Endothelial Colony-forming Cells Research Articles

Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium.

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Comparative transcriptomic analysis of circulating endothelial cells in sickle cell stroke.

Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.

Clusterin knockdown has effects on intracellular and secreted von Willebrand factor in human umbilical vein endothelial cells.

Alterations in von Willebrand factor (VWF) have an important role in human health and disease. Deficiency of VWF is associated with symptoms of bleeding and excesses of VWF are associated with thrombotic outcomes. Understanding the mechanisms that drive VWF regulation can lead to a better understanding of modulation of VWF levels in humans. We identified clusterin (CLU) as a potential candidate regulator of VWF based on a single cell RNA sequencing (scRNA-seq) analysis in control endothelial cells (ECs) and von Willebrand disease (VWD) endothelial colony-forming-cells (ECFCs). We found that patients with deficiencies of VWF (von Willebrand disease, VWD) had decreased CLU expression and ECs with low VWF expression also had low CLU expression. Based on these findings, we sought to evaluate the role of CLU in the regulation of VWF, specifically as it relates to VWD. As CLU is primarily thought to be a golgi protein involved in protein chaperoning, we hypothesized that knockdown of CLU would lead to decreases in VWF and alterations in Weibel-Palade bodies (WPBs). We used both siRNA- and CRISPR-Cas9-based approaches to modulate CLU in human umbilical vein endothelial cells (HUVECs) and evaluated VWF protein levels, VWF mRNA copy number, and WPB quantity and size. We demonstrated that siRNA-based knockdown of CLU resulted in decreases in VWF content in cellular lysates and supernatants, but no significant change in WPB quantity or size. A CRISPR-Cas9-based knockdown of CLU demonstrated similar findings of decreases in intracellular VWF content but no significant change in WPB quantity or size. Our data suggests that CLU knockdown is associated with decreases in cellular VWF content but does not affect VWF mRNA levels or WPB quantity or size.

Novel Glycomimetics Protect against Glycated Low-Density Lipoprotein-Induced Vascular Calcification In Vitro via Attenuation of the RAGE/ERK/CREB Pathway.

Heparan sulphate (HS) can act as a co-receptor on the cell surface and alterations in this process underpin many pathological conditions. We have previously described the usefulness of mimics of HS (glycomimetics) in protection against β-glycerophosphate-induced vascular calcification and in the restoration of the functional capacity of diabetic endothelial colony-forming cells in vitro. This study aims to investigate whether our novel glycomimetic compounds can attenuate glycated low-density lipoprotein (g-LDL)-induced calcification by inhibiting RAGE signalling within the context of critical limb ischemia (CLI). We used an established osteogenic in vitro vascular smooth muscle cell (VSMC) model. Osteoprotegerin (OPG), sclerostin and glycation levels were all significantly increased in CLI serum compared to healthy controls, while the vascular calcification marker osteocalcin (OCN) was down-regulated in CLI patients vs. controls. Incubation with both CLI serum and g-LDL (10 µg/mL) significantly increased VSMC calcification vs. controls after 21 days, with CLI serum-induced calcification apparent after only 10 days. Glycomimetics (C2 and C3) significantly inhibited g-LDL and CLI serum-induced mineralisation, as shown by a reduction in alizarin red (AR) staining and alkaline phosphatase (ALP) activity. Furthermore, secretion of the osteogenic marker OCN was significantly reduced in VSMCs incubated with CLI serum in the presence of glycomimetics. Phosphorylation of cyclic AMP response element-binding protein (CREB) was significantly increased in g-LDL-treated cells vs. untreated controls, which was attenuated with glycomimetics. Blocking CREB activation with a pharmacological inhibitor 666-15 replicated the protective effects of glycomimetics, evidenced by elevated AR staining. In silico molecular docking simulations revealed the binding affinity of the glycomimetics C2 and C3 with the V domain of RAGE. In conclusion, these findings demonstrate that novel glycomimetics, C2 and C3 have potent anti-calcification properties in vitro, inhibiting both g-LDL and CLI serum-induced VSMC mineralisation via the inhibition of LDLR, RAGE, CREB and subsequent expression of the downstream osteogenic markers, ALP and OCN.

Endothelial dysfunction and cardiovascular risk in post-COVID-19 patients after 6- and 12-months SARS-CoV-2 infection.

SARS-CoV-2 infection causes severe endothelial damage, an essential step for cardiovascular complications. Endothelial-colony forming cells (ECFCs) act as a biomarker of vascular damage but their role in SARS-CoV-2 remain unclear. The aim of this study was to assess whether the number of ECFCs and angiogenic biomarkers remained altered after 6 and 12-months post-infection and whether this imbalance correlated with the presence of long-COVID syndrome and other biological parameters measured. Seventy-two patients were recruited at different time-points after overcoming COVID-19 and thirty-one healthy controls. All subjects were matched for age, gender, BMI, and comorbidities. ECFCs were obtained from peripheral blood and cultured with specific conditions. The results confirm the presence of a long-term sequela in post-COVID-19 patients, with an abnormal increase in ECFC production compared to controls (82.8% vs. 48.4%, P < 0.01) that is maintained up to 6-months (87.0% vs. 48.4%, P < 0.01) and 12-months post-infection (85.0% vs. 48.4%, P < 0.01). Interestingly, post-COVID-19 patients showed a significant downregulation of angiogenesis-related proteins compared to controls indicating a clear endothelial injury. Troponin, NT-proBNP and ferritin levels, markers of cardiovascular risk and inflammation, remained elevated up to 12-months post-infection. Patients with lower numbers of ECFC exhibited higher levels of inflammatory markers, such as ferritin, suggesting that ECFCs may play a protective role. Additionally, long-COVID syndrome was associated with higher ferritin levels and with female gender. These findings highlight the presence of vascular sequela that last up to 6- and 12-months post-infection and point out the need for preventive measures and patient follow-up.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

Energizing the Lung Vasculature in the Premature Infant to Promote Alveolar Formation

Background/Objective: Chronic lung disease of prematurity, bronchopulmonary dysplasia (BPD) occurs when infants born with underdeveloped immature lungs are forced to navigate the expansion of future air spaces, with irregular vascular formation proceeding development of BPD. Lung development has distinct and dynamic metabolic requirements. We recently identified by mass spectrometry that nicotinamide adenine dinucleotide (NAD+), generated from vitamin B3 ‘Nicotinamide’ (NAM), was significantly reduced in a hyperoxia murine model of BPD. As NAM/NAD+ are dynamic regulators of tissue regenerating neovascularization in otherdisease processes, we hypothesized that NAM/NAD+ metabolic deficiencies contribute to compromised angiogenesis formation and alveolar formation.&#x0D; Methods: Impact of NAM supplementation on circulating human neonatal endothelial colonyforming cells (ECFCs) were assessed for differential capillary formation properties of angiogenesis (Matrigel), migration (wound healing), proliferation (WST1 and crystal violetstaining), and mitochondrial function in normoxia and hyperoxia (85% oxygen) conditions.&#x0D; Results: Hyperoxia suppresses ECFC angiogenesis, while NAM supplementation in hyperoxia significantly rescued vascular networks, branched nodes, and branch points (p&lt;0.001). Wound healing assays suggest that NAM promotes cell migration in normoxia and hyperoxia (p&lt;0.0001). Although NAM increased WST1 activity in hyperoxia, crystal violet analysis determined that NAM had no impact on ECFC proliferation. Lastly, NAM significantly reduced hyperoxia induced mitochondrial oxidative stress in a dose dependent manner (p&lt;0.05).&#x0D; Conclusion and Clinical Implications: Lung development has specific metabolic needs during different stages of development that are disrupted by premature birth. Replenishment of NAM promotes angiogenesis and migration in hyperoxia while reducing mitochondrial activity. Future studies are necessary to explore the role of NAM/NAD+ axis in the developing lung.

Colony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profile

Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.

Temporal Changes in the Surface Chemistry and Topography of Reactive Ion Plasma-Treated Poly(vinyl alcohol) Alter Endothelialization Potential.

Synthetic small-diameter vascular grafts (<6 mm) are used in the treatment of cardiovascular diseases, including coronary artery disease, but fail much more readily than similar grafts made from autologous vascular tissue. A promising approach to improve the patency rates of synthetic vascular grafts is to promote the adhesion of endothelial cells to the luminal surface of the graft. In this study, we characterized the surface chemical and topographic changes imparted on poly(vinyl alcohol) (PVA), an emerging hydrogel vascular graft material, after exposure to various reactive ion plasma (RIP) surface treatments, how these changes dissipate after storage in a sealed environment at standard temperature and pressure, and the effect of these changes on the adhesion of endothelial colony-forming cells (ECFCs). We showed that RIP treatments including O2, N2, or Ar at two radiofrequency powers, 50 and 100 W, improved ECFC adhesion compared to untreated PVA and to different degrees for each RIP treatment, but that the topographic and chemical changes responsible for the increased cell affinity dissipate in samples treated and allowed to age for 230 days. We characterized the effect of aging on RIP-treated PVA using an assay to quantify ECFCs on RIP-treated PVA 48 h after seeding, atomic force microscopy to probe surface topography, scanning electron microscopy to visualize surface modifications, and X-ray photoelectron spectroscopy to investigate surface chemistry. Our results show that after treatment at higher RF powers, the surface exhibits increased roughness and greater levels of charged nitrogen species across all precursor gases and that these surface modifications are beneficial for the attachment of ECFCs. This study is important for our understanding of the stability of surface modifications used to promote the adhesion of vascular cells such as ECFCs.

High-Throughput Measure of Mitochondrial Superoxide Levels as a Marker of Coronary Artery Disease to Accelerate Drug Translation in Patient-Derived Endothelial Cells Using Opera Phenix® Technology.

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, MitoSOXTM 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.

Producing and Testing Prototype Tissue-Engineered 3D Tri-Leaflet Valved Stents on Biodegradable Poly-ε-Caprolactone Scaffolds.

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.

Increased Activated Protein C Response Rates to Thrombin Formation in Asymptomatic Factor V Leiden Carriers Are Driven By the Endothelium

Background The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Recently we have established an ex vivo model for personalized assessment of the protein C (PC) pathway combining endothelial colony forming cells (ECFCs) and autologous plasma. Aim of this study was to investigate potential modulating factors of the APC response in the endothelium unraveling the enigma of the variable expressivity of the FVL phenotype. Methods ECFCs were isolated from FVL carriers with (FVL VTE+) or without (FVL VTE-) a history of unprovoked VTE and healthy controls (non-FVL) (n=7 each). Quality control of cultured cells included cobblestone morphology as examined by light microscopy, positive staining for CD31, CD309, CD201, and CD141, CD34, and negative staining for CD45 analyzed by flow cytometry. We either added autologous or pooled normal defibrinated plasma to the confluent cell culture and inititated thrombin formation by addition of tissue factor (1 pmol/L) and CaCl 2 (16.6 mmol/L) ( Fig. 1A). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs) and the ratio between the area under the curve (AUC) of APC formation and the AUC of thrombin formation was calculated as measure for APC response to thrombin. Moreover, we quantified thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression in cell-based enzyme-linked immunosorbent assays (cell-ELISAs) and measured activation of purified PC on ECFCs in the APC-OECA. Results ECFC morphology in light microscopy and characteristic surface marker expression in flow cytometry did not differ between non-FVL, FVL VTE-, and FVL VTE+ subgroups. In the ECFC-based ex vivo model, the APC response in plasma on autologous cells was significantly higher in asymptomatic FVL VTE- carriers compared to non-FVL carriers (P = .0072) and compared to the FVL VTE+ subgroup (P = .0292) whereas it did not differ significantly between non-FVL and FVL VTE+ carriers. EPCR expression on ECFCs measured by cell-ELISA was higher in non-FVL carriers compared to FVL VTE+ subjects (P = .0374) but did not differ significantly when comparing non-FVL versus FVL VTE-, and FVL VTE+ versus FVL VTE-. Similarly, we showed that TM expression as well as activation of purified PC on ECFCs did not differ between cohorts. However, when the APC response was assessed on FVL VTE- and FVL VTE+ ECFCs using non-FVL plasma, the difference in the APC response between asymptomatic FVL carriers and FVL carriers with a history of VTE persisted (P = .0111) ( Fig. 1B). Conclusion Consistent with previous in vivo experiments, APC response rates to thrombin formation were increased on ECFCs obtained from asymptomatic FVL carriers compared to those from FVL carriers with a history of VTE in autologous plasma. Although TM and EPCR expression did not differ between both groups, we showed that the endothelial APC response in asymptomatic FVL carriers remained increased when using plasma from healthy controls. Our observations suggest that the increased APC response to thrombin in asymptomatic FVL carriers is driven by the endothelium. Further studies are warranted to elucidate the underlying mechanism for a better understanding of endogenous protective factors in FVL carriers. These findings may pave the way towards personalized, precision medicine approaches for managing the thrombotic risk in FVL and other disorders related to the PC pathway.

Abstract 18638: Myocardial Infarction Induces Dysfunction of Endothelial Colony Forming Cells With Independence of Common Cardiovascular Comorbidities

Introduction: Endothelial Colony Forming Cells (ECFCs) present a dysfunctional and senescent phenotype in ischemic heart disease. However, the effect of myocardial infarction (MI), independently of common cardiovascular comorbidities (CCVC), on ECFCs functionality has not been evaluated up to date. Hypothesis: MI induces dysfunction of ECFCs with independence of CCVC Methods: To determine the potential effect of MI on ECFCs, human ECFCs (hECFCs) were isolated from peripheral blood from healthy volunteers (n=6), patients immediately after MI (n=6) (AMI-hECFCs) and patients 6 months after MI (n=6) (CMI-hECFCs). To determine the effect of MI on ECFCs impairment, isolated from the effect of common cardiovascular comorbidities that are found in patients, 6 healthy pigs were subjected to 90 minutes of myocardial ischemia by coronary balloon occlusion followed by reperfusion. Porcine ECFCs (pECFCs) (n=6) were isolated from blood samples before the MI and 1 month after the procedure (CMI-pECFCs) (n=6). For all groups, the following parameters were determined: days until colonies appearance, morphometry, percentage of senescent cells, and migratory, proliferatory and angiogenic capacities. Results: Both CMI-hECFCs and CMI-pECFCs showed a delayed time until colonies appearance (p-ANOVA=0,0117 and p-ANOVA=0,0159 respectively) but no differences were found in AMI-hECFCs. AMI- and CMI-hECFCs showed changes in their area and shape, but only shape changes were observed in CMI-pECFCs. In terms of senescence, both AMI- and CMI-hECFCs, but not CMI-pECFCs, presented an increment of senescence (p-ANOVA= 0,0011). In terms of functionality, AMI- and CMI-hECFCs and CMI-pECFCs showed a significant reduction of angiogenesis and proliferation, and only CMI-hECFCs showed an impaired migratory capacity. Conclusions: MI Induces dysfunction of ECFCs with independence of CCVC

Abstract 16746: Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Atherosclerosis is the leading cause of mortality in the United States and most other developed countries. Endothelial cell (EC) activation is a hallmark of the initiation and progression of atherosclerosis; identification of molecular markers of dysfunctional endothelium represents a biological challenge to developing new therapeutic targets. CD45 is a marker of hematopoietic cells, which is rarely expressed in ECs. However, recent studies indicated that CD45 is indispensable in driving the endothelial-to-mesenchymal transition (EndMT) following myocardial infarction. EndMT has also been implicated in the pathogenesis of atheroma progression. Thus, we aim to investigate whether endothelial CD45 contributes to the progression of atherosclerosis by facilitating the EndMT. Methods and results: In vitro , we used CRISPR/inactive Cas9 transcriptional activation system to express CD45 in human endothelial colony-forming cells (ECFCs). Using FACS analyses, we found that CD45+ECFCs upregulate transforming growth factors (TGFβ 1-3 ) that implicate EndMT but downregulate FGFR1 expression, which exacerbates EndMT. qRT-PCR and WB showed that CD45+ECFCs downregulate transcription factors KLFs. In vivo , we generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-CD45iKO) in ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We found that ApoE-/- / EC-iCD45KO mice reduced plaque macrophages, foam cells, expression of adhesion molecule, and lesion development compared to ApoE-/- controls. Interestingly, ApoE-/- / EC-iCD45KO mice showed the reduction of α-smooth muscle actin, which is typical of EndMT, in the aortic plaque. Single-cell RNA-seq analyses have been performed to characterize that endothelial CD45 regulates the expression of pro-inflammatory, EndMT, and endothelial cell markers genes on the aortic ECs. Conclusion: These findings demonstrate that endothelial CD45 potentiates atherosclerosis by inducing TGF signaling and mesenchymal phenotype, causing FGFR1 downregulation and attenuating KLFs expression to drive EndMT, highlighting endothelial CD45 may be a new drug target for the treatment of atherosclerosis.

Terminus-Selective Covalent Immobilization of Heparin on a Thermoresponsive Surface Using Click Chemistry for Efficient Binding of Basic Fibroblast Growth Factor.

Cell therapy using endothelial cells (ECs) has great potential for the treatment of congenital disorders, such as hemophilia A. Cell sheet technology utilizing a thermoresponsive culture dish is a promising approach to efficiently transplant donor cells. In this study, a new method to prepare terminus-selective heparin-immobilized thermoresponsive culture surfaces is developed to facilitate the preparation of EC sheets. Alkynes are introduced to the reducing terminus of heparin via reductive amination. Cu-catalyzed azide-alkyne cycloaddition (CuAAC) facilitates efficient immobilization of the terminus of heparin on a thermoresponsive surface, resulting in a higher amount of immobilized heparin while preserving its function. Heparin-immobilized thermoresponsive surfaces prepared using CuAAC exhibit good adhesion to human endothelial colony-forming cells (ECFCs). In addition, upon further binding to basic fibroblast growth factor (bFGF) on heparin-immobilized surfaces, increased proliferation of ECFCs on the surface is observed. The confluent ECFC monolayer cultured on bFGF-bound heparin-immobilized thermoresponsive surfaces exhibits relatively high fibronectin accumulation and cell number and detaches at 22°C while maintaining the sheet-like structure. Because heparin has an affinity for several types of bioactive molecules, the proposed method can be applied to facilitate efficient cultures and sheet formations of various cell types.

Potentials of Endothelial Colony-Forming Cells: Applications in Hemostasis and Thrombosis Disorders, from Unveiling Disease Pathophysiology to Cell Therapy.

Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells circulating in a limited number in peripheral blood. They can give rise to mature endothelial cells (ECs) and, with intrinsically high proliferative potency, contribute to forming new blood vessels and restoring the damaged endothelium in vivo. ECFCs can be isolated from peripheral blood or umbilical cord and cultured to generate large amounts of autologous ECs in vitro. Upon differentiation in culture, ECFCs are excellent surrogates for mature ECs showing the same phenotypic, genotypic, and functional features. In the last two decades, the ECFCs from various vascular disease patients have been widely used to study the diseases' pathophysiology ex vivo and develop cell-based therapeutic approaches, including vascular regenerative therapy, tissue engineering, and gene therapy. In the current review, we will provide an updated overview of past studies, which have used ECFCs to elucidate the molecular mechanisms underlying the pathogenesis of hemostatic disorders in basic research. Additionally, we summarize preceding studies demonstrating the utility of ECFCs as cellular tools for diagnostic or therapeutic clinical applications in thrombosis and hemostasis.

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 μM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

PB1411 Study of Endothelial Dysfunction in Patients with Unprovoked Venous Thromboembolism through Endothelial Colony-Forming Cell In Vitro Characterization

PB1411 Study of Endothelial Dysfunction in Patients with Unprovoked Venous Thromboembolism through Endothelial Colony-Forming Cell In Vitro Characterization

Thrombin receptor PAR1 silencing in endothelial colony–forming cells modifies stemness and vasculogenic properties

BackgroundThe 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. ObjectivesWe aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony–forming cells (ECFCs). Methods and ResultsPAR1 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). ConclusionIn conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.

MCPIP-1 knockdown enhances endothelial colony-forming cell angiogenesis via the TFRC/AKT/mTOR signaling pathway in the ischemic penumbra of MCAO mice

Cerebral ischemia is a serious disease characterized by brain tissue ischemia and hypoxic necrosis caused by the blockage of blood vessels within the central nervous system. Although stem cell therapy is a promising approach for treating ischemic stroke, the inflammatory, oxidative, and hypoxic environment generated by cerebral ischemia greatly reduces the survival and therapeutic effects of transplanted stem cells. Endothelial colony-forming cells (ECFCs) are a class of precursor cells with strong proliferative potential that can migrate and differentiate directly into mature vascular endothelial cells. Consequently, ECFCs can exert significant therapeutic and reparative effects in diseases associated with vascular injury. Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) exerts multiple biological effects; however, no studies have yet reported its role in the angiogenic function of ECFCs. In this study, we performed Proteome Profiler™ Human Angiogenesis Antibody arrays and tandem mass tag protein profiling to investigate the effect of MCPIP-1 on ECFCs. We demonstrated that MCPIP-1 knockdown enhanced the proliferation, migration, and in vivo and in vitro angiogenic capacity of ECFCs by upregulating the transferrin receptor-activated AKT/m-TOR signaling pathway to promote cellular trophic factor secretion. Furthermore, we found that the lateral ventricular transplantation of ECFCs with lentiviral MCPIP-1 knockdown into mice with middle cerebral artery occlusion increased serum vacular endothelial growth factor(VEGF), angiopoietin-1, and HIF-1a levels, enhanced neovascularization and neurogenesis in the ischemic penumbra, reduced the size of cerebral infarcts, and promoted neurological recovery. Together, these findings suggest new avenues for enhancing the therapeutic efficacy of ECFCs.