Search Results (44)

Endothelial integrity is essential for cardiovascular health, and circulating endothelial progenitor cells, particularly endothelial colony-forming cells (ECFCs), are key contributors to vascular repair and maintenance. Long non-coding RNAs (lncRNAs) have emerged as novel epigenetic regulators of endothelial physiology and pathology. Building on our previous work identifying the lncRNA KLRK1-AS1 as a positive modulator of ECFC wound healing, we aimed to elucidate its role in endothelial biology. Cord blood-derived ECFCs were subjected to siRNA-mediated silencing of KLRK1-AS1, followed by blinded evaluations of monolayer morphology, barrier stability using ECIS impedance measurements, assessments of proliferation, and spheroid-based angiogenic activity. SiRNA-mediated silencing of KLRK1-AS1 induced detectable alterations in ECFC monolayer morphology (p = 0.047), while proliferation remained unaffected. Notably, KLRK1-AS1 knockdown significantly compromised endothelial barrier integrity, resulting in a 44% reduction in impedance after 48 h (p < 0.001), suggesting weakened intercellular contacts. In contrast, loss of KLRK1-AS1 enhanced angiogenic behaviour, demonstrated by an increased number of sprouts (+62%, p = 0.031). Together, these findings indicate that KLRK1-AS1 supports a quiescent, stable endothelial phenotype, with intact barrier function, while its depletion shifts ECFCs toward a more angiogenic, activated state. Our results identify KLRK1-AS1 as a previously unrecognised regulator of endothelial function. Full article

Individuals born after intrauterine growth restriction (IUGR) are at increased risk of long-term cardiovascular complications, including elevated blood pressure, endothelial dysfunction, and arterial stiffness. Endothelial progenitor cells (EPCs), particularly endothelial colony-forming cells (ECFCs), play a critical role in maintaining vascular homeostasis. Previously, Simoncini et al. observed that in a rat model of IUGR, six-month-old males exhibited elevated systolic blood pressure (SBP) and microvascular rarefaction compared with control (CTRL) rats. These vascular alterations were accompanied by reduced numbers and impaired function of bone marrow-derived ECFCs, which were associated with oxidative stress and stress-induced premature senescence (SIPS). In contrast, IUGR females of the same age and from the same litter did not exhibit higher SBP or microvascular rarefaction, raising the question of whether ECFC dysfunction in IUGR female rats can be present without vascular alterations. So, we investigated ECFCs isolated from six-month-old female IUGR offspring (maternal 9% casein diet) and CTRL females (23% casein diet). To complete the vascular assessment, we performed in vivo and in vitro investigations. No alteration in pulse wave velocity (measured by echo-Doppler) was observed; however, IUGR females showed decreased aortic collagen and increased elastin content compared with CTRL. Regarding ECFCs, those from IUGR females maintained their endothelial identity (CD31⁺/CD146⁺ ratio among viable CD45⁻ cells) but exhibited slight alterations in progenitor marker expression (CD34) compared with those of CTRL females. Functionally, IUGR-ECFCs displayed a delayed proliferation phase between 6 and 24 h, while their ability to form capillary-like structures remained unchanged, however their capacity to form capillary-like structures was preserved. Regarding the nitric oxide (NO) pathway, a biologically relevant trend toward reduced NO levels and decreased endothelial nitric oxide synthase expression was observed, whereas oxidative stress and SIPS markers remained unchanged. Overall, these findings indicate that ECFCs from six-month-old female IUGR rats exhibit only minor functional alterations, which may contribute to vascular protection against increase SBP, microvascular rarefaction, and arterial stiffness. Full article

Angiogenesis is a fundamental biological process involved in the formation of new blood vessels from the pre-existing vascular network. In addition to physiological processes, angiogenesis is also implicated in pathological conditions such as tumour growth and metastatic progression. Research on marennine, a water-soluble blue-green pigment produced by the marine diatom Haslea ostrearia, has highlighted various promising biological activities. In vivo studies have suggested the potential of marennine in cancer treatment. However, these studies were conducted with crude extracts, the exact composition of which remained poorly defined. In this context, our study aimed to explore the effects of marennine on angiogenesis and tumour proliferation by using a Precipitated Extracellular Marennine (PEMn) extract. Our results confirmed the antiproliferative properties of PEMn on several cancer cell lines associated with angiogenic tumours. We then analysed its impact on the key steps of the angiogenic process, including Endothelial Colony-Forming Cells (ECFCs) proliferation, migration, and tubulogenesis. In parallel, we investigated the underlying mechanisms of its action, notably by assessing its effects on cell cycle regulation, senescence, and apoptosis. PEMn significantly inhibited tumour cell proliferation, induced ECFC senescence and apoptosis, impaired migration and tubulogenesis, and downregulated VEGFR-1 expression, highlighting its potential as a novel marine-derived antiangiogenic compound. These findings provide deeper insights into the mechanisms of action of marennine, identifying this bioactive natural compound as a novel bioactive compound in cancer treatment. Full article

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is essential for physiological processes such as development and wound healing, but its dysregulation contributes to a range of pathological conditions including cancer, diabetic retinopathy, and chronic inflammation. In recent years, marine-derived compounds have emerged as promising multitarget agents with anti-angiogenic potential. Posidonia oceanica, a Mediterranean seagrass traditionally used in folk medicine, is increasingly recognized for its pharmacological properties, including antioxidant, anti-inflammatory, and anti-invasive activities. This study investigated the effects of a hydroethanolic extract from P. oceanica leaves (POE) on human Endothelial Colony-Forming Cells (ECFCs), a subpopulation of endothelial progenitor cells with high proliferative and vessel-forming capacity, and a relevant model for studying pathological angiogenesis. ECFCs were treated with POE (4–8 µg/mL), and cell viability, morphology, migration, invasion, tube formation, oxidative stress, and activation markers were evaluated. POE did not alter ECFC morphology or viability, as confirmed by Trypan Blue and MTT assays. However, functional assays revealed that POE significantly impaired ECFC migration, invasion, and in vitro angiogenesis in a dose-dependent manner. Under VEGF (Vascular endothelial growth factor) stimulation, POE reduced intracellular ROS accumulation and downregulated key redox-regulating genes (hTRX1, hTRX2, PRDX2, AKR1C1, AKR1B10). Western blot analysis showed that POE inhibited VEGF-induced phosphorylation of KDR, mTOR and p-ERK, while p-AKT remained elevated, indicating selective disruption of VEGF downstream signaling. Furthermore, POE reduced the expression of pro-inflammatory and pro-coagulant markers (VCAM-1, ICAM-1, TF) and partially reversed TNF-α–induced endothelial activation. These findings suggest that POE exerts anti-angiogenic effects through a multitargeted mechanism, supporting its potential as a natural therapeutic agent for diseases characterized by aberrant angiogenesis. Full article

Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair, we evaluated two vascular reparative cell populations, CD34⁺ cells derived from hiPSC (hiPSC-CD34⁺) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs), alone and in combination, in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34⁺ cells (1 × 10⁴) or iPSC- ECFCs (1 × 10⁵) alone or in combination (1.1 × 10⁵) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection, mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34⁺ cells or hiPSC-ECFCs. Retinal thickness, assessed by OCT, was restored to near-nondiabetic levels in mice treated with hiPSC-CD34⁺ cells alone and the combination group, whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection, hiPSC-CD34⁺ cells were localized to perivascular regions, whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes, and this was interpreted as a combination-specific, non-additive host responses (m⁶A, PI3K–AKT–mTOR, glycolysis, endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34⁺ cells and hiPSC-ECFCs, both individually and in combination, showed benefit; however, iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA. Full article

Ticagrelor, a reversible platelet P2Y₁₂ receptor antagonist, exerts various pleiotropic actions, some of which are at least partially mediated through adenosine. We studied the ticagrelor and adenosine effect on the angiogenic properties of progenitor CD34⁺-derived endothelial colony-forming cells (ECFCs). Angiogenesis studies were performed in vitro using capillary-like tube formation and spheroid-based angiogenesis assays. The effects of adenosine receptor antagonists, including DPCPX (A₁ antagonist), SCH58621 (A_2A antagonist), MRS1706 (A_2B inverse agonist and antagonist), MRS1220 (A₃ antagonist) and adenosine deaminase (ADA), were also investigated. Ticagrelor, adenosine, and their combination increased capillary-like tube formation and spheroid sprout formation by ECFCs in a dose-dependent manner. This effect was significantly reduced by SCH58621, MRS1706, and their combination, as well as by ADA. By contrast, DPCPX and MRS1220 did not exhibit any inhibitory effects. Similar results were obtained when mature human umbilical vein endothelial cells (HUVECs) were studied. These results show that ticagrelor stimulates angiogenesis by progenitor and mature endothelial cells in an adenosine-dependent pathway in which the adenosine receptors A_2A and A_2B play major roles. The significance of these results at the clinical level in patients with atherothrombotic events and treated with ticagrelor needs to be investigated. Full article

Antioxidants are essential for mitigating oxidative stress and maintaining vascular health. Endothelial colony-forming cells (ECFCs) are pivotal in endothelial regeneration and angiogenesis and serve as a model to study the diversity of endothelial cells across various organs. This study evaluated the effects of peel, pulp, and seed extracts from Diospyros digyna Jacq. fruit (black persimmon) on human cord blood-derived ECFCs (CB-ECFCs) to determine how the distinct antioxidant profiles of the fruit’s different parts influence cellular functions. The extracts did not affect endothelial marker expression, cell proliferation, or nitric oxide production, indicating no cytotoxic or inflammatory effects. However, functional assays revealed that the seed extract significantly enhanced tube formation, increasing closed tubular networks by 1.5-fold. All extracts promoted cell migration, with the seed extract demonstrating the most substantial effect, surpassing even vascular endothelial growth factor (VEGF). Additionally, the seed extract exhibited the strongest reduction in cellular senescence, both before and after oxidative stress induction with H₂O₂. These findings underscore the potential of black persimmon extracts, especially from the seed, to enhance the regenerative capabilities of CB-ECFCs and reduce cellular senescence without affecting the normal endothelial phenotype. This positions them as promising candidates for developing endothelial cell therapies and advancing vascular regeneration. Full article

Background and Aims: Myeloperoxidase (MPO) plays a critical role in the innate immune response and has been suggested to be a surrogate marker of oxidative stress and inflammation, with elevated levels implicated in cardiovascular diseases, such as atherosclerosis and heart failure, as well as in conditions like rheumatoid arthritis and cancer. While MPO is well-known in leukocytes, its expression and function in human endothelial cells remain unclear. This study investigates MPO expression in patient-derived endothelial colony-forming cells (ECFCs) and its potential association with CAD and mitochondrial function. Methods: ECFCs were cultured from the peripheral blood of 93 BioHEART-CT patients. MPO expression and associated functions were examined using qRT-PCR, immunochemistry, flow cytometry, and MPO activity assays. CAD presence was defined using CT coronary angiography (CACS > 0). Results: We report MPO presence in patient-derived ECFCs for the first time. MPO protein expression occurred in 70.7% of samples (n = 41) which had nuclear co-localisation, an atypical observation given its conventional localisation in the granules of neutrophils and monocytes. This suggests potential alternative roles for MPO in nuclear processes. MPO mRNA expression was detected in 66.23% of samples (n = 77). CAD patients had a lower proportion of MPO-positive ECFCs compared to non-CAD controls (57.45% vs. 80%, p = 0.04), a difference that persisted in the statin-naïve sub-cohort (53.85% vs. 84.62%, p = 0.02). Non-CAD patients with MPO expression showed upregulated mitochondrial-antioxidant genes (AIFM2, TXNRD1, CAT, PRDX3, PRDX6). In contrast, CAD patients with MPO gene expression had heightened mROS production and mitochondrial mass and decreased mitochondrial function compared to that of CAD patients without MPO gene expression. Conclusions: MPO is present in the nucleus of ECFCs. In non-CAD ECFCs, MPO expression is linked to upregulated mitochondrial-antioxidant genes, whereas in CAD ECFCs, it is associated with greater mitochondrial dysfunction. Full article

Background: Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). Methods: qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. Results: HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. Conclusions: HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects. Full article

Improved human-relevant preclinical models of coronary artery disease (CAD) are needed to improve translational research and drug discovery. Mitochondrial dysfunction and associated oxidative stress contribute to endothelial dysfunction and are a significant factor in the development and progression of CAD. Endothelial colony-forming cells (ECFCs) can be derived from peripheral blood mononuclear cells (PBMCs) and offer a unique potentially personalised means for investigating new potential therapies targeting important components of vascular function. We describe the application of the high-throughput and confocal Opera Phenix^® High-Content Screening System to examine mitochondrial superoxide (mROS) levels, mitochondrial membrane potential, and mitochondrial area in both established cell lines and patient-derived ECFCs simultaneously. Unlike traditional plate readers, the Opera Phenix^® is an imaging system that integrates automated confocal microscopy, precise fluorescent detection, and multi-parameter algorithms to visualize and precisely quantify targeted biological processes at a cellular level. In this study, we measured mROS production in human umbilical vein endothelial cells (HUVECs) and patient-derived ECFCs using the mROS production probe, MitoSOX^TM Red. HUVECs exposed to oxidized low-density lipoprotein (oxLDL) increased mROS levels by 47.7% (p < 0.0001). A pooled group of patient-derived ECFCs from participants with CAD (n = 14) exhibited 30.9% higher mROS levels compared to patients with no CAD when stimulated with oxLDL (n = 14; p < 0.05). When tested against a small group of candidate compounds, this signal was attenuated by PKT-100 (36.22% reduction, p = 0.03), a novel P2X7 receptor antagonist. This suggests the P2X7 receptor as a valid target against excess mROS levels. As such, these findings highlight the potential of the MitoSOX-Opera Phenix technique to be used for drug discovery efforts in CAD. Full article

Transcatheter pulmonary valve replacement is a minimally-invasive alternative treatment for right ventricular outflow tract dysfunction and has been rapidly evolving over the past years. Heart valve prostheses currently available still have major limitations. Therefore, one of the significant challenges for the future is the roll out of transcatheter tissue engineered pulmonary valve replacement to more patients. In the present study, biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds in the form of a 3D leaflet matrix were successfully seeded with human endothelial colony-forming cells (ECFCs), human induced pluripotent stem cell-derived MSCs (hMSCs), and porcine MSCs (pMSCs) for three weeks for the generation of 3D tissue-engineered tri-leaflet valved stent grafts. The cell adhesion, proliferation, and distribution of these 3D heart leaflets was analyzed using fluorescence microscopy and scanning electron microscopy (SEM). All cell lineages were able to increase the overgrown leaflet area within the three-week timeframe. While hMSCs showed a consistent growth rate over the course of three weeks, ECFSs showed almost no increase between days 7 and 14 until a growth spurt appeared between days 14 and 21. More than 90% of heart valve leaflets were covered with cells after the full three-week culturing cycle in nearly all leaflet areas, regardless of which cell type was used. This study shows that seeded biodegradable PCL nanofiber scaffolds incorporated in nitinol or biodegradable stents will offer a new therapeutic option in the future. Full article

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8–NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment. Full article

The study of endothelial dysfunction (ED) is crucial to identify the pathogenetic mechanism(s) and provide indications for patient management in cardiovascular diseases. It is currently hindered by the limited availability of patient-specific primary endothelial cells (ECs). Endothelial colony-forming cells (ECFCs) represent an optimal non-invasive tool to overcome this issue. Therefore, we investigated the use of ECFCs as a substrate in thrombogenesis and thrombin generation assay (TGA) to assess ED. Both assays were set up on human umbilical vein endothelial cells (HUVECs) and then tested on ECFCs obtained from healthy donors. To prove the ability of the assays to detect endothelial activation, ECs stimulated with TNFα were compared with unstimulated ECs. EC activation was confirmed by the upregulation of VCAM-1 and Tissue Factor expression. Both assays discriminated between unstimulated and activated HUVECs and ECFCs, as significantly higher platelet deposition and fibrin formation in thrombogenesis assay, and thrombin generation in TGA, were observed when TNFα-activated ECs were used as a substrate. The amount of fibrin and thrombin measured in the two assays were directly correlated. Our results support the combined use of a thrombogenesis assay and TGA performed on patient-derived ECFCs to provide a personalized global assessment of ED relevant to the patient’s hemostatic profile. Full article

Individuals born after intrauterine growth restriction (IUGR) are at risk of developing cardiovascular diseases (CVDs). Endothelial dysfunction plays a role in the pathogenesis of CVDs; and endothelial colony-forming cells (ECFCs) have been identified as key factors in endothelial repair. In a rat model of IUGR induced by a maternal low-protein diet, we observed an altered functionality of ECFCs in 6-month-old males, which was associated with arterial hypertension related to oxidative stress and stress-induced premature senescence (SIPS). Resveratrol (R), a polyphenol compound, was found to improve cardiovascular function. In this study, we investigated whether resveratrol could reverse ECFC dysfunctions in the IUGR group. ECFCs were isolated from IUGR and control (CTRL) males and were treated with R (1 μM) or dimethylsulfoxide (DMSO) for 48 h. In the IUGR-ECFCs, R increased proliferation (5′-bromo-2′-deoxyuridine (BrdU) incorporation, p < 0.001) and improved capillary-like outgrowth sprout formation (in Matrigel), nitric oxide (NO) production (fluorescent dye, p < 0.01), and endothelial nitric oxide synthase (eNOS) expression (immunofluorescence, p < 0.001). In addition, R decreased oxidative stress with reduced superoxide anion production (fluorescent dye, p < 0.001); increased Cu/Zn superoxide dismutase expression (Western blot, p < 0.05); and reversed SIPS with decreased beta-galactosidase activity (p < 0.001), and decreased p16^ink4a (p < 0.05) and increased Sirtuin-1 (p < 0.05) expressions (Western blot). No effects of R were observed in the CTRL-ECFCs. These results suggest that R reverses long-term ECFC dysfunctions related to IUGR. Full article

Cellular therapy has shown promise as a strategy for the functional restoration of ischemic tissues through promoting vasculogenesis. Therapy with endothelial progenitor cells (EPCs) has shown encouraging results in preclinical studies, but the limited engraftment, inefficient migration, and poor survival of patrolling endothelial progenitor cells at the injured site hinder its clinical utilization. These limitations can, to some extent, be overcome by co-culturing EPCs with mesenchymal stem cells (MSCs). Studies on the improvement in functional capacity of late EPCs, also referred to as endothelial colony-forming cells (ECFCs), when cultured with MSCs have mostly focused on the angiogenic potential, although migration, adhesion, and proliferation potential also determine effective physiological vasculogenesis. Alteration in angiogenic proteins with co-culturing has also not been studied. We co-cultured ECFCs with MSCs via both direct and indirect means, and studied the impact of the resultant contact-mediated and paracrine-mediated impact of MSCs over ECFCs, respectively, on the functional aspects and the angiogenic protein signature of ECFCs. Both directly and indirectly primed ECFCs significantly restored the adhesion and vasculogenic potential of impaired ECFCs, whereas indirectly primed ECFCs showed better proliferation and migratory potential than directly primed ECFCs. Additionally, indirectly primed ECFCs, in their angiogenesis proteomic signature, showed alleviated inflammation, along with the balanced expression of various growth factors and regulators of angiogenesis. Full article