In vitro effects of waterpipe smoke condensate on endothelial cell function: a potential risk factor for vascular disease.

AIM: Despite its increasing popularity, little is known about the health effects of waterpipe smoking (WPS), particularly on the cardiovascular system. To investigate the role of WPS as a risk factor for vascular disease, we evaluated its effect on endothelial cell function, which is an early event in vascular disease pathogenesis. We assessed the changes in cell viability, ROS generation, inflammatory and vasodilatory markers and in vitro angiogenesis of human aortic endothelial cells in response to waterpipe smoke condensate exposure. METHODS AND RESULTS: Mainstream waterpipe smoke condensate (WSC) was generated using a standard laboratory machine protocol. Compared to control, WSC induced cell cycle arrest, apoptosis, and oxidative stress in human primary endothelial cells. In addition, we assayed for impaired endothelium-dependent vasodilation and induced inflammation by studying the effect of WPS on the content and activity of AMPK, eNOS proteins and NF-κB p65 ser536 phosphorylation, respectively. WSC inhibited AMPK/eNOS phosphorylation and induced phosphorylation of p65. Moreover, we evaluated endothelial cells repair mechanism related properties that include migration/invasion and in vitro tube formation upon treatment with WSC. WSC reduced the motility and inhibited angiogenic potential of HAEC cells. CONCLUSIONS: WPS induced endothelial cell dysfunction as evident by exerting oxidative stress, inflammation, and impaired endothelial vasodilatory function and repair mechanisms. All together these data provide evidence for the potential contribution of WPS to endothelial dysfunction and thus to vascular disease.

RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth.

The Ras/mitogen-activated protein kinase (MAPK) signalling cascade regulates various biological functions, including cell growth, proliferation and survival. As such, this pathway is often deregulated in cancer, including melanomas, which frequently harbour activating mutations in the NRAS and BRAF oncogenes. Hyperactive MAPK signalling is known to promote protein synthesis, but the mechanisms by which this occurs remain poorly understood. Here, we show that expression of oncogenic forms of Ras and Raf promotes the constitutive activation of the mammalian target of rapamycin (mTOR). Using pharmacological inhibitors and RNA interference, we find that the MAPK-activated protein kinase RSK (p90 ribosomal S6 kinase) is partly required for these effects. Using melanoma cell lines carrying activating BRAF mutations, we show that ERK/RSK signalling regulates assembly of the translation initiation complex and polysome formation, as well as the translation of growth-related messenger RNAs containing a 5'-terminal oligopyrimidine (TOP) motif. Accordingly, we find that RSK inhibition abrogates tumour growth in mice. Our findings indicate that RSK may be a valuable therapeutic target for the treatment of tumours characterized by deregulated MAPK signalling, such as melanoma.

In vitro cytotoxicity and mutagenicity of mainstream waterpipe smoke and its functional consequences on alveolar type II derived cells.

INTRODUCTION: While waterpipe tobacco smoking has become a global phenomenon, its potential health consequences are poorly understood. In this manuscript, we report the in vitro mutagenicity of waterpipe smoke condensate (WSC), the alteration in cellular parameters of lung alveolar cells in response to WSC exposure and discuss the implication of cellular responses in the pathophysiology of chronic obstructive pulmonary disease (COPD). METHODS: The mainstream WSC was generated using a standard laboratory machine protocol. We assessed its mutagenicity using Ames test. In addition, we studied the effect of WSC on the proliferation and cell cycle of alveolar type II cells and vascular endothelial cells. We also assessed the effect of WSC on the expression of genes involved in cell cycle arrest and inflammation. RESULTS: Within the range of tested doses, WSC did not elicit sufficient response to be considered mutagenic in any of the strains tested (TA98, TA100, TA102, and TA97a) but were found to be toxic for strains TA97a and TA102 at the highest tested doses. However, WSC induced cell cycle arrest and cellular senescence mediated by the p53-p21 pathway. Also our study indicated that WSC induced an increase in the transcriptional expression of matrix metalloproteinases, MMP-2 and MMP-9 and an immune response regulator, Toll Like Receptor-4. CONCLUSION: The data reported here represent the first in vitro demonstration of the effect of waterpipe smoke on cellular parameters providing evidence of the potential involvement of WPS in the pathogenesis of COPD through impairing cellular growth and inducing inflammation.