Objective-Lineage-negative bone marrow cells (lin-BMCs) are enriched in endothelial progenitor cells and mediate vascular repair. Aging-associated senescence and apoptosis result in reduced number and functionality of lin-BMCs, impairing their prorepair capacity. The molecular mechanisms underlying lin-BMC senescence and apoptosis are poorly understood. MicroRNAs (miRNAs) regulate many important biological processes. The identification of miRNA-mRNA networks that modulate the health and functionality of lin-BMCs is a critical step in understanding the process of vascular repair. The aim of this study was to characterize the role of the miR-146a-Polo-like kinase 2 (Plk2) network in regulating lin-BMC senescence, apoptosis, and their angiogenic capability.Approach and Results-Transcriptome analysis in lin-BMCs isolated from young and aged wild-type and ApoE(-/-)(apolipoprotein E) mice showed a significant age-associated increase in miR-146a expression. In silico analysis, expression study and Luciferase reporter assay established Plk2 as a direct target of miR-146a. miR-146a overexpression in young lin-BMCs inhibited Plk2 expression, resulting in increased senescence and apoptosis, via p16Ink4a/p19Arf and p53, respectively, as well as impaired angiogenic capacity in vitro and in vivo. Conversely, suppression of miR-146a in aged lin-BMCs increased Plk2 expression and rejuvenated lin-BMCs, resulting in decreased senescence and apoptosis, leading to improved angiogenesis.Conclusions-(1) miR-146a regulates lin-BMC senescence and apoptosis by suppressing Plk2 expression that, in turn, activates p16Ink4a/p19Arf and p53 and (2) modulation of miR-146a or its target Plk2 may represent a potential therapeutic intervention to improve lin-BMC-mediated angiogenesis and vascular repair.

Introduction: Lineage negative bone marrow cells (Lin - BMCs) mediate vascular repair. Aging-associated apoptosis and senescence result in reduced number and impair Lin - BMCs’s pro-repair capacity. Molecular mechanisms underlying lin - BMCs apoptosis and senescence remain poorly understood. MicroRNAs (miRNAs) regulate many important biological processes. The identification of miRNA-mRNA networks that modulate the health and functionality of lin - BMCs is a critical step in understanding the process of vascular repair. Hypothesis: miR-200b-cJun network plays a vital role in regulating Lin - BMCs apoptosis, senescence and self renewal in angiogenesis. Methods: Lin - BMCs were isolated from young (3-week-old) wild type (WT), young apoE -/- , aged WT (29-month-old) and aged apoE -/- (12 month-old) C57BL/6 mice. Global miRNA and gene expression profiling were analyzed. Results: Transcriptome analysis in Lin - BMCs isolated from young and aged WT and apoE -/- mice showed a significant aging-associated increase in miR-200b expression. We found that C-Jun was predicted to be a miR-200b target and c-Jun expression was reversely correlated with miR-200b levels. Luciferase reporter assays confirmed c-Jun as a direct target of miR-200b. MiR-200b overexpression in young lin-BMCs inhibited c-Jun expression, resulting in increased senescence and apoptosis in vitro and impaired angiogenic capacity in vivo . In contrast, suppression of miR-200b or overexpression of c-Jun in aged lin - BMCs increased c-Jun expression rejuvenated Lin - BMCs with decreased senescence and apoptosis, leading to improved angiogenesis in vitro and in vivo . Conclusions: Using genomic and functional studies, we discovered that miR-200b regulates apoptosis and senescence of age-associated lin - BMCs by suppressing c-Jun expression, thus negatively affecting vascular repair capacity of those cells in atherogenesis. Modulation of miR-200b and/or its target c-Jun may suggest a potential therapeutic intervention to improve Lin - BMCs-mediated angiogenesis and vascular repair.

Background: Bone marrow (BM) lineage negative fraction (lin- BMCs) enriched for mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs), appear to mediate cardiac repair via direct di...

Rationale: Endothelial progenitor cells (EPCs) contribute to the regeneration of endothelium. Aging-associated senescence results in reduced number and function of EPCs, potentially contributing to increased cardiac risk, reduced angiogenic capacity, and impaired cardiac repair effectiveness. The mechanisms underlying EPC senescence are unknown. Increasing evidence supports the role of microRNAs in regulating cellular senescence. Objective: We aimed to determine whether microRNAs regulated EPC senescence and, if so, what the underlying mechanisms are. Methods and Results: To map the microRNA/gene expression signatures of EPC senescence, we performed microRNA profiling and microarray analysis in lineage-negative bone marrow cells from young and aged wild-type and apolipoprotein E–deficient mice. We identified 2 microRNAs, microRNA-10A* (miR-10A*), and miR-21, and their common target gene Hmga2 as critical regulators for EPC senescence. Overexpression of miR-10A* and miR-21 in young EPCs suppressed Hmga2 expression, caused EPC senescence, as evidenced by senescence-associated β–galactosidase upregulation, decreased self-renewal potential, increased p16 Ink4a /p19 Arf expression, and resulted in impaired EPC angiogenesis in vitro and in vivo, resembling EPCs derived from aged mice. In contrast, suppression of miR-10A* and miR-21 in aged EPCs increased Hmga2 expression, rejuvenated EPCs, resulting in decreased senescence-associated β–galactosidase expression, increased self-renewal potential, decreased p16 Ink4a /p19 Arf expression, and improved EPC angiogenesis in vitro and in vivo. Importantly, these phenotypic changes were rescued by miRNA-resistant Hmga2 cDNA overexpression. Conclusions: miR-10A* and miR-21 regulate EPC senescence via suppressing Hmga2 expression and modulation of microRNAs may represent a potential therapeutic intervention in improving EPC-mediated angiogenesis and vascular repair.

Background: With advancing age the regenerative properties of somatic stem cells deteriorate in part due to cellular senescence, eventually leading to the diminished repair capability. Endothelial progenitor cells (EPCs) play an important role in angiogenesis. Several miRNAs have been reported to be critical modulators for EPC plasticity. The aim of this study was to determine whether miRNAs regulated age-related senescence in lineage negative bone marrow cells (lin - BMCs) enriched for EPCs. Methods: Lin - BMCs were isolated from young (3-week-old) wild type (WT) and apoE -/- C57BL/6 mice and aged WT (29-month-old) and apoE -/- (12 month-old) mice. Global miRNA and gene expression profiling were analyzed. Results: Using genome-wide miRNA analysis, we found that miR-146a was increased 6.3-fold and 3.4-fold in aged WT and apoE -/- lin - BM cells, respectively, relative to their young counterparts. Microarray analysis showed that the Polo-like kinase 2 (Plk2), a member of the ‘polo’ family of serine/threonine protein kinases that are critical regulators of cell cycle progression or apoptosis, was decreased 18-fold in aged lin - BMC as compared to young cells. Luciferase assay showed miR-146a substantially inhibited WT Plk2-3’-UTR-luciferase activity in 293T cells, but not mutant Plk2-3’-UTR-luciferase reporter. Overexpression of lentiviral-derived miR-146a resulted in reduction of Plk2 expression, increased senescence-associated (SA) β-gal expression and decreased vascular tube formation in young EPCs, whereas miR-146a antagomir rejuvenated aged EPCs, causing increased Plk2 expression, decreased SA β-gal expression and enhanced vascular tube formation. Furthermore, knockdown of Plk2 by its siRNA resulted in increased senescence and diminished vascular tube formation. Conclusions: Using genomic and functional studies, we have discovered miR-146a controls age-associated EPC senescence via regulating Plk2 expression, thus negatively affecting their vascular repair capacity, which may impact atherogenesis. These findings advance our understanding of EPC aging and provide molecular targets for genetic modification to increase the therapeutic efficacy of EPCs in preventing and repairing vascular damage in atherosclerosis.

AIMS:Vein graft endothelial damage is a key step in the development of neointimal hyperplasia, leading to vein graft failure. We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia. METHODS AND RESULTS:Carotid artery interposition grafting was performed with syngeneic inferior vena cavae in mice with severe combined immunodeficiency (SCID). Lineage-negative human umbilical cord blood (hUCB) cells (or medium alone) were injected into vein-grafted mice intra-operatively and 2 weeks post-operatively. In vein grafts from hUCB cell-injected mice, we found human HLA-expressing endothelial cells, as well as increased levels of VEGF and FGF-2. Furthermore, hUCB cells secreted VEGF and FGF-2 in vitro. The markedly enhanced endothelial regeneration, likely resulting from both direct engraftment and paracrine actions of hUCB cells, inhibited inflammatory response, diminished intimal cell proliferation, and reduced neointimal hyperplasia in the vein grafts. CONCLUSIONS:hUCB cells may accelerate vein graft re-endothelialization via both direct differentiation into endothelial cells and release of paracrine factors to enhance endothelial regeneration and reduce inflammation. These data highlight a potential therapeutic role for cellular therapy in vessel injury.

P ostnatal neovascularization involves endothelial progenitor cells (EPCs) derived from the bone marrow. EPCs play important roles in vascular homeostasis and compensatory vasculogenesis with their plasticity to differentiate into endothelial cells (ECs) and as a source of paracrine pro-angiogenic factors. EPCs are usually defined retrospectively by their ability to differentiate into ECs. Because diverse progenitor cell types have been shown to differentiate into ECs, there is no universally accepted molecular definition for EPCs. Several types of EPCs have been studied: (1) EPCs defined by cell surface markers, including CD34, CD133/CD34, CD133/ vascular endothelial growth factor receptor-2 or CD133/ CD34/vascular endothelial growth factor receptor-2; (2) early outgrowth EPCs, of myeloid origin, which stimulate angio-genic responses through paracrine secretion of angiogenic factors ; and (3) late outgrowth EPCs, of nonhematopoietic origin, possessing the capacity of de novo vessel formation through direct incorporation into the growing vasculature. 1 Regardless of what markers/techniques are used to define EPCs, it is conceivable that these cells are enriched in the lineage-negative bone marrow cells (lin − BMCs). Remarkably, multiple studies have shown that the number and angiogenic function of EPCs Objective: We aimed to determine whether microRNAs regulated EPC senescence and, if so, what the underlying mechanisms are. Ink4a / p19 Arf expression, and improved EPC angiogenesis in vitro and in vivo. Importantly, these phenotypic changes were rescued by miRNA-resistant Hmga2 cDNA overexpression. of microRNAs may represent a potential therapeutic intervention in improving EPC-mediated angiogenesis and vascular repair.