ABCG2-Expressing Clonal Repopulating Endothelial Cells Serve to Form and Maintain Blood Vessels.

BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

Origin, prospective identification, and function of circulating endothelial colony-forming cells in mice and humans.

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony-forming cells (C-ECFCs), also known as blood outgrowth endothelial cells, with proliferative and vasculogenic activity can be cultured; however, the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing revealed murine C-ECFCs emerged in the early postnatal period, displayed high vasculogenic potential with enriched frequency of clonal proliferative cells compared with tissue-resident ECFCs, and were not committed to or derived from the BM hematopoietic system but from tissue-resident ECFCs. In humans, C-ECFCs were present in the CD34bright cord blood mononuclear subset, possessed proliferative potential and in vivo vasculogenic function in a naive or cultured state, and displayed a single cell transcriptome sharing some umbilical venous endothelial cell features, such as a higher protein C receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

Self-regulation of TNF-α Induces Dysfunction of Endothelial Colony-forming Cells from Patients with Venous Thromboembolic Disease.

BACKGROUND: Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculogenesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-α. OBJECTIVE: To analyze the TNF-α signaling pathway and its relationship with the expression of cell-cycle regulators. METHODS: Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene transcripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF-κB and activated targets of each pathway: NF-κB (Ser536); IκBα (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software. RESULTS: In ECFCs from VTD patients, TNF-α receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regulators (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase. CONCLUSIONS: Our results strongly suggest that these molecular alterations in the pathway of TNF-α and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events.

Novel Markers of Angiogenesis in the Setting of Cognitive Impairment and Dementia.

BACKGROUND: Aberrant angiogenesis may play a role in the development of Alzheimer's disease and related dementia. OBJECTIVE: To explore the relationship between angiogenesis activity and evidence of neurodegeneration among older adults. METHODS: Cross-sectional study of 49 older adults clinically characterized as cognitively normal, mild cognitive impairment, or early Alzheimer's disease. In addition to neuroimaging, we completed assays on peripheral blood, including: vascular endothelial growth factor, tumor necrosis factor, fibroblast growth factor, and amyloid-ß peptide 40. We used advanced polychromatic flow cytometry to phenotype circulating mononuclear cells to assess angiogenesis activity. RESULTS: Although we documented differences in cognitive performance, structural changes on neuroimaging, and burden of amyloid and tau on positron emission tomography, angiogenesis activity did not vary by group. Interestingly, VEGF levels were shown to be increased among subjects with mild cognitive impairment. In ANCOVA models controlling for age, sex, intracranial volume, and monocyte subpopulations, angiogenesis activity was correlated with increased white matter hyperintensities. CONCLUSION: We demonstrate a significant association between angiogenesis activity and cerebrovascular disease. To better understand the potential of angiogenesis as an intervention target, longitudinal studies are needed.

Clonally selected primitive endothelial cells promote occlusive pulmonary arteriopathy and severe pulmonary hypertension in rats exposed to chronic hypoxia.

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells (ECs) contribute to severe pulmonary arterial hypertension (PAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). The remodelled pulmonary arteries of PAH patients harboured CD117+ ECs. Rat lung CD117+ ECs underwent four generations of clonal expansion to enrich hyperproliferative ECs. The resulting clonally enriched ECs behaved like ECs, as measured by in vitro and in vivo angiogenesis assays. The same primitive ECs showed a limited ability for mesenchymal lineage differentiation. Endothelial differentiation and function were enhanced by blocking TGF-ß signalling, promoting bone morphogenic protein (BMP) signalling. The transplantation of the EC clones caused arterio-occlusive PH in rats exposed to chronic hypoxia. These EC clones engrafted in the pulmonary arteries. Yet cessation of chronic hypoxia promoted lung cell apoptosis and resolution of vascular lesions. In conclusion, this is to the best of our knowledge, the first report that clonally enriched primitive ECs promote occlusive pulmonary arteriopathy and severe PH. These primitive EC clones further give rise to cells of endothelial and mesenchymal lineage as directed by BMP and TGF-ß signaling.

Mutant p53 drives clonal hematopoiesis through modulating epigenetic pathway.

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

Peripheral blood-derived mesenchymal stem cells demonstrate immunomodulatory potential for therapeutic use in horses.

Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans.

Identification of Circulating Endothelial Colony-Forming Cells from Murine Embryonic Peripheral Blood.

Human umbilical cord blood contains highly proliferative circulating endothelial colony-forming cells (ECFC). These cells have promising therapeutic potential for various cardiovascular diseases by possessing robust in vitro clonal expansion potential and the ability to form functional blood vessels in vivo upon transplantation into recipient immunodeficient mice. However whether similar cells also exist in murine blood remains unresolved, which impedes the study of circulating ECFC biology using murine models. Here we describe a method to identify and culture murine embryonic peripheral blood-derived circulating ECFC through co-culture with OP9 stromal cells. Using this method, embryonic circulating ECFC can be identified by the formation of sheet-like or network-like endothelial colonies upon OP9 stromal cell monolayers.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

It is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering.

Whole organ engineering paradigms typically involve repopulating acellular organ scaffolds with recipient-compatible cells, to generate a neo-organ that may provide key physiological functions. In the case of whole lung engineering, functionally endothelialized pulmonary vasculature is critical for establishing a fluid-tight barrier at the level of the alveolus, so that oxygen and carbon dioxide can be exchanged in the organ. We have previously developed a protocol to efficiently seed endothelial cells into the microvascular channels of decellularized lung scaffolds, but fully functional endothelial coverage, in terms of barrier function and resistance to thrombosis, was not achieved. In this study, we investigated whether various small molecules could favorably impact endothelial functionality after seeding into decellularized lung scaffolds. We demonstrated that the Epac-selective cAMP analog 8CPT-2Me-cAMP improves endothelial barrier function in repopulated lung scaffolds. When treated with the Epac agonist, barrier function of human umbilical vein endothelial cells (HUVECs) improved, and was maintained for at least three days, whereas the effect of other tested molecules lasted for only 5 h. Treatment with the Epac agonist re-organized actin structure, and appeared to increase the continuity of junction proteins such as VE-cadherin and ZO1. Blockade of actin polymerization abolished the effect of the Epac agonist on barrier function and actin reorganization, confirming a strong actin-mediated effect. Similarly, after treatment with Epac agonist, the barrier function in iPSC-derived endothelial colony forming cells (ECFCs) was increased and the enhanced barrier was maintained for at least 60 h. After culture in lung scaffolds for 5 days, iPSC-ECFCs maintained their phenotype by expressing CD31, eNOS, vWF, and VE-Cadherin. Treatment with the Epac agonist significantly improved the barrier function of iPSC-ECFC-repopulated lung for at least 6 h. Taken together, these findings demonstrated that Epac-selective 8CPT-2Me-cAMP activation enhanced vascular barrier in iPSC-ECFC-engineered lungs, and may be useful to improve endothelial functionality for whole organ tissue engineering.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

Endothelial Colony-Forming Cell Function Is Reduced During HIV Infection.

BACKGROUND: Human immunodeficiency virus (HIV) may be related to cardiovascular disease through monocyte activation-associated endothelial dysfunction. METHODS: Blood samples from 15 HIV-negative participants (the uninfected group), 8 HIV-positive participants who were not receiving antiretroviral therapy (ART) (the infected, untreated group), and 15 HIV-positive participants who were receiving ART (the infected, treated group) underwent flow cytometry of endothelial colony-forming cells (ECFCs) and monocyte proportions. IncuCyte live cell imaging of 8 capillary proliferative capacity parameters were obtained from cord blood ECFCs treated with participant plasma. RESULTS: The ECFC percentage determined by flow cytometry was not different between the study groups; however, values of the majority of capillary proliferative capacity parameters (ie, cell area, network length, network branch points, number of networks, and average tube width uniformity) were significantly lower in infected, untreated participants as compared to values for uninfected participants or infected, treated participants (P < .00625 for all comparisons). CD14+CD16+ intermediate monocytes and soluble CD163 were significantly and negatively correlated with several plasma-treated, cord blood ECFC proliferative capacity parameters in the combined HIV-positive groups but not in the uninfected group. CONCLUSIONS: Cord blood ECFC proliferative capacity was significantly impaired by plasma from infected, untreated patients, compared with plasma from uninfected participants and from infected, treated participants. Several ECFC functional parameters were adversely associated with monocyte activation in the HIV-positive groups, thereby suggesting a mechanism by which HIV-related inflammation may impair vascular reparative potential and consequently increase the risk of cardiovascular disease during HIV infection.

Hematopoietic-restricted Ptpn11E76K reveals indolent MPN progression in mice.

Juvenile Myelomonocytic Leukemia (JMML) is a pediatric myeloproliferative neoplasm (MPN) that has a poor prognosis. Somatic mutations in Ptpn11 are the most frequent cause of JMML and they commonly occur in utero. Animal models of mutant Ptpn11 have probed the signaling pathways that contribute to JMML. However, existing models may inappropriately exacerbate MPN features by relying on non-hematopoietic-restricted Cre-loxP strains or transplantations into irradiated recipients. In this study we generate hematopoietic-restricted models of Ptpn11E76K-mediated disease using Csf1r-MCM and Flt3Cre. We show that these animals have indolent MPN progression despite robust GM-CSF hypersensitivity and Ras-Erk hyperactivation. Rather, the dominant pathology is pronounced thrombocytopenia with expanded extramedullary hematopoiesis. Furthermore, we demonstrate that the timing of tamoxifen administration in Csf1r-MCM mice can specifically induce recombinase activity in either fetal or adult hematopoietic progenitors. We take advantage of this technique to show more rapid monocytosis following Ptpn11E76K expression in fetal progenitors compared with adult progenitors. Finally, we demonstrate that Ptpn11E76K results in the progressive reduction of T cells, most notably of CD4+ and naïve T cells. This corresponds to an increased frequency of T cell progenitors in the thymus and may help explain the occasional emergence of T-cell leukemias in JMML patients. Overall, our study is the first to describe the consequences of hematopoietic-restricted Ptpn11E76K expression in the absence of irradiation. Our techniques can be readily adapted by other researchers studying somatically-acquired blood disorders.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Mycophenolic acid induces senescence of vascular precursor cells.

OBJECTIVE: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. METHODS: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. RESULTS: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated ß-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. CONCLUSION: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties.

The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157+ VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.

Tissue regeneration using endothelial colony-forming cells: promising cells for vascular repair.

Repairing and rebuilding damaged tissue in diseased human subjects remains a daunting challenge for clinical medicine. Proper vascular formation that serves to deliver blood-borne nutrients and adequate levels of oxygen and to remove wastes is critical for successful tissue regeneration. Endothelial colony-forming cells (ECFC) represent a promising cell source for revascularization of damaged tissue. ECFCs are identified by displaying a hierarchy of clonal proliferative potential and by pronounced postnatal vascularization ability in vivo. In this review, we provide a brief overview of human ECFC isolation and characterization, a survey of a number of animal models of human disease in which ECFCs have been shown to have prominent roles in tissue repair, and a summary of current challenges that must be overcome before moving ECFC into human subjects as a cell therapy.

Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice.

BACKGROUND: Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN). RESULTS: E9.5 YS EMPs from VavCre+;PTPN11D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11E76K ;ROSAYFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN. CONCLUSIONS: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc.

Epigenetic Activation of Pro-angiogenic Signaling Pathways in Human Endothelial Progenitors Increases Vasculogenesis.

Human endothelial colony-forming cells (ECFCs) represent a promising source of adult stem cells for vascular repair, yet their regenerative capacity is limited. Here, we set out to understand the molecular mechanism restricting the repair function of ECFCs. We found that key pro-angiogenic pathways are repressed in ECFCs due to the presence of bivalent (H3K27me3/H3K4me3) epigenetic marks, which decreases the cells' regenerative potential. Importantly, ex vivo treatment with a combination of epigenetic drugs that resolves bivalent marks toward the transcriptionally active H3K4me3 state leads to the simultaneous activation of multiple pro-angiogenic signaling pathways (VEGFR, CXCR4, WNT, NOTCH, SHH). This in turn results in improved capacity of ECFCs to form capillary-like networks in vitro and in vivo. Furthermore, restoration of perfusion is accelerated upon transplantation of drug-treated ECFCs in a model of hindlimb ischemia. Thus, ex vivo treatment with epigenetic drugs increases the vascular repair properties of ECFCs through transient activation of pro-angiogenic signaling pathways.