Atherogenesis study in vitro - Adhesion assay (of monocytic cells to endothelial cells) with THS2.2

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Introduction video on the study concepts and results

Why?

Smoking is a major risk factor for the development of cardiovascular diseases1. Cigarette smoke (CS) contains constituents that cross the alveolar barrier into the blood stream, elicit systemic effects, and affect peripheral organs and tissues such as blood vessels and the heart distal from the lungs. Over time, smokers develop low grade inflammatory, (e.g., release of cytokines and eicosanoids) and pro-oxidative (e.g., oxidized low-density lipoprotein) features in the blood that can affect normal vascular regulatory functions including vasotone regulation, endothelial permeability, and coagulation. These changes favor and accelerate the appearance of atherosclerotic plaques1,3.

Cigarette smoke (CS) has been shown to modify the adhesive properties of the endothelium, leading to higher rates of leukocyte–endothelial cell adhesion4.

Understanding the mechanism(s) by which CS changes monocyte–endothelial cell adhesive properties in vivo is challenging because plasma/serum, which is in direct contact with the endothelium, contains a mixture of soluble mediators (eg, cytokines, lipids, and acute phase proteins) as well as CS compounds and/or metabolites that have crossed the lung barrier.

How?

The establishment of in vitro studies allows to understand direct and indirect effects of CS. Direct effects may be mediated through the effect of CS-derived chemical compounds on endothelial cells whereas indirect effects are likely mediated by soluble mediators released by CS-exposed monocytic cells on endothelial cells (see 5 and references therein).

A systems biology/toxicology approach combined with a functional in vitro adhesion assay was used to assess the impact of a candidate heat-not-burn technology-based MRTP (cMRTP), Tobacco Heating System 2.2 (THS2.2), on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs) compared with a reference cigarette (3R4F).

HCAECs were treated for 4 h with conditioned media of human monocytic Mono Mac 6 (MM6) cells preincubated with low or high concentrations of aqueous extracts from THS2.2 aerosol or 3R4F smoke for 2 h (indirect treatment), unconditioned media (direct treatment), or fresh aqueous aerosol/smoke extracts (fresh direct treatment).

Study Design and Conduct

Study design

The items and concentrations that have been tested in this study are the following (also refer to the figure):

  • THS2.2 (at diverse concentrations ranging from 0.045 to 2.25 puffs/ml)
  • 3R4F (at diverse concentrations ranging from 0.045 to 0.225 puffs/ml)

Choice of treatment types

  • Indirect (I): mimics exposure of the endothelial cell monolayer to inflammatory mediators released by other cells exposed to CS as well as low concentrations of stable CS-derived constituents or metabolites.
  • Direct (D): consists in treating endothelial cells with unconditioned medium (i.e. without the step in which monocytic cells condition the media in the indirect setting). The unconditioned medium only contains stable aerosol or smoke constituents. Using it to treat endothelial cells is important to discriminate the effects of stable constituents and those of monocyte-soluble mediators.
  • Fresh direct (FD): consists in treating endothelial cells with freshly generated aerosol- or smoke-bubbled medium containing both stable and unstable constituents.

A systems toxicology study

A systems biology/toxicology approach combined with a functional in vitro adhesion assay was used to assess the impact of a candidate heat-not-burn technology-based MRTP (cMRTP), Tobacco Heating System 2.2  (THS2.2), on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs) compared with a reference cigarette (3R4F).

The addition of molecular profiling to the functional assays allowed to highlight similar mechanisms at play in the adhesion of monocytic to endothelial cells in the THS2.2 treatment at high concentrations compared with the 3R4F treatment at low concentrations.


References

  1. Messner, B. & Bernhard, D. Smoking and cardiovascular disease: mechanisms of endothelial dysfunction and early atherogenesis. Arteriosclerosis, thrombosis, and vascular biology 34, 509-515 (2014)
  2. Favero, G. et al. Endothelium and its alterations in cardiovascular diseases: life style intervention. BioMed research international 2014, 801896 (2014)
  3. Yanbaeva, D. G.et al. Systemic effects of smoking. Chest 131, 1557-1566 (2007)
  4. Winkelmann, B. R. et al. Smoking and atherosclerotic cardiovascular disease: Part I: atherosclerotic disease process. Biomarkers in medicine 3, 411-428 (2009)
  5. Poussin, C. et al. Mechanism of an indirect effect of aqueous cigarette smoke extract on the adhesion of monocytic cells to endothelial cells in an in vitro assay revealed by transcriptomics analysis. Toxicology in vitro : an international journal published in association with BIBRA 28, 896-908 (2014)

Adhesion assay (THS2.2) Results

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Publications and Reports

Poussin, C. et al. Toxicological sciences 147: 370-385 (2015)

Systems Biology Reveals Cigarette Smoke-Induced Concentration-Dependent Direct and Indirect Mechanisms That Promote Monocyte-Endothelial Cell Adhesion.

Cigarette smoke (CS) affects the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte–endothelial cell adhesion is promoted. Primary human coronary artery endothelial cells (HCAECs) were treated for 4 h with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 h (indirect treatment, I), (2) unconditioned media similarly prepared without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells–HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor a (TNFa), a major inducer, was actually shed by unstable CS compound-activated TNFa-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. Aqueous CS extract directly and indirectly promotes monocytic cell–endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms.

Poussin, C. et al. Toxicology 339: 73-86 (2016)

Systems toxicology-based assessment of the candidate modified risk tobacco product THS2.2 for the adhesion of monocytic cells to human coronary arterial endothelial cells.

Alterations of endothelial adhesive properties by cigarette smoke (CS) can progressively favor the development of atherosclerosis which may cause cardiovascular disorders. Modified risk tobacco products (MRTPs) are tobacco products developed to reduce smoking-related risks. A systems biology/toxicology approach combined with a functional in vitro adhesion assay was used to assess the impact of a candidate heat-not-burn technology-based MRTP, Tobacco Heating System (THS) 2.2, on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs) compared with a reference cigarette (3R4F). HCAECs were treated for 4h with conditioned media of human monocytic Mono Mac 6 (MM6) cells preincubated with low or high concentrations of aqueous extracts from THS2.2 aerosol or 3R4F smoke for 2h (indirect treatment), unconditioned media (direct treatment), or fresh aqueous aerosol/smoke extracts (fresh direct treatment). Functional and molecular investigations revealed that aqueous 3R4F smoke extract promoted the adhesion of MM6 cells to HCAECs via distinct direct and indirect concentration-dependent mechanisms. Using the same approach, we identified significantly reduced effects of aqueous THS2.2 aerosol extract on MM6 cell-HCAEC adhesion, and reduced molecular changes in endothelial and monocytic cells. Ten- and 20-fold increased concentrations of aqueous THS2.2 aerosol extract were necessary to elicit similar effects to those measured with 3R4F in both fresh direct and indirect exposure modalities, respectively. Our systems toxicology study demonstrated reduced effects of an aqueous aerosol extract from the candidate MRTP, THS2.2, using the adhesion of monocytic cells to human coronary endothelial cells as a surrogate pathophysiologically relevant event in atherogenesis.