Origins and functional differences of blood endothelial cells.

The interests in blood endothelial cells arise from their therapeutic potential in vascular repair and regeneration. Our understanding of blood endothelial cells that exist in the circulation has been evolving significantly from the original concept of endothelial progenitor cells. Many studies have uncovered heterogeneities of blood endothelial subtypes where some cells express both endothelial and hematopoietic antigens, and others possess either mature or immature endothelial markers. Due to the lack of definitive cell marker identities, there have been momentums in the field to adopt a technical-oriented labeling system based on the cells' involvement in postnatal neovascularization and cell culture derivatives. Our review streamlines nomenclatures for blood endothelial subtypes and standardizes understanding of their functional differences. Broadly, we will discuss about myeloid angiogenic cells (MACs), endothelial colony-forming cells (ECFCs), blood outgrowth endothelial cells (BOECs) and circulating endothelial cells (CECs). The strategic location of blood endothelial cells confers them essential roles in supporting physiological processes. MACs exert angiogenic effects through paracrine mechanisms, while ECFCs are recruited to sites of vascular injury to participate directly in new vessel formation. BOECs are an in vitro derivative of ECFCs. CECs are shed into the bloodstream from damaged vessels, hence reflective of endothelial dysfunction. With clarity on the functional attributes of blood endothelial subtypes, we present recent advances in their applications in disease modelling, along with serving as biomarkers of vascular tissue homeostasis.

Endothelial-immune crosstalk contributes to vasculopathy in nonalcoholic fatty liver disease.

The top cause of mortality in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, mechanisms of NAFLD-associated vasculopathy remain understudied. Here, we show that blood outgrowth endothelial cells (BOECs) from NAFLD subjects exhibit global transcriptional upregulation of chemokines and human leukocyte antigens. In mouse models of diet-induced NAFLD, we confirm heightened endothelial expressions of CXCL12 in the aortas and the liver vasculatures, and increased retention of infiltrated leukocytes within the vessel walls. To elucidate endothelial-immune crosstalk, we performed immunoprofiling by single-cell analysis, uncovering T cell intensification in NAFLD patients. Functionally, treatment with a CXCL12-neutralizing antibody is effective at moderating the enhanced chemotactic effect of NAFLD BOECs in recruiting CD8+ T lymphocytes. Interference with the CXCL12-CXCR4 axis using a CXCR4 antagonist also averts the impact of immune cell transendothelial migration and restores endothelial barrier integrity. Clinically, we detect threefold more circulating damaged endothelial cells in NAFLD patients than in healthy controls. Our work provides insight into the modulation of interactions with effector immune cells to mitigate endothelial injury in NAFLD.

The Future of Kawasaki Disease Diagnosis: Liquid Biopsy May Hold the Key.

Kawasaki disease (KD) is a febrile illness characterised by systemic inflammation of small- and medium-sized blood vessels, which commonly occurs in young children. Although self-limiting, there is a risk of developing coronary artery lesions as the disease progresses, with delay in diagnosis and treatment. Unfortunately, the diagnosis of KD continues to remain a clinical dilemma. Thus, this article not only summarises the key research gaps associated with KD, but also evaluates the possibility of using circulating endothelial injury biomarkers, such as circulating endothelial cells, endothelial microparticles and vascular endothelial cell-free DNA, as diagnostic and prognostic tools for KD: a "liquid biopsy" approach. The challenges of translating liquid biopsies to use in KD and the opportunities for improvement in its diagnosis and management that such translation may provide are discussed. The use of endothelial damage markers, which are easily obtained via blood collection, as diagnostic tools is promising, and we hope this will be translated to clinical applications in the near future.

Levels of Plasma Endothelin-1, Circulating Endothelial Cells, Endothelial Progenitor Cells, and Cytokines after Cardiopulmonary Bypass in Children with Congenital Heart Disease: Role of Endothelin-1 Regulation.

Congenital heart disease (CHD) can be complicated by pulmonary arterial hypertension (PAH). Cardiopulmonary bypass (CPB) for corrective surgery may cause endothelial dysfunction, involving endothelin-1 (ET-1), circulating endothelial cells (CECs), and endothelial progenitor cells (EPCs). These markers can gauge disease severity, but their levels in children's peripheral blood still lack consensus for prognostic value. The aim of our study was to investigate changes in ET-1, cytokines, and the absolute numbers (Ɲ) of CECs and EPCs in children 24 h before and 48 h after CPB surgery to identify high-risk patients of complications. A cohort of 56 children was included: 41 cases with CHD-PAH (22 with high pulmonary flow and 19 with low pulmonary flow) and 15 control cases. We observed that Ɲ-CECs increased in both CHD groups and that Ɲ-EPCs decreased in the immediate post-surgical period, and there was a strong negative correlation between ET-1 and CEC before surgery, along with significant changes in ET-1, IL8, IL6, and CEC levels. Our findings support the understanding of endothelial cell precursors' role in endogenous repair and contribute to knowledge about endothelial dysfunction in CHD.

Angiogenesis and Ovarian Cancer: What Potential Do Different Subtypes of Circulating Endothelial Cells Have for Clinical Application?

Ovarian cancer (OC) remains the most fatal disease of gynaecologic malignant tumours. The neovasculature in the tumour microenvironment principally comprises endothelial cells. Haematogenous cancer metastases are significantly impacted by tumour neovascularisation, which predominantly depends on the tumour-derived endothelial vasculogenesis. There is an urgent need for biomarkers for the diagnosis, prognosis and prediction of drug response. Endothelial cells play a key role in angiogenesis and other forms of tumour vascularisation. Subtypes of circulating endothelial cells may provide interesting non-invasive biomarkers of advanced OC that might have the potential to be included in clinical analysis for patients' stratification and therapeutic management. In this review, we summarise the reported studies on circulating endothelial subtypes in OC, detailing their isolation methods as well as their potential diagnostic, prognostic, predictive and therapeutic utility for clinical application. We highlight key biomarkers for the identification of circulating endothelial cell subtypes and their targets for therapies and critically point out future challenges.

Are circulating endothelial cells the next target for transcriptome-level pathway analysis in ARDS?

Acute respiratory distress syndrome (ARDS) has had no mortality-improving pharmacological intervention despite 50 years of high-caliber research due to its heterogeneity (Huppert LA, Matthay MA, Ware LB. Semin Respir Crit Care Med 40: 31-39, 2019). For the field to advance, better definitions for ARDS subgroups that more uniformly respond to therapies are needed (Bos LDJ, Scicluna BP, Ong DSY, Cremer O, van der Poll T, Schultz MJ. Am J Respir Crit Care Med 200: 42-50, 2019; Dickson RP, Schultz MJ, T van der P, Schouten LR, Falkowski NR, Luth JE, Sjoding MW, Brown CA, Chanderraj R, Huffnagle GB, Bos LDJ, Biomarker Analysis in Septic ICU Patients (BASIC) Consortium. Am J Respir Crit Care Med 201: 555-563, 2020; Sinha P, Calfee CS. Am J Respir Crit Care Med 200: 4-6, 2019; Calfee CS, Delucchi K, Parsons PE, Thompson BT, Ware LB, Matthay MA, NHLBI ARDS Network. Lancet Respir Med 2: 611-620, 2014; Hendrickson CM, Matthay MA. Pulm Circ 8: 1-12, 2018). A plethora of high-quality clinical research has uncovered the next generation of soluble biomarkers that provide the predictive enrichment necessary for trial recruitment; however, plasma-soluble markers do not specify the damaged organ of origin nor do they provide insight into disease mechanisms. In this perspective, we make the case for querying the transcriptome of circulating endothelial cells (CECs), which when shed from vessels after inflammatory insult, become heralds of site-specific inflammatory damage. We review the application of CEC quantification to multiple disease phenotypes (including myocardial infarction, vasculitides, cancer, and ARDS), in each case supporting the association of CEC number with disease severity. We also argue for the utility of single-cell RNA transcriptomics to the understanding of cell-specific contributions to disease pathophysiology and its potential to uncover novel insight on signals contributing to CEC shedding in ARDS.

Longitudinal profile of circulating endothelial cells in post-acute coronary syndrome patients.

IntroductionPatients who have experienced an acute coronary syndrome (ACS) are at risk of a recurrent event, but their level of risk varies. Because of their close temporal relationship with vascular injury, longitudinal measurements of circulating endothelial cells (CECs) carry potential to improve individual risk assessment.MethodsWe conducted an explorative nested case-control study within our multicenter, prospective, observational biomarker study (BIOMArCS) of 844 ACS patients. Following an index ACS, high-frequency blood sampling was performed during 1-year follow-up. CECs were identified using flow cytometric analyses in 15 cases with recurrent event, and 30 matched controls.ResultsCases and controls had a median (25th-75thpercentile) age of 64.1 (58.1-75.1) years and 80% were men. During the months preceding the endpoint, the mean (95%CI) CEC concentration in cases was persistently higher than in controls (12.8 [8.2-20.0] versus 10.0 [7.0-14.4] cells/ml), although this difference was non-significant (P = 0.339). In controls, the mean cell concentration was significantly (P = 0.030) lower in post 30-day samples compared to samples collected within one day after index ACS: 10.1 (7.5-13.6) versus 17.0 (10.8-26.6) cells/ml. Similar results were observed for CEC subsets co-expressing CD133 and CD309 (VEGFR-2) or CD106 (VCAM-1).ConclusionDespite their close relation to vascular damage, no increase in cell concentrations were found prior to the occurrence of a secondary adverse cardiac event.

Changes and significance of vascular endothelial injury markers in patients with diabetes mellitus and pulmonary thromboembolism.

BACKGROUND: To investigate the changes and clinical significance of vascular endothelial injury markers in type 2 diabetes mellitus (T2DM) complicated with pulmonary embolism (PE). METHODS: This prospective study enrolled patients with T2DM hospitalized in one hospital from January 2021 to June 2022. Soluble thrombomodulin (sTM) (ELISA), von Willebrand factor (vWF) (ELISA), and circulating endothelial cells (CECs) (flow cytometry) were measured. PE was diagnosed by computed tomography pulmonary angiography (CTPA). RESULTS: Thirty participants were enrolled in each group. The plasma levels of sTM (151.22 ± 120.57 vs. 532.93 ± 243.82 vs. 1016.51 ± 218.00 pg/mL, P < 0.001) and vWF (9.63 ± 2.73 vs. 11.50 ± 2.17 vs. 18.02 ± 3.40 ng/mL, P < 0.001) and the percentage of CECs (0.17 ± 0.46 vs. 0.30 ± 0.08 vs. 0.56 ± 0.18%, P < 0.001) gradually increased from the control group to the T2DM group to the T2DM + PE group. sTM (OR = 1.002, 95%CI: 1.002-1.025, P = 0.022) and vWF (OR = 1.168, 95%CI: 1.168-2.916, P = 0.009) were associated with T2DM + PE. sTM > 676.68 pg/mL for the diagnosis of T2DM + PE achieved an AUC of 0.973, while vWF > 13.75 ng/mL achieved an AUC of 0.954. The combination of sTM and vWF above their cutoff points achieved an AUC of 0.993, with 100% sensitivity and 96.7% specificity. CONCLUSIONS: Patients with T2DM show endothelial injury and dysfunction, which were worse in patients with T2DM and PE. High sTM and vWF levels have certain clinical predictive values for screening T2DM accompanied by PE.

Circulating Endothelial Cells: A New Possible Marker of Endothelial Damage in Kawasaki Disease, Multisystem Inflammatory Syndrome in Children and Acute SARS-CoV-2 Infection.

BACKGROUND: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). METHODS: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. RESULTS: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. CONCLUSIONS: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Flow cytometric analysis of circulating endothelial cells and endothelial progenitor cells in pediatric solid tumors: prognostic impact on treatment response and survival.

BACKGROUND AND AIM: Solid tumors, including pediatric malignancies, depend on angiogenesis for tumor growth, invasion, and metastases. We aimed to evaluate the prognostic impact of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) on treatment response and survival of pediatric patients with solid tumors. METHODS: A prospective study included 70 patients with different pediatric solid tumors treated with different types of chemotherapy and 20 age and sex-matched healthy children as controls. Blood samples collected at diagnosis then on day 7 and day 21 after chemotherapy. CECs and EPCs were evaluated using flow cytometry. RESULTS: The mean levels of CECs and EPCs of patients at diagnosis were significantly higher than controls (85.29 ± 24.78 and 26.1 ± 9.11 versus 20.08 ± 6.65; and EPCs; 2.78 ± 1.48, respectively; P < 0.001 for both). The highest levels of CECs were observed in patients with rhabdomyosarcoma (RMS). An overall increase was reported in CECs, and after the first cycle of chemotherapy, that was significantly correlated to treatment response and overall survival. CONCLUSION: Pediatric patients with solid tumors have elevated levels of CECs and EPCs with more elevation after chemotherapy. The magnitude of increase of CECs occurred on day 7 after chemotherapy may be considered as an early predictor of response to therapy and outcome in pediatric patients with solid tumors.

Multimodal assessment of acute cardiac toxicity induced by thoracic radiotherapy in cancer patients. Study protocol.

BACKGROUND: Today, cancer ranks as one of the leading causes of death. Despite the large number of novel available therapies, radiotherapy (RT) remains as the most effective non-surgical method to cure cancer patients. In fact, approximately 50% of all cancer patients receive some type of RT and among these 60% receive RT-treatment with a curative intent. However, as occurs with any other oncological therapy, RT treated patients may experience toxicity side effects that range from moderate to severe. Among these, cardiotoxicity represents a significant threat for premature death. Current methods evaluate cardiotoxic damage based on volumetric changes in the Left Ventricle Ejected Fraction (LVEF). Indeed, a 10% drop in LVEF is commonly used as indicator of cardiotoxicity. More recently, a number of novel techniques have been developed that significantly improve specificity and sensitivity of heart's volumetric changes and early detection of cardiotoxicity even in asymptomatic patients. Among these, the Strain by Speckle Tracking (SST) is a technique based on echocardiographic analysis that accurately evaluates myocardial deformation during the cardiac cycle (ventricular and atrial function). Studies also suggest that Magnetic Resonance Imaging (MRI) is a high-resolution technique that enables a better visualization of acute cardiac damage. METHODOLOGY: This protocol will evaluate changes in SST and MRI in cancer patients that received thoracic RT. Concomitantly, we will assess changes in serum biomarkers of cardiac damage in these patients, including: high-sensitivity cardiac Troponin-T (hscTnT), N-Terminal pro-Brain Natriuretic Peptide (NTproBNP) and Circulating Endothelial Cells (CECs), a marker of endothelial dysfunction and vascular damage. DISCUSSION: The presented protocol is to our knowledge the first to prospectively and with a multimodal approach, study serological and image biomarkers off early cardiac damage due to radiotherapy. With a practical clinical approach we will seek early changes that could potentially be in the future be linked to clinical mayor events with consequences for cancer survivors.

Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19.

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CECs) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Together, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19.

Prognostic value of CEC count in HER2-negative metastatic breast cancer patients treated with bevacizumab and chemotherapy: a prospective validation study (UCBG COMET).

BACKGROUND: Proof of concept studies has reported that circulating endothelial cell (CEC) count may be associated with the outcome of HER2-negative metastatic breast cancer (mBC) patients treated by chemotherapy and the anti-VEGF antibody bevacizumab. We report the results obtained in an independent prospective validation cohort (COMET study, NCT01745757). METHODS: The main baseline criteria were HER2-negative mBC, performance status 0-2 and no prior chemotherapy for metastatic disease. CECs were detected by CellSearch® from 4 ml of blood at baseline and after 4 weeks of weekly paclitaxel and bevacizumab therapy. CEC counts (considered both as a continuous variable and using the previously described 20 CEC/4 ml cutoff) were associated with clinical characteristics and progression-free survival (PFS). RESULTS: CEC count was obtained in 251 patients at baseline and in 207 patients at 4 weeks. Median baseline CEC count was 22 CEC/4 ml (range 0-2231). Baseline CEC counts were associated with performance status (p = 0.02). No statistically significant change in CEC counts was observed between baseline and 4 weeks of therapy. High baseline CEC count was associated with shorter PFS in univariate and multivariate analyses (continuous: p < 0.001; dichotomized: HR 1.52, 95% CI [1.15-2.02], p = 0.004). CEC counts at 4 weeks had no prognostic impact. CONCLUSION: This study confirms that CEC count may be associated with the outcome of mBC patients treated with chemotherapy and bevacizumab. However, discrepancies with previous reports in terms of both the timing of CEC count and the direction of the prognostic impact warrant further clinical investigation.

Determination of Circulating Endothelial Cells and Endothelial Progenitor Cells Using Multicolor Flow Cytometry in Patients with Thrombophilia.

BACKGROUND/AIMS: Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) have been described as markers of endothelial damage and dysfunction in several diseases, including deep venous thrombosis. Their role in patients with known thrombophilia has not yet been evaluated. Both EPCs and CECs represent extremely rare cell populations. Therefore, it is essential to use standardized methods for their identification and quantification. METHODS: In this study, we used multicolor flow cytometry to analyze the number of EPCs and CECs in patients with thrombophilia with or without a history of thrombosis. Patients with hematological malignancies after high-dose chemotherapy and patients with acute myocardial infarction were used as positive controls. RESULTS: EPC and CEC immunophenotypes were determined as CD45dim/-CD34+CD146+CD133+ and CD45dim/-CD34+CD146+CD133-, respectively. Increased levels of endothelial cells were observed in positive control groups. No significant changes in the number of EPCs or CECs were detected in patients with thrombophilia compared to healthy controls. CONCLUSION: Our optimized multicolor flow cytometry method allows unambiguous identification and quantification of endothelial cells in the peripheral blood. Our results support previous studies showing that elevated levels of CECs could serve as an indicator of endothelial injury or dysfunction. Normal levels of CECs or EPCs were found in patients with thrombophilia.

High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients.

Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.

Circulating platelet aggregates damage endothelial cells in culture.

BACKGROUND: Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). METHODS: Endothelial cells were grown in glass tubes and tagged with/without 0.5 µm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. RESULTS AND CONCLUSION: No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.

Enrichment and Detection of Circulating Tumor Cells and Other Rare Cell Populations by Microfluidic Filtration.

The current standard methods for isolating circulating tumor cells (CTCs) from blood involve EPCAM-based immunomagnetic approaches. A major disadvantage of these strategies is that CTCs with low EPCAM expression will be missed. Isolation by size using filter membranes circumvents the reliance on this cell surface marker, and can facilitate the capture not only of EPCAM-negative CTCs but other rare cells as well. These cells that are trapped on the filter membrane can be characterized by immunocytochemistry (ICC) , enumerated and profiled to elucidate their clinical significance. In this chapter, we discuss advances in filtration systems to capture rare cells as well as downstream ICC methods to detect and identify these cells. We highlight our recent clinical study demonstrating the feasibility of using a novel method consisting of automated microfluidic filtration and sequential ICC for detection and enumeration of CTCs, as well as circulating mesenchymal cells (CMCs), circulating endothelial cells (CECs), and putative circulating stem cells (CSCs). We hypothesize that simultaneous analysis of circulating rare cells in blood of cancer patients may lead to a better understanding of disease progression and development of resistance to therapy.

Cellular response of the blood-brain barrier to injury: Potential biomarkers and therapeutic targets for brain regeneration.

Endothelial cells are the main component of the blood-brain barrier (BBB), a vital structure for maintaining brain homeostasis that is seriously disrupted in various neurological pathologies. Therefore, vascular-targeted therapies may bring advantages for the prevention and treatment of brain disorders. In this sense, novel methods to identify and evaluate endothelial damage have been developed and include the detection of circulating endothelial cells, endothelial progenitor cells, endothelial microparticles and exosomes. These cells and cellular structures have been documented in numerous diseases, and increasingly in neurodegenerative disorders, which have led many to assume that they can either be possible biomarkers or tools of repair. Therefore, the purpose of this review is to discuss available data on BBB endothelial damage occurring in two pathologies of the central nervous system, Alzheimer's disease and stroke, which exemplify conditions where chronic and acute vascular damage occur, respectively. The ultimate goal is to identify useful biomarkers and/or therapeutic tools in the healthy and diseased brain that can be used for the treatment of neurodegenerative diseases where BBB permeability and integrity are impaired.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Left ventricular dysfunction and subclinical atherosclerosis in children with classic congenital adrenal hyperplasia: a single-center study from upper Egypt.

UNLABELLED: Few studies assessed carotid artery intima-media thickness (CA-IMT) and left ventricular (LV) function in children with congenital adrenal hyperplasia (CAH) as compared to adults. This study aimed to assess carotid artery structural changes and myocardial function with CAH. The study included 32 children with classic CAH and 32 healthy children matched for age, gender, pubertal status, and socioeconomic status. Blood levels of high-sensitivity C-reactive protein (hs-CRP) and circulating endothelial cells (CECs) were measured. LV mass (LVM) and function were assessed using conventional echocardiography. Duplex ultrasonography was used to measure CA-IMT. Compared to controls, patients had higher hs-CRP and CEC concentrations (p < 0.001) and increased CA-IMT (p < 0.001), indicating vascular endothelial injury and subclinical atherosclerosis; higher LVM index (LVMI) (p < 0.001), indicating LV hypertrophy; and lower ratio of E/A wave and prolonged mitral deceleration time (DcT) and isovolumic relaxation times (IVRTs) (p < 0.001), indicating LV dysfunction. Abnormalities were marked in uncontrolled children on medical treatment. Testosterone levels were positively correlated with CA-IMT, LVMI, and DcT values. CONCLUSION: This study indicates that children with CAH and enhanced androgen levels are at increased risk of vascular endothelial injury, subclinical atherosclerosis, and LV dysfunction. These findings highlight early monitoring of children with CAH for cardiovascular abnormalities.