Vascular Dysfunction in Paediatric IBD
Condition: Inflammatory Bowel Disease; Crohn Disease; Ulcerative Colitis
MeSH terms: Crohn Disease; Intestinal Diseases; Inflammatory Bowel Diseases
Study type: Observational

Summary: 
      Inflammatory Bowel Diseases (IBD) is a group of relapsing and remitting gut inflammatory
      conditions acquired due to genetic susceptibility and/or environmental triggers. The disease
      manifestations are being increasingly seen in young children and the life-long debilitation
      has a severe effect on quality of life. Limited evidence suggests, although rare, in some
      young IBD individuals vascular complications may ensue. This leads to increased risk of
      vascular problems such as thrombosis, arterial disease and stroke.

      In the present project we aim to study and highlight potential vascular changes in young
      Inflammatory Bowel Disease (IBD) patients and compare these changes with age and gender
      matched controls. Vasculature will be measured in multiple ways including blood analysis in
      the laboratory and non-invasive, physiological measures of arterial health (e.g. ultrasound
      arterial scan). Our overall goal is to identify biomarkers indicative of increased risk of
      vascular dysfunction as this will open new avenues for early therapeutic intervention.
    

Detailed description: 
      Plan of Investigation:

      Patients: 130 children and adolescents (8-21y) with an expected ratio of 60% (n=78) Crohn's
      disease (CD), 35% (n=45) Ulcerative colitis (UC) 5% (n=6) Indeterminate colitis (IC). 78 age
      and sex-matched controls will be investigated. Sample size calculations are based on
      Circulating Endothelial Cells (CECs) as the primary end-point, as suggested by an on-going
      study by one of the co-applicants, on healthy children and children with Kawasaki disease
      (Brogan P et al, ref published).

      40 subjects/ group are required to detect a doubling average of CECs in CD and UC vs. control
      with 90% power, significance 0.05 and this should be achievable by our initial recruitment.
      Non-normality and the need to use non-parametric or transform prior to analysis, increases
      the number to 47; hence we will aim to recruit 50 to the UC group. This will provide adequate
      power, and is feasible based on the clinical cohort available to us.

      This patient cohort is an appropriate candidate group for the present investigation as

        1. Evidence indicates that vascular changes that occur between 10-20 years of age are a
           critical determinant of future vasculature health;

        2. Initiation into smoking habits is most likely in teenage years and

        3. Other established traditional risk factors for atherosclerosis (such as hypertension,
           type II diabetes, or even fully established atherosclerotic disease) that would act as
           confounding variables, are not yet present in adolescence.

      Data will be collated onto a de-identified excel sheet and will include age, sex, age at
      diagnosis, coronary artery status at presentation, growth, body mass index, blood pressure,
      family history of CVD, and smoking status.

      Aim 1: Do Children with IBD have evidence of a MP mediated prothrombotic tendency? MPs will
      be identified by flow cytometry as previously described by our group19. Briefly,
      platelet-poor plasma (PPP) will be obtained from blood and stored at -80 °C for future batch
      testing. 200 μL of PPP will allow sedimentation of MPs which will be resuspended in Annexin V
      (AnV) binding buffer prior to staining with FITC- or phycoerythrin -AnV (BD PharMingen).
      Endothelial, platelet and neutrophil-derived MPs (EMPs/PMPs/NMPs) and Tissue-Factor (TF) will
      be enumerated by detection with anti-human (CD62E, CD41 and CD11b activation epitope which
      binds to activated neutrophils respectively, plus relevant isotype controls). Latex beads
      (1.1 μm) are used to gate MPs < 1.1 μm. The thrombotic potential of MPs will be quantified by
      suspending MPs in control microparticle-free plasma (MPFP) containing trypsin inhibitor
      [inhibits contact activation] followed by exposure to calcium-fluorogenic substrate
      (Z-G-G-R-AMC). Kinetics of thrombin generation will be recorded up to 90min post-stimulation.
      Lag time min, peak thrombin nM, velocity index nM/min and endogenous thrombin potential (ETP)
      nM × min will be calculated. To investigate the relative contribution of PS and TF to
      MP-mediated thrombin generation, MPs will be pre-incubated with increasing concentrations of
      recombinant AnV protein or a blocking anti-TF (or isotype control) prior to thrombin
      analysis.

      Aim 2. Do Children with IBD have evidence of Endothelial Injury? In addition to investigating
      arterial health (below), we will quantify vascular injury by measuring CECs. CECs will be
      isolated by immunomagnetic bead extraction (based on an international consensus protocol) and
      counted using a Nageotte chamber/fluorescence microscopy. CEC enumeration is defined as
      Ulex-europaeus-lectin bright cells of >10 μm in size, with 5 magnetic beads attached19. Data
      will be analysed in the context of EMP data (Aim 1) as the combination will give an insight
      into the degree of endothelial injury in IBD versus control.

      Aim 3. Do Children with IBD have evidence of Structural Arterial Disease? Pulse Wave Velocity
      (PWV) will be an indicator of arterial structural health. Pressure waveforms will be recorded
      simultaneously at two sites (carotid-femoral) using the VICORDER analysis software (Skidmore
      Medical Limited);

      Aim 4. What is the relationship between indices of inflammation and established mediators of
      vascular Injury? Levels of hs-CRP, serum amyloid A (SAA), TNF-α, IL-1α, IL-1β, IL-6, MCP-1,
      VEGF, fasting lipids and angiopoietin 1/2 will be correlated with validated clinical
      assessment of disease activity [Paediatric UC Activity Index20 (PUCAI) & Paediatric CD
      Activity Index (PCDAI)] in addition to conventional markers (CRP, ESR, D-Dimers and platelets
      in active and inactive disease). In many cases, conventional circulating markers do not
      correlate with endoscopic findings in active disease; the non-conventional markers may show a
      higher sensitivity in detecting those with on-going active inflammation.

      Methodology:

        1. Clinical data collection: Subject clinical data will be collated onto a de-identified
           excel spread sheet from a paper data collection proforma as used in the pilot study.
           Clinical data collected will include age, sex, age at IBD diagnosis, coronary artery
           status at presentation and currently, growth and body mass index, blood pressure, family
           history of cardiovascular disease, and smoking status.

        2. Chronic inflammation will be assessed using Meso Scale Discovery (MSD, Maryland, USA)
           multi-array electrochemiluminescence detection of plasma: hs-CRP, SAA, TNF-α, IL-6, 8
           and 10, MCP-1, VEGF, angiopoietin 1 and 2 (the latter mediating endothelial detachment
           in systemic vasculitis (66). This technique allows many parameters to be assayed from
           very small amounts of blood and was used (as per manufacturer instructions) with great
           success in the pilot study; d. Endothelial activation/injury will be assessed by: CEC
           count quantified by immunomagnetic bead (IB) isolation from whole blood using anti-CD146
           coated beads, then stained with FITC-conjugated Ulex and enumerated using fluorescent
           microscopy and a Nageotte counting chamber as previously described by our group and
           others (46;67); EMPs expressing CD105, E-selectin, ICAM-1, VCAM-1 CD144, CD31, but
           negative for the platelet markers CD42a, or CD62P will be quantified from platelet poor
           plasma using a BD FACSArray flow cytometer as previously described by our group (44); e.
           Endothelial repair potential will be assessed in a total of 45 subjects (15 from each
           group: KD CAA+, CAA-, and healthy controls) by assessing (i) EPC colony forming unit
           (CFU) capacity (68), and (ii) EPC potential to incorporate into HUVEC vascular
           structures in matrigel (69), and now routinely performed by our group (see below). EPCs
           are prepared from PBMCs isolated from 5ml of blood are plated into culture dishes coated
           with human fibronectin and maintained in EGM-2 supplemented with 20% fetal calf serum
           and 40ng/ml VEGF. After 4 days in culture, non-adherent cells are removed by washing
           with phosphate buffered saline and the adherent cells maintained in culture until day 7.
           Early EPC colony forming units are then counted. Amplified EPCs harvested as described
           above are then labelled with Di -I-LDL and replated with HUVEC on top of a solidified
           matrigel layer and incubated at 37 °C for 24 h. Fluorescent microscopy is used to assess
           incorporation of EPCs into the HUVEC capillary lattice, and HUVEC tubule formation
           (structure exhibiting length four times its width) will be compared using EPC from KD
           (+/- CAA) to healthy controls. Five independent fields are assessed for each well, and
           the mean number of tubules/×100 field are determined. f. Vascular stiffness and carotid
           IMT will be assessed by: (i) carotid-femoral and carotid-radial pulse wave velocity
           (PWV) using the Vicorder device (Skidmore Medical Limited); All these techniques are
           routinely performed by our group in accordance with AHA recommendations (70), as
           illustrated by our pilot data and our previously published studies (71-73).
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Patients will be identified by the Clinical Nurse Specialists (CNS's) for Inflammatory
        Bowel Disease (IBD), on-call Gastroenterology registrars and consultants as well as the
        Pre-admission Nurses (PAN) within Great Ormond Street Hospital (GOSH). The lead consultants
        for IBD would also alert us of suitable patients for the study, as well as any suitable
        patients attending their regular gastroenterology clinics.
      
Biospecimen description: 
      Platelet poor plasma (PPP)