Publications

Summary Endothelial colony-forming cells (ECFCs) are progenitor cells committed to endothelial lineages and have robust vasculogenic properties. Mesenchymal stem cells (MSCs) have been described to support ECFC-mediated angiogenic processes in various matrices. However, MSC-ECFC interactions in hind limb ischemia (HLI) are largely unknown. Here we examined whether co-administration of ECFCs and MSCs bolsters vasculogenic activity in nude mice with HLI. In addition, as we have previously shown that endoglin is a key adhesion molecule, we evaluated its involvement in ECFC/MSC interaction. Foot perfusion increased on day 7 after ECFC injection and was even better at 14 days. Co-administration of MSCs significantly increased vessel density and foot perfusion on day 7 but the differences were no longer significant at day 14. Analysis of mouse and human CD31, and in situ hybridization of the human ALU sequence, showed enhanced capillary density in ECFC+MSC mice. When ECFCs were silenced for endoglin, coinjection with MSCs led to lower vessel density and foot perfusion at both 7 and 14 days (p<0.001). Endoglin silencing in ECFCs did not affect MSC differentiation into perivascular cells or other mesenchymal lineages. Endoglin silencing markedly inhibited ECFC adhesion to MSCs. Thus, MSCs, when combined with ECFCs, accelerate muscle recovery in a mouse model of hind limb ischemia, through an endoglindependent mechanism. Supplementary Material to this article is available online at www.thrombosis-online.com.

Abstract Upregulation of hypoxia-inducible transcription factor-1α (HIF-1α), through prolyl-hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF-1α were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow-derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF-1α and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF-1α or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF-1α. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1α and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies. Stem Cells 2014;32:231–243

P155 Pathomechanism of TSP/HAM is not yet clearly understood. Infected/activated T-cells infiltrating lesions are thought to be implicated in physiopathology. Using a model of interaction of glial cells with HTLV-I T-lymphocytes we previously observed an altered expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in astrocytes objectived by induction of MMP9 expression. This one was further detected in some cerebrospinal fluids from TSP/HAM patients. Objectives: The aim of the present study was to compare the expression of MMP9, TIMP1, TIMP3 and Neopterin level in the CSF from 32 TSP/HAM patients and 29 HTLV-I asymptomatic carriers (AC). Results: Using zymography, MMP9 was detected in 84% TSP/HAM versus 24% AC. Using Western-Blotting TIMP3 was detected in 75% TSP/HAM versus 15% AC. The more frequent detection of MMP9 (×3.5) and TIMP3 (×5) in the CSF from TSP/HAM patients presumably reflects intense cell remodelling and inflammation within CNS. As expected, neopterin level, a marker of cell-immune mediated inflammation, was higher in TSP/HAM (63% patients >10 nmol/L) than in AC (0% patients >10nmol/L). In TSP/HAM, inflammation revealed by high neopterin level (>10nmol/L) correlated the presence of MMP9 (94%) and TIMP3 (100%). Moreover, global examination of the HTLV-I positive group suggested that MMP9 in CSF may be a more discriminative marker than neopterin for TSP/HAM diagnosis since MMP9 positivity was found in 81% TSP/HAM while neopterin pathological level (>3 nmol/L) was found in 71% TSP/HAM. Conclusion: Since detection of MMP9 by zymography is less challenging than neopterin detection by HPLC and results conclusive, we propose MMP9 as a possible alternative marker helping in TSP/HAM diagnosis.

<b>Background:</b> IPF is associated to an important pulmonary vascular and tissular remodeling. Their mechanisms might involve the coagulation cascade through its role on matrix degradation. Microparticules (MP) are key fibrinolytic mediators so far unexplored in IPF. <b>Objectives:</b> To determine plasmatic levels of plasminogen activators (uPA and tPA) and inihibitor (PAI-1) in parallel of presence, levels and fibrinolytic activity of circulating MP. <b>Methods:</b> uPA, tPA and PAI-1 mRNAs in peripheral blood cells were quantitated using RT-PCR and plasmatic soluble levels by ELISA. MP cellular sub-populations were assessed using flow cytometry and their fibrinolytic activity through plasminogen activation kinetics (spectrophotometry) and zymography. <b>Results:</b> No difference was observed between IPF (n=54) and age-matched healthy controls (n=13) mRNAs of whole blood cells. In contrast, soluble uPA, tPA and PAI-1 in plasma were increased in IPF (p=0.0042, p=0.03 and 0.0017, respectively vs controls) as well as both endothelial and platelet-derived MP (respectively with p=0.0003 and 0.0014 vs controls). In addition, MP were active in generating plasmin (zymography) and this activity was increased vs controls (p=0.0042). <b>Conclusion:</b> Marked increases in circulating fibrinolytic mediators are present in IPF. Their potential contribution to fibrogenesis and vascular remodeling still needs to be determined. These data might add to the present hints to fibrogenesis and aberrant neoangiogenesis present in IPF.

Received: 19 July 2023 Accepted after revision: 31 August 2023 Accepted Manuscript online:01 September 2023

Introduction:A portable multi-parameter blood coagulation analysis device (EnzySystem) is being developed to improve the diagnosis and management of patients with haemophilia A (PwHA).It simultaneously measures factor VIII activity and thrombin generation in 100 μL of capillary whole blood within 1 h.This study surveys use case scenarios for the EnzySystem in Europe.Methods: Use-case analysis was performed with healthcare professionals (HCPs) and patients to determine their preferences regarding the use of the EnzySystem.Data was acquired using questionnaires and (online) interviews.Results: Most respondents (61%) were excited about the portable testing device.Both near-patient use (37%) and home-use (48%) were identified as attractive applications of such a device.The interest in (clinical) laboratory use (15%) was limited.In Western and Northern Europe, diagnosis and clinical laboratory testing are considered in place.Haemophilia treatment centres are accessible to all patients despite the great distances in some Northern European countries.PwHA would use the home-testing device to guide haemophilia A management in a (severe) bleeding situation and in unstable conditions.HCPs would prefer to gain experience with testing in a near-patient setting before advising patients to use the device at home.HCPs see added value of a near-patient device for use in remote areas as well as in emergency situations when laboratory testing is unavailable.80% of the HCPs would consider the portable testing device for their daily routine.In some Eastern European countries, the use of the device for diagnosis of haemophilia A is mentioned as not all PwHA have been diagnosed and haemophilia treatment centres are not accessible to all patients.Discussion/Conclusion: From the interim data analysis it can be concluded that the recommended use case scenario for Western and Northern European countries is the management of haemophilia A with a home-use device.Additionally, it was suggested to first raise awareness using the near-patient device.For some Eastern European countries, the near-patient device for diagnosis is put forward.

Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.