The PiPS-derived endothelial cells expressed a panel of endothelial markers at gene and protein levels and they formed vascular tubes in vitro and in vivo Matrigel plaque assays. To clarify the mechanism of PiPS cell differentiation into endothelial lineage, data from a series of experiments indicate a novel gene SETSIP was crucial in endothelial differentiation. SETSIP was expressed in parallel with endothelial genes, and its overexpression induced endothelial marker expression, while its downregulation by shRNA resulted in suppression of these genes. Interesting, this protein was translocated to the cell nucleus during endothelial differentiation, while ChIP assays showed that it bound direct to VE-cadherin promoter. Functionally, PiPS-derived endothelial cells displayed clearly endothelial properties in two animal models. When seeded on decellularised vessels in a special constructed bioreactor and implanted in SCID mice, the cells displayed well attachment, stabilisation, patency and typical vascular structure. Also, these cells showed endothelial engraftment to form typical vascular architecture when they were injected into the infarcted tissues in an ischaemia model. Thus, we developed a new method to generate PiPS cells from human fibroblasts that can differentiate into a variety of cell types without tumour risk, in which endothelial cells can be produced via OCT4-VEGF-SETSIP pathway useful for regenerating damaged tissue and vessels in vivo.

The generation of induced pluripotent stem (iPS) cells from somatic cells can be a useful tool for regenerative medicine. The artificial nature of iPS cells raises concerns whether iPS and embryonic stem (ES) cells, are molecularly and functionally comparable. In this study we generated iPS cells using a genomic integration free method, and using the advance of proteomics, we aimed to elucidate any differences in the protein profile between iPS and ES cells, in terms of pluripotency and differentiation potential to vascular cell lineages. The results demonstrated that 180 proteins were differentially expressed, in which 66% showed a decreased expression pattern in iPS cells. Functional classification analysis revealed that these proteins were associated with mRNA processing, energy metabolism, cytoskeleton, proliferation, and differentiation. Further experiments demonstrated that iPS cells have a decreased proliferation capacity when compared to ES cells. Importantly, iPS cells also displayed a greater potential to differentiate into smooth muscle and endothelial cell lineages, when compared to ES cells. When iPS and ES cells were seeded on collagen IV-coated dishes and cultured with differentiation media, MEM supplemented with 10% serum, iPS cells displayed a differentiation morphology as early as day 3 in comparison to ES cells, and real-time PCR data confirmed that iPS express smooth muscle cell markers such as SMA, calponin, and SM22 in higher levels. When vascular endothelial growth factor (VEGF) was added to differentiation media, iPS cells expressed endothelial markers such as CD31, CD144, Flk-1 and Flt-1 as early as day 3 on differentiation, while ES cells showed limited expression of these markers during the early stages of differentiation. Therefore, identifying differentially regulated protein expression between the two cell types and the pathways they influence, will allow us to elucidate the mechanisms that lead to vascular cell differentiation, thus making iPS technology a successful tool for regenerative medicine and treatment of vascular diseases.