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Supplemental Digital Content is available in the text., Rationale: Vascular progenitor cells play key roles in physiological and pathological vascular remodeling—a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving vascular progenitor cells recruitment and differentiation could help the development of new strategies to improve tissue-engineered vessel grafts and design drug-targeted therapy for vessel regeneration. Objective: In this study, we sought to investigate the role of Dkk3 (dickkopf-3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on vascular progenitor cells cell migration and vascular regeneration and to identify its functional receptor that remains unknown. Methods and Results: Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Sca-1 (stem cell antigen-1) marker. Dkk3 induced the chemotaxis of Sca-1+ cells in vitro in transwell and wound healing assays and ex vivo in the aortic ring assay. Functional studies to identify Dkk3 receptor revealed that overexpression or knockdown of chemokine receptor CXCR7 (C-X-C chemokine receptor type 7) in Sca-1+ cells resulted in alterations in cell migration. Coimmunoprecipitation experiments using Sca-1+ cell extracts treated with Dkk3 showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high-affinity Dkk3-CXCR7 binding with a dissociation constant of 14.14 nmol/L. Binding of CXCR7 by Dkk3 triggered the subsequent activation of ERK1/2 (extracellular signal-regulated kinases 1/2)-, PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)-, Rac1 (Ras-related C3 botulinum toxin substrate 1)-, and RhoA (Ras homolog gene family, member A)-signaling pathways involved in Sca-1+ cell migration. Tissue-engineered vessel grafts were fabricated with or without Dkk3 and implanted to replace the rat abdominal aorta. Dkk3-loaded tissue-engineered vessel grafts showed efficient endothelization and recruitment of vascular progenitor cells, which had acquired characteristics of mature smooth muscle cells. CXCR7 blocking using specific antibodies in this vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodeling. Conclusions: We provide a novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced vascular progenitor migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.

Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown. Methods: Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested in vitro using transwell and wound healing assays and ex vivo by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess in vivo the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta. Results: We demonstrated that Dkk3 induced the chemotaxis of Sca-1+ cells in vitro , and ex vivo in culture of aortic rings derived from Sca-1-GFP transgenic mice. Flow cytometry and WB analysis revealed that Sca-1+ cells expressed CXCR7, while overexpression or knockdown of CXCR7 resulted in alterations in cell migration. Interestingly, Co-IP experiments showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high affinity Dkk3-receptor binding with a dissociation constant of 14.14 nM. Activation of CXCR7 by Dkk3 triggered the ERK1/2, PI3K/AKT, Rac1 and RhoA signalling pathways. Furthermore, when Dkk3-loaded tissue-engineered vessels were grafted to the abdominal artery of rats, the grafts showed efficient endothelization and recruitment of VPCs, which had acquired characteristics of mature SMCs. CXCR7 blocking using specific antibodies in our vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodelling. Conclusions: We provide novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced VPCs migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.

Objectives: Tissue engineered vascular grafts with long-term patency are in great need in the clinics, for which smooth muscle cells (SMCs) are essential. Recently several laboratories have established methods to obtain SMCs from embryonic stem cells or iPS cells. However, the mechanisms of stem cell differentiation are not fully understood. And also it is hard to obtain a large number of SMCs with higher purity. To solve these issues, we take the advantage of mesenchymal stem cells (MSCs) from human umbilical cord that were cultivated and differentiated in vitro. Methods: MSCs from human umbilical cord were cultivated and differentiated to vascular lineages. Signal pathways involved in stem cell differentiation were studied using Western blot, siRNA, PCR, immunoprecipation and transfection. For vascular tissue engineering, stem cell-derived SMCs were applied to decelularized scaffold to create the vessels in a Bioreactor. Results: In response to TGFβ1 stimulation, MSCs were abundantly expressing SMC markers including αSMA, SM22, calponin and SMMHC at the gene and protein levels. Functionally, differentiated SMCs displayed a contraction ability in vitro and formed vessels in matrigel plug assay SCID mice. Micro-RNA (miR) array analysis showed the upregulation of miR-503 and the downregulation of miR-222-5p at early time points after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. Mechanistically, miR-503 was demonstrated to promote SMC differentiation through directly targeting Smad7, a negative regulator of TGFβ1 Smad-related signaling pathways. The expression of miR-503 was Smad4-dependent and Smad4 was enriched at the promoter region of miR-503. In addition, miR-222-5p inhibited SMC differentiation through targeting ROCK2 and αSMA 3’-UTR. Finally, SMCs differentiated from stem cells exhibited the ability to migrate into decellularized mouse aorta and gave rise to vascular graft with smooth muscle layer. Conclusions: We established a method to produce a large number of human SMCs from MSCs, which demonstrated the potential of utilizing the cells to generate vascular grafts. And we provide the mechanistic data demonstrating the impact of miRNA-503/222-5p on TGFβ1-mediated signaling in SMC differentiation from stem cells.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes., How the cardiac myocyte epigenome is rearranged during development, postnatal maturation and disease is not well understood. Here, the authors investigate the human cardiac myocyte epigenome during development and chronic heart failure and identify distinct epigenetic programs regulating these processes.

Supplemental Digital Content is available in the text., Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1+ vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1+ progenitor cells migration and neointimal formation and to understand the underlying mechanisms. Approach and Results— Sca-1+ progenitor cells from the vessel wall of Lepr+/+ and Lepr−/− mice were cultured and purified. The migration of Lepr+/+ Sca-1+ progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal–regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guidewire injury model, an increased expression of leptin in both injured vessels and serum was observed 24 hours post-surgery. RFP (red fluorescent protein)-Sca-1+ progenitor cells in Matrigel were applied to the adventitia of the injured femoral artery. RFP+ cells were observed in the intima 24 hours post-surgery, subsequently increasing neointimal lesions at 2 weeks when compared with the arteries without seeded cells. This increase was reduced by pre-treatment of Sca-1+ cells with a leptin antagonist. Guidewire injury could only induce minor neointima in Lepr−/− mice 2 weeks post-surgery. However, transplantation of Lepr+/+ Sca-1+ progenitor cells into the adventitial side of injured artery in Lepr−/− mice significantly enhanced neointimal formation. Conclusions— Upregulation of leptin levels in both the vessel wall and the circulation after vessel injury promoted the migration of Sca-1+ progenitor cells via leptin receptor–dependent signal transducer and activator of transcription 3- Rac1/Cdc42-ERK (extracellular signal–regulated kinase)-FAK pathways, which enhanced neointimal formation.

Atherosclerosis is a major cause for the death of human beings, and it takes place in large- and middle-sized arteries. The pathogenesis of the disease has been widely investigated, and new findings on vascular stem/progenitor cells could have an impact on vascular regeneration. Recent Advances: Recent studies have shown that abundant stem/progenitor cells present in the vessel wall are mainly responsible for cell accumulation in the intima during vascular remodeling. It has been demonstrated that the mobilization and recruitment of tissue-resident stem/progenitor cells give rise to endothelial and smooth muscle cells (SMCs) that participate in vascular repair and remodeling such as neointimal hyperplasia and arteriosclerosis. Interestingly, cell lineage tracing studies indicate that a large proportion of SMCs in neointimal lesions is derived from adventitial stem/progenitor cells.The influence of stem/progenitor cell behavior on the development of atherosclerosis is crucial. An understanding of the regulatory mechanisms that control stem/progenitor cell migration and differentiation is essential for stem/progenitor cell therapy for vascular diseases and regenerative medicine.Identification of the detailed process driving the migration and differentiation of vascular stem/progenitor cells during the development of atherosclerosis, discovery of the environmental cues, and signaling pathways that control cell fate within the vasculature will facilitate the development of new preventive and therapeutic strategies to combat atherosclerosis. Antioxid. Redox Signal. 00, 000-000.

Supplemental Digital Content is available in the text., 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.

Significance Which brain area drives hand/arm movements after learning or brain injury? When does motor cortical activity generate appropriate hand/arm movements? To address these issues, it is necessary to manipulate motor cortical activity in a controlled manner. Optogenetic tools allow neuronal activity to be manipulated in a variety of animals, but forelimb movements in nonhuman primates have not previously been optogenetically induced or modulated. Here, we improved a method of optogenetic cortical stimulation and induced overt forelimb movements in the common marmoset, a New World monkey. Photostimulation also modulated voluntary forelimb movements, with the modulated movement trajectories depending on the stimulation site and timing. Our results open doors for noninvasive interrogation of motor circuits in behaving nonhuman primates., Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2−expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

Cell diversity of the brain and how it is affected by starvation, remains largely unknown. Here, we introduce a single cell transcriptome atlas of the entire Drosophila first instar larval brain. We first assigned cell-type identity based on known marker genes, distinguishing five major groups: neural progenitors, differentiated neurons, glia, undifferentiated neurons and non-neural cells. All major classes were further subdivided into multiple subtypes, revealing biological features of various cell-types. We further assessed transcriptional changes in response to starvation at the single-cell level. While after starvation the composition of the brain remains unaffected, transcriptional profile of several cell clusters changed. Intriguingly, different cell-types show very distinct responses to starvation, suggesting the presence of cell-specific programs for nutrition availability. Establishing a single-cell transcriptome atlas of the larval brain provides a powerful tool to explore cell diversity and assess genetic profiles from developmental, functional and behavioral perspectives.