Background & Aims: We aimed to develop machine learning (ML) models for hepatocellular carcinoma (HCC) risk stratification in patients with cirrhosis and to test their ability to identify those with an annual HCC incidence >3%, for whom more intensive surveillance may be justified. Methods: Data from three prospective cohorts (ANRS CO12 CirVir, CO22 Hepather, APHP CIRRAL) were analyzed. All patients underwent semiannual ultrasound surveillance and were randomly split into training and validation sets. HCC incidence was evaluated using a competing risk framework. A single tree (ST) model was developed using conditional decision trees, while random forest (RF) models were built by aggregating 1,000 trees. A deep neural network (DNN)–based survival model was also applied. ML model performance was compared with established regression-based scores: aMAP (age–male–ALBI–platelets) and FASTRAK (FAST-MRI for HCC suRveillance in pAtients with high risK of liver cancer). Results: Among 4,867 patients with non-viral cirrhosis or resolved/controlled viral cirrhosis, 294 (9.2%) developed HCC over a median follow-up of 61.5 months (annual incidence: 1.99%). The ST model identified four key predictors, generating five distinct risk groups. These included patients with mildly impaired liver function or those with elevated GGT and low platelet counts. The RF and DNN approaches confirmed ST findings and delineated complex interactions among predictors. Performance metrics (C-index, Brier score, decision curve analysis) showed no significant advantage of ML models over aMAP and FASTRAK. Calibration was consistent across models. ML models identified higher proportions of patients with an annual HCC incidence >3% (ST 44%; DNN 37%; RF 30%) compared with aMAP (36%) and FASTRAK (29%). Conclusions: ML-based algorithms did not outperform traditional risk scores but provided novel insights into variable interactions and helped identify clinically relevant patient subgroups with differing HCC risk profiles. Impact and implications: Accurate stratification of hepatocellular carcinoma risk in cirrhosis is essential to optimize surveillance strategies, and this study provides a scientific rationale for exploring machine learning approaches to capture complex, non-linear interactions among clinical variables beyond traditional regression models. Although machine learning did not improve predictive performance over established scores, it revealed clinically meaningful risk subgroups defined by liver function, platelet count, and GGT, underscoring its value as an interpretative and hypothesis-generating tool. These results are particularly relevant for hepatologists and clinical researchers seeking to refine risk-adapted surveillance and to inform the design of future models or trials.

BACKGROUND & AIMS:Fibrolamellar carcinoma (FLC) is a rare primary liver cancer that predominantly affects young patients with normal known serum tumor biomarkers. An observation of elevated procalcitonin (PCT) in one patient prompted us to investigate PCT as a biomarker in a larger FLC cohort. METHODS:We measured serum PCT levels in 34 samples from 18 patients with FLC and in 64 patients with hepatocellular carcinoma (HCC), 24 with cholangiocarcinoma (CCA), and 20 with cirrhosis. Using RNA sequencing, we analyzed expression of CALCA, which encodes PCT, in 27 FLC tumors, 331 HCC tumors, 39 CCA tumors, 71 hepatoblastomas, 34 hepatocellular adenomas, and 55 non-tumor liver samples. Spatial transcriptomics was performed on three FLC tumors, and PCT immunohistochemistry was performed on 13 FLC tumors and 34 other primary or secondary liver cancers. RESULTS:In eight patients with FLC from the European cohort, median serum PCT was significantly elevated (55.2 μg/L) compared to patients with HCC (0.14 μg/L), CCA (0.16 μg/L), and cirrhosis (0.11 μg/L; p = 0.0005). These findings were validated in a US cohort of 10 patients with FLC compared to HCC and CCA (p = 0.0002). Across these cohorts, elevated serum PCT was observed in 83% of FLC cases vs. 3% of HCC and CCA cases (p <0.0001). In four patients, changes in serum PCT levels correlated with radiologic response according to RECIST 1.1. RNA sequencing demonstrated significant overexpression of CALCA in FLC compared to other primary liver tumors (p <0.0001), and spatial transcriptomics localized CALCA expression specifically to tumor cells. Immunohistochemistry confirmed PCT overexpression in 77% of FLC tumors but not in other liver cancers. CONCLUSION:Procalcitonin is a sensitive and specific serum biomarker for FLC among primary liver cancers, with potential utility for diagnosis and monitoring of treatment response. IMPACT AND IMPLICATIONS:This study is justified by the lack of reliable serum biomarkers for fibrolamellar carcinoma (FLC) and demonstrates that procalcitonin is specifically overexpressed by FLC tumor cells and detectable in the blood of the patients with FLC. These findings are important for clinicians and researchers, as they identify a readily available biomarker that may facilitate diagnosis, improve disease monitoring, and support clinical trial design in a rare cancer that predominantly affects young patients. In clinical practice, serum PCT measurement could be incorporated as a non-invasive adjunct to imaging for the diagnosis and longitudinal assessment of FLC, although prospective studies in larger and more diverse cohorts are needed to refine diagnostic cut-offs and confirm specificity.

Acute mesenteric ischemia (AMI) is a severe and life-threatening condition with a mortality rate of up to 50%. Its treatment, which depends on the etiology, focuses on preserving intestinal viability through prompt restoration of blood flow. Although it is well established that intestinal ischemia-reperfusion results in significant local tissue damage, it is less recognized that it can also lead to remote tissue injuries, particularly in the lungs. Acute lung injury following intestinal ischemia-reperfusion is a severe complication that affects nearly 30% of patients with acute mesenteric ischemia and significantly contributes to mortality. The underlying pathophysiology of this injury is complex and multifactorial, yet it remains poorly understood. Neutrophil-endothelial interactions, regulated by both systemic and local mediators, play a pivotal role. Among the contributing factors, the intestinal ischemia-reperfusion process itself appears to be the most significant. Reperfusion of the ischemic intestine allows the release of mediators generated during ischemia into the systemic circulation. This triggers a cascade of biological events, including elevated levels of proinflammatory cytokines, overproduction of reactive oxygen species (ROS), nitric oxide imbalance, neutrophil activation, mitochondrial damage, and the initiation of cell death pathways. Here, we review the current knowledge on the various pathophysiological pathways explored in clinical and animal models of acute lung injury induced by intestinal ischemia-reperfusion, with the aim of providing therapeutic insights.

Introduction:Fibrolamellar carcinoma (FLC) is a rare primary liver cancer that predominantly affects young patients with normal known serum tumor biomarkers (alpha-fetoprotein (AFP) and CA19-9). An observation of a markedly elevated procalcitonin (PCT) in one patient prompted us to investigate the potential role of PCT as a biomarker in a larger cohort of FLC. Methods:We measured serum PCT levels in 34 samples from 18 patients with metastatic FLC and in 64 patients with hepatocellular carcinoma (HCC), 24 with cholangiocarcinoma (CCA), and 20 with cirrhosis. Using RNA sequencing, we analyzed CALCA expression, the gene encoding PCT, in 27 FLC tumors, 331 HCC tumors, 39 CCA tumors, 71 hepatoblastomas, 34 hepatocellular adenomas, and 55 non-tumor liver samples. Spatial transcriptomics was performed on three FLC and PCT immunohistochemistry was conducted on 13 FLC and 34 other primary or secondary liver cancers. Results:In 8 FLC from the European cohort, median serum PCT was significantly elevated (55.2 μg/l) compared to patients with HCC (0.14 μg/l), CCA (0.16 μg/l), and cirrhosis (0.11 μg/l; P=0.0005). These findings were independently validated in a U.S. cohort of 10 FLC patients compared to HCC and CCA (P=0.0002). Across these cohorts, elevated serum PCT was observed in 83% of FLC cases versus 3% of HCC and CCA cases (P<0.0001). In four patients with longitudinal measurements, changes in PCT levels correlated with radiologic response according to RECIST 1.1. RNA sequencing demonstrated significant overexpression of CALCA in FLC compared to other primary liver tumors (P<0.0001), and spatial transcriptomics localized CALCA expression specifically to tumor cells. Immunohistochemistry confirmed PCT overexpression in 77% of FLC but not in other liver cancers. Conclusion:Procalcitonin is a sensitive and specific biomarker for FLC at both the serum and tumor levels among primary liver cancers, with potential utility in diagnosis and monitoring of treatment response.

CONTEXT We recently reported that the presence of glutamic acid decarboxylase antibodies (GADA) was not associated with large-for-gestational-age infants in women with hyperglycemia in pregnancy (HIP). OBJECTIVE We explored the association between the presence of GADA and other HIP-related adverse pregnancy outcomes. METHODS This observational prospective study, conducted at a university hospital in a suburb of Paris, France, included 1182 consecutive women with HIP measured for GADA at HIP care initiation between 2012 and 2017. Post hoc analyses for outcomes included gestational weight gain, insulin therapy, cesarean delivery, hypertensive disorders, small-for-gestational-age infant, prematurity, and neonatal hypoglycemia. RESULTS Of the 1182 women studied, 87 (7.4%) had positive (≥ 1 IU/mL) GADA. Although socioeconomic, clinical, and biological characteristics were similar across women in the positive and negative GADA groups, higher fasting plasma glucose values during early HIP screening were observed in the former (5.5 ± 1.5 vs 5.2 ± 0.7 mmol/L respectively, P < .001). At HIP care initiation, fructosamine levels were higher in women with positive GADA (208 ± 23 vs 200 ± 18 µmol/L; P < .05). In the homeostatic model assessment, insulin resistance (HOMA-IR) and beta secretion (HOMA-B) rates were similar in both groups. Gestational weight gain and the rates of all adverse outcomes were similar in both groups except for cesarean delivery (18.4 and 27.3% for positive and negative GADA, respectively; adjusted odds ratio 0.49 [95% CI, 0.26-0.92], P = .026). CONCLUSION Universal measurement of GADA in women with HIP highlighted that 7.4% had positive GADA. No association was observed between GADA and HIP-related adverse pregnancy outcomes, except a lower risk of cesarean delivery.

Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H2O2-assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (Mn and Mw) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H2O2-based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

BACKGROUND:Fast acting insulin analogues are known to improve arterial stiffness. The combination of metformin with insulin represents a widely used therapeutic strategy in diabetes. We hypothesized that insulin treatment in patients with type 2 diabetes (T2D) with long-acting, fast-acting or basal bolus insulin as an add-on to metformin would provide additional improvement of arterial stiffness.METHODS:The INSUlin Regimens and VASCular Functions (INSUVASC) study is a pilot, randomized, open label three-arms study that included 42 patients with type 2 diabetes (T2D) in primary prevention, after a failure to oral antidiabetic agents. Arterial stiffness measurements were performed at fasting and after a standardized breakfast. During the first visit (V1) pre-randomization, participants took only metformin to perform the tests. The same tests were repeated after 4 weeks of insulin treatment during the second visit (V2).RESULTS:Data were available for final analysis in 40 patients, with a mean age of 53.6±9.7 years and a mean duration of diabetes of 10.6±5.6 years. Twenty-one were females (52.5%), hypertension and dyslipidemia were present in 18 (45%) and 17 patients (42.5%), respectively. After insulin treatment, the metabolic control was associated to a decrease in oxidative stress and improvement of endothelial functions, with a post prandial diastole duration increased and a decrease of the peripheral arterial stiffness, with a better post prandial pulse pressure ratio and ejection duration after insulin. In hypertensive patients, insulin treatment provided positive effects by decreasing the pulse wave velocity and improving reflection time.CONCLUSIONS:A short time treatment by insulin in addition to metformin improved myocardial perfusion. Moreover, insulin treatment in hypertensive patients provides a better hemodynamic profile in large arteries.

PDF file - 194K, Subgroup analysis according to the Levels of serum proteoglycans and VEGF.

PDF file - 72K, Clinical and biological variables associated with overall mortality and recurrence-free survival in patients with early HCC based on Cox univariable and multivariable models.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 μM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

BACKGROUND:The combination of atezolizumab and bevacizumab (AtezoBev) is the current first-line treatment for patients with hepatocellular carcinoma (HCC). Our aim was to evaluate the prognostic role of alpha-foetoprotein (AFP) early response and its combination with albumin-bilirubin (ALBI) in these patients.METHODS:Patients with HCC under AtezoBev with AFP > 20 ng/ml were included in three centres. The optimal threshold of AFP variation after 3 weeks of treatment was identified for overall survival (OS) and radiological response (RR) using RECIST 1.1 and mRECIST and its ability to predict progression-free survival (PFS) and OS was tested using univariate and multivariate analysis in derivation and validation cohorts.RESULTS:Seventy-five patients with AFP values >20 ng/ml were included. Fifty-eight patients were male with a median age of 63.5 years; 73% had cirrhosis and HCC stage was classified as BCLC B (18.7%) or C (81.3%). In the derivation cohort (n = 38), a decline in AFP ≥ 20% at 3 weeks (AFP early response) was associated with RR using mRECIST criteria (OR: 13.09 95% CI: 1.44-19.34 p = .02), PFS (HR: 0.42; 95% CI: 0.19-0.93, p = .03) and OS (HR: 0.35; 95% CI: 0.15-0.83, p = .01). AFP early response was confirmed as predictor of RR (p = .02 for mRECIST) and OS (p = .03) in the validation cohort (n= 37). In the whole cohort, the combination of ALBI and AFP early response was significantly associated with OS (p = .046) and PFS (p = .012) with a poor prognosis in patients belonging to the ALBI2-AFP non-responders class.CONCLUSION:AFP early response at 3 weeks predicts oncological outcomes in HCC patients treated with AtezoBev and combination with ALBI grade refines prognostic discrimination.

BACKGROUND AND AIMS:Non-O blood group promotes deep vein thrombosis and liver fibrosis in both general population and hepatitis C. We aimed to evaluate the influence of Non-O group on the outcome of Child-Pugh A cirrhotic patients.METHODS:We used two prospective cohorts of Child-Pugh A cirrhosis due to either alcohol or viral hepatitis. Primary end point was the cumulated incidence of 'Decompensation' at 3 years, defined as the occurrence of ascites , hydrothorax, encephalopathy, gastrointestinal bleeding related to portal hypertension, or bilirubin >45 μmol/L. Secondary end points were the cumulated incidences of (1) 'Disease Progression' including a « decompensation» or « the occurrence of one or more parameters » among: prothrombin time (PT) <45%, albumin <28 g/L, Child-Pugh worsening (B or C vs A or B, C vs B), hepatorenal syndrome, and hepato-pulmonary syndrome, (2) other events such as non-malignant portal vein thrombosis (nmPVT), and (3) overall survival.RESULTS:Patients (n = 1789; 59.9% Non-O group; 40.1% group O) were followed during a median of 65.4 months. At 3 years cumulated incidence of Decompensation was 8.3% in Non-O group and 7.2% in group O (P = .27). Cumulated incidence of Disease Progression was 20.7% in Non-O group and 18.9% in group O (P = .26). Cumulated incidence of nmPVT was 2.7% in Non-O group and 2.8% in group O (P = .05). At 3 years overall survival was 92.4% in Non-O group and 93.4% in group O (P = 1).CONCLUSION:Non-O group does not influence disease outcome in Child-Pugh A cirrhotic patients. Clinicals trial number NCT03342170.

BACKGROUND & AIMS:Identifying individuals at higher risk of developing hepatocellular carcinoma (HCC) is pivotal to improve the performance of surveillance strategies. Herein, we aimed to evaluate the ability of single nucleotide polymorphisms (SNPs) to refine HCC risk stratification. METHODS:Six SNPs in PNPLA3, TM6SF2, HSD17B13, APOE, and MBOAT7 affecting lipid turnover and one variant involved in the Wnt-β-catenin pathway (WNT3A-WNT9A rs708113) were assessed in patients with alcohol-related and/or HCV-cured cirrhosis included in HCC surveillance programmes (prospective CirVir and CIRRAL cohorts). Their prognostic value for HCC occurrence was assessed using Fine-Gray models combined into a 7-SNP genetic risk score (GRS). The predictive ability of two clinical scores (a routine non-genetic model determined by multivariate analysis and the external aMAP score) with/without the GRS was evaluated by C-indices. The standardised net benefit was derived from decision curves. RESULTS:Among 1,145 patients, 86 (7.5%) developed HCC after 43.7 months. PNPLA3 and WNT3A-WNT9A variants were independently associated with HCC occurrence. The GRS stratified the population into three groups with progressively increased 5-year HCC incidence (Group 1 [n = 627, 5.4%], Group 2 [n = 276, 10.7%], and Group 3 [n = 242, 15.3%]; p <0.001). The multivariate model identified age, male sex, diabetes, platelet count, gamma-glutamyltransferase levels, albuminemia and the GRS as independent risk factors. The clinical model performance for 5-year HCC prediction was similar to that of the aMAP score (C-Index 0.769). The addition of the GRS to both scores modestly improved their performance (C-Indices of 0.786 and 0.783, respectively). This finding was confirmed by decision curve analyses showing only fair clinical net benefit. CONCLUSIONS:Patients with cirrhosis can be stratified into HCC risk classes by variants affecting lipid turnover and the Wnt-β-catenin pathway. The incorporation of this genetic information modestly improves the performance of clinical scores. IMPACT AND IMPLICATIONS:The identification of patients at higher risk of developing liver cancer is pivotal to improve the performance of surveillance. Risk assessment can be achieved by combining several clinical and biological parameters used in routine practice. The addition of patients' genetic characteristics can modestly improve this prediction and will ultimately pave the way for precision medicine in patients eligible for HCC surveillance, allowing physicians to trigger personalised screening strategies.

Aim. Recent studies have shown that women with hyperglycaemia in pregnancy and insulin resistance have a greater risk of adverse pregnancy outcomes than women with normoglycaemic pregnancies. This study aimed to determine adverse pregnancy outcomes of women with hyperglycaemia in pregnancy only as a function of insulin resistance. Methods. From a prospective cohort study, we included 1,423 women with hyperglycaemia in pregnancy whose insulin resistance was evaluated using homoeostatic model assessment for insulin resistance (HOMA-IR) when care was first provided for this condition. We compared the adverse pregnancy outcomes for different tertiles of HOMA-IR (intertertile range 1.9 and 3.3). Results. Increasing HOMA-IR tertiles were positively associated with the rate of insulin therapy (tertile 1, 2 and 3: 32.7, 47.0 and 58.7%, P < 0.0001), caesarean section (23.7, 26.0 and 32.2%, respectively, P < 0.01), gestational hypertension (1.3, 2.8 and 5.4% respectively, P < 0.01), preeclampsia (1.5, 2.8 and 4.5% respectively, P < 0.05), large-for-gestational-age infant (13.3, 10.4 and 17.6% respectively, P < 0.05), and neonatal hypoglycaemia (0.8, 1.5 and 3.2% respectively, P < 0.05). Women in the 3rd HOMA-IR tertile were more likely to have insulin therapy (odds ratio 2.09 (95% interval confidence 1.61-2.71)), hypertensive disorders (2.26 (1.42 -3.36)), and large-for-gestational-age infant (1.42 (1.01-1.99)) than those in the 1st and 2nd tertiles combined in multivariable logistic regression analyses adjusted for gestational age at HOMA-IR measurement, glycaemic status, age, body mass index, family history of diabetes, parity and ethnicity. Conclusion. Despite suitable care and increased rates of insulin therapy during pregnancy, higher insulin resistance in women with hyperglycaemia in pregnancy was associated with a greater risk of adverse pregnancy outcomes. (C) 2022 Elsevier Masson SAS. All rights reserved.

Purpose:Sulfatase 2 (SULF2) is an enzyme related to heparan sulfate modifications. Its expression, as for some heparan sulfate proteoglycans expression, has been linked to hepatocellular carcinoma (HCC) at mRNA level and immunohistochemistry staining on biopsy samples. This study aims to evaluate the prognostic value of serum levels of SULF2 in patients with alcoholic cirrhosis with or without HCC.Patients and Methods:Two hundred and eighty-seven patients with alcoholic cirrhosis were enrolled in this study: 164 without HCC, 57 with early HCC, and 66 with advanced HCC at inclusion. We analyzed the association between SULF2 serum levels and prognosis using Kaplan-Meier method and univariate and multivariate analysis using a Cox model.Results:Child-Pugh C Patients have higher serum levels of SULF2 than Child-Pugh A patients. Serum levels of SULF2 were also higher in patients with advanced HCC compared with the other groups. In patients with advanced HCC, high serum levels of SULF2 were associated with less favorable overall survival. Combination of SULF2 with Glypican 3 (GPC3) and Syndecan 1 (SDC1) serum levels enhanced the ability to discriminate worst prognostic in advanced HCC.Conclusion:SULF2 along with GPC3 and SDC1 serum levels have been shown to be associated with a prognostic value in advanced HCC.

Protein accumulation is the hallmark of various neuronal, muscular, and other human disorders. It is also often seen in the liver as a major protein-secretory organ. For example, aggregation of mutated alpha1-antitrypsin (AAT), referred to as PiZ, is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is found in chronic hepatitis B (CHB) infection. We investigated the interaction of both proteotoxic stresses in humans and mice. Animals overexpressing both PiZ and HBs (HBs-PiZ mice) had greater liver injury, steatosis, and fibrosis. Later they exhibited higher hepatocellular carcinoma load and a more aggressive tumor subtype. Although PiZ and HBs displayed differing solubility properties and distinct distribution patterns, HBs-PiZ animals manifested retention of AAT/HBs in the degradatory pathway and a marked accumulation of the autophagy adaptor p62. Isolation of p62-containing particles revealed retained HBs/AAT and the lipophagy adapter perilipin-2. p62 build-up led to activation of the p62-Nrf2 axis and emergence of reactive oxygen species. Our results demonstrate that the simultaneous presence of two prevalent proteotoxic stresses promotes the development of liver injury due to protein retention and activation of the p62-Nrf2 axis. In humans, the PiZ variant was over-represented in CHB patients with advanced liver fibrosis (unadjusted odds ratio = 9.92 [1.15-85.39]). Current siRNA approaches targeting HBs/AAT should be considered for these individuals. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Background and Aims: Ischemia refers to reduction of blood flow and oxygen in tissue due to the obstruction of vessels and it can lead to necrosis. Pro-angiogenic treatment is a potential approach for ischemia therapy. In angiogenesis processes, the chemokines (CXCL12, CCL5) binds to their specific receptors but also to co-receptors that are proteoglycans, containing the "core protein" with covalently attached glycosaminoglycan chains (GAG).These bindings lead to stimulation of local new blood vessels formation particularly via induction of endothelial cell proliferation and migration. However, the GAG pro-angiogenic mechanism of action is still unknown. In this context, we hypothesized that GAG are involved in the modulation of CXCL12-induced angiogenesis.

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro‐angiogenic agents. We analysed the miR‐126/CXCL12 axis and compared impacts of both miR‐126‐3p and miR‐126‐5p strands effects in CXCL12‐induced angiogenesis. Indeed, the two strands of miR‐126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D‐angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR‐126‐3p/‐5p or antimiR‐126‐3p/‐5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro‐angiogenic effects in vitro and ex vivo associated with overexpression of miR‐126‐3p in HUVEC and rat's aortas. Second, we showed that 2D‐angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR‐126‐3p inhibition. Finally, we observed that SPRED‐1 (one of miR‐126‐3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro‐angiogenic potential in vitro. Our results proved for the first time: 1‐the role of CXCL12 in modulation of miR‐126 expression; 2‐the involvement of miR‐126 in CXCL12 pro‐angiogenic effects; 3‐the involvement of SPRED‐1 in angiogenesis induced by miR‐126/CXCL12 axis.