Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of atherosclerosis, partly through the activation of the transcription factor NFkB. Several data indicate that magnesium deficiency caused by prolonged insufficient intake and/or defects in its homeostasis may be a missing link between diverse cardiovascular risk factors and atherosclerosis. Here we report that endothelial cells cultured in low magnesium rapidly activate NFkB, an event which is prevented by exposure to the anti-oxidant trolox. It is well known that NFkB activation correlates with marked alterations of the cytokine network. In the present study, we show that exposure of endothelial cells to low magnesium increases the secretion of RANTES, interleukin 8 and platelet derived growth factor BB, all important players in atherogenesis. Moreover, we describe the increased secretion of matrix metalloprotease-2 and -9 and of their inhibitor TIMP-2. Interestingly, by zymography we show that metalloprotease activity predominated over the inhibitory effect of TIMP-2. These results indicate that low magnesium promotes endothelial dysfunction by inducing pro-inflammatory and pro-atherogenic events.

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non‐CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133+ CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5–2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E‐cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer‐associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem‐cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.

Evidence has accumulated to suggest that magnesium might play a role in controlling angiogenesis. Since microvascular endothelial cells are protagonists in this process, we investigated the behavior of these cells cultured in low extracellular magnesium or silenced for its transporter Transient Receptor Potential Melastatin (TRPM)7, essential for cellular magnesium homeostasis. In particular, we focused on some crucial steps of the angiogenic process, i.e. proliferation, migration, protease production and organization in tridimensional structures. Silencing TRPM7 mimics the effects of low extracellular magnesium on human microvascular endothelial cells (HMEC). Indeed, while no effects were observed on the production of metalloproteases and on tridimensional organization on matrigel, both magnesium deficiency and silencing of TRPM7 impair cell migration and inhibit growth by arresting the cells in the G0/G1 and G2/M phases of the cell cycle. Since low extracellular magnesium markedly decreases TRPM7 in HMEC, we suggest that TRPM7 downregulation might mediate low magnesium-induced inhibition of cell growth and migration. Human endothelial cells from the umbilical vein are growth inhibited by low magnesium and growth stimulated after TRPM7 silencing. An impairment of ERK phosphorylation in HMEC silencing TRPM7 is responsible, in part, for the different proliferative behavior of these two cell types. We broadened our studies also to endothelial colony-forming cells and found that they are sensitive to fluctuations of the concentrations of extracellular magnesium, while their proliferation rate is not modulated by TRPM7 silencing. Our results point to magnesium and TRPM7 as a modulators of the angiogenic phenotype of microvascular endothelial cells.

TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function.

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