Atherogenesis study in vitro - Transendothelial migration assay with THS2.2

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Why?

Monocytes entering the subendothelial space of injured vessels play a key role in the pathogenesis of atherosclerosis. During this process, monocytes respond to chemotactic factors and begin adhering to and migrating through a layer of endothelial cells. Once in the subintimal space, monocytes may differentiate into macrophages. Uptake of oxidized low-density lipoproteins transforms macrophages into foam cells that accumulate and contribute to the development of atherosclerotic lesions.

Human and animal studies have demonstrated that cigarette smoking is associated with pathological effects on endothelial cells and adversely interferes with all stages of plaque development.

THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to investigate chemotaxis and transendothelial migration (TEM) of monocytes in conventional and impedance-based systems. THP-1 cells migrated through a monolayer of HCAECs in response to C-X-C motif ligand 12 (CXCL12), a chemokine involved in diverse cellular functions including chemotaxis and survival of stem cells.

How?

We investigated the effect from a new candidate modified risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), on the migratory behavior of monocytes in comparison with combustible 3R4F reference cigarettes. THP-1 cells and HCAECs were used to analyze chemotaxis and TEM. To assess the influence of aerosol extract from THS2.2 and smoke extract from 3R4F on toxicity and inflammation, flow cytometry and ELISA assays were performed.

Extracts from 3R4F cigarettes and THS2.2 were applied to experiments within 30 minutes of preparation, and various functional assays were run to assess the relative effect of the extracts on cytotoxicity, chemotaxis and transendothelial migration.

For more details on the study, please refer to the Results and to the Publications sections.

Transendothelial migration assay (THS2.2) Results

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Publications and Reports

van der Toorn, M. et al. Food and chemical toxicology 80: 277-286 (2015)

A prototypic modified risk tobacco product exhibits reduced effects on chemotaxis and transendothelial migration of monocytes compared with a reference cigarette.

Monocyte adhesion and migration to the subendothelial space represent critical steps in atherogenesis. Here, we investigated whether extracts from the aerosol of a prototypic modified risk tobacco product (pMRTP), based on heating rather than combusting tobacco, exhibited differential effects on the migratory behavior of monocytes compared with that from the reference cigarette, 3R4F. THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to investigate chemotaxis and transendothelial migration (TEM) of monocytes in conventional and impedance-based systems. THP-1 cells migrated through a monolayer of HCAECs in response to C-X-C motif ligand 12 (CXCL12), a chemokine involved in diverse cellular functions including chemotaxis and survival of stem cells. Treatment of THP-1 cells with extracts from 3R4F or pMRTP induced concentration-dependent increases in cytotoxicity (7-aminoactinomycin D), and inflammation (IL-8 and TNF-α). CXCL12-mediated chemotaxis and TEM were decreased in extract-treated THP-1 cells. Extracts from 3R4F were ~21 times more potent than those from pMRTP in all examined endpoints. Extracts from 3R4F and pMRTP induced concentration-dependent responses in assays of inflammation, cytotoxicity, chemotaxis, and TEM. Furthermore, our findings indicate that extracts from a pMRTP are significantly less cytotoxic and induce less inflammation than those from the reference cigarette, 3R4F.

van der Toorn, M. et al. Food and chemical toxicology 80: 277-286 (2015)

Aerosol from a candidate modified risk tobacco product has reduced effects on chemotaxis and transendothelial migration compared to combustion of conventional cigarettes.

Reduction of harmful constituents by heating rather than combusting tobacco is a promising new approach to reduce harmful effects associated with cigarette smoking. We investigated the effect from a new candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, on the migratory behavior of monocytes in comparison with combustible 3R4F reference cigarettes. The monocytic cell line (THP-1) and human coronary arterial endothelial cells (HCAECs) were used to analyze chemotaxis and transendothelial migration (TEM). To assess the influence of aerosol extract from THS2.2 and smoke extract from 3R4F on toxicity and inflammation, flow cytometry and ELISA assays were performed. The results show that treatment of THP-1 cells with extract from 3R4F or THS2.2 induced concentration-dependent increases in cytotoxicity and inflammation. The inhibitory effects of THS2.2 extract for chemotaxis and TEM were ∼18 times less effective compared to 3R4F extract. Furthermore, extract from 3R4F or THS2.2 induced concentration-dependent decreases in the integrity of HCAEC monolayer. For all examined endpoints, the extract from 3R4F showed more than one order of magnitude stronger effects than that from THS2.2 extract. These data indicate the potential of a heat not burn tobacco product to reduce the risk for cardiovascular disease compared to combustible cigarettes.