Cardiovascular Effects of Heated Tobacco Products (HTP)
Condition: Heated Tobacco Products; 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 heated tobacco
      products (HTP) on vascular function, microcirculation and thrombosis is assessed.
    

Detailed description: 
      The World Health Organization estimates that smoking is one of the leading causes of
      premature death worldwide with an estimated 5-8 million lives lost annually due to tobacco
      usage (1).

      Heated tobacco products (HTP) is a new form of tobacco products. HTP usually consists of a
      pod with tobacco that is mixed with glycerol which is inserted into a heating chamber. HTP is
      not combusted but only heated (2). Previous studies into smoking cessation with regular
      cigarettes and electronic cigarettes have suggested a risk for double usage instead of
      cessation, augmenting a nicotine addiction (3-5). There is a risk that HTP use simply
      enhances nicotine usage and smoking addiction.

      There is limited data on the health effects of HTP. A majority of the studies available have
      reported conflicts of interest to manufacturers of HTP (11). It has been shown that aerosols
      from HTP contain toxic compounds and free radicals just as in regular cigarette smoke, albeit
      in lower concentrations (6-8). Furthermore, aerosols from HTP can spread in a room, enabling
      passive exposure (9). It has been shown that there is a decrease in harmful biomarkers in
      smokers that switch to HTP after 5 days of usage but also of a higher HTP consumption
      compared to regular smoking (12). There are few studies on effects of HTP in humans.
      Nabavizadeh et al has shown impaired endothelial function in rats after exposure to IQOS
      (10).

      _____________

      Subjects and criteria:

      Thirty male or female occasional tobacco users (age 18-40, maximum 10 cigarettes per month or
      10 pouches of snus per month) will be included. They have to be healthy, having no
      preexisting conditions or take any medications. All subjects will have to complete a normal
      health declaration.

      Methods:

      In randomized cross-over fashion subjects will either inhale vapor (1 puff per minute for 30
      minutes, total 30 puffs) from a HTP of the brand IQOS (IQOS 3 Multi, Philip Morris AB) or
      perform sham-smoking of HTP. Measurements of arterial stiffness is performed previous, during
      and 60 minutes following exposures. Blood samples will be collected at baseline for cotinine,
      measurement with T-TAS, endothelial progenitor cells (EPC), NETs and extracellular vesicles
      (EV). Blood samples will be drawn up to 3 hours post-exposure (EPC, T-TAS,EV, NETs).
      Microcirculation is evaluated at baseline with skin capillaroscopy and Laser-Speckle contrast
      imaging (LSCI) and at 1 hour post-exposure.

      Measurement of vascular function Arterial stiffness (Sphygmocor) Increased arterial stiffness
      is recognized as a major factor in vascular aging and a risk factor for cardiovascular
      disease (13). Arterial stiffness will be assessed by pulse wave analysis and pulse wave
      velocity.

      Photopletysmography (PPG) Finger photoplethysmography is another method that provides
      information on the arterial bloodflow which allows measurement of pulse propagation time
      (PPT) (14).

      Measurement of microcirculation Microcirculation will be assessed by several methods. Skin
      perfusion is investigated through Laser Speckle contrast Imaging , which is an optical
      technique for assessment of skin flux, i.e movement of circulating red blood cells. This
      method measures overall skin flux in superficial arterioles, capillaries and venules over
      wide skin areas and with a high frequency. Iontophoresis is a non-invasive method for drug
      application across the skin using a small electric current. Acetylcholine (ACh, Sigma-Aldrich
      AB, Stockholm, Sweden) and sodium nitroprusside (SNP, Hospira, Inc., Lake Forest, IL, USA),
      both diluted in 9% physiological sodium chloride solutions, are used to investigate
      endothelium-dependent and endothelium-independent microvascular reactivity, respectively.
      Electrode chambers (LI611 Drug Delivery Electrode, Perimed, Järfälla, Sweden) are attached to
      the volar side of the left forearm, avoiding hair, broken skin and visible veins, and filled
      with a small volume of either ACh (2%) or SNP (2%). A battery-powered iontophoresis
      controller (Perilont LI 760; Perimed, Järfälla, Sweden) provides a single dose of 0.02 mA for
      200 seconds for drug iontophoresis. ACh is delivered using an anodal charge and SNP with a
      cathodal charge. LSCI (PeriCam PSI NR; Perimed, Järfälla, Sweden) is used to assess skin
      microvascular flux continuously before, during and 15 minutes after iontophoresis.

      Skin capillaroscopy is another method for evaluating the microcirculation. A USB microscope
      (CapillaryScope 500 pro, Dino-Lite®) is used to visualize nail fold capillaries in the finger
      (preferably 4th digit of left hand). Capillaries with good optical signals, i.e. with visible
      red blood cell movements and plasma gaps, are chosen for examination. Capillary blood flow is
      registered continuously at rest, during and after one-minute arterial occlusion at the
      proximal phalanx of the digit with a suprasystolic cuff pressure. Calculation of capillary
      blood cell velocity (CBV, mm/s) is done off line and will generate following four variables:
      rCVB (mean CBV at rest), pCVB (peak CBV following one-minute arterial occlusion), time to
      peak (time (s) from release of cuff pressure to peak flow, and post-occlusive reactive
      hyperemia (precentral increase of CBV from rest to peak flow).

      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 2 hour and 4 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 Measurement is done to ensure adherence that subjects have not used
      tobacco in the last 7 days. Levels of cotinine will be measured in serum using a commercial
      available ELISA technique.

      Measurement of EPCs The number of EPCs will be measured in whole blood by flow cytometry.
      EPCs are measured as CD34+ KDR+ (KDR: vascular endothelial growth factor receptor 2) double
      positive cells. Briefly, 20 µl of whole blood is incubated with CD34-FITC (Beckman Coulter,
      Brea, CA, USA) and CD309 (Becton Dickinson, Franklin Lakes, New Jersey, USA). Conjugate
      isotype-matched immuno-globulin (IgG1-FITC, IgG1-PE) with no reactivity against human
      antigens are used as a negative control. After 30 minutes of incubation in a dark
      environment, BD cell-fix is added to fixate the samples. Twenty thousand events of leukocytes
      are collected (based on classical forward scatter/side scatter (size/granularity)
      characteristics of blood leukocytes) and results will be presented as a number of EPC events.

      Measurement of Extracellular Vesicles 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-EV (LEV), Beckman Coulter, Dublin, Ireland) and
      CD144-APC (Endothelial-EV (EEV), AH diagnostics, Stockholm, SWE). PEVs are also labeled with
      CD154-PE (CD40L, abcam, Cambridge, UK) and EEVs with CD62E (E-selectin, Beckman Coulter,
      Dublin, Ireland). EVs are measured by flow cytometry on a Beckman Gallios instrument (CA,
      USA). The EV-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. EVs 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 EVs is calculated by means of the following formula: (EV counted x standard beads ⁄
      L) ⁄ standard beads counted, (FlowCount, Beckman Coulter).

      __________________

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Intervention name(s): HTP inhalation
Intervention type(s): Other
Intervention description(s): Sham inhalation for 30 minutes
Other intervention name(s): HTP inhalation for 30 minutes
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Single (Investigator)
Primary purpose: Other