Circulating Endothelial Cells Research Articles

Protective cardiovascular benefits of exercise training as measured by circulating endothelial cells and high-density lipoprotein in adults

ObjectiveThis study aims to determine the protective cardiovascular effect of aerobic exercise training by measuring cluster of differentiation 146 (CD146), circulating endothelial cell (CEC), and high-density lipoprotein-cholesterol (HDL-C) levels in adults. MethodsThis study was an experimental pre-post-test without a control group. Forty-five participants were divided into three groups based on aerobic exercise training intensity: low, moderate, and high. Whole blood samples were measured for HDL-C levels. In addition, CEC was isolated from Peripheral Blood Mononuclear Cells (PBMC) samples, then identified by CD146 marker using flow cytometry. ResultsCEC percentage and HDL-C increase after aerobic exercise training. There was a significant difference in CEC percentage between the intensity groups. However, there was no difference in HDL-C levels. ConclusionAerobic exercise training can protect cardiovascular health by stimulating CEC mobilization, identified by CD146. In addition, an HDL-C level increase also contributes to cardiovascular protection by decreasing inflammation levels, inhibiting low-density lipoprotein-cholesterol oxidation, improving endothelial regeneration capabilities, and lowering blood glucose.

Sulodexide as pharmacotherapy for protection of endothelium and suppression of thrombosis in COVID-19

The article presents an overview of publications describing the mechanisms of the development of the pathological process in patients infected with SARS-CoV-2 coronavirus. The authors analysed publications showing that damage to the endothelium and endothelial glycocalyx is the main factor influencing the development of coronavirus COVID-19 infection. It is the endothelium inflammation caused by a virus that leads to the dysfunction of the vascular system and the development of coagulopathy. Using scanning electron microscopy and flow cytofluorimetry, we showed that patients with COVID-19 of moderate severity (CT2) had significant desquamation of endothelial cells (concentration of circulating endothelial cells (CEC) in blood is 300-400 cells/ml whereas the normal range is no more than 10 cells/ ml). Such desquamation should cause exposure of the pro-inflammatory and thrombogenic subendothelial matrix, which, as a results, leads to the development of thrombotic disorders of the circulatory system. Therefore, it is only natural to try to counteract disease progression by protecting the endothelial glycocalyx from damage. Sulodexide, a mixture of fast-moving heparin fraction (80%) and the glycocalyx element dermatan sulfate (20%) obtained from the mucous membrane of the small intestine of pigs, is very promising in this regard. This drug can significantly reduce the inflammatory process, protect the glycocalyx and endothelium from damage, resulting in lowering the degree of thrombus formation in patients with coronavirus COVID-19 infection, which relieves the course of the disease and improves its outcome. The experimental data presented in the review, although obtained in not large enough population of patients, allow us to consider sulodexide a promising drug that protects the endothelium and suppresses thrombosis in COVID-19.

Investigation of the Attachment of Circulating Endothelial Cells to a Cell Probe: Combined Experimental and Numerical Study

Circulating endothelial cells (CECs) are a reliable biomarker for cardiovascular diseases (CVDs). A major unresolved challenge limiting the widespread use of CECs for the diagnosis and monitoring of CVDs is their unreliable detection. This problem is mainly attributed to the low sample volume (5–10 mL) of commonly used ex vivo CEC isolation methods. To overcome this limitation, the BMProbe for the in vivo isolation of CECs is proposed. It consists of a twisted medical flat wire with a polymer‐coated surface functionalized with anti‐CD105 antibodies. A combined experimental and numerical study is performed to investigate which flow conditions lead to an increased cell attachment to the probe's surface. Endothelial cells are solved in a dextran solution and circulated in a flow system containing the BMProbes. Microscopic images of the attached CECs are taken. In addition, the experiments are simulated using a computational fluid dynamics (CFD) flow solver to quantify the flow conditions at the probe's surface. The microscopic images are superimposed with the CFD data to investigate the influence of wall shear rate and wall normal rate on the attachment of CECs to the probe. Most of all attached cells (85.5%) are found in areas of negative wall normal rate.

Assessment of Reversibility in Pulmonary Hypertension Related to Congenital Heart Disease by Using Biomarkers and Clinical Features

Background: Reversibility of pulmonary hypertension (PH) is closely related to the treatment options for and prognosis of children with congenital heart disease. Objective: We combined patient-specific clinical features including diagnosis, age and echocardiographic results, and biomarkers of pulmonary vascular dysfunction to explore the noninvasive methods that can be used to accurately evaluate the reversibility of pulmonary hypertension in congenital heart disease (PH-CHD). Methods: Based on the preoperative systolic pulmonary arterial pressure (sPAP), 70 CHD patients were divided into normal, PH-CHD suspected, and confirmed groups. Additionally, biomarkers of circulating endothelial cells (CECs), endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS) were detected. Patients were categorized into reversible (RPH) and irreversible (IRPH) groups according to the sPAP 6 months after surgery. Risk stratification was performed according to the clinical features and biomarkers. Results: CECs and ET-1 levels in the confirmed group were significantly higher. eNOS was higher in the confirmed and suspected groups than that in the normal group. CECs in the IRPH group were significantly higher compared to the RPH group. No such intergroup differences were observed with respect to ET-1 and eNOS levels. The ROC curve showed that the risk stratification was of high diagnostic value to evaluate reversibility. Conclusion: The CECs, eNOS, and ET-1 were closely related with PH-CHD. CECs and risk stratification have high practical value in assessing the reversibility of PH-CHD.

Нарушения эндотелий-зависимой вазодилатации у больных с микрососудистой стенокардией

Background: disorders of both endothelium-dependent and endothelium-independent vasodilation play a role in the pathogenesis of microvascular angina (MVA), the contribution of which may be different. Aim: to study the processes of endothelium-dependent vasodilation (EDV) in patients with primary MVA. Patients and Methods: an open prospective study included 60 patients (mean age 57.3±6.4 years) with MVA, confirmed clinical picture (chest pain), coronary angiography data (the absence of coronary artery stenosis), positive stress test (pain syndrome and/or ST segment depression by ≥2 mm by treadmill exercise stress test), positron emission tomography (PET) myocardial perfusion imaging (MPI) with tests (cold pressor test and with adenosine). Patients with diseases that can lead to secondary microvascular dysfunction were not included in the study. To evaluate the processes of EDV, all patients underwent the following evaluations: peripheral arterial tonometry (PAT), assessment of circulating endothelial cells (CEC), high-sensitivity C-reactive protein (hsCRP), endothelin-1 (ET-1), total antioxidant status. Adding that, the results of a cold pressor test during PET were analyzed. Results: the majority of the examined patients were women — 81.7%. Most of the female patients included in the study (93.9%) were postmenopausal. In all patients with MVA, according to the data of PET MPI with cold pressor test, signs of EDV disorder were detected as a decrease in coronary blood flow through the three coronary arteries. During PET, the index of reactive hyperemia was reduced (<1,67) in all examined patients and amounted to 1.43±0.15. According to the results of laboratory studies, there was an increase in the level of ET-1 and CEC — 3,335 [1,545; 3,952] fmol/L and 14±8 cells/3×105 leukocytes, respectively. The CRP level was 4,44 [1.02; 4.45] mg/L. The total antioxidant capacity was reduced in 53 (88%) patients, the average values of the indicator were 289,03±52,14 μmol/L. Conclusion: according to laboratory and instrumental studies, signs of endothelial dysfunction, namely disorders of EDV, were revealed in all examined patients with primary MVA. KEYWORDS: microvascular angina, peripheral arterial tonometry, endothelium-dependent vasodilation, positron emission tomography. FOR CITATION: Leonova I.A., Zakharova O.V., Boldueva S.A. Disorders of endothelium-dependent vasodilation in patients with microvascular angina. Russian Medical Inquiry. 2022;6(8):427–432 (in Russ.). DOI: 10.32364/2587-6821-2022-6-8-427-432.

Study on Circulating Tumor Cells and Various Clinical Features and Blood-Related Indexes in Patients with Renal Cancer

Objective: To investigate the value of CTC clinical features and blood-related test indicators in renal cancer patients by detecting the number of circulating tumor cells (CTC) in patients with renal cancer. Methods: To analyze 59 patients with renal cell carcinoma (RCC) admitted to the Department of Urology, Affiliated Hospital of Chengde Medical College from May 2018 to October 2019. According to the CTC count (5 pcs/3.5 ml), they were divided into CTC positive group and CTC negative group. The age, gender, tumor location, tumor size, hematuria, CEC (circulating endothelial cells), CTC Cluster (aggregate), gene mutation, platelet (PLT), albumin (ALB), hemoglobin (HB), alkaline phosphatase (AKP), lactate dehydrogenase (LDH) were compared between the two groups of patients and the correlation of the number of CTCs. Results: There were significant differences in tumor size, CEC, and gene mutations between the two groups (P Conclusion: The number of CTC in RCC patients is correlated with some clinical features (tumor size, CEC, gene mutation) and some related test indicators (HB, PLT, LDH), and can be combined with the above related indicators to predict the occurrence, metastasis and prognosis of renal cancer.

Correlation between circulating endothelial cell level and acute respiratory distress syndrome in postoperative patients.

BACKGROUNDAcute respiratory distress syndrome (ARDS) is injury of alveolar epithelial cells and capillary endothelial cells caused by various factors, including endogenous and exogenous lung factors, leading to diffuse pulmonary interstitial and alveolar edema, and acute respiratory failure. ARDS involves alveolar epithelial cells and pulmonary interstitial capillary endothelial cells. Circulating endothelial cells (CECs) are the only marker that directly reflects vascular endothelial injury in vivo. There have been few studies on the correlation between peripheral blood CECs and ARDS at home and abroad. The lungs are the organs with the highest capillary density and the most endothelial cells, thus, it is speculated that when ARDS occurs, CECs are stimulated and damaged, and released into the circulatory system. AIMTo explore the correlation between CEC level and severity of ARDS in patients postoperatively.METHODSBlood samples were collected from all patients on day 2 (d2) and day 5 (d5) after surgery. The control group comprised 32 healthy volunteers. Number of CECs was measured by flow cytometry, and operation time was recorded. Changes in various indexes of patients were monitored, and diagnosis of ARDS was determined based on ARDS Berlin definition. We comprised d2 CECs in different groups, correlation between operation time and d2 CECs, ARDS of different severity by d2 CECs, and predictive value of d2 CECs for ARDS in postoperative patients.RESULTSThe number of d2 CECs in the ARDS group was significantly higher than that in the healthy control group (P < 0.001). The number of d2 CECs in the ARDS group was significantly higher than that in the non-ARDS group (P < 0.001). The number of d2 CECs in the non-ARDS group was significantly higher than that in the healthy control group (P < 0.001). Operation time was positively correlated with number of CECs on d2 (rs = 0.302, P = 0.001). The number of d2 CECs in the deceased group was significantly higher than that in the improved group (P < 0.001). There was no significant difference in number of d2 CECs between patients with mild and moderate ARDS. The number of d2 CECs in patients with severe ARDS was significantly higher than that in patients with mild and moderate ARDS (P = 0.041, P = 0.037). There was no significant difference in number of d5 and d2 CECs in the non-ARDS group after admission to intensive care. The number of d5 CECs was higher than the number of d2 CECs in the ARDS improved group (P < 0.001). The number of d5 CECs was higher than the number of d2 CECs in the ARDS deceased group (P = 0.002). If the number of CECs was > 1351/mL, sensitivity and specificity of predicting ARDS were 80.8% and 78.1%, respectively. CONCLUSIONChanges in number of CECs might predict occurrence and adverse outcome of ARDS after surgery, and higher numbers of CECs indicate worse prognosis of ARDS.

Abstract 13035: A Potent Protective Vascular Endothelium Property of Garcinia mangostana L. Extract in High-Risk Framingham Score Patients With Type 2 Diabetes: A Prospective Randomized Control Trial

Background: Endothelial dysfunction is the hallmark of vascular complications in cardiovascular disease with type 2 diabetes as the comorbid. Garcinia mangostana L., or mangosteen, has been known as the queen of fruits from Southeast Asia that owns anti-inflammatory and anti-oxidant properties for years. Objective: This study was aimed to evaluate the role of G. mangostana extract as a potent protective vascular endothelium in patients with type 2 diabetes and high-risk Framingham score. Method: This is a prospective, randomized, single blind, placebo-controlled trial in subjects with type 2 DM and high-risk Framingham score. Subjects were randomly divided into two groups: the treatment group of G. mangostana extract, administered 2,175 mg/day in divided dosage for 90 days, and control group receiving placebo. The primary endpoint was the changes in endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) levels. We also evaluated the levels of NO, IL-1, IL-6, TNF-α, fasting blood glucose and HbA1c. Study had been approved by local ethics committee. Statistical analysis was done and p&lt;0.05 was considered significant. Results: From 90 subjects with high-risk Framingham score, there were 49 (54.4%) subjects with type 2 diabetes, which randomly divided into two groups. The treatment group (n=23) received G. mangostana extract and control group (n=26) received placebo. Statistical analysis showed that increase of EPC and decrease of CEC levels were significant in treatment group, compared to control (p=0.000 and p=0.002, respectively). The levels of NO increased significantly (p=0.027) and levels of IL-1, IL-6, TNF-α decreased significantly in comparison to placebo (p=0.003, p=0.002, p=0.000, respectively). Analysis also showed that HbA1c decreased significantly (p=0.002) compared to placebo; however, it was insignificant for fasting blood glucose reduction (p=0.336). Conclusion: Garcinia mangostana L. extract, as an adjuvant therapy, plays a role in improving endothelial dysfunction by significant increase in EPCs and decrease in CECs, in patients with type 2 diabetes and high-risk Framingham score.

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

BackgroundToday, 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.MethodologyThis 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.DiscussionThe 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.

Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case; individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the “neoplastic” vascular niche.

Endothelial-T cell crosstalk contributes to vascular injury in fatty liver disease

Abstract Cardiovascular complications are often the fundamental causes of death in non-alcoholic fatty liver disease (NAFLD) patients. While there are known systemic mediators in NAFLD that may induce vascular inflammation, the mechanism of endothelial dysfunction remain understudied. In this work, we harnessed the replicative potential of blood outgrowth endothelial cells (BOECs) to develop personalized cell lines from NAFLD patients and healthy controls. Our transcriptomic analysis showed that the top interactome network enriched in NAFLD BOECs comprised of several C-C and C-X-C chemokine ligands involved in immune cell chemotaxis. We previously reported T cell infiltration in mouse model of non-alcoholic steatohepatitis, and here, we confirmed enhanced endothelial chemokine signatures in arterial histological sections. To elucidate endothelial-immune crosstalk, we performed single-cell analysis on human peripheral blood mononuclear cells and found T cell intensification in NAFLD patients compared to healthy controls. Our immunoprofiling by flow cytometry further revealed that NAFLD patients possessed higher levels CD8+ memory cells. Functionally, T cells, instead of monocytes, adhered more pronouncedly to NAFLD BOECs. In evaluating the CXCL12-CXCR4 axis in chemotaxis, CXCR4 antagonist (AMD3100) substantially modulated the migration of patient-derived CD8+ T cells towards NAFLD BOECs, which was not observed in healthy endothelial-T cell chemotaxis coculture. Finally, we validated NAFLD-associated endothelial dysfunction by enumerating two folds more circulating endothelial cells, a biomarker of vascular injury, in the blood samples of NAFLD patients than healthy controls. Our work provides insights for translation to restore blood vessel health and potentially mitigate adverse vascular events in NAFLD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Industrial Alignment Fund Pre-Positioning grant from the Agency for Science, Technology and Research, Singapore Endothelial-T cell crosstalk in NAFLD

ELEVATED CIRCULATING ENDOTHELIAL CELLS AND SUCCESS IN ENDOTHELIAL COLONY-FORMING CELLS ISOLATION

Circulating endothelial cells (CEC) have been associated with vascular injury and are described as potential biomarkers for cardiovascular disease (1). Besides, current optimized methodologies enable the isolation of a well-characterized subtype of endothelial progenitor known as Endothelial colony-forming cells (ECFCs) (2,3). Although ECFC isolation methodologies are well described; some discrepancies remain in relation to their isolation efficiency. To evaluate the isolation efficacy ECFCs and CEC frequency in human peripheral blood. The Ethics Research Committee of the University of Campinas approved the experimental procedures and all donors signed the informed consent form. CEC enumeration was assessed by flow cytometry in the peripheral blood of 18 donors. 100 μL of blood (with a leukocyte concentration between 5 and 10 ×10³/μL) was incubated with monoclonal anti-human antibodies (CD45 PerCp, CD133 APC, CD31 FITC, and CD146 PE). FACS lysing solution was used for red blood cell removal. 300,000 events or the total volume of the tube were acquired using a FACSCalibur® flow cytometer (4). To determine the number of cells/μL, the percentage of events was multiplied per number of leucocytes and divided per 100. Cell culture from venous blood was used to isolate ECFCs which were confirmed by their cobblestone morphology and immunophenotyping for endothelial markers (CD31, CD146, CD309, and CD144). ECFC isolation was successful in 9 out of 18 donors (1 male, 8 females; mean age: 35.8 ±11.1 years), and not successful in the other half of donors (2 male, 7 females; mean age: 37.7 ±9.7 years). CEC frequency was significantly higher (p=0.004) in the group with successful ECFC isolation when compared to the group that did not yield ECFCs (2.9 ±1.2 CECs/μL against 1.2 ±0,8 CECs/μL). This suggests that the success in ECFC isolation might be related to the number of CECs, and therefore ECFC mobilization into circulation may also be linked to vascular injury. Increased number of CECs might be associated with the success of ECFCs isolation.

Expression and role of interleukin-1β and associated biomarkers in deep vein thrombosis.

Lower extremity deep vein thrombosis (DVT) is a common peripheral vascular disease, in which inflammation plays an important role. The aim of the present study was to investigate the expression and role of inflammatory factors in DVT. A rat model of venous thrombosis of the lower extremities was established through venous ligation surgery. The rats were examined at 2, 8, 24, 48 and 72 h after the induction of inferior venous stenosis and compared with control and sham surgery groups. The serum levels of interleukin-1β (IL-1β), tissue factor (TF) and xanthine oxidase (XOD) were measured using ELISAs. The morphology of the DVT tissue was observed by hematoxylin and eosin staining. Circulating endothelial cells (CECs) in peripheral blood were counted by flow cytometry. Reverse transcription-quantitative PCR and western blotting were used to detect mRNA and protein expression, respectively. The serum levels of IL-1β, TF and XOD exhibited no significant differences between the control and sham surgery groups. However, those in the rat model of DVT presented an upward trend from 2 to 24 h and peaked at 24 h, with a significant difference from the respective levels in the control and sham surgery groups. The histopathological analysis revealed the presence of red and mixed thrombi in the rats 2-48 h following the induction of inferior venous stenosis group with inflammatory cell infiltration in the vascular wall. Thrombus formation was evident after 72 h. While significant difference was observed in the number of CECs in the peripheral blood between the control and sham surgery groups, the number of peripheral blood CECs in the rats with inferior venous stenosis group increased from 8 to 72 h, with significant differences among these groups. The mRNA levels of IL-1β, TF, XOD and NF-κB in the tissues peaked at 24 h, with significant differences compared with those in the control and sham surgery groups. In addition, the protein expression level of NF-κB increased from 2 to 72 h. In conclusion, these results suggest that the high expression of IL-1β, TF, XOD and NF-κB may promote thrombus formation.

Missing data handling technique in joint modeling context

The maximum tolerated dose (MTD) is commonly practiced for dose selection in oncology. Higher doses work as the best effective treatment. Conventionally, the doses are selected in phase 2 from a phase. Perhaps, the quality of life gets compromised due to the high dose of chemotherapeutic regimes in the early phase of the trial. Alternative chemotherapy administration is Metronomic Chemotherapy (MC). Here very minimal doses are administered to avoid high toxicity. This work is about to handle missing data of circulating endothelial cells (CEC) and supports the optimal biological dose (OBD) for MC. It is performed with mimic data. A data simulation strategy is adopted. Simulation work is performed with R software. It helps to identify the suitable technique to handle missing data. The results conclude that the MC is efficacious by improving the Progression Free Survival (PFS) and Oveall Survival (OS) through controlled toxicity. Now the illustrated example can be extended to explore the impact of CEC for OS. This is a preliminary attempt towards MC to address some critical issues.

The role of adipocytokines and endothelial dysfunction with irritable bowel syndrome associated with obesity

The article contains the analysis of results concerning investigation of adipocytokines content and endothelial dysfunction in 97 patients with irritable bowel syndrome associated with obesity. The blood serum was examined for the content of adipocytokines (leptin, adiponectin, resistin), stable nitrogen monoxide metabolites (nitrites/nitrates), endothelin-1, intercellular adhesion molecule 1 (ICAM-1), and the number of circulating endothelial cells (CEC). Patients with IBS associated with obesity and prevailing diarrhea are found to develop a prominent imbalance of adipocytokines which is manifested in high levels of leptin and resisin and low level of adiponectin in the blood serum. Endothelial dysfunction evidenced by a high level of endothelin-1, the number of circulating endothelial cells, general NO and intercellular adhesion molecules 1, is characteristic for patients with IBS associated with obesity and prevailing diarrhea.

Development and Validation of a Random Forest Diagnostic Model of Acute Myocardial Infarction Based on Ferroptosis-Related Genes in Circulating Endothelial Cells.

The high incidence and mortality of acute myocardial infarction (MI) drastically threaten human life and health. In the past few decades, the rise of reperfusion therapy has significantly reduced the mortality rate, but the MI diagnosis is still by means of the identification of myocardial injury markers without highly specific biomarkers of microcirculation disorders. Ferroptosis is a novel reported type of programmed cell death, which plays an important role in cancer development. Maintaining iron homeostasis in cells is essential for heart function, and its role in the pathological process of ischemic organ damages remains unclear. Being quickly detected through blood tests, circulating endothelial cells (CECs) have the potential for early judgment of early microcirculation disorders. In order to explore the role of ferroptosis-related genes in the early diagnosis of acute MI, we relied on two data sets from the GEO database to first detect eight ferroptosis-related genes differentially expressed in CECs between the MI and healthy groups in this study. After comparing different supervised learning algorithms, we constructed a random forest diagnosis model for acute MI based on these ferroptosis-related genes with a compelling diagnostic performance in both the validation (AUC = 0.8550) and test set (AUC = 0.7308), respectively. These results suggest that the ferroptosis-related genes might play an important role in the early stage of MI and have the potential as specific diagnostic biomarkers for MI.

Daily Users of Marijuana with Sickle Cell Disease Report More Severe Pain Crisis Yet Have Similar Rates of Admission to Non-Users

▪Introduction: Pain in sickle cell disease (SCD) starts as episodes of recurrent pain and progresses to chronic pain with age. Pain is typically treated with opioids which are often insufficient. One reason for this may be that opioids likely do not effectively treat neuropathic or inflammatory pain in SCD. Cannabinoids, such as marijuana, alleviate neuropathic pain and reduce inflammation. Our SCD patients report marijuana improves their pain and studies in murine models of SCD show cannabinoids reduce mast cell activation, inflammation, and hyperalgesia. Medical marijuana is now legal in 28 states and 4 of them list SCD as a qualifying condition. However, there are minimal data on the use of marijuana in SCD. Thus, we performed a pilot study of adults with SCD who use daily marijuana. Our hypothesis was that daily marijuana users would have worse pain scores from non-daily users which drives their drug use. Our secondary hypotheses were that daily marijuana users may have decreased levels of opioid use, hospital utilization, quality of life measures (QoL) or inflammation.Methods: We conducted a cross-sectional pilot study of adults living with SCD who do and do not use daily marijuana. Subjects were enrolled at scheduled clinic visits, any subject not at their heathy baseline was excluded. Subjects were asked if they had used marijuana in the past 30 days, reasons for use, and rate of use. Subjects who reported daily use were compared to those who used less often or reported no marijuana use. Our primary outcome was pain as defined by Adult Sickle Cell Quality of Life measurement information system (ASCQ-Me) pain domains. ASCQ-Me has separate domains for pain crisis frequency, pain crisis severity, and 7-day pain interference which allows for measurement of both crisis pain and chronic pain. Our secondary outcomes were differences in hospital utilization, opioid use, QoL measures included in ASCQ-ME: sleep, stiffness, social impact and emotional impact, Patient Reported Outcome Measurement Information System domains for nausea and anxiety, and inflammation as demonstrated by WBC and platelet counts, C reactive protein (CRP), markers of mast cell and neutrophil activation and endothelial damage. Hospital utilization (total annual admissions and emergency room visits) was assessed by medical record review over a one-year period (1/1/2016 to 12/31/2016). Weekly opioid use was estimated by review of dispensed prescriptions using the Connecticut Prescription Monitoring Program converted into average weekly oral morphine equivalents. Flow cytometry was performed for levels of: neutrophils and activated neutrophils, monocytes, non-classical and inflammatory monocytes, circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). The R statistical software program was used to perform t-tests and Wilcoxon rank-sum tests where normality was not met to compare daily users to non-daily users. Significance was set at p<0.05.Results: We enrolled 29 subjects,16 (55%) of whom reported marijuana use in the past 30 days and of those 6 (21%) reported daily marijuana use. Pain relief was endorsed as a reason for marijuana use in 93% of users and 100% of daily users. There was no difference in mean age (32.0 SD 8.6 vs 32.4 SD 12.6 years p=1.0) or gender (% female: 67 vs 52% p=0.7) between groups. Daily marijuana users had higher pain crisis severity scores (61.7 vs 50.0 p=0.03). There was no significant difference in scores for pain crisis frequency, pain interference, or other QoL measures (Table). Daily users had similar median annual admissions and emergency room visits to non-daily users (1.0 (IQR 0.25-1.0) vs 2.0 (IQR 0 - 5.0) admissions p=0.3; 1.0 (IQR 0.25-1.75) vs 0 (IQR 0-1.5) ED visits p=0.6) and used similar doses of weekly opioids (274.5 [IQR 91.5-3447.9] vs 28.6 [IQR 3.03-135.6] mg weekly po morphine equivalents p=0.5). There were no differences in CRP, CBC, CECs or EPCs, or in levels of all neutrophils and monocytes types.Conclusion: In our pilot study, we found daily marijuana users reported more severe pain crisis, but had similar rates of hospital utilization and similar amounts of opioids dispensed. We posit that patients with more severe pain crisis may use daily marijuana to effectively treat their pain allowing them to have similar hospital and opioid utilization to those with less severe pain. Larger prospective studies should be performed to test these hypotheses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Angio-Phenotype of Pediatric Acute Lymphoblastic Leukemia and Pharmacodynamic Evaluation of the Antiangiogenic Activity of Corticosteroids.

Corticosteroids are essential in the treatment of pediatric acute lymphoblastic leukaemia (ALL). Corticosteroid anti-angiogenic activity is postulated but unproven.Objectives: To assess the kinetics of cellular and plasma angiogenic surrogate markers during 7-day prednisone pre-phase/induction therapy in the AIEOP ALL 2000 protocol.Methods: 17 diagnosed common ALL and 12 normal controls were enrolled. Total and viable circulating endothelial cells (CECs) and progenitors (CEPs), vascular endothelial growth factor receptors (VEGFR2, R3), endoglin (CD105), VECadherin (CD144) expression were analyzed by four color flow-cytometry on peripheral blood. Plasma circulating VEGF, VEGFRs and endoglin were analyzed by ELISA. Gene expression of VE-Cadherin, Nanog, RGS5, VEGFR2 and R3 were quantified by Real Time RT-PCR.Results: Total CECs (31 ±8cells/uL), viable CECs (12 ± 5 cells/uL) and CEPs (0.91 ±0.91 cells/uL) were found to be significantly higher in pediatric controls than in adult. At baseline patients demonstrated a statistically higher number of CECs (CD45− CD31+ CD146+ cells) and CEPs (CD133+ cells) p=0.01 and 0.03, compared to controls. CD105, VEGFR-3 and VEGFR-2 expression on leukemic blasts was 91%, 27% and 0% of patients, respectively.After 7 days of prednisone, a statistically significant reduction of total and viable CECs (p= 0.01) and CEPs ( p<0.0001) as well as endothelial cells expressing VEGFR2, R3, and CD105 (p=0.01, p=0.03, p=0.02) was observed. Plasma VEGFR2, VEGFR3, and endoglin showed a trend to decrease at day 8 and 33 but did not reach significance. A statistically significant reduction of blast was also observed.The pro-apototic effect of prednisone was greater on blast compared to lymphocytes.VEGFR-3 gene expression was significantly increased in patients at diagnosis compared to pediatric controls, and significantly reduced on day 8.Conclusions:Increase in CECs, CEPS confirm a pro-angiogenic activity at diagnosis in common ALL. The anti-leukemic effects of prednisone may be linked to its pro-apoptotic and anti-angiogenic activities. Total and viable CECs, CEPs, expression of endoglin and VEGFR3 on blast appear to be suitable angiogenic surrogate biomarkers.

The Vasculopathy of Juvenile Dermatomyositis: Endothelial Injury, Hypercoagulability, and Increased Arterial Stiffness.

Vasculopathy is considered central to the pathogenesis of juvenile dermatomyositis (DM) and is associated with severe extramuscular manifestations. We undertook this study to investigate the hypothesis that the vasculopathy of juvenile DM can be noninvasively tracked by examining biomarkers of endothelial injury, subclinical inflammation, hypercoagulability, and vascular arterial stiffness. The study population was a UK cohort of children with juvenile DM. Circulating endothelial cells (CECs) and microparticles (MPs) were identified using immunomagnetic bead extraction and flow cytometry, respectively. Plasma thrombin generation was determined using a fluorogenic assay. Cytokine and chemokine levels were measured by electrochemiluminescence. Arterial stiffness was assessed using pulse wave velocity (PWV). Results were expressed as the median and interquartile range (IQR), and statistical significance was assessed using nonparametric analyses. Ninety patients with juvenile DM and 79 healthy control subjects were included. The median age of the patients was 10.21 years (IQR 6.68-13.40), and the median disease duration was 1.63 years (IQR 0.28-4.66). CEC counts were higher in all patients with juvenile DM compared to controls (median 96 cells/ml [IQR (40-192] and 12 cells/ml [IQR 8-24], respectively; P < 0.0001). Circulating MP numbers were also significantly higher in patients with active juvenile DM compared to controls (median 204.7 × 103 /ml [IQR 87.9-412.6] and 44.3 × 103 /ml [IQR 15.0-249.1], respectively; P < 0.0001). MPs were predominantly of platelet and endothelial origin. Enhanced plasma thrombin generation was demonstrated in patients with active juvenile DM compared to those with inactive disease (P = 0.0003) and controls (P < 0.0001). Carotid-radial PWV adjusted for age was increased in patients with juvenile DM compared to controls (P = 0.003). We observed increased endothelial injury and increased levels of proinflammatory cytokines in patients with active juvenile DM. MP profiles reflected distinct disease activity status in juvenile DM and are markers of vascular pathology, platelet activation, and thrombotic propensity. Ongoing long-term vascular injury may result in increased arterial stiffness in patients with juvenile DM.

POS0424 DETERMINING CIRCULATING ENDOTHELIAL CELLS USING CELLSEARCH SYSTEM IN SYSTEMIC SCLEROSIS PATIENTS

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.