Endothelial Colony-forming Cells Migration Research Articles

One-Carbon Metabolism, a Nutrient Dependent Mediator of Angiogenesis and Alveolar Formation in the Developing Lung

Background/Objective: Lung maturation has discrete dynamic shifts in metabolic derivatives that give rise to stage specific metabolite signatures. Components of one-carbon metabolism (OCM) are significantly increased in the late saccular and alveolar stage while hyperoxia disrupts OCM metabolism during these lung developmental stages. We hypothesize that disrupted OCM metabolites in the immature lung disrupts vessel dependent alveolar formation thus contributing to alveolar dysplasia. Our studies explore the impact that restoration of OCM has on angiogenesis and alveolar formation.Methods: Isolated human neonatal circulating Endothelial Colony Forming Cells (ECFCs) were examined for proliferation (WST-1), migration (scratch assay), and angiogenesis (matrigel) in response to supplementation of OCM derivatives, essential amino acid methionine and nicotinamide. OCM enzyme levels were assessed via Western blotting, NNMT, NAMPT, DNMT. Lastly, alveolar formation in a hyperoxia murine model of prematurity (HMMP) with dietary supplement of methionine vs control was assessed, MLI, H&E, and vessel formation (endomucin).Results: Methionine and nicotinamide increased ECFC proliferation (p<0.05) and OCM enzymes. Nicotinamide increased ECFC angiogenesis (p<0.05). ECFC migration was not impacted by methionine or nicotinamide supplementation (p NS). Methionine dietary supplementation in a HMMP model improved distal alveolar formation.Conclusion and Clinical Implications: Lung maturation is dependent on shifting metabolic needs during development. Replenishment of disrupted key OCM metabolites in HMMP promotes vessel and alveolar formation. Future studies are necessary to explore the role of precision nutritive supplementation in alveolar development of premature infants.

Impaired repair properties of endothelial colony-forming cells in patients with granulomatosis with polyangiitis.

In patients with ANCA‐associated vasculitis, interactions between neutrophils and endothelial cells cause endothelial damage and imbalance. Endothelial colony‐forming cells (ECFCs) represent a cellular population of the endothelial lineage with proliferative capacity and vasoreparative properties. This study aimed to evaluate the angiogenic capacity of ECFCs of patients with granulomatosis with polyangiitis (GPA). The ECFCs of 13 patients with PR3‐positive GPA and 14 healthy controls were isolated and characterized using fluorescence‐activated cell sorting, capillary tube formation measurement, scratching assays and migration assays with and without plasma stimulation. Furthermore, three patients with active disease underwent post‐treatment recollection of ECFCs for longitudinal evaluation. The ECFCs from the patients and controls showed similar capillary structure formation. However, the ECFCs from the patients with inactive GPA exhibited early losses of angiogenic capacity. Impairments in the migration capacities of the ECFCs were also observed in patients with GPA and controls (12th h, p = 0.05). Incubation of ECFCs from patients with GPA in remission with plasma from healthy controls significantly decreased migration capacity (p = 0.0001). Longitudinal analysis revealed that treatment significantly lowered ECFC migration rates. This study revealed that ECFCs from the patients with PR3‐positive GPA in remission demonstrated early losses of tube formation and reduced migration capacity compared to those of the healthy controls, suggesting impairment of endothelial function.

Endothelial Progenitor Cell-Based in vitro Pre-Endothelialization of Human Cell-Derived Biomimetic Regenerative Matrices for Next-Generation Transcatheter Heart Valves Applications.

Hemocompatibility of cardiovascular implants represents a major clinical challenge and, to date, optimal antithrombotic properties are lacking. Next-generation tissue-engineered heart valves (TEHVs) made from human-cell-derived tissue-engineered extracellular matrices (hTEMs) demonstrated their recellularization capacity in vivo and may represent promising candidates to avoid antithrombotic therapy. To further enhance their hemocompatibility, we tested hTEMs pre-endothelialization potential using human-blood-derived endothelial-colony-forming cells (ECFCs) and umbilical vein cells (control), cultured under static and dynamic orbital conditions, with either FBS or hPL. ECFCs performance was assessed via scratch assay, thereby recapitulating the surface damages occurring in transcatheter valves during crimping procedures. Our study demonstrated: feasibility to form a confluent and functional endothelium on hTEMs with expression of endothelium-specific markers; ECFCs migration and confluency restoration after crimping tests; hPL-induced formation of neo-microvessel-like structures; feasibility to pre-endothelialize hTEMs-based TEHVs and ECFCs retention on their surface after crimping. Our findings may stimulate new avenues towards next-generation pre-endothelialized implants with enhanced hemocompatibility, being beneficial for selected high-risk patients.

Gonadotropins as novel active partners in vascular diseases: Insight from angiogenic properties and thrombotic potential of endothelial colony‐forming cells

BackgroundThe impact of estrogen and testosterone on atherosclerotic cardiovascular disease is well known, but the role of the gonadotropins follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) to some extent remain less studied. ObjectivesTo explore the angiogenic potential of gonadotropins on endothelial colony‐forming cells (ECFCs). MethodsWe examined the effects of various doses of gonadotropins on ECFCs obtained from cord blood by assessing colony number, proliferation, migration, and sprouting ability. Moreover, we studied thrombin generation in ECFCs exposed to gonadotropins by performing a thrombin generation assay. Finally, we determined the levels of circulating gonadotropins in 30 men, to exclude the effect of estrogen, with lower extremity arterial disease (LEAD), in comparison with age‐ and sex‐matched controls. ResultsExposure to FSH, LH, or PRL resulted in an increase in ECFC migration but showed no effect on proliferation or ECFC commitment from cord blood mononuclear cells. Using a three‐dimensional fibrin gel assay, we showed that ECFC sprouting was significantly enhanced by gonadotropins. Exposure to FSH also increased the thrombin generation of ECFCs exposed to FSH. Finally, FSH and LH levels in men with LEAD were higher than those in controls. ConclusionGonadotropins increase ECFC‐related angiogenesis and may be involved in thrombin generation in cardiovascular disease. Gonadotropins may act as biomarkers; moreover, we hypothesize that gonadotropin‐blocking strategies may be a novel interesting therapeutic approach in atherosclerotic cardiovascular disease.

MicroRNA-139-5p upregulation is associated with diabetic endothelial cell dysfunction by targeting c-jun.

Dysfunction of endothelial cells (ECs) and their progenitor cells is an important feature of diabetic vascular disease. MicroRNA (miR)-139-5p is involved in inhibiting the metastasis and progression of diverse malignancies. However, the role of miR-139-5p in ECs still remains unclarified. Here we demonstrated that miR-139-5p expression was elevated in endothelial colony-forming cells (ECFCs) isolated from patients with diabetes, ECs derived from the aorta of diabetic rodents, and human umbilical vein endothelial cells (HUVECs) cultured in high glucose media. MiR-139-5p mimics inhibited tube formation, migration, proliferation, and down-regulated expression of c-jun, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF)-B, in ECFCs and HUVECs, respectively; moreover, miR-139-5p inhibitors reversed the tendency. Further, gain- and-loss function experiments and ChIP assay indicated that miR-139-5p regulate functions of ECFCs by targeting c-jun-VEGF/PDGF-B pathway. In vivo experiments (Matrigel plug assay and hindlimb ischemia model) showed that miR-139-5p downregulation further promoted ECFC-mediated angiogenesis and blood perfusion. In conclusion, diabetes-mediated high miR-139-5p expression inhibits the c-jun-VEGF/PDGF-B pathway, thus decreasing ECFCs migration, tube formation and proliferation, which subsequently reduces ECs survival. Therefore, miR-139-5p might be an important therapeutic target in the treatment of diabetic vasculopathy in the future.

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo.

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Calcium Signaling in Endothelial Colony Forming Cells in Health and Disease.

Endothelial colony forming cells (ECFCs) represent the only known truly endothelial precursors. ECFCs are released in peripheral circulation to restore the vascular networks dismantled by an ischemic insult or to sustain the early phases of the angiogenic switch in solid tumors. A growing number of studies demonstrated that intracellular Ca2+ signaling plays a crucial role in driving ECFC proliferation, migration, homing and neovessel formation. For instance, vascular endothelial growth factor (VEGF) triggers intracellular Ca2+ oscillations and stimulates angiogenesis in healthy ECFCs, whereas stromal derived factor-1α promotes ECFC migration through a biphasic Ca2+ signal. The Ca2+ toolkit endowed to circulating ECFCs is extremely plastic and shows striking differences depending on the physiological background of the donor. For instance, inositol-1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum is downregulated in tumor-derived ECFCs, while agonists-induced store-operated Ca2+ entry is up-regulated in renal cellular carcinoma and is unaltered in breast cancer and reduced in infantile hemangioma. This remodeling of the Ca2+ toolkit prevents VEGF-induced pro-angiogenic Ca2+ oscillations in tumor-derived ECFCs. An emerging theme of research is the dysregulation of the Ca2+ toolkit in primary myelofibrosis-derived ECFCs, as this myeloproliferative disorder may depend on a driver mutation in the calreticulin gene. In this chapter, I provide a comprehensive, but succinct, description on the architecture and role of the intracellular Ca2+ signaling toolkit in ECFCs derived from umbilical cord blood and from peripheral blood of healthy donors, cancer patients and subjects affected by primary myelofibrosis.

Human Adipose Mesenchymal Stem Cells Show More Efficient Angiogenesis Promotion on Endothelial Colony-Forming Cells than Umbilical Cord and Endometrium.

Angiogenesis is a complicated process in which perivascular cells play important roles. Multipotent mesenchymal stem/stromal cells (MSCs) from distinct tissues have been proved to be proangiogenic and share functional properties and gene expression profiles with perivascular cells. However, different tissues derived MSCs may exhibit different potential for clinical applications. Accordingly, comparative studies on different MSCs are essential. Here, we characterized MSCs from adipose (ADSCs), umbilical cord (UCMSCs), and endometrium (EMSCs) in terms of the surface antigen expression, differentiation ability, and the ability of angiogenesis promotion on endothelial colony-forming cells (ECFCs) both in vitro and in vivo. No significant differences in immunophenotype and differentiation were observed. In addition, three types of MSCs all located around tubular-like structures formed by ECFCs in coculture system on matrigel. But ECFCs seeded on ADSCs monolayer formed more organized capillary-like network than that on UCMSCs or EMSCs. When suspended with ECFCs in matrigel and implanted into nude mice, ADSCs promoted more functional vessel formation after 7 days. Moreover, in murine hindlimb ischemia model, cotransplantation of ECFCs with ADSCs was significantly superior to UCMSCs and EMSCs in promoting perfusion recovery and limb salvage. Furthermore, ADSC-conditioned medium (CM) contained more proangiogenic factors (such as vascular endothelial growth factor-A, platelet-derived growth factor BB, and basic fibroblast growth factor) and less inhibitory factor (such as thrombospondin-1), when compared with UCMSC-CM and EMSC-CM. And ADSC-CM more durably stabilized the vascular-like structures formed by ECFCs on matrigel and promoted ECFCs migration more efficiently. In summary, MSCs from adipose show significantly efficient promotion on angiogenesis both in vitro and in vivo than UCMSCs and EMSCs. Hence, ADSCs may be recommended as a more suitable source for treating hindlimb ischemia.

Endothelial Colony-Forming Cells (ECFC) As an Autologous Model for Studying Endothelial Pathophysiology in Sickle Cell Anemia and Myeloproliferative Neoplasms

Background: Endothelial colony-forming cells (ECFC) are an important source of autologous endothelial cells to study its implication in the pathophysiology of diseases with risk of vaso-occlusive events. Currently, our research group began to elucidate the ECFC mechanisms that contribute to the complex clinical vascular manifestations in two diseases, sickle cell anemia (SCA) and myeloproliferative neoplasms (MPN).Aims: In this study, we analyzed functional in vitro assays of endothelial cells. The adhesion to red blood cell (RBC), migration and angiogenesis process of ECFC isolated from patients with SCA and MPN, as well as from healthy individuals (CTR) were evaluated in seeking an expanded understanding of the biology of the endothelial cell and its role in vascular events.Methods: ECFC were obtained through the isolation and culture of human peripheral blood mononuclear cells. ECFC were isolated from 8 patients with SCA under regular transfusion, 6 patients with MPN and 10 CTR. Human umbilical vein endothelial cells (HUVEC) were used as additional controls. Flow cytometry of ECFC indicated that all cells were highly positive for endothelial cell markers CD31, CD144, CD146 and KDR with no indication of CD45 (leukocyte antigens), CD133 (endothelial progenitor cell marker) and CD34 (hematopoietic progenitor cell marker). RBCs from healthy individuals were obtained after centrifugation of whole blood. Cellular adhesion was evaluated after incubation of ECFCs with RBCs in the presence or absence of inflammatory stimulus (TNF-α). Endothelial adhesion molecules were analyzed by flow cytometry (ICAM-1, VCAM-1, E and P-selectin). ECFC migration was assessed using a scratch-wound healing assay and wound regression was analyzed by time-lapse videos. Angiogenesis capacity was evaluated through three-dimensional ECFC cultures in Matrigel. Network parameters (segments, junctions and meshes) were characterized during 24h after seeding. All experiments were performed in triplicate.Results: In total, 48 ECFC colonies were established, 10 from SCA, 25 MPN and 13 CTR. We observed a higher percentage of adhered RBCs to ECFCs isolated from patients with SCA (14.0%) and MPN (23.4%) without TNF-ɑ stimulus, when compared to ECFC from CTR, (8.4%, p< 0.04 and p< 0.001, respectively), and HUVEC (9.4%, p< 0.05 and p< 0.02, respectively). No differences were detected in the expression of adhesion molecules in ECFC.Mean wound regression rates at 14h were 79.9% for SCA, 84.4% for MPN, and 88.8% for CTR. The high variability among colonies in each group could explain why this difference was not statistically significant. Finally, HUVECs had a shorter time for wound closure, with complete wound regression at 10h.The angiogenesis analysis at 15h ECFC from SCA and MPN had, respectively, 20% and 50% less network parameters than CTR. Then beyond 15h post-seeding, the network parameters regressed until reaching a plateau. At 24h the segments began to disappear progressively, leading to a marked reduction in 40h. Unlike ECFC, HUVEC presented a high network formation.Conclusions: Our findings reveal distinct functional properties and behavior between the ECFCs from two diseases with vascular manifestations, SCA and MPN. ECFC do provide access to patient vascular endothelium and enable us to validate the use of these cells as investigative models. In contrast, HUVECs showed a unique behavior, which differed from both diseased and controls ECFCs. This highlights the differences between autologous in vitro and established cell lines as experimental models for vascular diseases. This raises the question of which is the most representative model of human endothelial pathophysiology in vascular diseases. DisclosuresOzelo:BioMarin: Honoraria, Speakers Bureau; Grifols: Honoraria; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Research Funding, Speakers Bureau; Bioverativ: Honoraria, Research Funding.

Transgelin induces dysfunction of fetal endothelial colony-forming cells from gestational diabetic pregnancies.

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through the functional impairment of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). Previously, we showed that fetal ECFCs exposed to GDM have decreased vasculogenic potential and altered gene expression. In this study, we evaluate whether transgelin (TAGLN), which is increased in GDM-exposed ECFCs, contributes to vasculogenic dysfunction. TAGLN is an actin-binding protein involved in the regulation of cytoskeletal rearrangement. We hypothesized that increased TAGLN expression in GDM-exposed fetal ECFCs decreases network formation by impairing cytoskeletal rearrangement resulting in reduced cell migration. To determine if TAGLN is required and/or sufficient to impair ECFC network formation, TAGLN was reduced and overexpressed in ECFCs from GDM and uncomplicated pregnancies, respectively. Decreasing TAGLN expression in GDM-exposed ECFCs improved network formation and stability as well as increased migration. In contrast, overexpressing TAGLN in ECFCs from uncomplicated pregnancies decreased network formation, network stability, migration, and alignment to laminar flow. Overall, these data suggest that increased TAGLN likely contributes to the vasculogenic dysfunction observed in GDM-exposed ECFCs, as it impairs ECFC migration, cell alignment, and network formation. Identifying the molecular mechanisms underlying fetal ECFC dysfunction following GDM exposure is key to ascertain mechanistically the basis for cardiovascular disease predisposition later in life.

Distinct Effect of Invasive and Non‐invasive Breast Cancer Cells on Vasculogenic Capacity of Endothelial Progenitors

Blood vessel formation plays an essential role in tumor growth and metastasis. Cancer cells secrete various pro‐angiogenic factors that activate preexisting vascular endothelial cells as well as induce recruitment of bone‐marrow‐derived endothelial progenitors; endothelial colony forming cells (ECFC). The interactions between cancer cells and endothelial cells have been largely investigated, but it has been rarely studied whether the vasculogenic capacity of ECFC are differently affected by invasive and non‐invasive phenotype of the cancer cells. Thus, we investigated to determine the vasculogenic potentials of invasive and non‐invasive breast cancer cells. MDA‐MB‐231 and MCF7 were applied as invasive and non‐invasive breast cancer cells. Bone marrow‐derived ECFC were isolated from human peripheral blood. Tubular structure number and length as well as spheroid sprout number and length formed by ECFC was increased when treated with MDA‐MB‐231‐derived conditioned medium (CM) compared with MCF7‐derived CM. Migration of ECFC was also increased by treatment of MDA‐MB‐231‐derived CM compared to MCF7. To mimic tumor microenvironment, ECFC were directly co‐cultured with cancer cells. Tube formation of ECFC was enhanced under the co‐culture condition with MDA‐MB‐231 compared to ECFC alone control. Interestingly, ECFC co‐cultured with MCF7 significantly decreased tube formation. Our results suggest that MDA‐MB‐231 and MCF7 promote ECFC vasculogenesis in different manners, which may be the important determinants to characterize invasive and non‐invasive phenotype of the cancer cells.Support or Funding InformationThis research was supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016r1a6a1a03007648), and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017R1A2B4005463).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a novel angiogenic peptide from periostin.

Angiogenic peptides have therapeutic potential for the treatment of chronic ischemic diseases. Periostin, an extracellular matrix protein expressed in injured tissues, promotes angiogenesis and tissue repair. We previously reported that in vivo administration of both recombinant full-length protein and the first FAS I domain of periostin alleviated peripheral artery occlusive disease by stimulating the migration of humane endothelial colony forming cells (ECFCs) and subsequent angiogenesis. In the present study, we ascertained the peptide sequence responsible for the periostin-induced angiogenesis. By serial deletion mapping of the first FAS I domain, we identified a peptide sequence (amino acids 142–151) of periostin for stimulation of chemotactic migration, adhesion, proliferation and endothelial tube formation of human ECFCs in vitro. Chemotactic migration of ECFCs induced by the periostin peptide was blocked by pre-incubation with an anti-β5 integrin neutralizing antibody. Treatment of ECFCs with the periostin peptide led to phosphorylation of both AKT and ERK, and pretreatment of ECFCs with the MEK-ERK pathway inhibitor U0126 or the PI3K-AKT pathway inhibitors, LY294002 or Wortmannin, blocked the periostin peptide-stimulated migration of ECFCs. These results suggest that the synthetic periostin peptide can be applied for stimulating angiogenic and therapeutic potentials of ECFCs.

Abstract 168: YAP and TAZ Mediate Mechanical Control of Vasculogenesis

Physical cues such as the mechanical properties of the extracellular matrix are known to modulate blood vessel formation, but the underlying mechanisms remain poorly understood. Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) were recently identified as key mediators of cellular mechanotransduction [1], but the roles of YAP and TAZ in neovascular mechanobiology have not been investigated. We hypothesized that YAP and TAZ mediate neovascular mechanotransduction of extracellular matrix rigidity by vasculogenic endothelial colony forming cells (ECFCs) cultured on/in matrices of controllable rigidity. YAP and TAZ depletion abrogated ECFC migration (Fig. 1A), and YAP/TAZ expression and nuclear localization (i.e., activation) was significantly upregulated in migrating cells (Fig. 1B). Combinatorial depletion also abrogated tubular network formation and 3D vasculogenesis in collagen matrices of variable rigidity but constant matrix density (data not shown). In 2D, PDMS rigidity regulated YAP/TAZ subcellular localization (data not shown). In 3D, vasculogenesis was enhanced in stiff (G’= 1049 Pa) collagen oligomer matrices compared to soft (G’ = 305 Pa) monomer matrices formulated at constant collagen matrix density. YAP and TAZ were nuclear localized (activated) in the stiff oligomer matrices, but cytosolic in soft monomer matrices (Fig. 1C). Mechanistically, YAP and TAZ depletion reduced cytoskeletal stress fiber formation (data not shown), implicating YAP and TAZ in a feedback mechanism to control cytoskeletal remodeling. Delivered in oligomer matrices in vivo , ECFCs formed a functional human neovascular plexus and exhibited strong expression and nuclear localization of YAP and TAZ and expression of downstream genes, Cyr61 and CTGF (data not shown). In summary, YAP and TAZ are essential for mechanical control of ECFC migration and vasculogenesis. [1] Dupont et al. 2011 Nature. 474:179-185.

30 NADPH oxidases influence the angiogenic function of endothelial colony-forming cells

Introduction Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Interestingly, recent evidence supports a key role for NADPH oxidases in underlying angiogenic processes of these and other endothelial cells. Aims To study the effects of Nox NADPH oxidases on pro-angiogenic function of ECFCs. Methods Human ECFCs isolated from umbilical cord blood were treated with pro-oxidant PMA and assessed in vitro , both basally and after siRNA knockdown of Nox4, a key endothelial isoform, alongside primary mature human aortic endothelial cells (HAoECs). Results PMA (500 nM for 8 h) increased cell migration (control 18.6 ± 2.8, PMA 32.7 ± 6.6% closure; n = 6, P Conclusion ECFC migration is enhanced by low concentrations of superoxide, and to a greater extent as compared to mature endothelial cells, and appears to be at least partly dependent upon Nox4 NADPH oxidase. These findings may have significant implications for potential ECFC-based therapies for ischaemic disease, which is associated with an oxidative microenvironment.

Vitamin D prevents endothelial progenitor cell dysfunction induced by sera from women with preeclampsia or conditioned media from hypoxic placenta.

ContextPlacenta-derived circulating factors contribute to the maternal endothelial dysfunction underlying preeclampsia. Endothelial colony forming cells (ECFC), a sub-population of endothelial progenitor cells (EPCs), are thought to be involved in vasculogenesis and endothelial repair. Low vitamin D concentrations are associated with an increased risk for preeclampsia.ObjectiveWe hypothesized that the function of human fetal ECFCs in culture would be suppressed by exposure to preeclampsia-related factors–preeclampsia serum or hypoxic placental conditioned medium– in a fashion reversed by vitamin D.Design, Setting, PatientsECFCs were isolated from cord blood of uncomplicated pregnancies and expanded in culture. Uncomplicated pregnancy villous placenta in explant culture were exposed to either 2% (hypoxic), 8% (normoxic) or 21% (hyperoxic) O2 for 48 h, after which the conditioned media (CM) was collected.Outcome MeasuresECFC tubule formation (Matrigel assay) and migration were examined in the presence of either maternal serum from preeclampsia cases or uncomplicated pregnancy controls, or pooled CM, in the presence or absence of 1,25(OH)2 vitamin D3.Results1,25(OH)2 vitamin D3 reversed the adverse effects of preeclampsia serum or CM from hypoxic placenta on ECFCs capillary-tube formation and migration. Silencing of VDR expression by VDR siRNA, VDR blockade, or VEGF pathway blockade reduced ECFC functional abilities. Effects of VDR or VEGF blockade were partially prevented by vitamin D.ConclusionVitamin D promotes the capillary-like tubule formation and migration of ECFCs in culture, minimizing the negative effects of exposure to preeclampsia-related factors. Further evaluation of the role of vitamin D in ECFC regulation and preeclampsia is warranted.

Therapeutic angiogenesis in a murine model of limb ischemia by recombinant periostin and its fasciclin I domain

Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against β3 and β5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.

Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors

Objective: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro- or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates.Method: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups.Results: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation.Conclusions: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates.

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

ObjectiveEndothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.Methods and ResultsLipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.ConclusionE-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.

Mechanistic study of the proangiogenic effect of osteoprotegerin

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVβ3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway.

A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20μg/mL of TSP-HepI peptide for 18h enhanced cell migration (p<0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.