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Abstract Background Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. Methods Twin-bearing ewes had surgery performed at 88–90 days’ gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. Results Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular–astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. Conclusions ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR.

Background: Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. Methods: Twin-bearing ewes had surgery performed at 88–90 days' gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. Results: Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular–astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. Conclusions: ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR. [ABSTRACT FROM AUTHOR]

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

The “senescence-associated secretory phenotype” (SASP) supports a pro-tumoral and pro-angiogenic microenvironment through increased secretion of inflammatory and growth factors. We investigated oleuropein aglycone (OLE, 10 µM, 5 weeks) effect on capillary morphogenesis induced by MRC5 fibroblast SASP in mature (hMVEC) and progenitor (ECFC) endothelial cells. In senescent fibroblasts OLE reduced NFkB signaling and the expression of several SASP factors. The levels of IL-8 and VEGF were decreased in senescent fibroblast-conditioned medium collected after OLE treatment (CM senOLE) compared to that from untreated fibroblasts (CM sen). Pre-incubation with CMsen enhanced invasive activity and capillary morphogenesis in endothelial cells. This angiogenic phenotype was significantly attenuated upon pre-incubation with CM senOLE, along with reduced MMP-2, MMP-9 and uPA secretion by endothelial cells. In conclusion, OLE can modulate angiogenesis indirectly acting on local senescent fibroblasts, a new mechanism possibly contributing to the beneficial effects of the Mediterranean diet against cancer and cardiovascular diseases.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

Background: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate., Objectives: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs)., Methods and Results: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001)., Conclusion: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Assessment of endothelial colony-forming cell (ECFC) number and vasculogenic properties is crucial for exploring vascular diseases and regeneration strategies. A previous survey of the Scientific and Standardization Committee on Vascular Biology of the International Society on Thrombosis and Haemostasis clarified key methodological points but highlighted a lack of standardization associated with ECFC culture., Objectives: The aim of this study was to provide expert consensus guidance on ECFC isolation and culture., Methods: We surveyed 21 experts from 10 different countries using a questionnaire proposed during the 2019 International Society on Thrombosis and Haemostasis Congress in Melbourne (Australia) to attain a consensus on ECFC isolation and culture., Results: We report here the consolidated results of the questionnaire. There was agreement on several general statements, mainly the technical aspects of ECFC isolation and cell culture. In contrast, on the points concerning the definition of a colony of ECFCs, the quantification of ECFCs, and the estimation of their age (in days or number of passages), the expert opinions were widely dispersed., Conclusion: Our survey clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of ECFC isolation and culture procedures for clinical laboratory practice and robustness of results. To this end, we propose a standardized protocol for the isolation and expansion of ECFCs from umbilical cord and adult peripheral blood., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The main pathogenic feature of preeclampsia is maternal endothelial dysfunction that results from impaired angiogenesis and reduced endothelial repair capacity. In addition, preeclampsia risk is associated with vitamin D deficiency. We hypothesized that vitamin D3 stimulates proangiogenic properties of endothelial colony-forming cells (ECFCs). ECFCs were obtained and cultured from cord blood and characterized by immunocytochemistry and flow cytometry. Proliferation, total length of tubule formation on Matrigel, expression of VEGF mRNA, and pro-matrix metalloproteinases (MMP)-2 activity were assessed after treatment of ECFCs with vitamin D3. Specificity of the observed effects was tested by blocking the vitamin D receptor (VDR) or the VEGF signaling pathway. ECFCs treated with 10 nM vitamin D3 showed a 1.27 times higher tubule formation compared with vehicle-treated controls (1.27 ± 0.19) as well as a 1.36 times higher proliferation rate (1.36 ± 0.06). Vitamin D3 induced pro-MMP-2 activity (1.29 ± 0.17) and VEGF mRNA levels (1.74 ± 0.73) in ECFCs. VDR blocking by pyridoxal- 5-phosphate (0.73 ± 0.19) or small interfering RNA (0.75 ± 0.17) and VEGF inhibition by Su5416 (0.56 ± 0.16) or soluble fms-like tyrosine kinase-1 (0.7 ± 0.14) reduced tubule formation and pro- MMP-2 activity (pyridoxal-5-phosphate: 0.84 ± 0.09; Su5416: 0.79 ± 0.11; or sFlt: 0.88 ± 0.13). This effect was neutralized by vitamin D3. Consequently, vitamin D3 significantly promoted angiogenesis in ECFCs in vitro possibly due to an increase in VEGF expression and pro-MMP-2 activity. Since angiogenesis is a crucial feature in the pathophysiology of preeclampsia these findings could explain the positive influence of vitamin D3 in reducing preeclampsia risk. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPC) play a central role in endothelial maintenance and repair. Endothelial colony-forming cells (ECFC) form a subpopulation of EPC. ECFC are readily attainable, can be easily isolated, possess a high proliferation potential, and are therefore a promising source of endothelial cells (EC) for future cardiovascular therapeutic applications. The extent to which these cells respond to shear stress as adult vascular EC remains to be elucidated. Here, we study the transcriptional response of ECFC induced by shear stress and compare it with the response of mature arterial and venous cells. ECFC, as well as human umbilical vein EC (HUVEC) and human umbilical artery EC (HUAEC), were subjected to low (0.5 Pa) and high (2.5 Pa) shear stress. The endothelial differentiation phenotype and transcriptional responses were analyzed using immunocytochemistry and quantitative polymerase chain reaction (Q-PCR). Performing absolute quantification of copy numbers by Q-PCR allows comparing the responses of cell types relative to each other. Our data show that isolated ECFC resemble mature EC in cobblestone morphology and endothelial marker expression. Absolute Q-PCR quantification revealed that although being truly endothelial, ECFC do not fully resemble HUVEC or HUAEC in the expression of specific differentiation markers. When subjected to shear stress, ECFC show a mature response to fluid flow, comparable to that of HUVEC and HUAEC. The capacity of endothelial progenitors to respond to fluid flow in a similar manner to HUVEC and HUAEC highlights the universal response of EC to fluid shear stress, independently of their endothelial differentiation status. This property supports the use of these cells as an EC source for tissue engineering applications. [ABSTRACT FROM AUTHOR]

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway. [ABSTRACT FROM AUTHOR]

Bicuspid aortic valve (BAV), the most common congenital heart defect, is associated with an increased prevalence of aortic dilation, aortic rupture and aortic valve calcification. Endothelial cells (ECs) play a major role in vessel wall integrity. Little is known regarding EC function in BAV patients due to lack of patient derived primary ECs. Endothelial colony forming cells (ECFCs) have been reported to be a valid surrogate model for several cardiovascular pathologies, thereby facilitating an in vitro system to assess patient-specific endothelial dysfunction. Therefore, the aim of this study was to investigate cellular functions in ECFCs isolated from BAV patients. Outgrowth and proliferation of ECFCs from patients with BAV (n = 34) and controls with a tricuspid aortic valve (TAV, n = 10) were determined and related to patient characteristics. Interestingly, we were only able to generate ECFCs from TAV and BAV patients without aortic dilation, and failed to isolate ECFC colonies from patients with a dilated aorta. Analyzing EC function showed that while proliferation, cell size and endothelial-to-mesenchymal transition were similar in TAV and BAV ECFCs, migration and the wound healing capacity of BAV ECFCs is significantly higher compared to TAV ECFCs. Furthermore, calcification is blunted in BAV compared to TAV ECFCs. Our results reveal ECs dysfunction in BAV patients and future research is required to unravel the underlying mechanisms and to further validate ECFCs as a patient-specific in vitro model for BAV. [ABSTRACT FROM AUTHOR]

Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases, as less than 10% of patients with an ischemic stroke are eligible for thrombolysis. We previously reported that erythropoietin priming of ECFCs increased their in vitro and in vivo angiogenic properties in mice with hindlimb ischemia. The present study used SPECT/CT to evaluate whether priming of ECFCs with erythropoietin could enhance their homing to the ischemic site after transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats and potentiate their protective or regenerative effect on blood-brain barrier (BBB) disruption, cerebral apoptosis, and cerebral blood flow (CBF)., Methods: Rats underwent a 1-h MCAO followed by reperfusion and then 1 d after MCAO received an intravenous injection of either PBS (control, n = 10), PBS-primed ECFCs (ECFC PBS , n = 13), or erythropoietin-primed ECFCs (ECFC EPO , n = 10). ECFC homing and the effect on BBB disruption, cerebral apoptosis, and CBF were evaluated by SPECT/CT up to 14 d after MCAO. The results were expressed as median ± interquartile range for ipsilateral-to-contralateral ratio of the activity in middle cerebral artery-vascularized territories in each hemisphere. Histologic evaluation of neuronal survival and astrocytic proliferation was performed on day 14., Results: Erythropoietin priming increased homing of ECFCs to the ischemic hemisphere (ECFC PBS , 111.0% ± 16.0%; ECFC EPO , 146.5% ± 13.3%). BBB disruption was significantly reduced (control, 387% ± 153%; ECFC PBS , 151% ± 46% [P < 0.05]; ECFC EPO , 112% ± 9% [P < 0.001]) and correlated negatively with ECFC homing (Pearson r = -0.6930, P = 0.0002). Cerebral apoptosis was significantly reduced (control, 161% ± 10%; ECFC PBS , 141% ± 9% [P < 0.05]; ECFC EPO ,118% ± 5% [P < 0.001]) and correlated negatively with ECFC homing (r = -0.7251, P < 0.0001). CBF was significantly restored with ECFCs and almost totally so with erythropoietin priming (control, 72% ± 2%; ECFC PBS , 90% ± 4% [P < 0.01]; ECFC EPO , 99% ± 4% [P < 0.001]) and correlated positively with ECFC homing (r = 0.7348, P < 0.0001). Immunoblocking against the CD146 receptor on ECFCs highlighted its notable role in ECFC homing with erythropoietin priming (ECFC EPO , 147% ± 14%, n = 4; ECFC EPO with antibody against CD146, 101% ± 12%, n = 4 [P < 0.05])., Conclusion: Priming with erythropoietin before cell transplantation is an efficient strategy to amplify the migratory and engraftment capacities of ECFCs and their beneficial impact on BBB disruption, apoptosis, and CBF., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)