The Acute Effects of E-cigarette Inhalation on Vascular Function, Microcirculation and Thrombosis
Condition: E-cigarette Use; Microcirculation; Thrombosis; Vascular Function; Blood Markers; Microvesicles; NETs
MeSH terms: Thrombosis
Study type: Interventional

Summary: 
      This is a human randomized controlled cross-over study where the effects of e-cigarette
      inhalation (with or without nicotine) on vascular function, microcirculation and thrombosis
      is assessed.
    

Detailed description: 
      Twenty-two healthy, female and male occasional smokers or snus users (age 18-55, 10
      cigarettes per month, 10 pouches of snus per month) will be included. Research subjects are
      required to refrain from intake of alcohol and caffeine during at least 24 hours prior to the
      exposure. Tobacco usage (including Swedish moist snuff) or e-cigarette usage is not permitted
      within 7 days prior to exposure.

      All subjects have to complete a normal health declaration.

      In a randomized cross-over fashion, all subjects will inhale vapor (30 puffs for 30 minutes)
      from one electronic cigarette, eVic-Primo SE (Shenzhen Joyetech Co., Ltd., China). in a
      specially prepared room with adequate ventilation.

      There are plenty e-liquid manufacturers and distributors on the market. Independent content
      analysis showed that nicotine variation across e-liquid batches is low and that the amount of
      nicotine as stated by the manufacturer often is correct. E- liquid without any flavourings
      (Valeo laboratories GmbH, Germany) without and with nicotine (19 mg/ml) will be used. Content
      analysis is free available on the manufacturers homepage (http://www.e-liquid-wholesale.com).
      A third-generation EC is used with settings (Temperature 230°C, Effect 32 W, Resistance 0,20
      Ω).

      Research subjects are required to refrain from intake of alcohol during at least 24 hours
      prior to the exposure sequences, as well as caffeine for 12 hours. The two occasions will be
      separated by at least one week. Subjects rest for 15 minutes preceding the exposure.

      Following the initial 15 minute rest the measurements for various cardiovascular endpoints
      will be performed at baseline and up to two hours following both exposures. This will be done
      with the following methods:

      Measurement of vascular function

      Photopletysmography (PPG) Arterial stiffness is an independent measure of vascular damage and
      risk, and is best assessed with the gold-standard technique pulse-wave velocity (PWV).
      However this method is technically demanding requiring special equipment (like SphygmoCor)
      and educated study personell. Finger photoplethysmography, however, is potentially providing
      the same information as PWV, and is ideal for continous measurment of arterial stiffness.

      pulse propagation time (PPT), the interval from the systolic to the diastolic peak of the
      pulse wave. PPT is an upcoming marker for arterial stiffness and vascular aging. At the same
      time ECG and heat sounds by a microphone will be recorded making the analysis easier to
      validate.

      Microcirculation Iontophoresis and laser speckle contrast imaging (LSCI) Microcirculation is
      assessed by a non-invasive method where two substances (acetylcholine and nitroprusside) is
      applied in a small chamber (0,5 ml) on the surface of the skin. Changes in microcirculation
      is measured with laser Doppler. The method is completely harmless and pain free. An uncommon
      but plausible side effect is local temporary skin rash.

      For every heart-beat a pressure wave is generated reaching even the most peripheral parts of
      the body. By measuring changes in light absorption it is possible to assess distensions (even
      very small) in the peripheral arteries and arterioles in the subcutaneous tissue, in for
      example the tip of the finger. Microcirculation can also be measured by laser speckle
      contrast imaging (LSCI) which is a non-invasive completely pain free method for assessing
      microcirculation of the skin.

      Blood pressure A semi-automatic oscillometric sphygmomanometer will be used to measure blood
      pressure and heart rate.

      Blood sampling Blood samples will be drawn into test tubes containing 1/10 0.129 M sodium
      citrate, EDTA and serum at baseline, at 0 hour, 1 hour and 2 hours after exposure. Plasma is
      later collected after centrifugation at 2 000g for 20 min in room temperature (RT) and then
      frozen at -70°C until analysis.

      Measurement of thrombus formation (T-TAS) T-TAS® (Total Thrombus-formation Analysis System)
      is a means of assessing thrombus formation during variable flow conditions using a small
      blood sample.

      Measurement of cotinine Levels of cotinine will be measured in serum using a commercial
      available ELISA technique.

      Through direct microscopy of the capillaries of the nail bed of one of the fingers capillary
      blood flow can be assessed (capillary blood cell velocity, CBV, mm/s). This is a completely
      pain free and non-invasive method that takes about 15 minutes to perform.

      Measurement of MV Plasma is thawed and centrifuged at 2000g for 20 minutes at RT. The
      supernatant is then re- centrifuged, at 13 000g for 2 minutes at RT. 20 μl of sample is
      incubated for 20 minutes in dark with phalloidin-Alexa-660 (Invitrogen, Paisley, UK),
      lactadherin-FITC (Haematologic Technologies, Vermont, USA), CD42a-PE (Platelet-MP (PMP), BD,
      Clone Alma-16), CD45- PC7 (Leukocyte-MV (LMV), Beckman Coulter, Dublin, Ireland) and
      CD144-APC (Endothelial-MV (EMV), AH diagnostics, Stockholm, SWE). PMVs are also labeled with
      CD154-PE (CD40L, abcam, Cambridge, UK) and EMVs with CD62E (E-selectin, Beckman Coulter,
      Dublin, Ireland). MVs are measured by flow cytometry on a Beckman Gallios instrument (CA,
      USA). The MV-gate is determined using Megamix beads (BioCytex, Marseille, France), which is a
      mix of beads of with diameters of 0.5 μm, 0.9 μm and 3.0 μm, respectively. MVs are defined as
      particles less than 1.0 μm in size, negative to phalloidin (in order to exclude cell membrane
      fragments) and positive to lactadherin. Conjugate isotype- matched immunoglobulin (IgG1-FITC,
      IgG1-PE, IgG1-APC and IgG1- PC7) with no reactivity against human antigens is used as a
      negative control to define the background noise of the cytometric analysis. The absolute
      number of MVs is calculated by means of the following formula: (MV counted x standard beads ⁄
      L) ⁄ standard beads counted, (FlowCount, Beckman Coulter).

      To determine whether MVs are released due to activation or apoptosis MVs are labeled with
      SYTO 13. SYTO 13 (Molecular Probes) is cell permeable and has a high fluorescent yield when
      bound to DNA or RNA. Our hypothesis is that MVs released from apoptotic cells express more
      DNA or RNA.

      Pro-coagulant effect of MVs In vitro experiments are performed to investigate the influence
      of MVs on thrombin generation, and the relative contribution of the negatively charged
      surface provided by MVs. Briefly plasma samples are thawed and after the centrifugation steps
      described above we further centrifuge the samples (twice 21 000g in 45 min, RT) to obtain an
      MV enrich pellet. The pellet is then added to commercial plasma (Haemochrom, Diagnostica,
      Essen, Germany) with no addition of TF or phospholipids. Thrombin generation in plasma is
      determined by using the calibrated automated thrombogram (CAT) as originally described by
      Hemker et al. The calculated area under the curve represents the total amount of thrombin
      generated over time and is called the endogenous thrombin potential (ETP). Time to the start
      of thrombin generation (lag time), maximal concentration of thrombin generation (peak
      thrombin) and time to maximal thrombin generation (time to peak thrombin) is also assessed in
      CAT analysis.
    

Intervention name(s): e-cigarette
Intervention type(s): Other
Intervention description(s): E-cigarette inhalation for 30 minutes using e-cigarette with and without nicotine
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Other