Cigarette smoke (CS) is the primary cause for morbidity and mortality worldwide. Alternative next generation products, such as electronic cigarette (E-cig) and Tobacco Heated Products (THP), are developed with the purpose to reduce the adverse effects of traditional cigarette (TC).

CS contains several lipo- and hydrophilic components thought to imbalance cardiovascular system homeostasis. Aim of our study was to define phenotypic changes and molecular pathway modulation caused by CS in vascular smooth muscle cells (VSMC). Firstly, we investigated the effects of CS condensate (CSC) lipophilic components from TC on mouse and human VSMC, then we extended the study to aqueous extracts (AEs) from TC, E-cig, and THP.

We performed cell proliferation and wound healing assays, gene expression, western blot, and mass spectrometry (MS)-based differential proteomic analyses. Bioinformatics combination of the acquired results allowed a holistic and interactomic overview on adverse effects of smoking on VSMC.

The addition of CSC on VSMC cultures significantly reduced the expression of SMC-specific markers and increased the expression of inflammation-related ones. Our data supported the hypothesis that the acquisition of inflammatory state and the conversion of VSMC into foam-like cells depends on downregulation of Myocardin/Kruppel-like factor 4 (KLF4) axis and may lead to the formation of atherosclerotic plaques. CSC also drives the VSMC phenotypic switch by inducing migration through the increased expression of metalloproteases (MMPs).

Also the exposure of VSMC to TC AE was observed to increase the expression of inflammatory markers. On the contrary, E-cig and THP AEs significantly induced the expression of contractile markers and genes involved in extracellular matrix (ECM) remodeling, for several of which we also proved the up-regulation of corresponding proteins. E-cig AE treatment was the most effective in inducing proliferation and migration of VSMC, while THP AE showed a minimal effect on VSMC migratory activity.

Finally, the first-shell interactomic analysis highlighted as both CSC and AEs effects on VSMCs converged on and act through KLF4, which resulted, along with MMPs, EGF-containing fibulin like extracellular matrix protein 1 (EFEMP1), protein kinase RNA-activated (PKR), and interleukin-1 beta (IL-1b) the main players in the VSMC phenotype switching induced by smoke extract treatments.