Novel biomarkers are needed to improve current imperfect risk prediction models for cancer-associated thrombosis (CAT). We recently identified an RNA-sequencing profile that associates with CAT in... Show moreNovel biomarkers are needed to improve current imperfect risk prediction models for cancer-associated thrombosis (CAT). We recently identified an RNA-sequencing profile that associates with CAT in colorectal cancer (CRC) patients, with REG4, SPINK4, and SERPINA1 as the top-3 upregulated genes at mRNA level. In the current study, we investigated whether protein expression of REG4, SPINK4 and alpha-1 antitrypsin (A1AT, encoded by SERPINA1) in the tumor associated with CAT in an independent cohort of CRC patients. From 418 patients with resected CRC, 18 patients who developed CAT were age, sex, and tumor stage-matched to 18 CRC patients without CAT. Protein expression was detected by immunohistochemical staining and scored blindly by assessing the H-score (percentage positive cells*scoring intensity). The association with CAT was assessed by means of logistic regression, using patients with an H-score below 33 as reference group. The odds ratios (ORs) for developing CAT for patients with A1AThigh, REG4high, SPINK4high tumors were 3.5 (95%CI 0.8–14.5), 2.0 (95%CI 0.5–7.6) and 2.0 (95%CI 0.5–7.4) when compared to A1ATlow, REG4low, SPINK4low, respectively. The OR was increased to 24.0 (95%CI 1.1–505.1) when two proteins were combined (A1AThigh/REG4high). This nested case–control study shows that combined protein expression of A1AT and REG4 associate with CAT in patients with colorectal cancer. Therefore, REG4/A1AT are potential biomarkers to improve the identification of patients with CRC who may benefit from thromboprophylaxis. Show less
Kapteijn, M.Y.; Zwaan, S.; Linden, E. ter; Laghmani, E.; Akker, R.F.P. van den; Rondon, A.M.R.; ... ; Buijs, J.T. 2023
Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of... Show moreGlioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy. Show less
Background Colorectal cancer patients have an increased risk of developing venous thromboembolism (VTE), resulting in increased morbidity and mortality. Because the exact mechanism is yet unknown,... Show moreBackground Colorectal cancer patients have an increased risk of developing venous thromboembolism (VTE), resulting in increased morbidity and mortality. Because the exact mechanism is yet unknown, risk prediction is still challenging; therefore, new biomarkers are needed. MicroRNAs (miRNAs) are small, relatively stable RNAs, that regulate a variety of cellular processes, and are easily measured in body fluids. Objective The aim of this study was to identify novel tumor-expressed miRNAs associated with VTE. Methods In a cohort of 418 colorectal cancer patients diagnosed between 2001 and 2015 at the Leiden University Medical Center, 23 patients (5.5%) developed VTE 1 year before or after cancer diagnosis. Based on availability of frozen tumor material, tumor cells of 17 patients with VTE and 18 patients without VTE were isolated using laser capture microdissection and subsequently analyzed on the Illumina sequencing platform NovaSeq600 using 150-bp paired-end sequencing. Cases and controls were matched on age, sex, tumor stage, and grade. Differential miRNA expression was analyzed using edgeR. Results A total of 547 miRNAs were detected. Applying a 1.5-fold difference and false discovery rate of <0.1, 19 tumor-miRNAs were differentially regulated in VTE cases versus controls, with hsa-miR-3652, hsa-miR-92b-5p, and hsa-miR-10,394-5p as most significantly downregulated. Seven of the 19 identified miRNAs were predicted to regulate the gonadotropin-releasing hormone receptor pathway. Conclusion We identified 19 differentially regulated tumor-expressed miRNAs in colorectal cancer-associated VTE, which may provide insights into the biological mechanism and in the future might have potential to serve as novel, predictive biomarkers. Show less
EssentialsFactor Xa (FXa)-targeting direct oral anticoagulants (DOACs) reduce venous thromboembolism (VTE) The effects of FXa-targeting DOACs on cancer progression remain to be studied In xenograft... Show moreEssentialsFactor Xa (FXa)-targeting direct oral anticoagulants (DOACs) reduce venous thromboembolism (VTE) The effects of FXa-targeting DOACs on cancer progression remain to be studied In xenograft models, a FXa-targeting DOAC did not inhibit breast cancer growth and metastasis A thrombin-targeting DOAC, dabigatran, also did not inhibit breast cancer growth and metastasis Background Factor Xa-targeting DOACs were recently found to reduce recurrent VTE efficiently in cancer patients when compared to the standard treatment with low-molecular-weight heparins (LMWHs). While the anticancer effects of LMWHs have been extensively studied in preclinical cancer models, the effects of FXa-targeting DOACs on cancer progression remain to be studied. Objective We investigated whether the FXa-targeting DOAC rivaroxaban and the thrombin-targeting DOAC dabigatran etexilate (DE) affected human breast cancer growth and metastasis in orthotopic xenograft models. Methods/results Mice that were put on a custom-made chow diet supplemented with rivaroxaban (0.4 or 1.0 mg/g diet) or dabigatran etexilate (DE) (10 mg/g diet) showed prolonged ex vivo coagulation times (prothrombin time [PT] and activated partial thromboplastin time [aPTT] assay, respectively). However, rivaroxaban and DE did not inhibit MDA-MB-231 tumor growth and metastasis formation in lungs or livers of 7-week-old fully immunodeficient NOD/SCID/x1b4;(-/-)(C) (NSG) mice. Comparable data were obtained for rivaroxaban-treated mice when using NOD-SCID mice. Rivaroxaban and DE treatment also did not significantly inhibit tumor growth and metastasis formation when using another human triple negative breast cancer (TNBC) cell line (HCC1806) in NOD-SCID mice. The FXa and thrombin-induced gene expression of the downstream target CXCL8 in both cell lines, but FXa and thrombin, did not significantly stimulate migration, proliferation, or stemness in vitro. Conclusion Although effectively inhibiting coagulation, the DOACs rivaroxaban and DE did not inhibit orthotopic growth and metastasis of human TNBC. It remains to be investigated whether DOACs exert antitumorigenic effects in other types of cancer. Show less
Factor Xa‐targeting DOACs were recently found to reduce recurrentVTE efficiently in cancer patients when compared to the standard treatment withlow‐molecular‐weight heparins (LMWHs). While the... Show moreFactor Xa‐targeting DOACs were recently found to reduce recurrentVTE efficiently in cancer patients when compared to the standard treatment withlow‐molecular‐weight heparins (LMWHs). While the anticancer effects of LMWHshave been extensively studied in preclinical cancer models, the effects of FXa‐targetingDOACs on cancer progression remain to be studied.We investigated whether the FXa‐targeting DOAC rivaroxaban and thethrombin‐targeting DOAC dabigatran etexilate (DE) affected human breast cancergrowth and metastasis in orthotopic xenograft models.Mice that were put on a custom‐made chow diet supplementedwith rivaroxaban (0.4 or 1.0 mg/g diet) or dabigatran etexilate (DE) (10 mg/g diet)showed prolonged ex vivo coagulation times (prothrombin time [PT] and activatedpartial thromboplastin time [aPTT] assay, respectively). However, rivaroxabanand DE did not inhibit MDA‐MB‐231 tumor growth and metastasis formationin lungs or livers of 7‐week‐old fully immunodeficient NOD/SCID/ƴC−/− (NSG) mice.Comparable data were obtained for rivaroxaban‐treated mice when using NOD‐SCIDmice. Rivaroxaban and DE treatment also did not significantly inhibit tumor growthand metastasis formation when using another human triple negative breast cancer(TNBC) cell line (HCC1806) in NOD‐SCID mice. The FXa and thrombin‐induced geneexpression of the downstream target CXCL8 in both cell lines, but FXa and thrombin,did not significantly stimulate migration, proliferation, or stemness in vitro.Although effectively inhibiting coagulation, the DOACs rivaroxaban andDE did not inhibit orthotopic growth and metastasis of human TNBC. It remains to beinvestigated whether DOACs exert antitumorigenic effects in other types of cancer. Show less
Heestermans, M.; Salloum-Asfar, S.; Streef, T.; Laghmani, E.; Salvatori, D.; Luken, B.M.; ... ; Vlijmen, B.J.M. van 2019
Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in... Show moreTissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibodymediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus. Show less
Chen, X.; Orriols, M.; Laghmani, E.; Hoogeboom, A.; Hogen-Esch, A.; Hiemstra, P.; ... ; Wagenaar, G. 2017
Rationale Apelin, a potent vasodilator and angiogenic factor, may be a novel therapeutic agent in neonatal chronic lung disease, including bronchopulmonary dysplasia Objectives To determine the... Show moreRationale Apelin, a potent vasodilator and angiogenic factor, may be a novel therapeutic agent in neonatal chronic lung disease, including bronchopulmonary dysplasia Objectives To determine the beneficial effect of apelin in neonatal rats with hyperoxia induced lung injury, a model for premature infants with bronchopulmonary clysplasia Methods The cardiopulmonary effects of apelin treatment (62 mu g/kg/d) were studied in neonatal rats by exposure to 100% oxygen, using two treatment strategies early concurrent treatment during con tinuous exposure to hyperoxia for 10 days and late treatment and recovery in which treatment was started on Day 6 after hyperoxic injury for 9 days and continued during the 9 day recovery period We investigated in both models the role of the nitric oxide cyclic guanosine monophosphate (cGMP) pathway in apelin treatment by specific inhibition of the nitric oxide synthase activity with N-omega nitro L arginine methyl ester (L NAME, 25 mg/kg/d) Measurements and Main Results Parameters investigated include survival, lung and heart histopathology, pulmonary fibrin deposition and inflammation, alveolar vascular leakage, lung cGMP levels, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue Prophylactic treatment with apelin improved alveolarization and angiogenesis increased lung cGMP levels, and reduced pulmonary fibrin deposition, inflammation, septum thickness, arteriolar wall thickness, and right ventricular hypertrophy These beneficial effects were completely absent in the presence of c NAME In the injury recovery model apelin also improved alveolarization and angiogenesis, reduced arteriolar wall thickness, and attenuated right ventricular hypertrophy Conclusions. Apelin reduces pulmonary inflammation, fibrin deposition, and right ventricular hypertrophy, and partially restores alveolarization in rat pups with neonatal hyperoxic lung Injury via a nitric oxide synthase-dependent mechanism Show less
