ABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cell Transdifferentiation/physiology , Endothelial Cells/cytology , Fibroblasts/drug effects , Animals , Bioreactors , Cells, Cultured , Culture Media , Epithelial-Mesenchymal Transition/physiology , Fibroblasts/cytology , Humans , Mice , Mice, Inbred NOD , MicroRNAs/physiology , Neovascularization, Physiologic , Recombinant Proteins/biosynthesis , STAT3 Transcription Factor/physiology , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
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-ß (transforming growth factor-ß)/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.
Subject(s)
Aortic Diseases/metabolism , Atherosclerosis/metabolism , Carotid Stenosis/metabolism , Cell Transdifferentiation , Fibroblasts/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Plaque, Atherosclerotic , Stem Cells/metabolism , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Adaptor Proteins, Signal Transducing , Animals , Aorta/metabolism , Aorta/pathology , Aortic Diseases/genetics , Aortic Diseases/pathology , Ataxin-1/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Carotid Arteries/metabolism , Carotid Arteries/pathology , Carotid Stenosis/genetics , Carotid Stenosis/pathology , Cells, Cultured , Chemokines , Disease Models, Animal , Female , Fibroblasts/pathology , Hemorrhage/genetics , Hemorrhage/metabolism , Hemorrhage/pathology , Hemorrhage/prevention & control , Humans , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Phenotype , Rabbits , Stem Cells/pathology , Transforming Growth Factor beta1/metabolism , Wnt Signaling PathwayABSTRACT
To date, Alzheimer's disease (AD) clinical trials have been largely unsuccessful. Failures have been attributed to a number of factors including ineffective drugs, inadequate targets, and poor trial design, of which the choice of endpoint is crucial. Using data from the Alzheimer's Disease Neuroimaging Initiative, we have calculated the minimum detectable effect size (MDES) in change from baseline of a range of measures over time, and in different diagnostic groups along the AD development trajectory. The Functional Activities Questionnaire score had the smallest MDES for a single endpoint where an effect of 27% could be detected within 3 years in participants with Late Mild Cognitive Impairment (LMCI) at baseline, closely followed by the Clinical Dementia Rating Sum of Boxes (CDRSB) score at 28% after 2 years in the same group. Composite measures were even more successful than single endpoints with an MDES of 21% in 3 years. Using alternative cognitive, imaging, functional, or composite endpoints, and recruiting patients that have LMCI could improve the success rate of AD clinical trials.
Subject(s)
Alzheimer Disease/drug therapy , Brain/drug effects , Brain/diagnostic imaging , Cognition/drug effects , Neuroimaging/methods , Humans , Magnetic Resonance Imaging , Treatment OutcomeABSTRACT
Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)-HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW-HA-stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2-induced HA synthesis and organization drives ESC-SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225-1238.
Subject(s)
Cell Differentiation , Cell Lineage , Hyaluronic Acid/metabolism , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Myocytes, Smooth Muscle/cytology , Animals , Enzyme Activation , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Extracellular Space/metabolism , Hyaluronan Receptors/metabolism , Hyaluronan Synthases/metabolism , Hyaluronic Acid/biosynthesis , MAP Kinase Signaling System , Mice , Models, Biological , Neointima/metabolism , Neovascularization, Physiologic , Protein Binding , Up-RegulationABSTRACT
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.
Subject(s)
Cell Movement , Chemokine CCL2/metabolism , Chemokine CXCL1/metabolism , Myocytes, Smooth Muscle/metabolism , Neointima/pathology , Stem Cells/cytology , Stem Cells/metabolism , Animals , Antigens, Ly/metabolism , Cell Movement/drug effects , Culture Media, Conditioned/pharmacology , Membrane Proteins/metabolism , Mice, Inbred C57BL , Receptors, CCR2 , Receptors, Interleukin-8B/metabolism , Signal Transduction/drug effects , cdc42 GTP-Binding Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
Several studies have reported a decline in incidence of dementia which may have large implications for the projected burden of disease, and provide important guidance to preventive efforts. However, reports are conflicting or inconclusive with regard to the impact of gender and education with underlying causes of a presumed declining trend remaining largely unidentified. The Alzheimer Cohorts Consortium aggregates data from nine international population-based cohorts to determine changes in the incidence of dementia since 1990. We will employ Poisson regression models to calculate incidence rates in each cohort and Cox proportional hazard regression to compare 5-year cumulative hazards across study-specific epochs. Finally, we will meta-analyse changes per decade across cohorts, and repeat all analysis stratified by sex, education and APOE genotype. In all cohorts combined, there are data on almost 69,000 people at risk of dementia with the range of follow-up years between 2 and 27. The average age at baseline is similar across cohorts ranging between 72 and 77. Uniting a wide range of disease-specific and methodological expertise in research teams, the first analyses within the Alzheimer Cohorts Consortium are underway to tackle outstanding challenges in the assessment of time-trends in dementia occurrence.
Subject(s)
Alzheimer Disease/epidemiology , Dementia/epidemiology , Gene-Environment Interaction , Aged , Aged, 80 and over , Alzheimer Disease/diagnosis , Alzheimer Disease/genetics , Cohort Studies , Dementia/diagnosis , Dementia/genetics , Female , Humans , Incidence , Male , Population Surveillance , Proportional Hazards Models , Prospective StudiesABSTRACT
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.
Subject(s)
Blood Vessel Prosthesis , Fibroblasts/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Lung/cytology , Myocytes, Smooth Muscle/cytology , Pluripotent Stem Cells/cytology , Adaptor Proteins, Signal Transducing , Cell Differentiation/physiology , Cell Nucleus/metabolism , Cell Separation/methods , Chemokines , Fetus/cytology , Fibroblasts/metabolism , Humans , Kruppel-Like Factor 4 , Membrane Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocytes, Smooth Muscle/metabolism , Transcriptional Activation/physiology , Wnt Signaling Pathway/physiology , beta Catenin/metabolismABSTRACT
OBJECTIVE: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. APPROACH AND RESULTS: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. CONCLUSIONS: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.
Subject(s)
Adult Stem Cells/cytology , Endothelial Cells/drug effects , Gene Expression Regulation, Developmental/physiology , Macrophages, Peritoneal/physiology , Myocytes, Smooth Muscle/drug effects , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/physiology , Adult Stem Cells/drug effects , Angiogenic Proteins/pharmacology , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Apoptosis , Cadherins/biosynthesis , Cadherins/genetics , Cell Line , Cell Lineage , Culture Media, Conditioned/pharmacology , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Gene Expression Regulation, Developmental/drug effects , Interleukin-6/pharmacology , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Neointima/pathology , Neovascularization, Physiologic/drug effects , Nitric Oxide Synthase Type III/physiology , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering/pharmacology , Radiation Chimera , Receptors, Tumor Necrosis Factor, Type I/drug effects , Receptors, Tumor Necrosis Factor, Type I/physiology , Recombinant Proteins/pharmacology , Signal Transduction , Thrombophilia/etiology , Thrombophilia/physiopathology , Tissue Scaffolds , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/pharmacology , Veins/transplantationABSTRACT
OBJECTIVE: This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. APPROACH AND RESULTS: A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1-positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1-positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1-positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1-positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E-deficient mice, which contributed to ≈ 30% of neointimal SMCs. CONCLUSIONS: We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.
Subject(s)
Atherosclerosis/pathology , Cell Lineage/physiology , Graft Occlusion, Vascular/pathology , Neointima/pathology , Stem Cells/pathology , Veins/transplantation , Adventitia/pathology , Animals , Antigens, Ly/metabolism , Apolipoproteins E/genetics , Cell Differentiation/physiology , Cells, Cultured , Collagen Type IV/metabolism , Disease Models, Animal , Integrins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Knockout , Stem Cells/metabolism , Transplantation, Autologous , Veins/pathologyABSTRACT
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 ß-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal-regulated kinase 1/2, or ß-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/ß-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.
Subject(s)
Adult Stem Cells/drug effects , Adventitia/drug effects , Cell Proliferation/drug effects , Chemotaxis/drug effects , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Myocytes, Smooth Muscle/drug effects , Sirolimus/pharmacology , beta Catenin/metabolism , Active Transport, Cell Nucleus/drug effects , Adult Stem Cells/enzymology , Adventitia/cytology , Adventitia/enzymology , Animals , Antigens, Ly/metabolism , Biomarkers/metabolism , Calcium-Binding Proteins/metabolism , Cells, Cultured , Constriction, Pathologic , Enzyme Activation , Membrane Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Myocytes, Smooth Muscle/enzymology , RNA Interference , Receptors, CXCR4/agonists , Receptors, CXCR4/metabolism , Signal Transduction/drug effects , Time Factors , Tissue Scaffolds , Transfection , beta Catenin/genetics , CalponinsABSTRACT
OBJECTIVE: To determine changes in the incidence of dementia between 1988 and 2015. METHODS: This analysis was performed in aggregated data from individuals >65 years of age in 7 population-based cohort studies in the United States and Europe from the Alzheimer Cohort Consortium. First, we calculated age- and sex-specific incidence rates for all-cause dementia, and then defined nonoverlapping 5-year epochs within each study to determine trends in incidence. Estimates of change per 10-year interval were pooled and results are presented combined and stratified by sex. RESULTS: Of 49,202 individuals, 4,253 (8.6%) developed dementia. The incidence rate of dementia increased with age, similarly for women and men, ranging from about 4 per 1,000 person-years in individuals aged 65-69 years to 65 per 1,000 person-years for those aged 85-89 years. The incidence rate of dementia declined by 13% per calendar decade (95% confidence interval [CI], 7%-19%), consistently across studies, and somewhat more pronouncedly in men than in women (24% [95% CI 14%-32%] vs 8% [0%-15%]). CONCLUSION: The incidence rate of dementia in Europe and North America has declined by 13% per decade over the past 25 years, consistently across studies. Incidence is similar for men and women, although declines were somewhat more profound in men. These observations call for sustained efforts to finding the causes for this decline, as well as determining their validity in geographically and ethnically diverse populations.
Subject(s)
Dementia/epidemiology , Age Distribution , Aged , Aged, 80 and over , Cohort Studies , Europe/epidemiology , Female , Humans , Incidence , Male , Sex Distribution , United States/epidemiologyABSTRACT
There exist a large number of cohort studies that have been used to identify genetic and biological risk factors for developing Alzheimer's disease (AD). However, there is a disagreement between studies as to how strongly these risk factors affect the rate of progression through diagnostic groups toward AD. We have calculated the probability of transitioning through diagnostic groups in six studies and considered how uncertainty around the strength of the effect of these risk factors affects estimates of the distribution of individuals in each diagnostic group in an AD clinical trial simulator. In this work, we identify the optimal choice of widely collected variables for comparing data sets and calculating probabilities of progression toward AD. We use the estimated transition probabilities to inform stochastic simulations of AD progression that are based on a Markov model and compare predicted incidence rates to those in a community-based study, the Cardiovascular Health Study.
ABSTRACT
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.
Subject(s)
Ataxin-1/genetics , Cell Movement/genetics , Hyperlipidemias/etiology , Hyperlipidemias/metabolism , Stem Cells/metabolism , Animals , Apolipoproteins E/deficiency , Ataxin-1/metabolism , Atherosclerosis/blood , Atherosclerosis/etiology , Atherosclerosis/metabolism , Atherosclerosis/pathology , Biomarkers , Cell Differentiation/genetics , Cholesterol, LDL/metabolism , Computational Biology/methods , Cytokines/metabolism , Disease Models, Animal , Gene Expression , Gene Expression Profiling , Hyperlipidemias/blood , Immunohistochemistry , Inflammation Mediators/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Knockout , Stem Cells/cytologyABSTRACT
PURPOSE: Pharmacokinetic interaction of sunitinib with diclofenac, paracetamol, mefenamic acid and ibuprofen was evaluated due to their P450 mediated metabolism and OATP1B1, OATP1B3, ABCB1, ABCG2 transporters overlapping features. METHODS: Male and female mice were administered 6 sunitinib doses (60 mg/kg) PO every 12 h and 30 min before the last dose were administered vehicle (control groups), 250 mg/kg paracetamol, 30 mg/kg diclofenac, 50 mg/kg mefenamic acid or 30 mg/kg ibuprofen (study groups), euthanized 6 h post last administration and sunitinib plasma, liver, kidney, brain concentrations analyzed. RESULTS: Ibuprofen halved sunitinib plasma concentration in female mice (p < 0.01) and showed 59 % lower concentration than male mice (p < 0.05). Diclofenac and paracetamol female mice showed 45 and 25 % higher plasma concentrations than male mice which were 27 % lower in mefenamic acid female mice. Paracetamol increased 2.2 (p < 0.05) liver and 1.4-fold (p < 0.05) kidney sunitinib concentrations in male mice that were lower in female mice (p < 0.01, p < 0.001, respectively). Ibuprofen increased 2.9-fold (p < 0.01) liver concentration in male mice that were higher than in female mice (p < 0.001). Female control mice had 35 % higher sunitinib brain concentration than male mice but the concentration decreased 37, 33, 10 and 57 % in the diclofenac, paracetamol, mefenamic acid and ibuprofen (p < 0.001), respectively. Tissue-plasma concentrations correlations were nonsignificant in control, paracetamol, mefenamic acid and ibuprofen groups but was significant in the diclofenac group in male mice (liver, brain) and female mice (liver, kidney). CONCLUSIONS: These results portray gender-based sunitinib pharmacokinetic differences and NSAIDs selective effects on male or female mice, with potential clinical translatability.
Subject(s)
Acetaminophen/pharmacology , Analgesics, Non-Narcotic/pharmacology , Angiogenesis Inhibitors/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Diclofenac/pharmacology , Ibuprofen/pharmacology , Indoles/pharmacokinetics , Mefenamic Acid/pharmacology , Pyrroles/pharmacokinetics , Animals , Carrier Proteins/metabolism , Drug Interactions , Female , Male , Mice , Mice, Inbred ICR , Sex Characteristics , Sunitinib , Tissue DistributionABSTRACT
The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through ß-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/ß-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.
Subject(s)
Blood Vessel Prosthesis , Embryonic Stem Cells/cytology , Kruppel-Like Transcription Factors/metabolism , Muscle, Smooth, Vascular/cytology , Proto-Oncogene Proteins c-kit/metabolism , Tissue Engineering/methods , Wnt Signaling Pathway , Animals , Antigens, Ly/analysis , Antigens, Ly/metabolism , Cell Differentiation , Cell Line , Cells, Cultured , Embryonic Stem Cells/metabolism , Kruppel-Like Factor 4 , Membrane Proteins/analysis , Membrane Proteins/metabolism , Mice , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Proto-Oncogene Proteins c-kit/analysisABSTRACT
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.
Subject(s)
Cell Differentiation , Chemotaxis , Embryonic Stem Cells/physiology , HSP47 Heat-Shock Proteins/metabolism , Myocytes, Smooth Muscle/physiology , Animals , Cells, Cultured , Chemokines/physiology , Gene Expression , Mice , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolismABSTRACT
In major cancer centers, heavy patients load and multiple registration stations could cause significant wait time, and can be result in patient complains. Real-time patient journey data and visual display are useful tools in hospital patient queue management. This paper demonstrates how we capture patient queue data without deploying any tracing devices; and how to convert data into useful patient journey information to understand where interventions are likely to be most effective. During our system development, remarkable effort has been spent on resolving data discrepancy and balancing between accuracy and system performances. A web-based dashboard to display real-time information and a framework for data analysis were also developed to facilitate our clinics' operation. Result shows our system could eliminate more than 95% of data capturing errors and has improved patient wait time data accuracy since it was deployed.
Subject(s)
Ambulatory Care/organization & administration , Continuity of Patient Care/organization & administration , Data Mining/methods , Hospitalization , Medical Records Systems, Computerized/organization & administration , Waiting Lists , Workflow , Computer Systems , Efficiency, Organizational , SingaporeABSTRACT
AIMS: Accumulating evidence suggest that numerous microRNAs (miRNAs) play important roles in cell proliferation, apoptosis, and differentiation, as well as various diseases that accompany inflammatory responses. Inflammation is known to be a major contributor to atherogenesis. Previous studies provide promising evidence in support of the role of miRNAs in cardiovascular disease. However, mechanistic data on these small molecules in atherosclerosis (AS) are still missing. The present study aims to investigate the potential role of miRNAs in AS. METHODS AND RESULTS: The miRNA transcriptase was verified by TaqMan real-time polymerase chain reaction assay. Thoracic aorta samples were obtained from Apolipoprotein E knockout mice, and plasma samples were from coronary artery disease (CAD) patients. The results showed that the miR-155 level was the most significantly elevated both in AS mice and CAD patients relative to the normal control. The functional role of miR-155 in the atherosclerotic path physiological process was also observed in vivo and in vitro. The observations suggested that miR-155 is a part of a negative feedback loop, which down-modulates inflammatory cytokine production and decreases AS progression. miR-155 was also found to mediate the inflammatory response and mitogen-activated protein kinase (MAPK) pathway by targeting mitogen-activated protein kinase kinase kinase 10. CONCLUSIONS: miR-155 contributes to the prevention of AS development and progression. It may also be involved in the posttranscriptional regulation of the inflammatory response and MAPK pathway by targeting mitogen-activated protein kinase kinase kinase 10.